Cell sorting device and flow cytometer

Abstract

The invention provides a cell sorting device and a flow cytometer, the device comprises a micro-fluidic chip and a trigger component, the micro-fluidic chip comprises a substrate, a main channel, a sheath flow channel assembly and a plurality of sorting channels, the main channel, the sheath flow channel assembly and the sorting channels are arranged on the substrate, and the sheath flow channel assembly and the sorting channels are communicated with the main channel; the sorting channel comprises a sorting inlet connected with the main channel and a sorting pipe connected with the sorting inlet, the sorting pipe is provided with a plurality of straight parts connected end to end and winding parts, the straight parts are provided with containing structures close to the winding parts, and the curvatures of the multiple winding parts are gradually reduced from front to back. The triggering part comprises sound wave transduction assemblies which are arranged at the two ends of the straight movement part and incline towards the containing structure, the triggering part further comprises a plurality of optical fiber pressure devices which are arranged on the substrate and connected with the main channel, the optical fiber pressure devices are located on the opposite sides of the sorting inlets, and the flow of the sorting inlets is sequentially increased from front to back. According to the cell sorting device, secondary sorting can be carried out on cell populations, and the cell sorting efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and in particular to a cell sorting device and a flow cytometer. Background Art

[0002] In biological research, sorting heterogeneous cell samples into different subpopulations enables subsequent physical and biochemical analysis.

[0003] In the prior art, flow cytometers are usually used for cell sorting. Cells with different characteristics are separated by injecting a cell mixture into a microfluidic chip and applying various external forces to the fluid in the chip. Currently available cell sorting devices can usually sort cells once according to their size, and the sorted cell population is only within a certain range. If single cell sorting is to be performed again within this range, a different chip needs to be used for the operation. Summary of the invention In view of the deficiencies of the prior art, the object of the present invention is to provide a cell sorting device, aiming to solve the technical problems mentioned in the background technology.

[0004] In order to achieve the above object, the present invention is implemented by the following technical solutions: A cell sorting device comprises a microfluidic chip and a trigger component arranged on the microfluidic chip, wherein the microfluidic chip comprises a substrate, and a main channel, a sheath flow channel assembly and a plurality of sorting channels arranged on the substrate, wherein the sheath flow channel assembly and the sorting channel are both connected to the main channel, the sorting channel comprises a sorting inlet connected to the main channel, and a sorting tube connected to the sorting inlet, wherein the sorting tube has a plurality of straight sections and a winding section connected end to end, wherein a containing structure is arranged at a position of the straight section near the winding section, and the curvature of the plurality of the winding sections gradually decreases from front to back, the trigger component comprises an acoustic wave transducer assembly arranged at both ends of the straight section and inclined toward the containing structure, the trigger component further comprises a plurality of optical fiber pressure devices arranged on the substrate and connected to the main channel, the optical fiber pressure devices are located on opposite sides of the sorting inlet, the flow rates of the plurality of sorting inlets increase from front to back, and the amplitudes of the plurality of acoustic wave transducer assemblies increase from front to back. According to one aspect of the above technical solution, the sheath flow channel assembly includes a first sheath flow pipeline and a second sheath flow pipeline respectively arranged on both sides of the main channel, the first sheath flow pipeline is located on the same side of the sorting channel, and the ratio of the outlet flow of the second sheath flow pipeline to the first sheath flow pipeline is 2~5.

[0005] According to one aspect of the above technical solution, the containing structure includes a containing portion connected to one side of the straight portion and arranged in a semicircular shape, and a blocking portion respectively connected to the containing portion and the other side of the straight portion, and the containing portion is used to contain a single cell.

[0006] According to one aspect of the above technical solution, the acoustic wave transducer assembly includes a first acoustic wave transducer and a second acoustic wave transducer respectively arranged at both ends of the straight portion, and the sinusoidal signals of the first acoustic wave transducer and the second acoustic wave transducer are consistent and have a value of 10V~20V.

[0007] According to one aspect of the above technical solution, the inclination angles of the first sound wave transducer and the second sound wave transducer are 15° to 30°.

[0008] According to one aspect of the above technical solution, the ratio of the flow rate of the accommodating portion to the flow rate of the nearest winding portion is 1.43-1.51.

[0009] According to one aspect of the above technical solution, the ratio of the flow rate of the sorting inlet to the flow rate of the main channel is 0.3~0.6.

[0010] According to one aspect of the above technical solution, the pressure of the optical fiber compressor is 10PN~12PN, and the pressure of the optical fiber compressor gradually increases from front to back.

[0011] According to one aspect of the above technical solution, the microfluidic chip is also provided with a sheath flow inlet, a sample flow inlet, and a waste liquid port, the sheath flow inlet is connected to the sheath flow channel assembly, the sample flow inlet is connected to the starting point of the main channel, the waste liquid port is connected to the end point of the main channel, and the microfluidic chip is also provided with an air outlet connected to the sorting tube.

[0012] A flow cytometer comprises the cell sorting device as described above.

[0013] Compared with the prior art, the present invention has the following beneficial effects: By setting up a microfluidic chip, wherein the microfluidic chip includes a substrate, a main channel, a sheath flow channel component and multiple sorting channels, when cell sorting is required, a sample flow is injected into the main channel, wherein the sample flow is a mixture of a suspension and a cell population, and a flow rate is given to the sample flow in the main channel to make the sample flow move forward. At the same time, a suspension is also added to the sheath flow channel, and a flow rate is given to the sheath flow channel component. When the suspension in the sheath flow channel component meets the sample flow in the main channel, a shear force is applied to the sample flow, so that the sample flow is arranged in a straight line in sequence. Then, when the sample flow passes through the optical fiber pressure device, it is subjected to the pressure of the optical fiber and deviates toward the sorting inlet. Since the offset is proportional to the particle size, smaller particles are more likely to be separated. The particles will have a larger displacement than the larger particles and will be closer to the first sorting entrance. Since the flow rate of the sorting entrance increases from front to back, and the movement of the particles follows the direction of the flow rate, the smaller particles will enter the first sorting entrance. The flow rate of the first sorting entrance is not enough to absorb the larger particles, so the larger particles will continue to flow. Until reaching the second sorting entrance, since the flow rate of the sorting entrance increases from front to back, the flow rate of the second sorting entrance can absorb particles of a larger size, and can absorb particles of each interval size range in turn, so that a classification is carried out. It should be noted that the particles here are also cells, and the flow rate of the sorting entrance can be adjusted by the diameter of the sorting entrance. Then the sorting tube has a plurality of straight sections and winding sections connected end to end. A containing structure is provided near the winding section of the straight section. The particle flow follows a rule and moves toward the direction with smaller flow resistance first. Since the winding section is curved, the flow resistance is greater than the containing structure at the straight section. The particles will move toward the containing structure first. At this time, the acoustic wave transducer assembly acts on the straight section. Since the larger the particle diameter, the greater the acoustic wave force it receives, the acoustic wave emitted by the acoustic wave transducer assembly is first set to a smaller value, so that the larger particles in the straight section that have been classified once can be removed by the acoustic wave. The larger particles are driven to be closer to the containing structure, and the smaller particles are not displaced much. When the containing structure is blocked by a particle (i.e., a single cell), the flow resistance at the containing structure will be greater than the flow resistance of the meandering part, and the remaining particles will continue to move backward. The power at the acoustic wave transducer assembly can be gradually increased to gradually sort all the single cells in order from large to small. The reason why the curvature of multiple meandering parts gradually increases from front to back is that the greater the curvature of the meandering part, the greater the flow resistance, and the greater the resistance to the sample flow, preventing the sample from flowing to the rear section without sufficient kinetic energy to continue moving forward.

[0014] The present invention can perform secondary sorting on cell groups, thereby improving the cell sorting efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1This is a diagram showing the internal structure of a cell sorting device in the first embodiment of the present invention; Figure 2 for Figure 1 Schematic diagram of the structure when the cell group has not reached the sorting entrance; Figure 3 for Figure 1 Schematic diagram of the structure when the cell group has reached the sorting entrance; Figure 4 for Figure 1 Schematic diagram of the structure where the middle cell group is located at the sorting tube; Description of main component symbols:

[0016] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0018] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0020] See also Figures 1 to 4, shown is a cell sorting device in the first embodiment of the present invention, comprising a microfluidic chip and a trigger component arranged on the microfluidic chip, the microfluidic chip comprising a substrate 10, and a main channel 20, a sheath flow channel assembly and a plurality of sorting channels 50 arranged on the substrate 10, the sheath flow channel assembly and the sorting channels 50 are both connected to the main channel 20, the sorting channels 50 comprise a sorting inlet 52 connected to the main channel 20, and a sorting tube connected to the sorting inlet 52, the sorting tube having a plurality of straight sections 53 connected end to end, and a winding section 54 The straight portion 53 is provided with a containing structure near the winding portion 54, and the curvature of the multiple winding portions 54 gradually decreases from front to back. The trigger component includes an acoustic wave transducer assembly 60 arranged at both ends of the straight portion 53 and inclined toward the containing structure. The trigger component also includes a plurality of optical fiber pressers 30 arranged on the base 10 and connected to the main channel 20, and the optical fiber pressers 30 are located on the opposite side of the sorting inlet 52. The flow rates of the multiple sorting inlets 52 increase successively from front to back, and the amplitudes of the multiple acoustic wave transducer assemblies 60 increase successively from front to back.

[0021] It can be understood that the present invention sets up a microfluidic chip, wherein the microfluidic chip includes a substrate 10, a main channel 20, a sheath flow channel assembly and a plurality of sorting channels 50. When cell sorting is required, a sample flow is injected into the main channel 20, wherein the sample flow is a mixture of a suspension and a cell population. The sample flow in the main channel 20 is advanced by giving a flow rate. At the same time, a suspension is also added to the sheath flow channel, and a flow rate is given to the sheath flow channel assembly. When the suspension in the sheath flow channel assembly meets the sample flow in the main channel 20, a shear force is applied to the sample flow, so that the sample flow is arranged in a straight line. Then, when the sample flow passes through the optical fiber pressure device 30, it is subjected to the pressure of the optical fiber and deviates toward the sorting inlet 52. Since the offset is proportional to the particle size, smaller particles are more likely to be separated. The particles of smaller sizes will have a larger displacement than the particles of larger sizes, and will be closer to the first sorting entrance 52; and because the flow rate of the sorting entrance 52 increases from front to back, and the movement of the particles follows the direction of the flow rate, the particles of smaller sizes will enter the first sorting entrance 52, and the flow rate of the first sorting entrance 52 will not absorb the particles of larger sizes, so the particles of larger sizes will continue to flow; until reaching the second sorting entrance 52, because the flow rate of the sorting entrance 52 increases from front to back, the flow rate of the second sorting entrance 52 can absorb particles of a larger size, and can absorb particles of particle sizes in each interval range in turn, so that a classification is performed. It should be noted that the particles here are also cells, and the flow rate of the sorting entrance 52 can be adjusted by the pipe diameter of the sorting entrance 52; Then the sorting tube has a plurality of straight sections 53 and winding sections 54 connected end to end. A containing structure is provided near the straight section 53 near the winding section 54. The particle flow follows a rule and moves toward the direction with smaller flow resistance first. Since the winding section 54 is curved, the flow resistance is greater than the containing structure at the straight section 53. The particles will move toward the containing structure first. At this time, the sound wave transducer assembly 60 acts on the straight section 53. Since the larger the particle diameter, the greater the sound wave force received. Therefore, the sound wave emitted by the sound wave transducer assembly 60 is first set to a smaller value, so that the particles at the straight section 53 that have been sorted once can be transported to the straight section 53 by the sound wave. The larger particles among the particles are driven to be closer to the containing structure, and the smaller particles are not displaced by a large distance. When the containing structure is blocked by a particle (i.e., a single cell), the flow resistance at the containing structure will be greater than the flow resistance of the winding portion 54, and the remaining particles will continue to move backward. The power at the acoustic wave transducer assembly 60 can be gradually increased to gradually sort all the single cells in order from large to small. The reason why the curvature of the multiple winding portions 54 gradually increases from front to back is that the greater the curvature of the winding portion 54, the greater the flow resistance, and the greater the resistance to the sample flow, preventing the sample from flowing to the rear section without sufficient kinetic energy to continue moving forward.

[0022] The present invention can perform secondary sorting on cell groups, thereby improving the cell sorting efficiency.

[0023] Specifically, in this embodiment, the sheath flow channel assembly includes a first sheath flow pipeline 41 and a second sheath flow pipeline 42 which are arranged on both sides of the main channel 20, the first sheath flow pipeline 41 is located on the same side of the sorting channel 50, and the ratio of the outlet flow of the second sheath flow pipeline 42 to the first sheath flow pipeline 41 is 2~5.

[0024] Understandable, see Figure 2 Through the shearing action of the first sheath flow line 41 and the second sheath flow line 42, the cell group in the sample flow can be squeezed into a straight line arrangement. The present invention defaults that the ratio of the flow rate of the main channel 20 to the flow rate of the first sheath flow line 41 is 1:1, and the ratio of the flow rate of the main channel 20 to the flow rate of the second sheath flow line 42 is 1:5. By changing the ratio of the outlet flow rates of the second sheath flow line 42 and the first sheath flow line 41, it can be found that the larger the flow rate of the second sheath flow line 42, the closer the cell group in the sample flow can be to the sorting channel 50, so that the cell group arranged in a straight line can enter the sorting inlet 52 more easily.

[0025] Furthermore, the ratio of the flow rate of the sorting inlet 52 to the flow rate of the main channel 20 is 0.3-0.6; the pressure of the optical fiber pressure device 30 is 10PN-12PN.

[0026] Understandable, see Figure 3The present invention is illustrated with two sorting entrances 52. By setting the flow of the sorting entrance 52 to increase from front to back, it can be seen that the particle group with a smaller size range will first enter the first sorting entrance 52, and the particle group with a larger size range will continue to move forward and enter the second sorting entrance 52. Combined with the effect of the optical fiber pressure device 30, the adjustment of the particle group can be more precise. Preferably, the pressure of the optical fiber pressure device 30 gradually increases from front to back, so as to further improve the accuracy of the initial sorting. It should be noted that the ratio of the flow of the sorting entrance 52 to the flow of the main channel 20 can be adjusted by the size of the sorting entrance 52.

[0027] Furthermore, the containing structure includes a containing portion 55 connected to one side of the straight portion 53 and arranged in a semicircular shape, and a blocking portion 56 respectively connected to the containing portion 55 and the other side of the straight portion 53, and the containing portion 55 is used to contain a single cell.

[0028] It can be understood that the size of the portion where the receiving portion 55 and the barrier portion 56 are connected needs to be at least larger than the smallest single cell so that the receiving portion 55 can accommodate the single cell. When the single cell enters the receiving portion 55, the barrier portion 56 will be blocked, so that the flow resistance of the receiving structure is greater than the flow resistance of the winding portion 54, and the remaining cell group will continue to flow toward the winding portion 54, waiting to reach the receiving structure next time for the next single cell to be sorted.

[0029] Furthermore, the acoustic wave transducer assembly 60 includes a first acoustic wave transducer 61 and a second acoustic wave transducer 62 respectively arranged at both ends of the straight portion 53, the sinusoidal signals of the first acoustic wave transducer 61 and the second acoustic wave transducer 62 are consistent, and the amplitude is 10V~20V; the inclination angle of the first acoustic wave transducer 61 and the second acoustic wave transducer 62 is 15°~30°.

[0030] Understandable, see Figure 4Due to the inverse piezoelectric effect, surface acoustic waves are generated under the action of the first acoustic transducer 61 and the second acoustic transducer 62, thereby causing the internal pressure of the sample flow to change, forming a periodically distributed pressure, and thereby causing the cell group in the sample flow to shift. The inclination angle in this embodiment can further enhance the degree of shift, so that the cell group can move further toward the containment structure. Since the larger the single cell, the greater the degree of shift, the amplitude of the sinusoidal signal of the first acoustic transducer 61 and the second acoustic transducer 62 is set to allow the larger single cell to pass through, while the smaller single cell has a smaller shift distance, so that the larger single cell can enter the containment structure, and the smaller single cell group can enter the next containment structure. By increasing the amplitude of the sinusoidal signal of the first acoustic transducer 61 and the second acoustic transducer 62 in sequence, all single cells can be sorted in order from large to small. Furthermore, the ratio of the flow rate of the accommodating portion 55 to the flow rate of the nearest meandering portion 54 is 1.43-1.51.

[0031] It can be understood that by making the flow rate of the receiving portion 55 and the flow rate of the nearest winding portion 54 have the most suitable ratio, single cells can be better allowed to enter the receiving portion 55, thereby avoiding the situation where the ratio is too small so that single cells cannot enter the receiving portion 55, and avoiding the situation where the ratio is too large so that excess single cells enter the receiving portion 55 and cause sorting errors.

[0032] Furthermore, the microfluidic chip is also provided with a sheath flow inlet 40, a sample flow inlet 21, and a waste liquid port 22. The sheath flow inlet 40 is connected to the sheath flow channel assembly, the sample flow inlet 21 is connected to the starting point of the main channel 20, and the waste liquid port 22 is connected to the end point of the main channel 20. The microfluidic chip is also provided with an outlet 51 connected to the sorting tube.

[0033] It can be understood that the above structures are conventional structures in the microfluidic chip, which are used to put in sheath flow, put in sample flow, and collect excess sample flow, etc., and will not be described in detail here.

[0034] This example verifies the capture rate of cell sorting of the present invention by selecting the values ​​of various parameters in various components.

[0035]

[0036] It should be noted that the cell capture rates of each sorting inlet 52 and the cell capture rates at each containing structure are carried out on the basis of more than 50 simulation experiments, and there are 10 containing structures in the first sorting channel 50 in the simulation model (the schematic diagram in the figure shows only 5); the signal amplitudes of the first acoustic wave transducer and the second acoustic wave transducer increase in an arithmetic progression.

[0037] It can be seen from the above table that, through the structure of the present invention, whether the first sorting is performed at the sorting inlet 52 or the second sorting is performed at the containing structure, the cell sorting accuracy can reach more than 90%, which provides a better idea for the technical personnel in the field of microfluidic chip development.

[0038] As for the preparation method of the microfluidic chip, it can be prepared by conventional exposure and development methods.

[0039] In summary, the cell sorting device in the above-mentioned embodiment of the present invention can perform a primary sorting of cell groups by setting the optical fiber pressure device and the sorting inlet flow, and can perform a secondary sorting of cell groups by setting the flow ratio between the containing part and the winding part, and setting the amplitude of the acoustic wave transducer, thereby improving the cell sorting efficiency. The second embodiment of the present invention further provides a flow cytometer, which has the cell sorting device as described in the first embodiment. It should be noted that the basic components such as microscope and oscilloscope in the flow cytometer are all prior art, and the present invention has not made any improvements, so they will not be described here.

[0040] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0041] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cell sorting device, characterized in that: The invention comprises a microfluidic chip and a trigger component arranged on the microfluidic chip. The microfluidic chip comprises a substrate, and a main channel, a sheath flow channel assembly and a plurality of sorting channels arranged on the substrate. The sheath flow channel assembly and the sorting channel are both connected to the main channel. The sorting channel comprises a sorting inlet connected to the main channel and a sorting tube connected to the sorting inlet. The sorting tube has a plurality of straight sections and winding sections connected end to end. A containing structure is arranged near the winding section on the straight section. The curvature of the winding sections gradually decreases from front to back. The trigger component comprises an acoustic wave transducer assembly inclined toward the containing structure and arranged at both ends of the straight section. The trigger component also comprises a plurality of optical fiber pressure devices arranged on the substrate and connected to the main channel. The optical fiber pressure devices are located on the opposite side of the sorting inlet. The flow rates of the plurality of sorting inlets increase from front to back, and the amplitudes of the plurality of acoustic wave transducer assemblies increase from front to back.

2. The cell sorting device according to claim 1, characterized in that: The sheath flow channel assembly includes a first sheath flow pipeline and a second sheath flow pipeline respectively arranged on both sides of the main channel, the first sheath flow pipeline is located on the same side of the sorting channel, and the ratio of the outlet flow of the second sheath flow pipeline to the first sheath flow pipeline is 2~5.

3. The cell sorting device according to claim 1, characterized in that: The containing structure comprises a containing part connected to one side of the straight part and arranged in a semicircular shape, and a blocking part respectively connected to the containing part and the other side of the straight part, and the containing part is used to contain a single cell.

4. The cell sorting device according to claim 1, characterized in that: The acoustic wave transducer assembly includes a first acoustic wave transducer and a second acoustic wave transducer respectively arranged at two ends of the straight portion. The sinusoidal signals of the first acoustic wave transducer and the second acoustic wave transducer are consistent, and the amplitude is 10V~20V.

5. The cell sorting device according to claim 4, characterized in that: The inclination angles of the first acoustic wave transducer and the second acoustic wave transducer are 15° to 30°.

6. The cell sorting device according to claim 1, characterized in that: The ratio of the flow rate of the accommodating portion to the flow rate of the nearest winding portion is 1.43-1.

51.

7. The cell sorting device according to claim 1, characterized in that: The ratio of the flow rate of the sorting inlet to the flow rate of the main channel is 0.3-0.

6.

8. The cell sorting device according to claim 1, characterized in that: The pressure of the optical fiber compressor is 10PN~12PN, and the pressure of the optical fiber compressor gradually increases from front to back.

9. The cell sorting device according to claim 1, characterized in that: The microfluidic chip is also provided with a sheath flow inlet, a sample flow inlet, and a waste liquid port. The sheath flow inlet is connected to the sheath flow channel assembly, the sample flow inlet is connected to the starting point of the main channel, the waste liquid port is connected to the end point of the main channel, and the microfluidic chip is also provided with an air outlet connected to the sorting tube.

10. A flow cytometer, characterized in that: A cell sorting device comprising the cell sorting device according to any one of claims 1 to 9.

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