Microfluidic chip for cell sorting

Abstract

The present disclosure relates to a microfluidic chip for cell sorting, comprising a sample flow channel, a pair of sheath liquid flow channels, a target cell flow channel and a driving fluid flow channel, wherein a sorting area is arranged on the sample flow channel; the sheath liquid flow channels are symmetrically distributed relative to the sample flow channel, and the intersection of the sheath liquid flow channels and the sample flow channel is located upstream of the sorting area; one end of the driving fluid flow channel and the target cell flow channel intersects 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 the opening and closing of the driving fluid flow channel is arranged at the end of the driving fluid flow channel close to the sorting area, and a second valve for controlling the opening and closing of the target cell flow channel is arranged at the end of the target cell flow channel close to the sorting area; by arranging the first valve, the driving potential energy is accumulated at the first valve closer to the cell, and a small enough pressure can be used to quickly flush the target cell into the target flow channel.

Description

Technical Field

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

[0002] Flow cytometry works by using monoclonal antibodies to rapidly perform multi-parameter quantitative analysis of single cells or other biological particles at the molecular level. It can analyze tens of thousands of cells at high speed and simultaneously measure multiple parameters from a single cell. Its advantages are speed, precision, and accuracy, making it the gold standard for contemporary quantitative cell analysis. Existing cell sorting methods primarily use flow cytometers equipped with sorting functions to label cells with different fluorescent markers. Existing flow cytometers often use microfluidic chips to sort cells. For example, a sorting device that simultaneously detects multiple fluorescent signals within cells contains a microfluidic chip comprising a detection region, a sorting region, a driving fluid inlet, a waste fluid reservoir, and a target cell reservoir. The detection region and the sorting region are connected by a first cell flow channel, the sorting region and the waste fluid reservoir are connected by a second cell flow channel, the driving fluid inlet is connected to the sorting region by a driving fluid flow channel, and the target cell reservoir and the sorting region are connected by a sorting flow channel. 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 pattern around the sorting area. This scheme uses the driving fluid flow channel to apply force to move target cells into the sorting flow channel and into the target cell pool, while non-target cells continue along the second cell flow channel into the waste liquid pool.

[0003] However, in the above scheme, since pressurized gas is only supplied to the driving fluid flow channel when the 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 into the driving fluid flow channel. As a result, when the target cells are sorted, the non-target cell fluid in the driving fluid flow channel enters the sorting flow channel together with the target cells, affecting the sorting accuracy. The second problem is that since the target cells are in a state of flow 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, then in order to achieve the target cells into the sorting channel, considering the loss of fluid overshoot during flow, a higher air pressure must be supplied. Under the action of air pressure, the driving fluid can quickly drive the fluid to the sorting port within a limited time, so that the cell fluid near the target cells enters the sorting channel. However, excessive air pressure can damage the cells and easily bring non-target cell fluid before and after the target cells into the sorting channel. If lower air pressure is used, the fluid flow rate is slow, and non-target cells have already left the sorting port or deviated from the sorting port by the time the fluid arrives, it is easy to fail to deliver the target cells 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] The present disclosure aims to overcome the problem of excessive sorting air pressure in microfluidic chips in the prior art causing damage to cells, and 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 paired sheath fluid flow channel, a target cell flow channel and a driving fluid flow channel, wherein a sorting zone is provided 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 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 flow channel and the target cell flow channel are respectively located on both sides of the extension direction of the sample flow channel; the end of the driving fluid flow channel close to the sorting area is provided with a first valve for controlling the opening and closing of the driving fluid flow channel, and the end of the target cell flow channel close to the sorting area is provided with a second valve for controlling the opening and closing of the target cell flow channel; the cell solution is introduced into the sorting area from the inlet end of the sample flow channel; the driving fluid in the driving fluid flow 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 into the target cell flow 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: keeping the first valve and the second valve in a closed state; pressurizing the driving fluid and injecting it into the driving fluid inlet driving fluid channel; pressurizing the cell solution and injecting it into the inlet end of the sample flow channel; using a cell recognition device to photograph a first cell located at a photographing position of the sample flow channel; identifying whether the first cell is a target cell; in response to being the target cell, after the first cell enters the sorting area, opening the first valve and the second valve at the same time, and closing 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 effect of the embodiment of the present disclosure is: 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 first valve can accumulate potential energy, 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 This 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 herein;

[0011] Figure 4 Schematic diagram of another embodiment of a microfluidic chip for cell sorting, including a sorting area, a target cell flow channel, and a driving fluid flow channel;

[0012] Figure 5 The figure is a schematic structural diagram of another embodiment of a sheath fluid channel and a sorting area of ​​a microfluidic chip for cell sorting disclosed herein. DETAILED DESCRIPTION

[0013] The drawings are for illustrative purposes only and should not be construed as limiting this patent. To better illustrate the embodiments, some components in the drawings may be omitted, enlarged, or reduced in size, and do not represent actual product dimensions. Those skilled in the art will understand that some well-known structures and their descriptions may be omitted from the drawings. The positional relationships depicted in the drawings are for illustrative purposes only and should not be construed as limiting this patent.

[0014] 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 there are terms such as "upper", "lower", "left", "right", "long", "short", etc. indicating orientations or positional relationships, they are based on the orientations or positional relationships shown in the drawings. This is only for the convenience of describing the present disclosure and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0015] The technical solution of the present disclosure is further described below through specific embodiments and in conjunction with the accompanying drawings:

[0016] Example 1

[0017] like Figure 1-2 The embodiment 1 of a microfluidic chip for cell sorting is shown, comprising a sample flow channel 1, a paired sheath fluid flow channel 8, a target cell flow channel 3 and a driving fluid flow channel 4, wherein a sorting region 201 is provided 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 region 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 region 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 either side of the extension direction of the sample channel 1; a first valve 6 for controlling the opening and closing of the driving fluid channel 4 is provided at the end of the driving fluid channel 4 near the sorting zone 201, and a second valve 7 for controlling the opening and closing of the target cell channel 3 is provided at the end of the target cell channel 3 near the sorting zone 201; the cell solution is introduced from the inlet end of the sample channel 1 into the sorting zone 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 zone 201 into the target cell channel 3. The structures of the first valve 6 and the second valve 7 of this embodiment can be found in the valve of a micro-fabricated elastic member with the publication number "CN100402850C".

[0018] Exemplarily, the driving fluid is a liquid, and air pressure is applied to the driving fluid at the driving fluid inlet. The first and second valves are in a normally closed state. The pressurized driving fluid is blocked by the first valve and trapped at the first valve, allowing the cell fluid to flow along the sample flow path. A recognition device for identifying target cells identifies cells at a capture position within the sample flow path. If a target cell is identified, the first and second valves are simultaneously opened after the cell at the capture position enters the sorting zone. The driving fluid then rushes into the sorting zone, forcing the target cells in the sorting zone into the target cell flow path, where they flow along the target cell flow path to the target cell outlet. By arranging the first valve so that the driving potential energy accumulates at the first valve closer to the cells, a lower air pressure (operating at a driving air pressure below 40 mbar) can be used to quickly (with a sorting pulse width of less than 100 ms), preventing damage to the cells by the driving fluid and preventing the driving fluid from acting on non-target cells, thereby improving sorting accuracy. The first and second valves are opened for a preset period of time and then closed to prevent the driving fluid from acting on non-target cells. Cells not acted upon by the driving fluid continue to flow along the sample flow channel to the waste port.

[0019] In a specific embodiment, the distance between the first valve 6 and the second valve 7 and the sorting area 201 is 15-17µm, for example, 16µm. 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 will easily 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 disordered, 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 at the driving fluid inlet, which may easily cause the target cells to be unable to be flushed into the target cell flow channel 3. Setting the first valve 6 at a distance of 15-17µm from the sorting zone 201 can ensure that the driving fluid rushing into the sorting zone from the driving fluid channel 4 is buffered to a certain extent, while preventing non-target cells from being brought into the target cell channel 3 and causing cell flow disturbance. Furthermore, the target cells can be rushed into the target cell channel 3 quickly enough under relatively low air pressure. The position of the second valve also requires careful design. If some target cells that have passed through the second valve linger near the side of the second valve away from the sorting zone, if the second valve 7 is too close to the sorting zone 201, the target cells that have passed through the second valve 7 in the target cell channel will easily flow back into the sorting zone once the second valve 7 is opened. If the second valve 7 is too far from the sorting zone, the opening time of the second valve 7 needs to be increased to ensure that the target cells pass through the second valve 7 before closing the second valve 7. This will result in an inconsistency between the opening time of the second valve 7 and the opening time of the first valve 6. If the second valve 7 remains open for a short period after the first valve 6 closes, target cells that have entered the target cell flow channel 3 but have not passed through the second valve, as well as target cells in the sorting zone 201, may lose external force and flow back, preventing the target cells from passing through the second valve 7. Ensuring that the distance between the second valve 7 and the first valve 6 and the sorting zone is substantially the same can ensure that the opening time of the second valve 7 is substantially consistent with the opening time of the first valve 6. Furthermore, when the distance between the second valve 7 and the sorting zone 201 is 15-176 µm, even if target cells in the target cell flow channel that have passed through the second valve 7 flow back when the second valve 7 opens, they will not have enough time to leave the target cell flow channel 3 and enter the sorting zone. When the first valve 6 is opened again, these target cells will be driven by the fluid into the side of the target cell flow channel 3 away from the second valve 7.

[0020] 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 smaller 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 is the same as the width of 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 is smaller in width and has a faster flow rate, while the slow flow section 10 has a slower flow rate. This 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.

[0021] 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 diffusion 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 diffusion section 502 is 2.5-4 times the width of the sample flow channel 1. Under normal circumstances, the flow rate of the waste liquid flow channel 5 is larger than that of the target cell flow channel, and the problem of how to avoid backflow has a greater impact on the sorting results. The width of the diffusion 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 rate in the connecting section 501 and the diffusion section 502 is increased, which can avoid the backflow of cells in the waste liquid flow channel 5. It is understandable that the cell fluid that is not driven into the target flow channel by the driven fluid will flow downstream along the sample flow channel into the waste liquid flow channel. The waste fluid may be waste fluid or cells that are different from the target cells but still need to be collected. For example, it is necessary to separate two types of cells, one of which is the target cells and the other is the non-target cells that need to be collected.

[0022] For example, the angle between the driving fluid channel 4 and the target cell channel 3 and the sample channel 1 is 90 degrees. In this configuration, when multiple sets of driving fluid channels 4, target cell channels 3, and sorting areas 201 are provided, the multiple sets of structures do not interfere with each other, enabling the simultaneous sorting of multiple different target cells using the same microfluidic chip.

[0023] In a specific embodiment, the distance from the intersection 2 to the sorting region 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 these dimensions, both the cell fluid and the sheath fluid can remain stable.

[0024] Example 2

[0025] Example 2 differs from Example 1 in that the distance between first valve 6 and sorting zone 201 is 17µm, and the distance between second valve 7 and sorting zone 201 is 15µm. The distance between intersection 2 and sorting port 201 is 150µm. The width and height of sample channel 1 are 85µm and 55µm, respectively.

[0026] The remaining working principles and effects of this embodiment are consistent with those of embodiment 1.

[0027] Example 3

[0028] Embodiment 3 of a microfluidic chip for cell sorting differs from Embodiments 1 or 2 in that, in the microfluidic chip of Embodiment 3, when the first valve 6 is closed, the driving fluid channel 4 maintains a first preset opening; when the second valve 7 is closed, the target cell channel 3 maintains a second preset opening. The first and second preset openings prevent cells from passing through the second valve 7 of the target cell channel. The first preset opening prevents the driving force of the driving fluid acting on cells in the sorting area and the target cell channel from being sufficient to force the cells past the second valve. The second preset opening allows only liquid to pass through the second valve of the target cell channel, disallowing the passage of cells. Thus, when the first and second valves are closed, the driving fluid still flows toward the outlet of the target cell channel, thereby imparting a certain thrust to the target cells that have passed through the second valve of the target cell channel, allowing the target cells to flow toward the outlet. Exemplarily, maintaining a first preset opening of the driving fluid flow channel when the first valve is in a closed state and maintaining a second preset opening of the target cell flow channel when the second valve is in a closed state can be achieved by existing methods, for example, by making the heights of the driving fluid flow 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.

[0029] The remaining features and working principles of this embodiment are consistent with those of embodiment 1 or embodiment 2.

[0030] Example 4

[0031] Example 4 of a microfluidic chip for cell sorting, such as Figure 3As 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 two driving fluid flow channels 4 are each provided, and the target cell flow channels 3 and the driving fluid flow channels 4 correspond one to one to form two sorting flow channel groups, each of which 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 type of 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. In this embodiment, there is one sorting area 201, and the two sorting flow channel groups share one sorting area 201.

[0032] 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 angle between 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 cell in the sample flow channel sorting area enters 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. 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.

[0033] Example 5

[0034] Example 5 of a microfluidic chip for cell sorting, such as Figure 4 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. Here, there are two sorting zones, each with one sorting channel group. The multiple sorting zones 201 are distributed along the flow direction of the cell fluid in the sample channel 1.

[0035] The working principle of this embodiment is as follows: Each sorting channel group corresponds to a target cell, and multiple sorting zones and multiple sorting channel groups can sort a variety of different target cells. For example, the microfluidic chip includes two sorting zones, and each sorting zone has two sorting channel groups. Then the microfluidic chip can sort a maximum of four target cells plus one cell that is not a target cell but can still be collected. The two sorting channel groups in the first sorting zone (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, and 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 type of 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). Non-target cells enter the waste liquid flow channel.

[0036] Example 6

[0037] Example 6 of a microfluidic chip for cell sorting, based on the above Example 5, as Figure 5 As shown, multiple pairs of sheath fluid channels 8 are provided, the number of which matches the number of sorting zones 201. The intersection of each pair of sheath fluid channels 8 and the sample channel 1 is located upstream of its corresponding sorting zone, and the sheath fluid channels 8 do not cross other sorting zones. Each pair of sheath fluid channels 8 can focus the cell flow from its corresponding intersection to the sorting zone. This can enhance the flow focusing effect, aligning cells in the sample channel with the center of the flow path, and minimizing the occurrence of side-by-side cells, thereby enabling the target cells to be accurately flushed into the corresponding target cell flow channel using the driving fluid.

[0038] It is understandable that, except for situations that are obviously contradictory, the features in the above embodiments can be combined arbitrarily.

[0039] Example 7

[0040] A cell sorting method based on the above microfluidic chip, based on the microfluidic chip of Example 1 above, is as follows:

[0041] 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 pellet in 400uL PBS to a final cell concentration of ~10 6cells / mL (the final concentration is based on the cell count 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, with a total volume of 1 mL. The driving fluid composition can be the same as the sheath fluid or buffer.

[0042] Keep the first valve 6 and the second valve 7 closed; continuously apply 30-40 mbar air pressure to the driving fluid to allow the driving fluid to enter the driving fluid channel 4. Use a syringe pump to inject the cell sample into the input end of the sample channel 1, and also use a syringe pump to inject the sheath fluid into the sheath fluid channels on both sides.

[0043] A cell recognition device is used to capture the first cell located at the capturing position of the sample flow channel 1; and to identify whether the first cell is a target cell. The capturing position is located upstream of the sorting position, and the specific position can be determined based on the cell flow rate and the valve response speed. The faster the valve response speed, the closer the capturing position can be to the sorting position. For example, after the first valve and the second valve are instructed to open, the first valve and the second valve are opened 3ms later. The distance between the capturing position and the sorting position must be at least 3ms to allow the cell to flow. If the cell at the capturing position is a target cell, a valve opening instruction is immediately given after identification as a target cell. 3ms later, the target cell flows into the sorting area, the first valve and the second valve in the sorting area are opened, and the target cell enters the target cell flow channel under the action of the driving fluid.

[0044] If the cells are target cells, the first valve 6 and the second valve 7 are opened and held for a preset duration before closing. This allows the target cells in the sorting area 201 to enter the target cell flow channel 3 under the drive of the driving fluid for a preset duration of 2 milliseconds. If the cells are non-target cells, the first valve 6 and the second valve 7 remain closed, and the sample flows to the waste flow channel 5. A collection tank is provided at the fluid outlet of the target cell flow channel 3. A collection tank can also be provided for the waste flow channel.

[0045] 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 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, in the experimental group 3 of the present disclosure, the driving fluid flow channel and the target cell flow channel are kept at a preset opening when the first valve and the second valve are closed. 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.

[0046] Table 1 Microfluidic chip control experiment

[0047]

[0048] From the data comparison between the control group 1 and 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. And 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.

[0049] Example 8

[0050] A cell sorting method based on the microfluidic chip, based on the above embodiment 7, wherein the target cells include a first type of target cells and a second type of target cells;

[0051] There is at least one target cell flow channel 3 and at least one driving fluid flow channel 4. 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 corresponding target cell flow channel 3 and a driving fluid flow channel 4. Each sorting flow channel group is used to sort one target cell. A first valve 6 is provided at one end of each driving fluid flow channel 4 near the sorting area 201. A second valve 7 is provided at one end of each target cell flow channel 3 near the sorting area 201.

[0052] Identifying whether the first cell is a target cell includes: identifying whether the first cell is a first type of target cell;

[0053] 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:

[0054] 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 flow channel in the first sorting flow channel group and the second valve 7 of the first target cell flow channel in the first sorting flow channel group are opened simultaneously, and are closed simultaneously after being maintained for a first preset time, so that the first cell in the sorting area enters the first target cell flow channel under the drive of the driving fluid during the first preset time;

[0055] The method also includes:

[0056] identifying whether the first cell is a second type of target cell;

[0057] 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 under the drive of the driving fluid during the second preset time.

[0058] In other words, the disclosed embodiment achieves the collection of multiple types of target cells by providing multiple sorting channel groups. There can be one shooting position, and after shooting, it can simultaneously identify whether the captured cell is a first type of target cell or a second type of target cell. If it is a first type of target cell, the first valve and the second valve of the first sorting channel group are opened; if it is a second type of target cell, the first valve and the second valve of the first sorting channel group are opened; if it is neither type of target cell, the valves remain closed.

[0059] Example 9

[0060] A cell sorting method based on the microfluidic chip, based on the above embodiment 7, wherein the target cells include a first type of target cells and a second type of target cells;

[0061] 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 with a corresponding relationship, and each sorting flow channel group is used to sort one target cell; each driving fluid flow channel 4 is provided with a first valve at one end close to the sorting area 201; each target cell flow channel 3 is provided with a second valve at one end close to the sorting area 201;

[0062] There is at least one sorting zone; the sorting zones correspond to the sorting flow channel groups one to one;

[0063] Using the cell recognition device to photograph the first cell located at the photographing position of the sample flow channel 1 includes: using the cell recognition device to photograph the first cell located at the first photographing position of the sample flow channel 1;

[0064] Identifying whether the first cell is a target cell includes:

[0065] identifying whether the first cell is a first type of target cell;

[0066] 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:

[0067] In response to the first type of target cell, after the first cell enters the first sorting zone, 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 simultaneously opened and simultaneously closed after being maintained for a first preset time, so that the first cell located in the first sorting zone enters the first target cell channel under the drive of the driving fluid during the first preset time;

[0068] The method also includes:

[0069] Using the cell recognition device to photograph a second cell located at a second photographing position in the sample flow channel 1;

[0070] identifying whether the second cell is a second type of target cell;

[0071] In response to the second type of target cell, after the first cell enters the second sorting zone, 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 simultaneously opened and 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 under the drive of the driving fluid during the second preset time;

[0072] 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.

[0073] That is, the embodiment of the present disclosure realizes the collection of multiple types of target cells by setting up multiple sorting areas. There can be one shooting position, in which case the 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.

[0074] There can be two imaging positions, one for each sorting zone. At the first imaging position corresponding to the first sorting zone, it is sufficient to identify whether the cell is a first type of target cell. If so, the first and second valves of the first sorting channel group are opened; otherwise, they remain closed. At the second imaging position corresponding to the second sorting zone, it is sufficient to identify whether the cell is a second type of target cell. If so, the first and second valves of the second sorting channel group are opened; otherwise, they remain closed.

[0075] Obviously, the above embodiments of the present disclosure are merely examples for the purpose of clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those skilled in the art, other variations 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, and improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection 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 area (201) is provided 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); the sheath fluid in the sheath fluid flow channel (8) is used to focus the cell flow in the sample flow channel (1); the driving fluid is used to focus the cell flow in the sample flow channel (1); One end of the 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 the driving fluid inlet, and the other end of the target cell flow channel is the 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); the end of the driving fluid flow channel (4) close to the sorting area (201) is provided with a control for controlling the opening and closing of the driving fluid flow channel (4). The first valve (6) is provided at one end of the target cell flow channel (3) 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; the 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 always in a pressurized state, and when the target cell is identified, the first valve and the second valve are opened, and the driving fluid drives the target cell in the cell solution in the sorting area (201) into the target cell flow channel (3); when the first valve (6) is in a closed state, the driving fluid flow 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 the cell from passing through the second valve (7) of the target cell flow channel. When the first valve and the second valve are closed, the driving fluid still flows to the outlet of the target cell flow channel.

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) with 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. The microfluidic chip according to claim 3, wherein 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 to which it corresponds, 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), wherein 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 angle between the rapid flow section of the driving fluid flow channel (4) and the sample flow channel (1) is 90 degrees; the 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 waste liquid flow channel (5) located downstream of the downstream sorting zone in the sample flow channel includes a connecting section (501) connected to the downstream sorting zone and a flow expansion section (502) connected to the connecting section (501), and 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. A cell sorting method based on the microfluidic chip according to any one of claims 1 to 10, characterized in that: The method comprises: Maintaining the first valve (6) and the second valve (7) in a closed state; injecting the driving fluid into the driving fluid inlet 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) under the drive of the driving fluid during the preset time.

12. The method according to claim 11, 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 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. The first valve (6) is provided at one end of each driving fluid flow channel (4) close to the sorting area (201); the second valve (7) is provided at one end of each target cell flow channel (3) close to the sorting area (201). The identifying whether the first cell is the target cell includes: 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, including: 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 flow channel in a first sorting flow channel group and a second valve (7) of a first target cell flow channel in the first sorting flow 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 flow channel under the drive of the driving fluid during the first preset time; The method further comprises: identifying whether the first 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 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 under the drive of the driving fluid during the second preset time.

13. The method according to claim 11, 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 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 driving fluid flow channel (4) is provided with the first valve at one end close to the sorting area (201); each target cell flow channel (3) is provided with the second valve at one end close to the sorting area (201); There is at least one sorting area; and each sorting area corresponds to each sorting flow channel group 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 includes: 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, including: In response to the target cell being the first type, after the first cell enters the first sorting zone, a first valve (6) of a first driving fluid channel in the first sorting channel group and a second valve (7) of a first target cell channel in the first sorting channel group are simultaneously opened, and are simultaneously closed after being kept for a first preset time, so that the first cell located in the first sorting zone enters the first target cell channel 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); Identify whether the second cell is a target cell of the second type; in response to the second type of target cell, after the first cell enters the second sorting zone, open 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 at the same time, and close them at the same time after maintaining them for a second preset time, so that the second cell located in the second sorting zone enters the second target cell channel under the drive of the driving fluid during the second preset time; the first shooting position is located in the first sorting zone or upstream of the first sorting zone; the second shooting position is located in the second sorting zone or upstream of the second sorting zone.

Images