Micro-fluidic chip for cell sorting

Abstract

The invention relates to a micro-fluidic chip for cell sorting, which comprises a sample flow channel, paired sheath fluid flow channels, a target cell flow channel and a driving fluid flow channel, and a sorting area is arranged on the sample flow channel; the sheath fluid flow channel is symmetrically distributed relative to the sample flow channel, and the intersection of the sheath fluid flow channel and the sample flow channel is located at the upstream of the sorting area; one end of the driving fluid flow channel and one end of the target cell flow channel intersect at the sorting area, the other end of the driving fluid flow channel is a driving fluid inlet, and the other end of the target cell flow channel is a target cell outlet; a first valve for controlling opening and closing of the driving fluid flow channel is arranged at one end, close to the sorting area, of the driving fluid flow channel, and a second valve for controlling opening and closing of the target cell flow channel is arranged at one end, close to the sorting area, of the target cell flow channel; by arranging the first valve, the driving potential energy is accumulated at the first valve closer to the cells, and the target cells can be quickly flushed into the target flow channel by using enough small pressure.

Description

Technical Field

[0001] The present disclosure relates to the field of cell sorting, and more specifically, to a microfluidic chip for cell sorting. Background Art

[0002] The working principle of flow cytometry is to perform multi-parameter, rapid quantitative analysis of single cells or other biological particles through monoclonal antibodies at the cellular molecular level. It can analyze tens of thousands of cells at high speed and can measure multiple parameters from one cell at the same time. It has the advantages of fast speed, high precision and good accuracy. It is also the gold standard technology for contemporary cell quantitative analysis. The existing cell sorting method mainly uses different fluorescent labeled cells and sorts them with a flow cytometer with a sorting function. Existing flow cytometers often use microfluidic chips to sort cells, such as an existing sorting device that simultaneously detects multiple fluorescent signals in cells. Its microfluidic chip includes a detection area, a sorting area, a driving fluid inlet, a waste liquid pool and a target cell pool. The detection area and the sorting area are connected by a first cell flow channel, the sorting area and the waste liquid pool are connected by a second cell flow channel, the driving fluid inlet and the sorting area are connected by a driving fluid flow channel, and the target cell pool and the sorting area are connected by a sorting flow channel, and air pressure is applied to the driving fluid. The first cell flow channel, the second cell flow channel, the driving fluid flow channel and the sorting flow channel are arranged in a cross shape around the sorting area. The scheme uses the driving fluid flow channel to apply power to send the target cells to the sorting flow channel and then enter the target cell pool, while the non-target cells continue to enter the waste liquid pool along the second cell flow channel.

[0003] However, in the above scheme, since pressurized gas is supplied to the driving fluid flow channel only when target cells are found, this will cause at least two problems. The first problem is that since the waste liquid port itself has a certain hydraulic resistance, when the pressure of the cell fluid is insufficient, turbulence will occur in the flow channel, and non-target cells will flow to the driving fluid flow channel, resulting in the non-target cell fluid in the driving fluid flow channel entering the sorting flow channel together with the target cells when sorting the target cells, affecting the sorting accuracy. The second problem is that since the target cells are in a flowing state in the flow channel, the distance from being identified as target cells to the sorting port is short. If the cell flow rate is fast, in order to achieve the delivery of the target cells into the sorting channel, considering the loss of fluid overshoot in the flow, a larger air pressure must be supplied so that the driving fluid can quickly drive the fluid to flow to the sorting port within a limited time under the action of air pressure, so that the cell fluid near the target cells can enter the sorting channel. However, excessive air pressure will damage the cells and easily bring non-target cell fluid before and after the target cells into the sorting channel. If a lower air pressure is used, the fluid flow rate is slow, and the non-target cells have left the sorting port or deviated from the sorting port when the fluid arrives, it is easy to cause the target cells to be unable to be delivered into the sorting channel, which will seriously affect the accuracy of cell sorting. If the cell flow rate is reduced, the sorting efficiency will be affected. Summary of the invention

[0004] In order to overcome the problem of excessive sorting gas pressure of the microfluidic chip in the prior art causing damage to cells, the present invention provides a microfluidic chip for cell sorting, which reduces the damage to cells caused by driving fluid pressure during sorting while ensuring accuracy.

[0005] In order to solve the above technical problems, according to the first aspect of the present disclosure, a microfluidic chip for cell sorting is provided, comprising a sample flow channel, a pair of sheath fluid flow channels, a target cell flow channel and a driving fluid flow channel, wherein a sorting zone is arranged on the sample flow channel; the sheath fluid flow channel is symmetrically distributed relative to the sample flow channel, the intersection point of the sheath fluid flow channel and the sample flow channel is located upstream of the sorting zone, and the sheath fluid in the sheath fluid flow channel is used to focus the cell flow in the sample flow channel; one end of the driving fluid flow channel and the target cell flow channel intersect at the sorting zone, the other end of the driving fluid flow channel is a driving fluid inlet, and the target cell flow channel is located upstream of the sorting zone. The other end of the channel is the target cell outlet, and the driving fluid channel and the target cell channel are respectively located on both sides of the extension direction of the sample channel; a first valve for controlling the opening and closing of the driving fluid channel is arranged at one end of the driving fluid channel close to the sorting area, and a second valve for controlling the opening and closing of the target cell channel is arranged at one end of the target cell channel close to the sorting area; the cell solution is introduced from the inlet end of the sample channel into the sorting area; the driving fluid in the driving fluid channel is used to cooperate with the first valve and the second valve to drive the target cells in the cell solution located in the sorting area to enter the target cell channel.

[0006] According to the second aspect of the present disclosure, a cell sorting method based on the above-mentioned microfluidic chip is also provided, which is as follows: keep the first valve and the second valve in a closed state; pressurize the driving fluid and inject it into the driving fluid flow channel at the driving fluid inlet; pressurize the cell solution and inject it into the inlet end of the sample flow channel; use a cell recognition device to photograph a first cell located at a photographing position of the sample flow channel; identify whether the first cell is a target cell; in response to being the target cell, after the first cell enters the sorting area, open the first valve and the second valve at the same time, and close them at the same time after maintaining them for a preset time, so that the first cell located in the sorting area enters the target cell flow channel under the drive of the driving fluid during the preset time.

[0007] Compared with the prior art, the beneficial effects of the embodiments of the present disclosure are as follows: by setting a first valve on the driving fluid flow channel and a second valve on the target cell flow channel, the driving fluid flow channel is allowed to be in a pressurized state at all times. When the target cell is identified, the first valve and the second valve are opened, and the potential energy accumulated by the driving fluid in the driving fluid flow channel at the first valve can quickly flush the target cell into the target cell flow channel. Since the potential energy can be accumulated at the first valve, even if the pressure of the fluid is lost during the transportation process of the driving fluid flow channel, after accumulation at the first valve, the fluid pressure can be close to or equal to the rated pressure, thereby flushing the target cell into the target cell flow channel. This can greatly improve the reaction speed, thereby achieving a higher cell sorting accuracy with a lower driving fluid pressure, while avoiding damage to the cells caused by excessive driving fluid pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Schematic diagram of the structure of a microfluidic chip for cell sorting disclosed in the present invention;

[0009] Figure 2 for Figure 1 A partial enlarged view of position A;

[0010] Figure 3 Schematic diagram of the structure for setting up empty slots for rapids section;

[0011] Figure 4 It is a schematic structural diagram of another embodiment of a target cell flow channel and a driving fluid flow channel of a microfluidic chip for cell sorting disclosed in the present invention;

[0012] Figure 5 It is a structural schematic diagram of another embodiment of a sorting area, a target cell flow channel and a driving fluid flow channel of a microfluidic chip for cell sorting disclosed in the present invention;

[0013] Figure 6 It is a structural schematic diagram of another embodiment of a sheath fluid channel and a sorting area of ​​a microfluidic chip for cell sorting disclosed in the present invention;. DETAILED DESCRIPTION

[0014] The drawings are only for illustrative purposes and cannot be construed as limiting the present invention. To better illustrate the present embodiment, some parts of the drawings may be omitted, enlarged, or reduced, and do not represent the size of the actual product. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the drawings. The positional relationships described in the drawings are only for illustrative purposes and cannot be construed as limiting the present invention.

[0015] The same or similar numbers in the drawings of the embodiments of the present disclosure correspond to the same or similar parts; in the description of the present disclosure, it should be understood that if the terms "upper", "lower", "left", "right", "long", "short" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, they are only for the convenience of describing the present disclosure and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limitations on this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0016] The technical solution of the present disclosure is further described in detail below through specific embodiments and in conjunction with the accompanying drawings:

[0017] Example 1

[0018] like Figure 1-2 The embodiment 1 of a microfluidic chip for cell sorting is shown, comprising a sample flow channel 1, a pair of sheath fluid flow channels 8, a target cell flow channel 3 and a driving fluid flow channel 4, wherein a sorting area 201 is arranged on the sample flow channel 1; the sheath fluid flow channel 8 is symmetrically distributed relative to the sample flow channel 1, and the intersection 2 of the sheath fluid flow channel 8 and the sample flow channel is located upstream of the sorting area 201, and the sheath fluid in the sheath fluid flow channel 8 is used to focus the cell flow in the sample flow channel 1; one end of the driving fluid flow channel 4 and the target cell flow channel 3 intersect at the sorting area 201, the other end of the driving fluid flow channel 4 is a driving fluid inlet, and the other end of the target cell flow channel is a target cell inlet. The target cell outlet, the driving fluid channel 4 and the target cell channel 3 are respectively located on both sides of the extension direction of the sample channel 1; the end of the driving fluid channel 4 close to the sorting area 201 is provided with a first valve 6 for controlling the opening and closing of the driving fluid channel 4, and the end of the target cell channel 3 close to the sorting area 201 is provided with a second valve 7 for controlling the opening and closing of the target cell channel 3; the cell solution is introduced from the inlet end of the sample channel 1 and enters the sorting area 201; the driving fluid in the driving fluid channel 4 is used to cooperate with the first valve 6 and the second valve 7 to drive the target cells in the cell solution located in the sorting area 201 to enter the target cell channel 3. The structures of the first valve 6 and the second valve 7 of this embodiment can be seen in the valve of the micro-manufactured elastic part with the publication number "CN100402850C".

[0019] Exemplarily, the driving fluid is a liquid, and air pressure is applied to the driving fluid at the driving fluid inlet. The first valve and the second valve are in a normally closed state, and the pressurized driving fluid is blocked by the first valve and is trapped at the first valve. At this time, the cell fluid flows along the sample flow channel. The identification device for identifying the target cell identifies the cells in the shooting position in the sample flow channel. If the target cell is identified, the first valve and the second valve are opened at the same time after the cell at the shooting position enters the sorting area. At this time, the driving fluid will rush into the sorting area, and the target cells in the sorting area will be rushed into the target cell flow channel, and the target cells will flow along the target cell flow channel to the target cell outlet. By setting the first valve, the driving potential energy is accumulated at the first valve closer to the cell, and a smaller air pressure (which can work below the driving air pressure of 40mbar) can be used to rush the target cell into the target flow channel quickly enough (the sorting pulse width can be less than 100ms), which can avoid the driving fluid from causing damage to the cell, and at the same time avoid the driving fluid from acting on non-target cells, so the sorting accuracy can be improved. The first valve and the second valve are closed after opening for a preset time to prevent the driving fluid from acting on non-target cells. The cells not acted upon by the driving fluid continue to flow along the sample flow channel to the waste port.

[0020] In a specific embodiment, the distance between the first valve 6 and the second valve 7 and the sorting area 201 is 15-17 μm, for example, 16 μm. If the first valve 6 is too close to the sorting area 201, at the moment of opening the first valve 6, the surging driving fluid is likely to flush the cell fluid containing non-target cells near the target cells into the target cell flow channel 3 or cause the cell fluid to be turbulent, thereby increasing the number of non-target cells in the target cell flow channel 3 and reducing the overall sorting accuracy. If the first valve 6 is too far away from the sorting area 201, the surging driving fluid will act more gently on the sorting area, but the speed of the driving fluid flowing to the sorting area 201 will decrease when the same air pressure is applied to the driving fluid inlet, which may easily cause the target cells to be unable to be flushed into the target cell flow channel 3. The first valve 6 is set at a distance of 15-17 μm from the sorting area 201, which can ensure that the driving fluid rushed into the sorting area from the driving fluid flow channel 4 is buffered to a certain extent, and non-target cells will not be brought into the target cell flow channel 3, and the cell flow will not be disturbed. The target cells can also be rushed into the target cell flow channel 3 quickly enough under a relatively small air pressure. The position of the second valve also needs to be carefully designed. When some target cells that have passed through the second valve are hovering near the side of the second valve away from the sorting area, if the second valve 7 is too close to the sorting area 201, the second valve 7 is easily opened to cause the target cells that have passed through the second valve 7 in the target cell flow channel to flow back to the sorting area. If the second valve 7 is too far from the sorting area, in order to ensure that the target cells pass through the second valve 7 before closing the second valve 7, it is necessary to increase the opening time of the second valve 7, which will cause the opening time of the second valve 7 to be inconsistent with the opening time of the first valve 6. When the second valve 7 is still in the open state for a short period of time after the first valve 6 is closed, the target cells that have entered the target cell flow channel 3 but have not passed through the second valve, and the target cells in the sorting area 201 are prone to lose the external force and flow back, resulting in the target cells being unable to pass through the second valve 7. The second valve 7 and the first valve 6 are basically at the same distance from the sorting area, so that the opening time of the second valve 7 can be basically consistent with the opening time of the first valve 6. In addition, when the distance between the second valve 7 and the sorting area 201 is 15-176 μm, even if the target cells in the target cell flow channel that have passed through the second valve 7 flow back when the second valve 7 is opened, they will not have time to leave the target cell flow channel 3 and enter the sorting area. When the first valve 6 is opened again, these target cells will be driven by the fluid to rush into the side of the target cell flow channel 3 away from the second valve 7.

[0021] In a specific embodiment, the driving fluid flow channel 4 and the target cell flow channel 3 both include a rapid flow section 9 connected to the sorting area 201 and a slow flow section 10 connected to the rapid flow section 9, and the width of the rapid flow section 9 is less than the width of the slow flow section 10. Exemplarily, the width of the rapid flow section 9 is consistent with the width of the sample flow channel 1, and the width of the expansion section 502 is 2.5-4 times the width of the sample flow channel 1. The flow direction of the driving fluid in the driving fluid flow channel 4 is from the slow flow section 10 to the rapid flow section 9. The rapid flow section 9 of the driving fluid flow channel has the same width as the sample flow channel 1, which can more accurately flush the target cells into the target cell flow channel 3. The flow direction of the fluid in the target cell flow channel 3 is from the rapid flow section 9 of the target cell flow channel to the slow flow section 10. The rapid flow section 9 has a small width and a fast flow rate, and the slow flow section 10 has a slow flow rate, which can prevent the cells in the slow flow section 10 of the target cell flow channel from flowing back to the rapid flow section 9 of the target cell flow channel.

[0022] Exemplarily, only non-target cells will flow to the sample flow channel located downstream of the most downstream sorting zone. The sample flow channel downstream of the most downstream sorting zone can be called a waste liquid flow channel. The waste liquid flow channel 5 includes a connecting section 501 connected to the sorting zone 201 and a flow expansion section 502 connected to the connecting section 501. The width of the connecting section 501 is consistent with the width of the sample flow channel 1, and the width of the flow expansion section 502 is 2.5-4 times the width of the sample flow channel 1. Generally, compared with the target cell flow channel, the flow rate of the waste liquid flow channel 5 is larger, and how to avoid the problem of backflow has a greater impact on the sorting results. The width of the flow expansion section 502 of the waste liquid flow channel 5 is set to be greater than the width of the connecting section 501, so that the difference in cell flow rates in the connecting section 501 and the flow expansion section 502 is increased, and the cells in the waste liquid flow channel 5 can be avoided from flowing back. It is understandable that the cell fluid that is not driven into the target channel by the driving fluid will flow downstream along the sample flow channel into the waste liquid flow channel. What enters the waste liquid flow channel may be waste liquid, or cells that are different from the target cells but still need to be collected. For example, two types of cells need to be separated, one is the target cells, and the other is the non-target cells that need to be collected.

[0023] Exemplarily, the angle between the driving fluid channel 4 and the target cell channel 3 and the sample channel 1 is 90 degrees. Under this structure, when multiple groups of driving fluid channels 4, target cell channels 3 and sorting areas 201 are set, the multiple groups of structures can not interfere with each other, and multiple different target cells can be sorted simultaneously using the same microfluidic chip.

[0024] In a specific embodiment, the distance from the intersection 2 to the sorting area 201 is 160 μm. The width of the sample flow channel 1 is 100 μm; the height of the sample flow channel 1 is 60 μm. Under the size of , both the cell fluid and the sheath fluid can always maintain a stable state.

[0025] Example 2

[0026] The difference between Example 2 and Example 1 is that the distance between the first valve 6 and the sorting area 201 is 17 μm, and the distance between the second valve 7 and the sorting area 201 is 15 μm. The distance between the intersection 2 and the sorting port 201 is 150 μm. The width of the sample flow channel 1 is 85 μm; the height of the sample flow channel 1 is 55 μm.

[0027] The rest of the working principles and effects of this embodiment are consistent with those of Embodiment 1.

[0028] Example 3

[0029] Embodiment 3 of a microfluidic chip for cell sorting is different from Embodiment 1 or 2 in that, in the microfluidic chip of Embodiment 3, when the first valve 6 is in a closed state, the driving fluid channel 4 maintains a first preset opening; when the second valve 7 is in a closed state, the target cell flow channel 3 maintains a second preset opening, and the first preset opening and the second preset opening prevent cells from passing through the second valve 7 of the target cell flow channel. The first preset opening makes it impossible for the driving force of the driving fluid acting on the cells in the sorting area and the target cell flow channel to be insufficient to flush the cells through the second valve, and the second preset opening allows only liquid to pass through the second valve of the target cell flow channel, but not cells to pass through. In this way, when the first valve and the second valve are closed, the driving fluid still flows to the outlet of the target cell flow channel, thereby being able to give a certain thrust to the target cells that have passed through the second valve of the target cell flow channel, allowing the target cells to flow to the outlet. Exemplarily, the driving fluid channel to maintain a first preset opening when the first valve is in a closed state and the target cell flow channel to maintain a second preset opening when the second valve is in a closed state can be achieved by existing methods, such as making the heights of the driving fluid channel and the target cell flow channel high enough, so that when the valve is in a closed state, the middle of the flow channel is in a closed state, and gaps are still left on both sides of the flow channel, thereby achieving the flow channel maintaining a preset opening when the valve is in a closed state.

[0030] The remaining features and working principles of this embodiment are consistent with those of Embodiment 1 or Embodiment 2.

[0031] Example 4

[0032] Embodiment 4 of a microfluidic chip for cell sorting, such as Figure 4As shown, in Example 4, the target cell flow channel 3 and the driving fluid flow channel 4 are each provided with at least one, and the remaining features are the same as those in Example 1. For example, two target cell flow channels 3 and driving fluid flow channels 4 are each provided, and the target cell flow channels 3 and the driving fluid flow channels 4 correspond to each other one by one, forming two sorting flow channel groups, and each sorting flow channel group includes a target cell flow channel 3 and a driving fluid flow channel 4 with a corresponding relationship. Each sorting flow channel group is used to sort a target cell; each of the driving fluid flow channels 4 is provided with the first valve 6 at one end close to the sorting area 201; each of the target cell flow channels 3 is provided with the second valve 7 at one end close to the sorting area 201. In this embodiment, there is one sorting area 201, and two sorting flow channel groups share one sorting area 201.

[0033] After the driving fluid flow channel and the target cell flow channel in a sorting flow channel group are opened, the target cell enters the target cell flow channel in the sorting flow channel group under the action of the driving fluid. It is understandable that the target cell flow channel and the driving fluid flow channel in the same sorting flow channel group need to be located on both sides of the sample flow channel respectively, and the angles of the driving fluid flow channel, the target cell flow channel and the sample flow channel can be determined according to the fluid flow rate in the sample flow channel and the fluid flow rate in the driving flow channel, so that the target cells in the sample flow channel sorting area enter the target cell flow channel in the sorting flow channel group under the action of the driving fluid. When multiple groups of sorting fluid channel groups are connected in a sorting area, the driving flow channel in which sorting flow channel group is opened can be selected according to the cell type in the sorting area, and each sorting flow channel group is used to sort a target cell, so that multiple types of cells can be sorted at the same time.

[0034] Example 5

[0035] Embodiment 5 of a microfluidic chip for cell sorting, such as Figure 5 As shown, the number of sorting zones 201 in Example 5 is consistent with the number of sorting channel groups and corresponds one to one, one sorting zone corresponds to one sorting channel group, and each sorting zone has at least one sorting channel group. There are 2 sorting zones here, and each sorting zone has 1 sorting channel group. The multiple sorting zones 201 are distributed along the flow direction of the cell fluid of the sample channel 1.

[0036] The working principle of this embodiment: Each sorting channel group corresponds to a target cell, and multiple sorting areas and multiple sorting channel groups can sort a variety of different target cells. For example, the microfluidic chip includes two sorting areas, each of which has two sorting channel groups. The microfluidic chip can sort up to 4 target cells plus a cell that is not a target cell but can still be collected. The two sorting channel groups in the first sorting area (sorting channel groups 1 and 2) respectively sort one target cell (if it is target cell A, the first valve and the second valve in sorting channel group 1 are opened, and target cell A enters the target cell channel of sorting channel group 1; if it is target cell B, the first valve and the second valve in sorting channel group 2 are opened. The second valve is opened, target cell A enters the target cell flow channel of sorting flow channel group 2), the two sorting flow channel groups in the second sorting area (sorting flow channel groups 3 and 4) respectively sort one target cell (if it is target cell C, the first valve and the second valve in sorting flow channel group 3 are opened, and target cell C enters the target cell flow channel of sorting flow channel group 3; if it is target cell D, the first valve and the second valve in sorting flow channel group 4 are opened, and target cell D enters the target cell flow channel of sorting flow channel group 4), and non-target cells enter the waste liquid flow channel.

[0037] Example 6

[0038] Embodiment 6 of a microfluidic chip for cell sorting, based on the above embodiment 5, as Figure 6 As shown, there are multiple pairs of sheath liquid channels 8, the number of which is consistent with the number of sorting areas 201. The intersection of each pair of sheath liquid channels 8 and the sample channel 1 is respectively located upstream of the corresponding sorting area, and the sheath liquid channel 8 will not cross other sorting areas. Each pair of sheath liquid channels 8 can focus the cell flow from its corresponding intersection to the sorting area. In this way, the flow focusing effect can be improved, so that the cells are located in the middle of the flow path in the sample flow channel, and the appearance of side-by-side cells is avoided as much as possible, so that the target cells can be accurately flushed into the corresponding target cell flow channel with the driving fluid.

[0039] It is understandable that, except for situations that are obviously contradictory, the features in the above embodiments can be combined arbitrarily.

[0040] Example 7

[0041] A cell sorting method based on the above microfluidic chip, based on the microfluidic chip of the above embodiment 1, is as follows:

[0042] Sample preparation: 1. To facilitate identification of target cells, stain the target cells. Wash the stained cells twice with PBS and centrifuge at 1200g for 5 minutes. 2. Resuspend the cells with 400uL PBS after centrifugation to a final concentration of ~10 6cells / mL (the final concentration is based on the cell counting result). 3. Mix the target cells and non-target cells and dilute them with sheath fluid (buffer + 10% glycerol) to a total concentration of 4x10 5 cells / mL, and the total volume is 1mL. The driving fluid composition can be consistent with the sheath fluid or buffer.

[0043] Keep the first valve 6 and the second valve 7 closed; continue to apply 30-40 mbar air pressure to the driving fluid to allow the driving fluid to enter the driving fluid channel 4. Inject the cell sample into the input end of the sample channel 1 through a syringe pump, and inject the sheath fluid into the sheath fluid channels on both sides through a syringe pump.

[0044] The cell recognition device is used to photograph the first cell located at the photographing position of the sample flow channel 1; and it is identified whether the first cell is a target cell. The photographing position is located upstream of the sorting position, and the specific position can be determined according to the cell flow rate and the valve reaction speed. The faster the valve reaction speed, the closer the photographing position can be to the sorting position. For example, after the first valve and the second valve are opened with an instruction to open, the first valve and the second valve are opened after 3ms, and the distance between the photographing position and the sorting position is at least 3ms for the cell to flow. If the cell at the photographing position is a target cell, a valve opening instruction is immediately given after it is identified as a target cell. After 3ms, the target cell flows into the sorting area, and the first valve and the second valve of the sorting area are opened, and the target cell enters the target cell flow channel under the action of the driving fluid.

[0045] If it is a target cell, the first valve 6 and the second valve 7 are opened and kept for a preset time and then closed, so that the target cell located in the sorting area 201 enters the target cell flow channel 3 under the drive of the driving fluid during the preset time, wherein the preset time is 2ms; if it is a non-target cell, the first valve 6 and the second valve 7 are kept closed, and the sample flows to the waste liquid flow channel 5. A collection pool is provided at the fluid outlet of the target cell flow channel 3. A collection pool may also be provided for the waste liquid flow channel.

[0046] By the above method, the measured data obtained under different working parameters using the microfluidic chips of Example 1 and Example 3 (experimental group 3 uses the microfluidic chip of Example 3, and the others use the microfluidic chip of Example 1) are as shown in the following table, wherein the control group is a microfluidic chip of the same structure but without the first valve 6 and the second valve 7, which is equivalent to the first valve 6 and the second valve 7 being kept in a normally open state. In addition, the experimental group 3 of the present disclosure keeps the driving fluid flow channel and the target cell flow channel at a preset opening when the first valve and the second valve are in a closed state. Among them, the total number of cells in the target cell flow channel is the number of all cells collected at the outlet of the target cell flow channel, including target cells and false positive cells. The total number of target cells is the number of target cells contained in the cells actually introduced. The total number of target cells is the number of target cells contained in the total number of cells in the target cell flow channel. The total flow rate is the sum of the two sheath fluids and the sample flow rate.

[0047] Table 1 Microfluidic chip control experiment

[0048]

[0049] From the data comparison between the control group 1 and the experimental groups 1, 2 and 3, it can be seen that under the same test conditions, the sorting accuracy of the present disclosure is much greater than that of the control group. From the data comparison between the control group 1 and the control group 2, it can be seen that the sorting accuracy can be significantly increased by increasing the air pressure. From the data of the present disclosure 1 and the present disclosure 2, it can be seen that the effect of the increase in air pressure on the accuracy is not obvious and has decreased, because the greater the air pressure, the greater the potential energy accumulated at the first valve, and the easier it is to flush non-target cells into the target cell flow channel. In summary, the embodiment of the present disclosure can also complete high-accuracy sorting using low air pressure, avoiding cell damage caused by increasing air pressure in order to increase the accurate flow rate. The data in the above table are the average values ​​after multiple tests in each group.

[0050] Example 8

[0051] A cell sorting method based on the above microfluidic chip, based on the above embodiment 7, the target cells include a first type of target cells and a second type of target cells;

[0052] There is at least one target cell flow channel 3 and at least one driving fluid flow channel 4, and the target cell flow channel 3 and the driving fluid flow channel 4 correspond to each other one by one to form at least one sorting flow channel group, each sorting flow channel group includes a target cell flow channel 3 and a driving fluid flow channel 4 having a corresponding relationship, and each sorting flow channel group is used to sort a target cell; a first valve 6 is provided at one end of each driving fluid flow channel 4 close to the sorting area 201; a second valve 7 is provided at one end of each target cell flow channel 3 close to the sorting area 201;

[0053] Identifying whether the first cell is a target cell includes: identifying whether the first cell is a first type of target cell;

[0054] In response to the first cell being a target cell, after the first cell enters the sorting area 201, the first valve 6 and the second valve 7 are opened simultaneously, and are closed simultaneously after being kept for a preset time, so that the first cell located in the sorting area enters the target cell flow channel 3 under the drive of the driving fluid during the preset time, including:

[0055] In response to the first type of target cell, after the first cell enters the sorting area 201, the first valve 6 of the first driving fluid channel in the first sorting channel group and the second valve 7 of the first target cell channel in the first sorting channel group are opened simultaneously, and are closed simultaneously after being maintained for a first preset time, so that the first cell located in the sorting area enters the first target cell channel 3 under the drive of the driving fluid during the first preset time;

[0056] The method also includes:

[0057] identifying whether the first cell is a second type of target cell;

[0058] In response to the second type of target cell, after the first cell enters the sorting area 201, the first valve 6 of the second driving fluid channel in the second sorting channel group and the second valve 7 of the second target cell channel in the second sorting channel group are opened simultaneously, and are closed simultaneously after being maintained for a second preset time, so that the first cell located in the sorting area enters the second target cell channel 3 driven by the driving fluid during the second preset time.

[0059] That is, the embodiment of the present disclosure realizes the collection of multiple types of target cells by setting multiple sorting flow channel groups. There can be one shooting position, and after shooting, it can be simultaneously identified whether the photographed cells are the first type of target cells or the second type of target cells. If they are the first type of target cells, the first valve and the second valve of the first sorting flow channel group are opened, and if they are the second type of target cells, the first valve and the second valve of the first sorting flow channel group are opened, and if they are neither, the valves are not opened.

[0060] Example 9

[0061] A cell sorting method based on the above microfluidic chip, based on the above embodiment 7, the target cells include a first type of target cells and a second type of target cells;

[0062] There is at least one target cell flow channel 3 and at least one driving fluid flow channel 4, and the target cell flow channel 3 and the driving fluid flow channel 4 correspond to each other one by one to form at least one sorting flow channel group, each sorting flow channel group includes a target cell flow channel 3 and a driving fluid flow channel 4 with a corresponding relationship, and each sorting flow channel group is used to sort a target cell; a first valve is provided at one end of each driving fluid flow channel 4 close to the sorting area 201; a second valve is provided at one end of each target cell flow channel 3 close to the sorting area 201;

[0063] There is at least one sorting zone; and the sorting zones correspond to the sorting flow channel groups one by one;

[0064] Using the cell recognition device to photograph a first cell located at a photographing position of the sample flow channel 1 includes: using the cell recognition device to photograph a first cell located at a first photographing position of the sample flow channel 1;

[0065] Identifying whether the first cell is a target cell includes:

[0066] identifying whether the first cell is a first type of target cell;

[0067] In response to the first cell being a target cell, after the first cell enters the sorting area 201, the first valve 6 and the second valve 7 are opened simultaneously, and are closed simultaneously after being kept for a preset time, so that the first cell located in the sorting area enters the target cell flow channel 3 under the drive of the driving fluid during the preset time, including:

[0068] In response to the first type of target cell, after the first cell enters the first sorting area 201, the first valve 6 of the first driving fluid channel in the first sorting channel group and the second valve 7 of the first target cell channel in the first sorting channel group are opened simultaneously, and are closed simultaneously after being kept for a first preset time, so that the first cell located in the first sorting area enters the first target cell channel 3 under the drive of the driving fluid during the first preset time;

[0069] The method also includes:

[0070] Using the cell recognition device to photograph a second cell located at a second photographing position of the sample flow channel 1;

[0071] identifying whether the second cell is a second type of target cell;

[0072] In response to the second type of target cell, after the first cell enters the second sorting area 201, the first valve 6 of the second driving fluid channel in the second sorting channel group and the second valve 7 of the second target cell channel in the second sorting channel group are opened simultaneously, and are closed simultaneously after being kept for a second preset time, so that the second cell located in the second sorting area enters the second target cell channel 3 under the drive of the driving fluid during the second preset time;

[0073] The first shooting position is located in the first sorting area or upstream of the first sorting area; the second shooting position is located in the second sorting area or upstream of the second sorting area.

[0074] That is, the embodiment of the present disclosure realizes the collection of multiple types of target cells by setting multiple sorting areas. There can be one shooting position, in which case two sorting areas share one shooting position, and the cells at the shooting position need to be identified as the first type of target cells, the second type of target cells, or neither. For example, after 3ms, the cells at the shooting position reach the first sorting area, and after 5ms, the cells at the shooting position reach the second sorting area. If the cells at the shooting position are the first target cells, the first valve and the second valve of the first sorting channel group are controlled to open after 3ms; if the cells at the shooting position are the second target cells, the first valve and the second valve of the second sorting channel group are controlled to open after 5ms. If neither is the case, the valve will not be opened.

[0075] There can be two shooting positions, one for each sorting area. At this time, it is sufficient to identify whether the cell is a first type of target cell at the first shooting position corresponding to the first sorting area. If so, the first valve and the second valve of the first sorting channel group are opened, otherwise they are not opened. It is sufficient to identify whether the cell is a second type of target cell at the second shooting position corresponding to the second sorting area. If so, the first valve and the second valve of the second sorting channel group are opened, otherwise they are not opened.

[0076] Obviously, the above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

1. A microfluidic chip for cell sorting, characterized in that: The invention comprises a sample flow channel (1), a pair of sheath fluid flow channels (8), a target cell flow channel (3) and a driving fluid flow channel (4), wherein a sorting zone (201) is arranged on the sample flow channel (1); the sheath fluid flow channel (8) is symmetrically distributed relative to the sample flow channel (1), and a junction (2) between the sheath fluid flow channel (8) and the sample flow channel is located upstream of the sorting zone (201); the sheath fluid in the sheath fluid flow channel (8) is used to focus the cell flow in the sample flow channel (1); one end of the driving fluid flow channel (4) and the target cell flow channel (3) meet at the sorting zone (201), the other end of the driving fluid flow channel (4) is a driving fluid inlet, and the other end of the target cell flow channel is a target cell outlet; the driving fluid flow channel ( 4) and the target cell flow channel (3) are respectively located on both sides of the extension direction of the sample flow channel (1); a first valve (6) for controlling the opening and closing of the driving fluid flow channel (4) is provided at one end of the driving fluid flow channel (4) close to the sorting area (201), and a second valve (7) for controlling the opening and closing of the target cell flow channel (3) is provided at one end of the target cell flow channel (3) close to the sorting area (201); a cell solution is introduced from the inlet end of the sample flow channel (1) into the sorting area (201); the driving fluid in the driving fluid flow channel (4) is used to cooperate with the first valve (6) and the second valve (7) to drive the target cells in the cell solution located in the sorting area (201) to enter the target cell flow channel (3).

2. The microfluidic chip according to claim 1, characterized in that: There is at least one target cell flow channel (3) and at least one driving fluid flow channel (4), and the target cell flow channel (3) and the driving fluid flow channel (4) correspond one to one to form at least one sorting flow channel group. Each sorting flow channel group includes a target cell flow channel (3) and a driving fluid flow channel (4) having a corresponding relationship, and each sorting flow channel group is used to sort a target cell; each of the driving fluid flow channels (4) is provided with the first valve (6) at one end close to the sorting area (201); and each of the target cell flow channels (3) is provided with the second valve (7) at one end close to the sorting area (201).

3. The microfluidic chip according to claim 2, characterized in that: There is at least one of the sorting area (201) and the sorting channel group, and the sorting area (201) and the sorting channel group correspond one to one.

4. According to the microfluidic chip of claim 3, the sheath liquid flow channel (8) is at least one pair, and the at least one pair of sheath liquid flow channels (8) corresponds one-to-one to the at least one sorting area (201); the first intersection of the first sheath liquid flow channel and the sample flow channel (1) is located upstream of the first sorting area corresponding thereto, and the sheath fluid in the first sheath liquid flow channel is used to focus the cell flow in the sample flow channel (1) from the first intersection to the first sorting area; the first sheath liquid flow channel is one pair among the multiple pairs of sheath liquid flow channels.

5. The microfluidic chip according to claim 1, characterized in that: The distance between the first valve (6) and the second valve (7) and the sorting area (201) is 15-17 μm.

6. The microfluidic chip according to claim 1, characterized in that: The driving fluid flow channel (4) and the target cell flow channel (3) both include a rapid flow section (9) connected to the sorting area (201) and a slow flow section (10) connected to the rapid flow section (9), and the width of the rapid flow section (9) is smaller than the width of the slow flow section (10); the first valve (6) and the second valve (7) are located in the rapid flow section of the driving fluid flow channel (4) and the rapid flow section of the target cell flow channel (3), respectively.

7. The microfluidic chip according to claim 6, characterized in that: The included angle between the rapid flow section of the driving fluid flow channel (4) and the sample flow channel (1) is 90 degrees; the included angle between the rapid flow section of the target cell flow channel (3) and the sample flow channel (1) is 90 degrees.

8. The microfluidic chip according to claim 1, characterized in that: The flow channel section (5) located downstream of the downstream sorting zone in the sample flow channel comprises a connecting section (501) connected to the downstream sorting zone (201) and a flow expansion section (502) connected to the connecting section (501), wherein the width of the connecting section (501) is smaller than the width of the flow expansion section (502); the downstream sorting zone is the most downstream sorting zone in the microfluidic chip.

9. The microfluidic chip according to claim 1, characterized in that: The distance from the intersection point (2) to the sorting area (201) is 140-170 μm.

10. The microfluidic chip according to claim 1, characterized in that: The sample flow channel (1) between the intersection (2) and the sorting area (201) has a width of 85-110 μm and a height of 45-70 μm.

11. The microfluidic chip according to any one of claims 1 to 10, characterized in that: When the first valve (6) is in a closed state, the driving fluid channel (4) maintains a first preset opening; when the second valve (7) is in a closed state, the target cell flow channel (3) maintains a second preset opening, and the first preset opening and the second preset opening prevent cells from passing through the second valve (7) of the target cell flow channel.

12. A cell sorting method based on the microfluidic chip according to any one of claims 1 to 11, characterized in that: The method comprises: Keeping the first valve (6) and the second valve (7) in a closed state; injecting the driving fluid into the driving fluid inlet driving fluid flow channel after pressurizing the driving fluid; Pressurizing the cell solution and injecting it into the inlet of the sample flow channel (1); Using a cell recognition device to photograph a first cell located at a photographing position of the sample flow channel (1); identifying whether the first cell is a target cell; In response to the target cell, after the first cell enters the sorting area (201), the first valve (6) and the second valve (7) are opened simultaneously, and are closed simultaneously after being maintained for a preset time, so that the first cell located in the sorting area enters the target cell flow channel (3) driven by the driving fluid during the preset time.

13. The method according to claim 12, characterized in that The target cells include a first type of target cells and a second type of target cells; There is at least one target cell flow channel (3) and at least one driving fluid flow channel (4), and the target cell flow channel (3) and the driving fluid flow channel (4) correspond to each other one by one to form at least one sorting flow channel group. Each sorting flow channel group includes a target cell flow channel (3) and a driving fluid flow channel (4) having a corresponding relationship, and each sorting flow channel group is used to sort a target cell; each driving fluid flow channel (4) is provided with the first valve (6) at one end close to the sorting area (201); each target cell flow channel (3) is provided with the second valve (7) at one end close to the sorting area (201); The identifying whether the first cell is the target cell comprises: identifying whether the first cell is the first type of target cell; In response to the target cell, after the first cell enters the sorting area (201), the first valve (6) and the second valve (7) are opened simultaneously, and are closed simultaneously after being kept for a preset time, so that the first cell located in the sorting area enters the target cell flow channel (3) under the drive of the driving fluid during the preset time, comprising: In response to the target cell being the first type, after the first cell enters the sorting area (201), a first valve (6) of a first driving fluid channel in a first sorting channel group and a second valve (7) of a first target cell channel in the first sorting channel group are opened simultaneously, and are closed simultaneously after being kept for a first preset time, so that the first cell located in the sorting area enters the first target cell channel (3) under the drive of the driving fluid during the first preset time; The method further comprises: identifying whether the first cell is the second type of target cell; In response to the target cell being the second type, after the first cell enters the sorting area (201), the first valve (6) of the second driving fluid channel in the second sorting channel group and the second valve (7) of the second target cell channel in the second sorting channel group are opened simultaneously, and are closed simultaneously after being maintained for a second preset time, so that the first cell located in the sorting area enters the second target cell channel (3) driven by the driving fluid during the second preset time.

14. The method according to claim 12, characterized in that The target cells include a first type of target cells and a second type of target cells; There is at least one target cell flow channel (3) and at least one driving fluid flow channel (4), and the target cell flow channel (3) and the driving fluid flow channel (4) correspond to each other one by one to form at least one sorting flow channel group, and each sorting flow channel group includes a target cell flow channel (3) and a driving fluid flow channel (4) having a corresponding relationship, and each sorting flow channel group is used to sort a target cell; each of the driving fluid flow channels (4) is provided with the first valve at one end close to the sorting area (201); and each of the target cell flow channels (3) is provided with the second valve at one end close to the sorting area (201); There is at least one sorting area; and the sorting areas correspond to the sorting flow channel groups one by one; The method of photographing a first cell located at a photographing position of the sample flow channel (1) using a cell recognition device comprises: photographing a first cell located at a first photographing position of the sample flow channel (1) using a cell recognition device; Identifying whether the first cell is the target cell comprises: identifying whether the first cell is a target cell of the first type; In response to the target cell, after the first cell enters the sorting area (201), the first valve (6) and the second valve (7) are opened simultaneously, and are closed simultaneously after being kept for a preset time, so that the first cell located in the sorting area enters the target cell flow channel (3) under the drive of the driving fluid during the preset time, comprising: In response to the target cell being the first type, after the first cell enters the first sorting area (201), a first valve (6) of a first driving fluid channel in a first sorting channel group and a second valve (7) of a first target cell channel in the first sorting channel group are opened simultaneously, and are closed simultaneously after being kept for a first preset time, so that the first cell located in the first sorting area enters the first target cell channel (3) under the drive of the driving fluid during the first preset time; The method further comprises: Using a cell recognition device to photograph a second cell located at a second photographing position of the sample flow channel (1); identifying whether the second cell is a target cell of the second type; In response to the target cell being the second type, after the first cell enters the second sorting zone (201), a first valve (6) of a second driving fluid channel in a second sorting channel group and a second valve (7) of a second target cell channel in the second sorting channel group are simultaneously opened, and are simultaneously closed after being maintained for a second preset time, so that the second cell located in the second sorting zone enters the second target cell channel (3) under the drive of the driving fluid during the second preset time; The first shooting position is located in the first sorting area or upstream of the first sorting area; the second shooting position is located in the second sorting area or upstream of the second sorting area.

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