Cell sorting device, micro-fluidic chip and cell sorting method

Abstract

The invention provides a cell sorting device, a micro-fluidic chip and a cell sorting method. The cell sorting device comprises a micro-fluidic chip, an image acquisition unit, a processing unit and an actuating unit. Wherein the micro-fluidic chip at least comprises a first liquid inlet, a second liquid inlet, a liquid flow channel, a first liquid outlet and a second liquid outlet, the liquid flow channel is provided with an image acquisition area, and the first liquid outlet flow channel is provided with a closure area. The image acquisition unit is arranged corresponding to the image acquisition area. And the processing unit is connected with the image acquisition unit, and is used for controlling the image acquisition unit to obtain the feature information of the to-be-sorted cells in the cell sap in the image acquisition area and comparing the feature information with preset cell information. And the actuating unit is connected with the processing unit, is arranged corresponding to the closure area of the first liquid outlet flow channel, and is used for controlling the on-off of the first liquid outlet flow channel under the control of the processing unit.

Description

Technical Field

[0001] This application relates to the field of biology, and specifically designs a cell sorting device, a microfluidic chip, and a cell sorting method. Background Art

[0002] In recent years, in the fields of biology and medicine such as early cancer screening, non-invasive prenatal diagnosis, and single-cell sequencing, microfluidic cell sorting technology has been increasingly widely used. In practical applications, it is often necessary to screen out extremely rare cells from blood with a relatively complex composition, and then analyze these rare cells.

[0003] Currently, the commonly used microfluidic cell sorting methods are passive cell sorting methods and active sorting methods. Common passive cell sorting methods include membrane filtration and spiral channel inertial focusing, etc. However, these passive cell sorting methods all have the problem of low sorting accuracy and cannot complete single-cell sorting. Active cell sorting devices include a cell detection module and a cell manipulation module. The cell detection module is used to detect cells, and the cell manipulation module is used to sort cells. Common cell detection methods are fluorescence-labeled imaging image detection and bright-field imaging image detection. Fluorescence-labeled image processing requires pretreatment of cells with fluorescent dyes in advance. However, using the fluorescence-labeled image processing method requires long sample preparation time, can reflect limited cell information, and using fluorescent dyes may affect cell metabolism, cell activity, and cells cannot be observed for a long time, affecting cell detection results. Compared with fluorescence-labeled image processing, bright-field imaging image processing does not require pretreatment of cells and can be directly observed under a microscope, and it is not easy to damage cells during the detection process. Common active sorting cell manipulation methods in bright-field imaging image processing include magnetic bead method, dielectrophoresis, acoustic fluidics, etc. However, when using these methods, the sorting device has a complex structure and high cost. Some methods even cause damage to cells, and these methods can basically only achieve single-cell sorting operations, and the efficiency of actively screening rare cells from a large number of cells is low. Summary of the Invention

[0004] This application provides a cell sorting device, a microfluidic chip, and a cell sorting method to improve the cell sorting rate and simplify the structure of the cell sorting device.

[0005] In a first aspect, the present application provides a cell sorting device, which includes a microfluidic chip, an image acquisition unit, a processing unit, and an actuation unit. Among them, the microfluidic chip includes a first liquid inlet, a second liquid inlet, a first liquid outlet, and a second liquid outlet. The first liquid inlet is a cell liquid inlet, the second liquid inlet is a non-cell liquid inlet, the first liquid inlet is communicated with the liquid inlet port of the liquid flow channel through a first liquid inlet channel, the second liquid inlet is communicated with the liquid inlet port of the liquid flow channel through a second liquid inlet channel, the first liquid outlet is communicated with the first liquid outlet port of the liquid flow channel through a first liquid outlet channel, and the second liquid outlet is communicated with the second liquid outlet port of the liquid flow channel through a second liquid outlet channel; along the flow direction of the cell liquid in the liquid flow channel, the second liquid outlet port is located between the liquid inlet port and the first liquid outlet port, and the second liquid inlet channel and the second liquid outlet channel are located on the same side of the line connecting the liquid inlet port and the first liquid outlet port; an image acquisition area is provided in the liquid flow channel, and a cut-off area is provided in the first liquid outlet channel. The image acquisition unit is arranged corresponding to the image acquisition area. The processing unit is connected to the image acquisition unit, and obtains the characteristic information of the cells to be sorted in the cell liquid in the image acquisition area by controlling the image acquisition unit, and compares the characteristic information with the preset cell information. The actuation unit is connected to the processing unit, and is arranged corresponding to the cut-off area of the first liquid outlet channel, and is used to control the on-off of the first liquid outlet channel under the control of the processing unit.

[0006] In the cell sorting device of the present application, the layered flow of cell liquid and non-cell liquid is realized in the microfluidic chip. The image acquisition unit acquires the image information of the cells to be sorted in the liquid flow channel, and the processing unit extracts the characteristic information of each cell in the image information, and then compares it with the preset target cell information. Among them, the first liquid outlet channel has two states: on and off. When the first liquid outlet channel is in the on state, the cell liquid enters the first liquid outlet channel. When the first liquid outlet channel is in the off state, the cell liquid enters the second liquid outlet channel. When the characteristic information of the cells to be sorted extracted is consistent with the preset target cell information, the actuation unit is controlled to act, and the first liquid outlet channel is controlled to be off, so that the cell liquid in the liquid flow channel flows out from the second liquid outlet through the second liquid outlet channel, and the target cells in the cell liquid are collected. When the characteristic information of the cells to be sorted extracted is inconsistent with the preset target cell information, the actuation unit is controlled to act, and the first liquid outlet channel is controlled to be on, so that the cell liquid in the liquid flow channel flows out from the first liquid outlet through the first liquid outlet channel, and the non-target cells in the cell liquid are collected. Among them, the image acquisition unit takes pictures of the cells to be sorted at a preset time, and the processing unit predicts the time when the cells to be sorted reach the cut-off area according to the photographing position, the flow rate of the cell liquid, and the distance between the photographing position and the cut-off area, and realizes the control of the on-off of the first liquid outlet channel by the actuation unit according to this time.

[0007] Among them, the first liquid inlet in the microfluidic chip is used to introduce cell fluid, and the second liquid inlet is used to introduce non-cell fluid. The first liquid outlet is used to collect cell fluid without target cells, and the second liquid outlet is used to collect cell fluid containing target cells. During the sorting process, the cell fluid and non-cell fluid in the liquid flow channel of the microfluidic chip can flow in layers. By controlling the flow rates of the cell fluid and non-cell fluid, the width of the cell fluid layer can be controlled, thereby controlling the number of cells to be sorted in the cross-section perpendicular to the flow direction in the cell fluid layer. When the width of the cell fluid layer in the liquid flow channel is large, the number of cells in each cross-section is large, and enrichment of target cells can be achieved. When the width of the cell fluid layer in the liquid flow channel is small, the number of cells in each cross-section is small, and single-cell sorting of target cells can be achieved.

[0008] In the microfluidic chip of the present application, since the second liquid outlet port is arranged between the liquid inlet port and the first liquid outlet port of the liquid flow channel, and the second liquid inlet channel and the second liquid outlet channel are located on the same side of the connection line between the liquid inlet port and the first liquid outlet port, when introducing cell fluid and non-cell fluid, the non-cell fluid layer introduced from the second liquid inlet forms on the side where the second liquid outlet port is provided. Thus, during the flow of the cell fluid, by controlling the position of the interface between the cell fluid and the non-cell fluid, the first liquid outlet channel can be made to allow the cell fluid to enter the first liquid outlet channel and the non-cell fluid to enter the second liquid outlet channel when the first liquid outlet channel is in a conducting state. When the first liquid outlet channel is disconnected by the actuating unit in the intercepting area, the cell fluid can follow the non-cell fluid into the second liquid outlet channel and flow out from the liquid outlet communicating with the second liquid outlet channel.

[0009] Therefore, the cell sorting device of the present application can achieve active cell enrichment and single-cell sorting, and has a simple structure. An unlabeled imaging device can be used to detect cells to be sorted, without the need for fluorescent labeling of cells, which has less impact on cell viability. Through unlabeled image processing, characteristic information of cells such as cell position, cell size, cell number, and cell flow rate can be obtained, and after comparison with the target cell information, control is performed, and the sorting accuracy is higher. In addition, the cell sorting device of the present application uses a single actuating unit, and under the combined action of the single actuating unit and the fluid stratified flow, deflection of the composite fluid containing cell fluid is achieved. By adjusting the flow rates of the cell fluid and the non-cell fluid, the width of the cell fluid layer formed by the cell fluid in the liquid flow channel and its position in the liquid flow channel are changed, so as to achieve active cell enrichment and single-cell sorting. The actuating unit is separated from the microfluidic chip, and different specifications of microfluidic chips can be replaced at any time, which is convenient to operate, has low cost, and little pollution.

[0010] In an optional implementation, in the microfluidic chip, the liquid flow channel is a linear liquid flow channel, the first liquid outlet flow channel is connected in parallel to the liquid flow channel, and the second liquid outlet flow channel is arranged at an angle to the liquid flow channel. The linear liquid flow channel is convenient for controlling the flow direction of the cell fluid and the non-cell fluid, and realizing the width control of the cell fluid layer. The first liquid outlet flow channel is arranged in parallel with the liquid flow channel, so that the fluid in the liquid flow channel can enter the first liquid outlet flow channel in a straight line, which is convenient for controlling the relative position of the interface between the cell fluid and the non-cell fluid and the first liquid outlet flow channel. When the cell fluid does not contain target cells, the first liquid outlet flow channel is controlled to be conductive, so as to control the cell fluid to flow out from the liquid outlet connected to it through the first liquid outlet flow channel.

[0011] In an optional implementation, the liquid flow channel is a rectangular liquid flow channel, and the liquid flow channel includes a first inner wall and a second inner wall arranged opposite to each other, the first inner wall and the second inner wall are both perpendicular to the width direction of the liquid flow channel, and the second liquid outlet port is arranged on the second inner wall; the first liquid outlet flow channel is a rectangular flow channel, and the first liquid outlet flow channel includes a third inner wall and a fourth inner wall arranged opposite to each other, the third inner wall and the fourth inner wall are both perpendicular to the width direction of the first liquid outlet flow channel, the plane where the fourth inner wall is located is located between the plane where the first inner wall is located and the plane where the second inner wall is located, and the third inner wall is located on the side of the fourth inner wall facing away from the second inner wall.

[0012] The liquid flow channel and the first liquid outlet flow channel are both long strip flow channels, and the cross-section along the extension direction thereof is rectangular or square. The rectangular flow channel is convenient for processing, and is convenient for the flow of liquid, and the flow rate of the liquid at various positions in the flow channel. When a composite fluid of a layered flow channel is formed in the liquid flow channel, the non-cellular liquid is usually in contact with the first inner wall and the second inner wall, and the cell liquid layer is sandwiched between the non-cellular liquid layers. The plane where the fourth inner wall is located is located between the planes where the first inner wall and the second inner wall are located, and can cooperate with the interface between the cell liquid layer and the non-cellular liquid layer. When the first liquid outlet flow channel is connected, the cell liquid flows into the first liquid outlet flow channel, and the non-cellular liquid enters the second liquid outlet flow channel from the second liquid outlet port; when the first liquid outlet flow channel is disconnected, the cell liquid containing the target cells flows out from the second liquid outlet flow channel, thereby increasing the concentration of the target cells flowing out from the second liquid outlet flow channel.

[0013] In an optional implementation, the plane where the third inner wall is located is flush with the plane where the first inner wall is located. The flush setting can reduce the processing difficulty of the microfluidic chip, while reducing the width of the non-cellular fluid on one side of the cell fluid layer, saving the amount of non-cellular fluid.

[0014] In an alternative implementation, the angle between the liquid flow channel from the liquid inlet port to the second liquid outlet port and the second liquid outlet flow channel is an obtuse angle, facilitating the inflow of fluid into the second liquid outlet flow channel and reducing the flow resistance.

[0015] In an alternative implementation, the first liquid inlet flow channel is arranged parallel to the liquid flow channel, and the second liquid inlet flow channel is arranged at an angle to the liquid flow channel.

[0016] In an alternative implementation, the microfluidic chip further includes a third liquid inlet port, which is a non-cell liquid inlet port. The third liquid inlet port is communicated with the liquid inlet port of the liquid flow channel through a third liquid inlet flow channel. The third liquid inlet flow channel and the second liquid inlet flow channel are located on both sides of the first liquid inlet flow channel. The three liquid inlet flow channels form a trident structure. When there are three liquid inlet ports, the first liquid inlet port is used to introduce cell liquid, and the second and third liquid inlet ports are used to introduce non-cell liquid. In this way, a composite fluid with a three-layer fluid structure can be formed in the liquid flow channel. In the width direction of the liquid flow channel, the three fluid layers are a non-cell liquid layer, a cell liquid layer, and a non-cell liquid layer respectively. At this time, a composite fluid with two interfaces can be formed in the liquid flow channel. Among them, the cell liquid layer can be limited by the upper and lower non-cell liquid layers. By controlling the flow rates of the three fluid layers, the width of the cell liquid layer can be adjusted, with a larger adjustment range and higher precision, and the precision of enrichment and single-cell sorting can be achieved.

[0017] In an alternative implementation, in the first liquid outlet flow channel, the width of the first liquid outlet flow channel corresponding to the intercepting area is smaller than the width of the remaining part of the first liquid outlet flow channel. The smaller width of the first liquid outlet flow channel in the intercepting area facilitates the implementation of extrusion control of the intercepting area, thereby realizing the on-off of the first liquid outlet flow channel. The shape of the first liquid outlet flow channel in the intercepting area can be a dumbbell structure, reducing the resistance of the fluid in the first liquid outlet flow channel.

[0018] In an alternative implementation, the microfluidic chip includes a substrate, a flow channel layer, and an elastic layer. The flow channel layer is arranged between the substrate and the elastic layer, and the elastic layer covers the flow channel layer. The elastic layer covers the upper part of the flow channel layer. When the intercepting area is squeezed by the actuating unit, the elastic layer can deform and enter the first liquid outlet flow, preventing the fluid from flowing in the first liquid outlet flow channel.

[0019] Among them, the width of the first liquid outlet channel can be 300 microns to 1 millimeter, and the width of the first liquid outlet channel in the intercepting area can be 100 microns to 300 microns. The width of the liquid inlet channel can be 300 microns to 1 millimeter. The width of the liquid flow channel can be 300 microns to 1 millimeter. The height of the liquid inlet channel, the height of the liquid flow channel, and the height of the first liquid outlet channel can all be 40μm - 60μm. The height of the liquid flow channel is the dimension of the liquid flow channel along the direction perpendicular to the bottom plate. The width of the liquid flow channel is the dimension in the plane parallel to the bottom plate and in the direction perpendicular to the liquid flow channel. The width and height of the first liquid outlet channel are understood in the same way and will not be elaborated here one by one.

[0020] In an optional implementation manner, the actuating unit is a piezoelectric actuating unit, including a fixed bracket, a piezoelectric driving sheet connected to the fixed bracket, and a micro needle disposed at the end of the piezoelectric driving sheet. The piezoelectric driving sheet drives the micro needle to reciprocate in the vertical direction during vibration. The micro needle is disposed corresponding to the intercepting area and presses against or away from the first liquid outlet channel during the reciprocating movement, so that the first liquid outlet channel is switched between a closed state and a connected state. The on-off of the first liquid outlet channel is controlled by using the piezoelectric actuating unit, and the actuating unit and the microfluidic chip can be separately arranged, which is convenient for replacing the microfluidic chip.

[0021] In a second aspect, the present application provides a cell sorting method. The cell sorting method includes:

[0022] Injecting cell liquid and non-cell liquid into the liquid flow channel of the microfluidic chip to form a composite fluid, the composite fluid includes a cell liquid layer and a non-cell liquid layer flowing in layers, and there is a first interface between the cell liquid layer and the non-cell liquid layer in the liquid flow channel; wherein, the microfluidic chip includes a first liquid outlet channel communicated with the first liquid outlet port of the liquid flow channel and a second liquid outlet channel communicated with the second liquid outlet port of the liquid flow channel; the second liquid outlet port is disposed between the liquid inlet port and the first liquid outlet port of the liquid flow channel; the first liquid outlet channel is set to two states of being conductive and disconnected, and the first interface is set such that when the first liquid outlet channel is conductive, all the cell liquid flows into the first liquid outlet channel;

[0023] Obtaining the characteristic information of the cells to be sorted in the cell liquid corresponding to the image acquisition area in the liquid flow channel, and comparing the characteristic information with the preset cell information. If there is characteristic information of the cells to be sorted that is consistent with the target cell information, then control the first liquid outlet channel to be disconnected so that the cells to be sorted flow into the second liquid outlet channel; if the characteristic information of all the cells to be sorted is inconsistent with the target cell information, then control the first liquid outlet channel to be conductive so that the cells to be sorted flow into the first liquid outlet channel and flow out.

[0024] The cell sorting method of the present application forms a first interface between a cell fluid layer and a non-cell fluid layer in a microfluidic chip, and controls the position of the first interface so that the cell fluid flows out of the first outlet flow channel when the first outlet flow channel is normally connected, and the cell fluid containing the target cells flows out of the second outlet flow channel when the first outlet flow channel is disconnected, so as to enrich and sort the cells. The cell sorting method of the present application can obtain characteristic information of cells such as cell position, cell size, cell number, cell flow rate, etc. through label-free image processing, and control it after comparing it with the target cell information, so as to achieve a higher sorting accuracy.

[0025] Among them, the liquid flow channel may include a first inner wall and a second inner wall arranged opposite to each other, the first liquid outlet channel includes a third inner wall and a fourth inner wall arranged opposite to each other, the third inner wall is located on the side of the fourth inner wall away from the second inner wall, and the second dividing interface is located between the plane where the first inner wall is located and the plane where the fourth inner wall is located.

[0026] In an optional implementation, in the width direction of the liquid flow channel, the composite fluid includes a first non-cellular liquid layer, a cell liquid layer and a second non-cellular liquid layer, the cell liquid layer is located between the first non-cellular liquid layer and the second non-cellular liquid layer, a first interface is formed between the first non-cellular liquid layer and the cell liquid layer, a second interface is formed between the cell liquid layer and the second non-cellular liquid layer, and the spacing between the first interface and the second interface is 30μm-1000μm.

[0027] In an optional implementation, in the cell sorting method, when enriching target cells, the distance between the first interface and the second interface is 70 μm-1000 μm.

[0028] In an optional implementation, in the cell sorting method, when single cell sorting is performed on target cells, the distance between the first interface and the second interface is 30 μm-70 μm.

[0029] In an optional implementation, the second liquid outlet channel is disposed on a side of the second boundary layer away from the first boundary layer.

[0030] In an optional implementation, the steps of the cell sorting method of the present application are repeated until the concentration of target cells in the cell fluid flowing out of the first outlet channel reaches a target value.

[0031] In a third aspect, the present application provides a microfluidic chip for the above-mentioned cell sorting device. The microfluidic chip of the present application includes: a first liquid inlet, a second liquid inlet, a first liquid outlet, and a second liquid outlet. The first liquid inlet is a cell liquid inlet, the second liquid inlet is a non-cell liquid inlet. The first liquid inlet is communicated with the liquid inlet port of the liquid channel through a first liquid inlet channel, the second liquid inlet is communicated with the liquid inlet port of the liquid channel through a second liquid inlet channel, the first liquid outlet is communicated with the first liquid outlet port of the liquid channel through a first liquid outlet channel, and the second liquid outlet is communicated with the second liquid outlet port of the liquid channel through a second liquid outlet channel. Along the flow direction of the cell liquid in the liquid channel, the second liquid outlet port is located between the liquid inlet port and the first liquid outlet port, and the second liquid inlet channel and the second liquid outlet channel are located on the same side of the line connecting the liquid inlet port and the first liquid outlet port. An image acquisition area is provided in the liquid channel, and a cut-off area is provided in the first liquid outlet channel.

[0032] The first liquid inlet and the second liquid inlet in the microfluidic chip of the present application are respectively used for introducing cell liquid and non-cell liquid. The first liquid outlet is used to collect cell liquid without target cells, and the second liquid outlet is used to collect cell liquid containing target cells. During the sorting process, the cell liquid and non-cell liquid in the liquid channel of the microfluidic chip can flow in layers. By controlling the flow rates of the cell liquid and non-cell liquid, the width of the cell liquid layer can be controlled, thereby controlling the number of cells to be sorted in the cross-section perpendicular to the flow direction in the cell liquid layer. When the width of the cell liquid layer in the liquid channel is large, the number of cells in each cross-section is large, and enrichment of target cells can be achieved. When the width of the cell liquid layer in the liquid channel is small, the number of cells in each cross-section is small, and single-cell sorting of target cells can be achieved.

[0033] In the microfluidic chip of the present application, since the second liquid outlet port is arranged between the liquid inlet port and the first liquid outlet port of the liquid channel and the second liquid inlet channel and the second liquid outlet channel are located on the same side of the line connecting the liquid inlet port and the first liquid outlet port, when introducing cell liquid and non-cell liquid, the non-cell liquid layer introduced from the second liquid inlet forms on the side where the second liquid outlet port is provided. Thus, during the flow of the cell liquid, by controlling the position of the interface between the cell liquid and the non-cell liquid, when the first liquid outlet channel is in a conducting state, the cell liquid can enter the first liquid outlet channel, and the non-cell liquid can enter the second liquid outlet channel. When the actuation unit disconnects the passage of the first liquid outlet channel in the cut-off area, the cell liquid can follow the non-cell liquid into the second liquid outlet channel and flow out from the liquid outlet communicated with the second liquid outlet channel.

[0034] The cell sorting device composed of the microfluidic chip of the present application realizes the layered flow of cell fluid and non-cell fluid in the microfluidic chip, uses an image acquisition unit to collect image information of the cells to be sorted in the liquid flow channel, uses a processing unit to extract the characteristic information of each cell in the image information, and then compares it with the preset target cell information. When the extracted characteristic information of the cells to be sorted is consistent with the preset target cell information, the actuating unit is controlled to act, the disconnection of the first liquid outlet flow channel is controlled, the cell fluid in the liquid flow channel flows out from the liquid outlet through the second liquid outlet flow channel, and the cells in the cell fluid are collected. When the extracted characteristic information of the cells to be sorted is inconsistent with the preset target cell information, the actuating unit is controlled to act, the conduction of the first liquid outlet flow channel is controlled, the cell fluid in the liquid flow channel flows out from the liquid outlet through the first liquid outlet flow channel, and the cells in the cell fluid are collected. Among them, the image acquisition unit takes pictures of the cells to be sorted according to the preset time, and the processing unit predicts the time when the cells to be sorted reach the interception area according to the photographing position and the flow rate of the cell fluid and the distance from the photographing position to the interception area, and the actuating unit controls the on-off of the first liquid outlet flow channel according to the time. Therefore, by using the microfluidic chip of the present application, in conjunction with the image acquisition unit and the actuating unit, the processing unit and other equipment, active cell enrichment and single cell sorting can be achieved, and the structure is simple. A label-free imaging device can be used to detect the cells to be sorted, and there is no need to fluorescently label the cells, which has less effect on cell activity. The characteristic information of cells such as cell position, cell size, cell number, and cell flow rate can be obtained through label-free image processing, and the control is performed after comparison with the target cell information, and the sorting accuracy is higher.

[0035] In an optional implementation, in the microfluidic chip, the liquid flow channel is a linear liquid flow channel, the first liquid outlet flow channel is connected to the liquid flow channel in parallel, and the second liquid outlet flow channel is arranged at an angle to the liquid flow channel.

[0036] In an optional implementation, the liquid flow channel is a rectangular parallelepiped liquid flow channel, comprising a first inner wall and a second inner wall arranged opposite to each other in a direction perpendicular to the liquid flow channel, and the second liquid outlet port is arranged on the second inner wall;

[0037] The first liquid outlet channel is a rectangular channel, and includes a third inner wall and a fourth inner wall that are oppositely arranged in a direction perpendicular to the first liquid outlet channel. The plane where the fourth inner wall is located is located between the plane where the first inner wall is located and the plane where the second inner wall is located, and the third inner wall is located on the side of the fourth inner wall that is away from the second inner wall.

[0038] In an optional implementation, the plane where the third inner wall is located is flush with the plane where the first inner wall is located.

[0039] In an alternative implementation, the microfluidic chip further includes a third liquid inlet, which is a non-cell liquid inlet. The third liquid inlet is communicated with the liquid inlet port of the liquid flow channel through a third liquid inlet flow channel, and the third liquid inlet flow channel and the second liquid inlet flow channel are located on both sides of the first liquid inlet flow channel.

[0040] In an alternative implementation, in the first liquid outlet flow channel, the width of the first liquid outlet flow channel corresponding to the throttling area is less than or equal to the width of the remaining part of the first liquid outlet flow channel.

[0041] For the technical effects that can be achieved in the second and third aspects above, reference can be made to the corresponding effect descriptions in the first aspect above, and details will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic structural diagram of a cell sorting device according to an embodiment;

[0043] Figure 2 Schematic structural diagram of a microfluidic chip according to an embodiment;

[0044] Figure 3 Schematic top view structural diagram of a flow channel in a microfluidic chip according to an embodiment;

[0045] Figure 4 Schematic structural diagram of a microfluidic chip according to another embodiment of the present application;

[0046] Figure 5 Schematic diagram of the positional relationship between a microfluidic chip and an actuating unit according to an embodiment;

[0047] Figure 6 Schematic diagrams of different working states of a piezoelectric driving sheet driving a micro needle;

[0048] Figure 7 Schematic structural diagram of an image acquisition unit;

[0049] Figure 8 Schematic diagram of another process for obtaining cell information;

[0050] Figure 9 Schematic diagram of fluid flow for cell enrichment and single cell sorting according to an embodiment;

[0051] Figure 10 Schematic diagram of fluid flow during single cell sorting according to an embodiment;

[0052] Figure 11 Schematic diagram of fluid flow during single cell sorting according to another embodiment;

[0053] Figure 12Schematic diagram of the process of cell enrichment using a microfluidic chip with two liquid inlets and two liquid outlets.

[0054] Reference numerals:

[0055] 1 - Microfluidic chip; 101 - Substrate; 102 - Channel layer; 103 - Elastic layer;

[0056] 111 - First liquid inlet; 112 - Second liquid inlet; 113 - Third liquid inlet;

[0057] 121 - First liquid inlet channel; 122 - Second liquid inlet channel; 123 - Third liquid inlet channel;

[0058] 13 - Liquid channel; 131 - Liquid inlet port; 132 - First liquid outlet port; 133 - Second liquid outlet port;

[0059] 134 - Image acquisition area; 135 - First inner wall; 136 - Second inner wall; 14 - First liquid outlet channel; 141 - Third inner wall; 142 - Fourth inner wall;

[0060] 143 - Interception area; 15 - Second liquid outlet channel; 161 - First liquid outlet; 162 - Second liquid outlet;

[0061] 171 - First interface; 172 - Second interface;

[0062] 2 - Image acquisition unit; 3 - Processing unit; 31 - Computer; 32 - Power amplifier; 11 - Target cells; 12 - Non - target cells;

[0063] 4 - Actuation unit; 41 - Fixed bracket; 42 - Piezoelectric driving sheet; 43 - Microneedle;

[0064] 201 - Two - dimensional diffraction grating; 202, 204 - Lenses; 203 - Diaphragm; 205 - Imaging plane. Detailed implementation manners

[0065] In order to make the objectives, technical solutions, and advantages of this application clearer, the following will further describe this application in detail with reference to the accompanying drawings.

[0066] The terms used in the following embodiments are only for the purpose of describing specific embodiments and are not intended to limit this application. As used in the description of this application and the appended claims, the singular forms "a", "an", "the above", "the", and "this" are also intended to include, for example, the form "one or more", unless there is a clear indication to the contrary in the context.

[0067] References to "one embodiment" or "some embodiments" or the like described in this specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the present application. Thus, statements such as "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments", etc., which appear in different places in this specification, do not necessarily all refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized. The terms "comprising", "including", "having" and their variants all mean "including but not limited to", unless otherwise specifically emphasized.

[0068] Common cell detection methods include fluorescence-labeled imaging image detection and bright-field imaging image detection. Fluorescence-labeled image processing requires pre-treatment of cells with fluorescent dyes in advance, which may affect cell metabolism, cell viability, and prevent long-term cell observation, thus affecting cell detection results. In contrast, bright-field imaging image processing does not require pre-treatment of cells and can directly observe cells under a microscope, and is less likely to damage cells during the detection process. However, common active cell sorting manipulation methods in existing bright-field imaging image processing include magnetic bead method, dielectrophoresis, acoustic flow control, etc. The sorting device for implementing the above sorting methods has a complex structure and high cost. Some methods may even damage cells, and these methods can basically only achieve single-cell sorting operations, with low efficiency in actively screening rare cells from a large number of cells.

[0069] In view of the above problems, the present application provides a cell sorting device that can use an active sorting method to achieve high-efficiency cell enrichment and single-cell sorting without damaging cells. When using this cell sorting device for cell sorting, there is no need to perform fluorescence labeling on cells, which has little impact on cell detection results and may obtain more cell information. Moreover, in this cell sorting device, the actuation unit is single. By controlling the single actuation unit, the layered flow of the fluid, and the fluid flow rate, active enrichment and single-cell sorting of target cells can be achieved, providing a more convenient and simple device and method for cell sorting.

[0070] Figure 1 It is a schematic structural diagram of a cell sorting device according to an embodiment. The cell sorting device includes a microfluidic chip 1, an image acquisition unit 2, a processing unit 3, and an actuation unit 4. Among them, the microfluidic chip 1 is used to achieve the layered flow of cell fluid and sub-cell fluid. The image acquisition unit 2 can achieve image acquisition of cells to be sorted. The processing unit 3 can process the acquired image information and convert it into cell feature information, and can also control the image acquisition unit 2 and the actuation unit 4. The actuation unit 4 can achieve on-off control of the flow channels in the microfluidic chip 1.

[0071] Figure 2Schematic structural diagram of a microfluidic chip according to an embodiment. Figure 3 Schematic top view structure diagram of a flow channel in a microfluidic chip according to an embodiment. As Figure 2 and Figure 3 shown, in an embodiment of the present application, the microfluidic chip 1 includes a first liquid inlet 111, a second liquid inlet 112, a third liquid inlet 113, a first liquid inlet flow channel 121, a second liquid inlet flow channel 122, a third liquid inlet flow channel 123, a liquid flow channel 13, a first liquid outlet flow channel 14, a second liquid outlet flow channel 15, a first liquid outlet 161, and a second liquid outlet 162.

[0072] The following will further elaborate on the structure of the microfluidic chip 1 in conjunction with Figure 2 and Figure 3 for a more detailed description.

[0073] As Figure 2 shown, in an embodiment of the present application, the microfluidic chip 1 has a three-layer structure, including a substrate 101, a flow channel layer 102, and an elastic layer 103. The flow channel layer 102 is disposed between the substrate 101 and the elastic layer 103, and the elastic layer 103 covers the flow channel layer 102.

[0074] The elastic layer 103 and the flow channel layer 102 can be made of polydimethylsiloxane (PDMS) material. The substrate 101 layer can be a resin substrate 101 or a glass substrate 101. The resin substrate can be a polydimethylsiloxane (PDMS) substrate 101 or a polyethylene glycol p-phthalate (PET) substrate 101.

[0075] As Figure 2 and Figure 3 shown, there are three liquid inlets in the microfluidic chip 1, namely the first liquid inlet 111, the second liquid inlet 112, and the third liquid inlet 113. Among them, the first liquid inlet 111 can be used as a cell liquid inlet, and the second liquid inlet 112 and the third liquid inlet 113 can be used as non-cell liquid inlets, such as sheath liquid inlets. The first liquid inlet 111 can be located between the second liquid inlet 112 and the third liquid inlet 113.

[0076] The first liquid inlet 111 is communicated with the liquid inlet port 131 of the liquid flow channel 13 through the first liquid inlet flow channel 121. The second liquid inlet 112 is communicated with the liquid inlet port 131 of the liquid flow channel 13 through the second liquid inlet flow channel 122. The third liquid inlet 113 is communicated with the liquid inlet port 131 of the liquid flow channel 13 through the third liquid inlet flow channel 123. The first liquid outlet 161 is communicated with the first liquid outlet port 132 of the liquid flow channel 13 through the first liquid outlet flow channel 14. The second liquid outlet 162 is communicated with the second liquid outlet port 133 of the liquid flow channel 13 through the second liquid outlet flow channel 15. Along the flowing direction of the cell sap in the liquid flow channel 13, the second liquid outlet port 133 is located between the liquid inlet port 131 and the first liquid outlet port 132, and the second liquid inlet flow channel 122 and the second liquid outlet flow channel 15 are located on the same side of the connection line between the liquid inlet port 131 and the first liquid outlet port 132; an image acquisition area 134 is provided in the liquid flow channel 13, and a cut-off area 143 is provided in the first liquid outlet flow channel 14.

[0077] The first liquid inlet flow channel 121 is located between the other two second liquid inlet flow channels 122 and the third liquid inlet flow channel 123. The first liquid inlet flow channel 121, the second liquid inlet flow channel 122, and the third liquid inlet flow channel 123 present a trident structure, that is, the first liquid inlet flow channel 121 for flowing the cell sap is clamped between the second liquid inlet flow channel 122 for flowing the non-cell sap and the third liquid inlet flow channel 123.

[0078] The first liquid inlet flow channel 121, the second liquid inlet flow channel 122, and the third liquid inlet flow channel 123 are all communicated with the liquid flow channel 13. The liquid flow channel 13 is provided with a liquid inlet port 131, a first liquid outlet port 132, and a second liquid outlet port 133. Among them, the three liquid inlet flow channels, the first liquid inlet flow channel 121, the first and second liquid inlet flow channels 122, and the third liquid inlet flow channel 123 are all connected to the liquid inlet port 131 of the liquid flow channel 13. That is, the cell sap and the non-cell sap flowing in from the first liquid inlet flow channel 121, the second liquid inlet flow channel 122, and the third liquid inlet flow channel 123 all enter the liquid flow channel 13 from the liquid inlet port 131 of the liquid flow channel 13. Among them, the liquid flow channel 13 can be a straight liquid flow channel, and the three first liquid inlet flow channels 121, the second liquid inlet flow channel 122, and the third liquid inlet flow channel 123 are also all straight flow channels. The first liquid inlet flow channel 121 located in the middle position is arranged in parallel with the liquid flow channel 13. The second liquid inlet flow channel 122 and the third liquid inlet flow channel 123 are arranged at an angle with the liquid flow channel 13.

[0079] Such as Figure 2 and Figure 3As shown, the liquid flow channel 13 is further provided with a first liquid outlet port 132 and a second liquid outlet port 133. The number of the first liquid outlet port 132 and the second liquid outlet port 133 is one each. The first liquid outlet port 132 is connected to the first liquid outlet flow channel 14. The second liquid outlet port 133 is connected to the second liquid outlet flow channel 15. Among them, both the first liquid outlet flow channel 14 and the second liquid outlet flow channel 15 are linear flow channels. The first liquid outlet flow channel 14 is arranged parallel to the liquid flow channel 13. The second liquid outlet flow channel 15 can be arranged on one side of the liquid flow channel 13 and is arranged obliquely to the liquid flow channel 13. To facilitate the flow of the liquid, the included angle between the liquid flow channel 13 between the liquid inlet port 131 and the second liquid outlet port 133 and the second liquid outlet flow channel 15 is an obtuse angle. The number of liquid outlet ports of the microfluidic chip 1 can be two, namely a first liquid outlet 161 and a second liquid outlet 162. The first liquid outlet 161 is connected to the first liquid outlet port 132 through a first liquid outlet flow channel 14, and the second liquid outlet 162 is connected to the second liquid outlet port 133 through a second liquid outlet flow channel 15.

[0080] It can be understood that the number of liquid outlet ports can be multiple in addition to two. The number of the liquid outlet ports 161 and 162 can be designed according to actual needs and will not be specifically limited here. Among them, the number of the second liquid outlet ports 133 can be one or multiple, and the number of the second liquid outlet flow channels 15 connected to the second liquid outlet ports 133 can be one or multiple, which will not be specifically limited here.

[0081] Among them, the first liquid inlet flow channel 121, the second liquid inlet flow channel 122, the third liquid inlet flow channel 123, the liquid flow channel 13, the first liquid outlet flow channel 14 and the second liquid outlet flow channel 15 can all be rectangular parallelepiped liquid flow channels 13. Taking the liquid flow channel 13 as an example, the length direction of the rectangular parallelepiped liquid flow channel is the extending direction of the liquid flow channel 13, such as Figure 2 the x direction shown in. The height direction of the liquid flow channel 13 is the direction from the substrate 101 to the elastic layer 103, such as Figure 2 the z direction shown in. The width direction of the liquid flow channel 13 is the direction perpendicular to the height direction and the length direction, such as Figure 2 the y direction shown in.

[0082] Continue to refer to Figure 2 and Figure 3 ., the rectangular parallelepiped liquid flow channel 13 includes a first inner wall 135 and a second inner wall 136 which are oppositely arranged. The first inner wall 135 and the second inner wall 136 are perpendicular to the width direction of the liquid flow channel 13, that is, the first inner wall 135 and the second inner wall 136 are perpendicular to Figure 3Similarly, the first liquid outlet channel 14 of the rectangular parallelepiped structure includes a third inner wall 141 and a fourth inner wall 142 that are arranged opposite to each other, and the third inner wall 141 and the fourth inner wall 142 are perpendicular to the width direction of the first liquid outlet channel 14, that is, the third inner wall 141 and the fourth inner wall 142 are perpendicular to the width direction of the first liquid outlet channel 14. Figure 3 The y direction shown in is vertical.

[0083] The first inner wall 135 of the liquid flow channel 13 and the third inner wall 141 of the first liquid outlet flow channel 14 are arranged on the same side. The second inner wall 136 of the liquid flow channel 13 and the fourth inner wall 142 of the first liquid outlet flow channel 14 are arranged on the same side, that is, the third inner wall 141 is located on the side of the fourth inner wall 142 away from the second inner wall 136. The second liquid outlet port 133 of the liquid flow channel 13 is arranged on one side of the second inner wall 136. The plane where the fourth inner wall 142 is located is located between the plane where the first inner wall 135 is located and the plane where the second inner wall 136 is located.

[0084] Reference Figure 3 , the width of the first liquid outlet channel 14 may be smaller than the width of the liquid channel 13. A constriction may be provided at the first liquid outlet port 132 of the liquid channel 13 to achieve a smooth transition between the liquid channel 13 and the first liquid outlet channel 14. To achieve smooth flow of the liquid, the plane where the third inner wall 141 is located is flush with the plane where the first inner wall 135 is located, so that the fluid in the liquid channel 13 flows smoothly into the first liquid outlet channel 14.

[0085] In the cell sorting device of the embodiment of the present application, the actuating unit 4 is used to control the opening and closing of the first liquid outlet flow channel 14. Therefore, the first liquid outlet flow channel 14 is provided with an intercepting area 143, and the actuating unit 4 can be set corresponding to the intercepting area 143 of the first liquid outlet flow channel 14. In order to achieve precise control, the width of the first liquid outlet flow channel 14 corresponding to the intercepting area 143 is smaller than the width of other parts of the first liquid outlet flow channel 14. The shape of the first liquid outlet flow channel 14 corresponding to the intercepting area 143 can be a dumbbell-shaped structure, that is, the width of the flow channel of this part first narrows from wide, and then widens from narrow. The maximum width size range of the first liquid outlet flow channel 14 is 300 microns to 1 mm, and the minimum width size range of the intercepting area 143 of the first liquid outlet flow channel 14 is 100 microns to 300 microns. When the width of the first liquid outlet flow channel 14 corresponding to the intercepting area 143 is consistent with the width of the rest of the first liquid outlet flow channel 14, the first liquid outlet flow channel 14 is a rectangular flow channel structure as a whole. When the width of the first liquid outlet channel 14 corresponding to the intercepting area 143 is slightly smaller than the width of the remaining parts, most of the first liquid outlet channel 14 is a rectangular channel, and the intercepting area 143 part can present a dumbbell-shaped channel structure. In addition, the first inner wall 135 and the second inner wall 136 of the first liquid outlet channel 14 are the inner walls of the first liquid outlet channel 14 in the non-intercepting area 143.

[0086] It can be understood that the interception area 143 can be arranged near the first liquid outlet port 132, in the middle of the first liquid outlet channel 14, or at the tail of the first liquid outlet channel 14, and no specific limitation is made here.

[0087] Figure 4 The following is a schematic structural diagram of a microfluidic chip 1 according to another embodiment of the present application. As Figure 4 shown, different from the microfluidic chip 1 in the embodiment Figure 3 shown, the number of microfluidic chips 1 in this embodiment is two, namely the first liquid inlet 111 and the second liquid inlet 112. Among them, the first liquid inlet 111 is used to introduce cell fluid, and the second liquid inlet 112 is used to introduce non-cell fluid. The first liquid inlet channel 121 communicated with the first liquid inlet 111 for introducing cell fluid is arranged in parallel with the liquid channel 13, and their internal dimensions are the same. The second liquid inlet channel 122 communicated with the non-cell fluid is arranged obliquely to the liquid channel 13, and there is a certain angle between them. The descriptions of the other parts can refer to Figure 3 the descriptions of the parts shown, and will not be repeated here.

[0088] Exemplarily, the microfluidic chip of the present application can be obtained through the following processing technology.

[0089] The preparation process of a microfluidic chip according to an embodiment of the present application includes: spin-coating a polydimethylsiloxane (PDMS) with a specified thickness on a glass substrate, curing it by heating to form an elastic PDMS film, and using a punching machine to process the liquid inlet and outlet as the elastic layer of the microfluidic chip; then making another PDMS film with a different thickness by the same process, and using a picosecond laser to process the hollow structures of the liquid inlet channel, liquid channel, first liquid outlet channel and second liquid outlet channel, and using this layer of PDMS film as the channel layer of the microfluidic chip. Using a glass slide as the substrate layer of the microfluidic chip; cleaning and drying the elastic layer and the channel layer, then bonding them after plasma treatment, and then cleaning and drying the bonded PMDS and the substrate and bonding them after plasma treatment to form a complete microfluidic chip.

[0090] The preparation process of the microfluidic chip according to another embodiment of the present application includes: spin-coating a photoresist on a silicon wafer to a specified thickness, and obtaining a photolithographic master template with various channel structures through photolithography; then spin-coating a specified thickness of PDMS on the master template, demolding after heating and curing. At this time, the PDMS layer can integrate the elastic layer and the channel layer into an integral structure. The PDMS layer is provided with grooves corresponding to the liquid inlet channel, the liquid channel, the first liquid outlet channel, and the second liquid outlet channel. A liquid inlet and a liquid outlet are processed on the PDMS layer using a punching machine; a glass slide is used as the substrate of the microfluidic chip; after cleaning and drying the punched PDMS layer and the substrate, plasma treatment is performed and then bonding is carried out to form a complete microfluidic chip.

[0091] Figure 5 It is a schematic diagram of the positional relationship between the microfluidic chip and the actuating unit according to an embodiment. As Figure 5 shown, the actuating unit 4 can be correspondingly arranged in the cut-off area 143 of the first liquid outlet channel 14 for controlling the on-off of the first liquid outlet channel 14. Among them, the actuating unit 4 can be a piezoelectric actuating unit 4, and the actuating unit 4 includes a fixed bracket 41, a piezoelectric driving sheet 42, and a micro needle 43. The piezoelectric driving sheet 42 can be a long strip-shaped sheet structure. One end of the piezoelectric driving sheet 42 is connected to the fixed bracket 41, and the other end is connected to the micro needle 43. The piezoelectric driving sheet 42 maintains a certain distance from the microfluidic chip 1 in the vertical direction. The micro needle 43 is arranged on the surface of the piezoelectric driving sheet 42 facing the microfluidic chip 1. The micro needle 43 can be a metal micro needle. The tip diameter of the end of the micro needle 43 can be 300 microns. After the piezoelectric driving sheet 42 is powered on, it can drive the micro needle 43 to reciprocate in the vertical direction, so that the micro needle 43 squeezes or separates from the microfluidic chip 1, so that the first liquid outlet channel 14 can be switched between a closed state and a connected state.

[0092] Figure 6 It is a schematic diagram of different working states of the piezoelectric driving sheet driving the micro needle. Figure 6 Figure (a) therein shows a schematic diagram of the structure when the micro needle 43 does not squeeze the elastic layer 103 and the first liquid outlet channel 14 is in a flowing state. Figure 6 Figure (b) therein shows a schematic diagram of the structure when the micro needle 43 squeezes the elastic layer 103 and the first liquid outlet channel 14 is in a disconnected state. As Figure 6As shown, when the piezoelectric driving sheet 42 does not act, the tip of the micro needle 43 is close to the upper surface of the elastic layer 103 of the microfluidic chip 1 and is aligned with the cut-off area 143 of the first liquid outlet channel 14 of the microfluidic chip 1, and the first liquid outlet channel 14 is in an open state. When the piezoelectric driving sheet 42 deforms, the end of the piezoelectric driving sheet 42 displaces and drives the micro needle 43 to vertically press the elastic layer 103 of the microfluidic chip 1, causing the elastic layer 103 of the microfluidic chip 1 to deform and block the first liquid outlet channel 14 at the cut-off area 143, blocking the flow of the fluid in the first liquid outlet channel 14, so that the first liquid outlet channel 14 is in a closed state, thereby causing the fluid containing the target cells to deflect and flow out from the second liquid outlet channel 15, for enrichment and single cell sorting of the target cells.

[0093] In the cell sorting device according to the embodiment of the present application, a piezoelectric actuation unit 4 can be provided and combined with the microfluidic chip 1 to form a piezoelectric micro valve. By controlling the action of the piezoelectric actuation unit 4, the conversion of the cell liquid in the first liquid outlet channel 14 and the second liquid outlet channel 15 can be controlled, so that the enrichment and single cell sorting of the target cells can be realized. The structure is simple and convenient to replace.

[0094] Refer to Figure 1 , when enriching and single cell sorting the target cells by using the cell sorting device of the present application, it is also necessary to collect the information of the cells to be sorted through the image acquisition unit 2, and compare the collected information with the preset target cell information through the processing unit 3, and at the same time, it is realized by controlling the action of the actuation unit 4.

[0095] Refer to Figure 1 and Figure 2 , when performing image acquisition, it is necessary to collect the information of the cells to be sorted in the liquid channel 13. Therefore, an image acquisition area 134 can be set in the liquid channel 13, and the image acquisition unit 2 can be set corresponding to the image acquisition area 134 to obtain the image information of the cells to be analyzed in the image acquisition area 134. Among them, the image acquisition area 134 can be set between the second liquid outlet port 133 and the liquid inlet port 131 of the liquid channel 13. Exemplarily, it can be located in the middle area between the liquid inlet port 131 and the second liquid outlet port 133, and is at a substantially equal distance from the liquid inlet port 131 and the second liquid outlet port 133. It can be understood that the setting of the image acquisition area 134 can be set according to the specific length of the liquid channel 13, and there is no need to limit the specific position, as long as the processing unit 3 can timely obtain the image information of a certain cell to be sorted and issue a control instruction to the actuation unit 4 to control the on-off of the first liquid outlet channel 14 so that the cell to be sorted can flow in a turning manner.

[0096] Among them, the image acquisition unit 2 acquires the image information of the cells to be analyzed. The processing unit 3 can analyze the acquired image information by using a processing module and convert the image information into the characteristic information of the cells to be analyzed. Among them, the characteristic information includes but is not limited to: the position information of the cells to be sorted in the microfluidic chip 1, cell morphology, cell quantity, cell flow velocity, etc. The image acquisition unit 2 can be a label-free image acquisition device. Exemplarily, the image acquisition unit 2 can include an inverted microscope, a label-free imaging detection microscopy module, and a high-speed camera. The microscope eyepiece interface is connected to the light inlet of the label-free imaging detection microscopy module, and the output port of the label-free imaging detection microscopy module is connected to the high-speed camera.

[0097] The processing unit 3 can also compare the characteristic information of the cells to be analyzed with the preset target cell information through a processing module, such as a computer 31. After identifying the target cells, a trigger signal is sent to the actuating unit 4 to control the action of the actuating unit 4. Among them, the processing unit 3 can also include a power amplifier 32. The trigger signal can be a voltage signal, and the trigger signal is amplified by the power amplifier 32 and then output to the actuating unit 4.

[0098] It can be understood that the processing module and the power amplifier module in the processing unit 3 can be set independently or integrated.

[0099] Figure 7 It is a schematic structural diagram of an image acquisition unit. As Figure 7 shown, the image acquisition unit 2 is a label-free image acquirer. The image acquirer can implement shear interference imaging. Among them Figure 7 Figure (a) can implement one kind of shear interference imaging, Figure 7 Figure (b) can implement another kind of shear interference imaging. Referring to Figure 7 Figure (a), for the image acquirer with this structure, a two-dimensional diffraction grating 201 is used to split the light beam transmitted through the object, and then it is imaged on the camera through the lenses 202 and 204 of a 4f system. A diaphragm 203 is used for spatial filtering in the middle. Finally, the split light is recombined and interfered on the image plane and captured by the imaging plane 205 of the camera. Referring to Figure 7 Figure (b), for this image acquirer, the two-dimensional diffraction grating 201 is directly placed on the detector chip of the camera. It can be in a close contact or encapsulated manner. The light beam is directly modulated by the two-dimensional diffraction grating and then an interference pattern is formed on the imaging plane 205 of the camera and captured by the detector chip.

[0100] The interferogram captured by the image collector is transmitted to the processing unit 3, where it will be restored to a bright-field image and a phase image by the phase retrieval algorithm. The restored images can be directly input into the trained neural network for cell segmentation. Thus, the contour of each cell is obtained. The cell parameters of each cell after segmentation are extracted, and then through another trained cell modeling model, it is determined whether the analyzed cell is a target cell. At the same time, according to the cell displacement distance between two consecutive frames of images, divided by the frame interval time, the cell flow velocity information is obtained. The cell identification information and the cell flow velocity information are both recorded by the processing unit 3 for the next cell sorting operation.

[0101] Figure 8 is a schematic flow diagram for obtaining another type of cell information. As Figure 8 shown, the image processing in the embodiments of the present application can also use ordinary bright-field imaging image processing for cell detection. For ordinary bright-field imaging, the microscope eyepiece can be directly connected to a high-speed camera. The ordinary bright-field imaging image processing process includes three steps: background elimination, threshold segmentation, and contour detection. Background elimination means comparing the current frame image with the background model, calculating the difference between pixels. Pixels with a difference greater than a certain threshold are considered foreground, while pixels with a smaller difference are regarded as background, which can make the cells in the figure clearer. Threshold segmentation divides the image into different regions. By taking an appropriate threshold and comparing each pixel in the image with the threshold, a binary black-and-white image is generated. This binary image can be used to highlight the cell size and contour for subsequent analysis and processing. Contour detection means using an edge detection algorithm to detect the contour of the cells and obtaining a series of point coordinates on the contour. According to the detected contour information, shape features such as perimeter, area, and roundness are calculated. According to the processed image, cell quantity information and cell position information are obtained. According to the cell displacement distance between two consecutive frames of images, divided by the frame interval time, the cell flow velocity information is obtained. The identification information and the cell flow velocity information are both recorded by the processing unit 3 for the next cell sorting operation.

[0102] Combining the above image acquisition, image processing, and control processes, the processes of cell enrichment and cell sorting using the cell sorting device according to the embodiments of the present application are explained as follows.

[0103] Figure 9 is a schematic diagram of fluid flow for cell enrichment and single-cell sorting in one embodiment. Figure 9 In figure (a), it is a schematic diagram of the liquid flow direction when the first liquid outlet channel 14 is in the conducting state. Figure 9 In figure (b), it is a schematic diagram of the liquid flow direction when the first liquid outlet channel 14 is in the closed state. Referring to Figure 9 , the method for cell enrichment using the microfluidic chip 1 according to the embodiments of the present application includes the following steps:

[0104] Step S11, with reference to Figure 3 and Figure 9 , introduce the cell fluid into the first liquid inlet 111, and at the same time introduce the non-cell fluid into the second liquid inlet 112 and the third liquid inlet 113 of the microfluidic chip 1 respectively. The cell fluid and the non-cell fluid form a composite fluid in the liquid flow channel 13. The composite fluid is divided into a non-cell fluid layer, a cell fluid layer, and a non-cell fluid layer in the width direction of the liquid flow channel 13. Among them, with reference to Figure 9 for the shown orientation, the interface between the cell fluid and the lower non-cell fluid is the first interface 171, and the interface between the cell fluid and the upper non-cell fluid is the second interface 172. Adjust the flow rates of the cell fluid and the non-cell fluid so that the first interface 171 is above the fourth inner wall 142 of the first liquid outlet channel 14, and keep the distance between the first interface 171 and the second interface 172 between 70 micrometers and 1 millimeter. Exemplarily, the distance between the first interface 171 and the second interface 172 can be 250 micrometers. At this distance, the fluid width of the cell fluid can be 250 micrometers, and more than 10 cells can be accommodated side by side in the width direction of the fluid. The piezoelectric driving sheet 42 drives the microneedle 43 to block the first liquid outlet channel 14 once, and 100 cells can be sorted to achieve the active enrichment of the target cells 11.

[0105] Step S12, with reference to Figure 1 , use the image acquisition unit 2 to acquire the image of the cells to be sorted in the image acquisition area 134, and analyze and process it by the processing unit 3 to obtain the cell characteristic information to be sorted, and compare it with the preset information of the target cells 11 to identify whether the cells to be sorted contain the target cells 11. If the target cells 11 are not included, make the first liquid outlet channel 14 in the liquid outlet conducting state, as shown in Figure 9 in figure (a), so that the cell fluid flows out from the first liquid outlet channel 14 and is collected. The non-cell fluid below the first interface 171 flows out from the second liquid outlet channel 15. If the cells to be sorted contain the target cells 11, send a control signal to the actuating unit 4. The actuating unit 4 controls the piezoelectric driving sheet 42 to act, controls the on-off of the first liquid outlet channel 14 in the intercepting area 143, drives the fluid containing the target cells 11 to flow to the second liquid outlet channel 15, and collects it into an external container through the second liquid outlet channel 15, as shown in Figure 9 in figure (a).

[0106] Figure 10 is a schematic diagram of fluid flow during the single-cell sorting process of an embodiment. With reference to Figure 10, the method for single-cell sorting using the microfluidic chip according to the embodiment of the present application is different from the cell enrichment method in that step S11 is different. Specifically, when performing single-cell sorting, by adjusting the flow rates of the cell fluid and the non-cell fluid, the distance between the first interface 171 and the second interface 172 is maintained between 30 microns and 70 microns. The remaining steps are the same as those in the cell enrichment process and will not be repeated here.

[0107] As Figure 5 and Figure 10 shown, when performing the single-cell sorting operation, the distance between the first interface 171 and the second interface 172 is maintained between 30 microns and 70 microns. Exemplarily, the distance between the two is 50 microns. At this time, the fluid width of the cell fluid is 50 microns, and 1-2 cells can pass through each time. The piezoelectric driving piece 42 drives the micro needle 43 to block the first liquid outlet channel 14 once, and 1 cell can be sorted, thereby realizing single-cell sorting.

[0108] The following will combine Figure 1 , Figure 5 , Figures 9 to 11 to illustrate the specific working process of the cell sorting device according to the embodiment of the present application. Taking the microfluidic chip 1 shown in Figure 9 as an example, the overall system working process of the cell sorting device according to the embodiment of the present application is as follows:

[0109] Step S1, as Figure 5 shown, align the tip of the micro needle 43 connected to the piezoelectric driving piece 42 with the first liquid outlet channel 14 in the intercepting area, and make the tip of the micro needle 43 closely adhere to the upper surface of the elastic layer 103 of the microfluidic chip. Align the unlabeled imaging image acquisition device with the image acquisition area 134 of the microfluidic chip 1. Among them, the tip of the micro needle 43 can be arc-shaped, and its outer diameter size is the same as or slightly smaller than the width of the first liquid outlet channel 14 in the intercepting area, so that when the tip of the micro needle 43 is pressed into the first liquid outlet channel 14, the flow of the liquid in the first liquid outlet channel 14 can be blocked.

[0110] Step S2, as Figure 9As shown in the figure, first, configure the cell solution containing the cells to be sorted and the cell-free solution without cells. The cell-free solution is usually a cell culture medium. Use a pressure pump to pump the cell solution and the cell-free solution into the microfluidic chip 1 from the liquid inlets of the microfluidic chip respectively. Among them, the cell solution is pumped in from the first liquid inlet 111, and the cell-free solution is pumped in from the second liquid inlet 112 and the third liquid inlet 113. Adjust the flow rates of the cell solution and the cell-free solution so that the composite fluid in the liquid flow channel 13 flows in layers. When the piezoelectric driving piece 42 does not act, the distance between the first interface 171 and the second interface 172 of the cell solution and the cell-free solution is 250 micrometers. And the first interface 171 is located above the fourth inner wall 142 of the first liquid outlet channel 14. Among them, the first liquid outlet 161 connected to the first liquid outlet channel 14 is connected to an external container for collecting the sorted non-target cells 12. The second liquid outlet 162 connected to the second liquid outlet channel 15 is connected to an external container for collecting the sorted target cells 11.

[0111] Step S3, as Figure 1 and Figure 9 shown in the figure, the label-free imaging image acquisition device in the image acquisition unit 2 acquires images of the image acquisition area 134 and transmits the acquired images to the processing unit 3. The processing unit 3 can be a computer 31. The computer 31 obtains information such as the number of cells, cell size, cell morphology, cell position, and cell flow rate through a preset algorithm. After detecting the target cells 11, the computer 31 predicts the time when the cells reach the micro-needle 43 through the above cell information. After predicting that the cells reach the optimal sorting area, the computer 31 sends a trigger signal to the control module. The control module can be composed of a single-chip microcomputer and a power amplifier 32, or can be integrated in the computer 31. After receiving the trigger signal, the control module 4 sends a preset pulse voltage signal to the power amplifier 32 to obtain a high-voltage pulse signal and applies it to the piezoelectric driving piece 42 of the actuating unit 4. Refer to Figure 3 and Figure 5, the piezoelectric drive sheet 42 drives the microneedle 43 to move, squeeze the elastic layer of the microfluidic chip, and block the circulation of the first liquid outlet channel 14. The first liquid outlet channel 14 is closed, so that the fluid containing the target cell 11 is deflected. At this time, the fluid containing the cells and the fluid without cells will flow into the second liquid outlet channel 15 together, and enter the external container from the liquid outlet 162 of the target cell 11. After the target cell 11 is deflected, the piezoelectric drive sheet 42 is reset to the non-actuated state, the first liquid outlet channel 14 is turned on, and the fluid containing the cells flows back to the first liquid outlet channel 14, and enters the external container from the liquid outlet 161 of the non-target cell 12. Repeat the above steps and wait for the next high-voltage pulse signal. Until all the liquids containing cells are fully selected, active cell enrichment is completed. Among them, the liquid flowing out from the liquid outlet 161 of the target cell 11 can be repeatedly pumped into the first liquid inlet 111 for repeated enrichment and sorting to achieve high-concentration and high-purity enrichment and sorting of the target cells 11 until the purity and quantity of the target cells 11 meet the requirements.

[0112] Step S4: In the single cell sorting stage, Figure 11 As shown, since during step S3, the liquid without cells continues to flow into the external container corresponding to the liquid outlet 161 of the target cell 11, this process has completed the dilution of the cells, so no additional cell dilution operation is required. In the subsequent single cell sorting operation, the target cell 11 collection liquid enriched in step S3 can be directly pumped back to the liquid storage tank container (not shown) at the first liquid inlet 111 of the cell liquid using a peristaltic pump for repeated separation. During this period, the distance between the first interface 171 and the second interface 172 is 50 microns, and the control process in step S3 is repeated to achieve single cell sorting.

[0113] Figure 12 Schematic diagram of the process of cell enrichment using a microfluidic chip with two liquid inlets and two liquid outlets. Figure 12 As shown, the first liquid inlet 111 and the second liquid inlet 112 in the microfluidic chip are respectively a cell liquid inlet and a non-cell liquid inlet. In the microfluidic chip of this structure, in the liquid flow channel 13, there is only one interface between the cell liquid and the non-cell liquid, namely, the first interface 171. Figure 12 In the orientation shown, the first interface 171 is also located above the fourth inner wall 142 of the first liquid outlet channel 14, so that the cell fluid can flow into the first liquid outlet channel 14. The microfluidic chip of this structure can be used to complete the active enrichment operation of the target cells 11 because the width of the liquid layer formed by the cell fluid in the liquid channel 13 can be adjusted accurately.

[0114] The above is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of changes or substitutions, which should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A cell sorting device, characterized in that, include: A microfluidic chip, comprising a first liquid inlet, a second liquid inlet, a first liquid outlet and a second liquid outlet, wherein the first liquid inlet is a cell fluid inlet, the second liquid inlet is a non-cell fluid inlet, the first liquid inlet is connected to a liquid inlet port of a liquid channel through a first liquid inlet channel, the second liquid inlet is connected to the liquid inlet port of the liquid channel through a second liquid inlet channel, the first liquid outlet is connected to a first liquid outlet port of the liquid channel through a first liquid outlet channel, and the second liquid outlet is connected to a second liquid outlet port of the liquid channel through a second liquid outlet channel; along the flow direction of the cell fluid in the liquid channel, the second liquid outlet port is located between the liquid inlet port and the first liquid outlet port, and the second liquid inlet channel and the second liquid outlet channel are located on the same side of a line connecting the liquid inlet port and the first liquid outlet port; the liquid channel is provided with an image acquisition area, and the first liquid outlet channel is provided with a flow interception area; An image acquisition unit, arranged corresponding to the image acquisition area; a processing unit connected to the image acquisition unit, for acquiring characteristic information of the cells to be sorted in the cell fluid in the image acquisition area by controlling the image acquisition unit, and comparing the characteristic information with preset cell information; An actuating unit is connected to the processing unit and is disposed corresponding to the interception area of the first liquid outlet flow channel, and is used to control the opening and closing of the first liquid outlet flow channel under the control of the processing unit.

2. The cell sorting device according to claim 1, wherein In the microfluidic chip, the liquid flow channel is a linear liquid flow channel, the first liquid outlet flow channel is connected to the liquid flow channel in parallel, and the second liquid outlet flow channel is arranged at an angle to the liquid flow channel.

3. The cell sorting device according to claim 2, wherein, The liquid flow channel is a rectangular parallelepiped liquid flow channel, and comprises a first inner wall and a second inner wall arranged opposite to each other in a direction perpendicular to the liquid flow channel, and the second liquid outlet port is arranged on the second inner wall; The first liquid outlet channel is a rectangular channel, and includes a third inner wall and a fourth inner wall that are oppositely arranged in a direction perpendicular to the first liquid outlet channel. The plane where the fourth inner wall is located is located between the plane where the first inner wall is located and the plane where the second inner wall is located, and the third inner wall is located on the side of the fourth inner wall that is away from the second inner wall.

4. The cell sorting device according to claim 3, wherein The plane where the third inner wall is located is flush with the plane where the first inner wall is located.

5. The cell sorting device according to any one of claims 1-4, characterized in that, An angle between the liquid flow channel from the liquid inlet port to the second liquid outlet port and the second liquid outlet flow channel is an obtuse angle.

6. The cell sorting device according to any one of claims 1-5, characterized in that, The first liquid inlet channel is arranged in parallel with the liquid channel, and the second liquid inlet channel is arranged at an angle to the liquid channel.

7. The cell sorting device according to any one of claims 1-5, characterized in that, The microfluidic chip also includes a third liquid inlet, which is a non-cellular fluid inlet. The third liquid inlet is connected to the liquid inlet port of the liquid channel through a third liquid inlet channel. The third liquid inlet channel and the second liquid inlet channel are located on both sides of the first liquid inlet channel.

8. The cell sorting device according to claim 7, wherein, The first liquid outlet channel is arranged in parallel with the liquid flow channel.

9. The cell sorting device according to any one of claims 1-8, characterized in that, In the first liquid outlet flow channel, the width of the first liquid outlet flow channel corresponding to the intercepting area is less than or equal to the width of the remaining part of the first liquid outlet flow channel.

10. The cell sorting device according to any one of claims 1-9, characterized in that, The microfluidic chip includes a substrate, a flow channel layer, and an elastic layer. The flow channel layer is disposed between the substrate and the elastic layer, and the elastic layer covers the flow channel layer.

11. The cell sorting device according to any one of claims 1-10, characterized in that, The actuating unit is a piezoelectric actuating unit, which includes a fixed bracket, a piezoelectric driving sheet connected to the fixed bracket, and a micro needle disposed at the end of the piezoelectric driving sheet. The piezoelectric driving sheet drives the micro needle to reciprocate in the vertical direction during vibration. The micro needle is arranged corresponding to the cut-off area and presses against or moves away from the first liquid outlet channel during the reciprocating movement, so that the first liquid outlet channel is switched between a closed state and a connected state.

12. A cell sorting method, characterized in that, Including: Inject cell fluid and non-cell fluid into the liquid flow channel of the microfluidic chip to form a composite fluid. The composite fluid includes a cell fluid layer and a non-cell fluid layer flowing in layers, and a first interface between the cell fluid layer and the non-cell fluid layer exists in the liquid flow channel. Wherein, the microfluidic chip includes a first liquid outlet channel communicated with the first liquid outlet port of the liquid flow channel and a second liquid outlet channel communicated with the second liquid outlet port of the liquid flow channel; the second liquid outlet port is disposed between the liquid inlet port and the first liquid outlet port of the liquid flow channel; the first liquid outlet channel is set to two states of conduction and disconnection, and the first interface is set such that when the first liquid outlet channel is conducted, all the cell fluid flows into the first liquid outlet channel. Obtain the characteristic information of the cells to be sorted in the cell fluid corresponding to the image acquisition area in the liquid flow channel, and compare the characteristic information with the preset cell information. If there is characteristic information of the cells to be sorted that is consistent with the target cell information, control the first liquid outlet channel to disconnect so that the cells to be sorted flow into the second liquid outlet channel; if the characteristic information of all the cells to be sorted is inconsistent with the target cell information, control the first liquid outlet channel to conduct so that the cells to be sorted flow out through the first liquid outlet channel.

13. The cell sorting method according to claim 12, wherein In the width direction of the liquid flow channel, the composite fluid includes a first non-cell fluid layer, a cell fluid layer, and a second non-cell fluid layer. The cell fluid layer is located between the first non-cell fluid layer and the second non-cell fluid layer. A first interface is formed between the first non-cell fluid layer and the cell fluid layer, and a second interface is formed between the cell fluid layer and the second non-cell fluid layer. The distance between the first interface and the second interface is 30μm - 1000μm.

14. The cell sorting method according to claim 13, wherein The cell sorting method is to enrich target cells, and the distance between the first interface and the second interface is 70μm - 1000μm.

15. The cell sorting method according to claim 13, wherein The cell sorting method is to perform single-cell sorting of target cells, and the distance between the first interface and the second interface is 30μm - 70μm.

16. The cell sorting method according to any one of claims 12-15, characterized in that, Repeat the steps of any one of claims 12 - 15 until the concentration of target cells in the cell fluid flowing out from the first liquid outlet channel reaches the target value.

17. A microfluidic chip for a cell sorting device according to any one of claims 1-11 or a sorting method according to any one of claims 12-16, characterized in that, It includes a first liquid inlet, a second liquid inlet, a first liquid outlet and a second liquid outlet, the first liquid inlet is a cell fluid inlet, the second liquid inlet is a non-cell fluid inlet, the first liquid inlet is connected to the liquid inlet port of the liquid channel through a first liquid inlet channel, the second liquid inlet is connected to the liquid inlet port of the liquid channel through a second liquid inlet channel, the first liquid outlet is connected to the first liquid outlet port of the liquid channel through a first liquid outlet channel, and the second liquid outlet is connected to the second liquid outlet port of the liquid channel through a second liquid outlet channel; along the flow direction of the cell fluid in the liquid channel, the second liquid outlet port is located between the liquid inlet port and the first liquid outlet port, and the second liquid inlet channel and the second liquid outlet channel are located on the same side of the line connecting the liquid inlet port and the first liquid outlet port; the liquid channel is provided with an image acquisition area, and the first liquid outlet channel is provided with a cutoff area.

18. The microfluidic chip according to claim 17, wherein, The liquid flow channel is a rectangular parallelepiped liquid flow channel, and the liquid flow channel comprises a first inner wall and a second inner wall which are arranged opposite to each other, the first inner wall and the second inner wall are both perpendicular to the width direction of the liquid flow channel, and the second liquid outlet port is arranged on the second inner wall; The first liquid outlet channel is a rectangular channel, and the first liquid outlet channel includes a third inner wall and a fourth inner wall that are arranged opposite to each other, and the third inner wall and the fourth inner wall are both perpendicular to the width direction of the first liquid outlet channel, and the plane where the fourth inner wall is located is located between the plane where the first inner wall is located and the plane where the second inner wall is located, and the third inner wall is located on the side of the fourth inner wall that is away from the second inner wall.

19. The microfluidic chip according to claim 17 or 18, characterized in that, The microfluidic chip also includes a third liquid inlet, which is a non-cellular fluid inlet. The third liquid inlet is connected to the liquid inlet port of the liquid channel through a third liquid inlet channel. The third liquid inlet channel and the second liquid inlet channel are located on both sides of the first liquid inlet channel.

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