A cell sorting device and a flow cytometer

Abstract

The present invention provides a cell sorting device and a flow cytometer. The device includes a microfluidic chip and a triggering component. The microfluidic chip includes a substrate, a main channel, a sheath flow channel assembly, and a plurality of sorting channels disposed on the substrate. The sheath flow channel assembly and the sorting channels are both connected to the main channel. The sorting channel includes 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 portions connected end to end and a winding portion. A receiving structure is provided at a position where the straight portion is close to the winding portion. The curvatures of the plurality of winding portions gradually decrease from front to back. The triggering component includes acoustic transducer assemblies disposed at both ends of the straight portion and inclined towards the receiving structure. The triggering component further includes a plurality of optical fiber pressure sensors disposed on the substrate and connected to the main channel. The optical fiber pressure sensors are located on the opposite side of the sorting inlet. The flow rates of the plurality of sorting inlets gradually increase from front to back. The present invention can perform secondary sorting on a cell population, improving the cell sorting efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and particularly relates 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 analyses.

[0003] In the prior art, a flow cytometer is usually used for cell sorting. By injecting a cell mixed fluid into a microfluidic chip and applying various external forces to the fluid in the chip, cells with different characteristics are separated. Currently, existing cell sorting devices can usually sort cells once according to cell size, and the sorted cell population is only within a certain range. If single-cell sorting is to be performed again within this range, different chips need to be used for operation. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a cell sorting device, aiming to solve the technical problems mentioned in the background art.

[0005] To achieve the above purpose, the present invention is realized by the following technical solutions:

[0006] A cell sorting device includes a microfluidic chip and a triggering component disposed on the microfluidic chip. The microfluidic chip includes a substrate, a main channel, a sheath flow channel assembly, and a plurality of sorting channels disposed on the substrate. The sheath flow channel assembly and the sorting channels are both connected to the main channel. The sorting channels include 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 portions connected end to end and a winding portion. A receiving structure is provided at a position where the straight portion is close to the winding portion. The curvatures of the plurality of winding portions gradually decrease from front to back. The triggering component includes acoustic wave transducer assemblies disposed at both ends of the straight portion and inclined towards the receiving structure. The triggering component further includes a plurality of optical fiber pressure sensors disposed on the substrate and connected to the main channel. The optical fiber pressure sensors are located on the opposite side of the sorting inlet. The flow rates of the plurality of sorting inlets gradually increase from front to back, and the amplitudes of the plurality of acoustic wave transducer assemblies gradually increase from front to back.

[0007] 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 disposed on both sides of the main channel. The first sheath flow pipeline is located on the same side of the sorting channels. The ratio of the outlet flow rate of the second sheath flow pipeline to that of the first sheath flow pipeline is 2 - 5.

[0008] According to one aspect of the above technical solution, the accommodating structure includes an accommodating portion that is connected to one side of the straight portion and is semicircularly arranged, and a blocking portion that is respectively connected to the other side of the accommodating portion and the straight portion. The accommodating portion is used to accommodate single cells.

[0009] 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 disposed at two ends of the straight portion. The sine signals of the first acoustic wave transducer and the second acoustic wave transducer are the same and have a value of 10V to 20V.

[0010] According to one aspect of the above technical solution, the inclination angles of the first acoustic wave transducer and the second acoustic wave transducer are 15° to 30°.

[0011] 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 meandering portion is 1.43 to 1.51.

[0012] 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 to 0.6.

[0013] According to one aspect of the above technical solution, the pressure of the optical fiber pressure device is 10PN to 12PN, and the pressure of the optical fiber pressure device gradually increases from front to back.

[0014] According to one aspect of the above technical solution, the microfluidic chip is further provided with a sheath flow inlet, a sample flow inlet, and a waste liquid outlet. The sheath flow inlet is communicated with the sheath flow channel assembly, the sample flow inlet is communicated with the starting point of the main channel, the waste liquid outlet is communicated with the end point of the main channel, and the microfluidic chip is further provided with an air outlet communicated with the sorting tube.

[0015] A flow cytometer includes the cell sorting device as described above.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] By setting up a microfluidic chip, which includes a substrate, a main channel, a sheath flow channel assembly, and multiple sorting channels. When cell sorting is required, a sample flow is injected into the main channel, where the sample flow is composed of a suspension and a cell population. By giving the sample flow in the main channel a flow rate, the sample flow advances. 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, a shear force will be applied to the sample flow, causing the sample flow to line up in a straight line in sequence. Then, when the sample flow passes through the fiber optic pressure device, it is subjected to the pressure of the fiber optic and will flow towards the sorting inlet with an offset. Since the offset is proportional to the particle size, smaller-sized particles will have a greater offset than larger-sized particles and will be closer to the first sorting inlet. Also, since the flow rate of the sorting inlet increases sequentially from front to back, and the movement of the particles follows the direction of the flow rate, smaller-sized particles will enter the first sorting inlet, and the flow rate of the first sorting inlet is not large enough to suck in larger-sized particles, so the larger-sized particles will continue to flow; until they reach the second sorting inlet. Since the flow rate of the sorting inlet increases sequentially from front to back, the flow rate of the second sorting inlet can absorb particles of a larger size range. In this way, particles of each particle size range can be absorbed in sequence, and thus a classification is carried out. It should be noted that the particles here are also cells, and the flow rate of the sorting inlet can be adjusted by the diameter of the sorting inlet;

[0018] Then the sorting tube has multiple straight sections connected end to end and a winding section. A receiving structure is provided at the straight section near the winding section. The particles flow according to a rule, first moving towards the direction with less flow resistance. Since the winding section is curved, its flow resistance is greater than that of the receiving structure at the straight section, so the particles will preferentially move towards the receiving structure. 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 received, the acoustic wave emitted by the acoustic wave transducer assembly is first set to a smaller value, so that the larger particles among the particles classified once at the straight section are driven closer to the receiving structure by the acoustic wave. The offset distance of the smaller particles is not large. After the receiving structure is blocked by a single particle (i.e., a single cell), the flow resistance at the receiving structure will be greater than that of the winding section, 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 single cells in descending order; The reason why the curvatures of the multiple winding sections gradually increase from front to back is that the greater the curvature of the winding section, the greater the flow resistance, and the greater the resistance received by the sample flow, preventing the sample flow from not having enough kinetic energy to continue moving forward in the later section.

[0019] The present invention can perform secondary sorting on a cell population, improving the sorting efficiency of cells. Brief Description of the Drawings

[0020] Figure 1 Internal structure diagram of the cell sorting device in the first embodiment of the present invention;

[0021] Figure 2 is Figure 1 Schematic diagram of the structure when the cell population has not reached the sorting inlet;

[0022] Figure 3 is Figure 1 Schematic diagram of the structure when the cell population has reached the sorting inlet;

[0023] Figure 4 is Figure 1 Schematic diagram of the structure when the cell population is located at the sorting tube;

[0024] Description of main component symbols:

[0025]

[0026] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0027] For ease of understanding the present invention, the present invention will be described more comprehensively 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, these embodiments are provided so that the disclosure of the present invention is thorough and complete.

[0028] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly on the other element or there can also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field 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.

[0030] Please refer to Figures 1 to 4, shown is a cell sorting device in the first embodiment of the present invention, including a microfluidic chip and a triggering component disposed on the microfluidic chip. The microfluidic chip includes a substrate 10, a main channel 20, a sheath flow channel assembly, and a plurality of sorting channels 50 disposed on the substrate 10. The sheath flow channel assembly and the sorting channels 50 are both connected to the main channel 20. The sorting channel 50 includes a sorting inlet 52 connected to the main channel 20 and a sorting tube connected to the sorting inlet 52. The sorting tube has a plurality of straight portions 53 connected end to end and a winding portion 54. An accommodating structure is provided at a position where the straight portion 53 is close to the winding portion 54. The curvatures of the plurality of winding portions 54 gradually decrease from front to back. The triggering component includes acoustic wave transducer assemblies 60 disposed at both ends of the straight portion 53 and inclined towards the accommodating structure. The triggering component further includes a plurality of optical fiber pressure sensors 30 disposed on the substrate 10 and connected to the main channel 20. The optical fiber pressure sensors 30 are located on the opposite side of the sorting inlet 52. The flow rates of the plurality of sorting inlets 52 increase sequentially from front to back, and the amplitudes of the plurality of acoustic wave transducer assemblies 60 increase sequentially from front to back.

[0031] It can be understood that by setting the microfluidic chip in the present invention, 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, where the sample flow is composed of a suspension and a cell population. By giving the sample flow in the main channel 20 a flow rate, the sample flow advances. 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 will be applied to the sample flow, causing the sample flow to be arranged in a straight line in sequence. Then, when the sample flow passes through the optical fiber pressure sensor 30, it is subjected to the pressure of the optical fiber and will deflect towards the sorting inlet 52. Since the deflection amount is proportional to the particle size, smaller-sized particles will have a greater deflection amount than larger-sized particles and will be closer to the first sorting inlet 52. Also, since the flow rates of the sorting inlets 52 increase sequentially from front to back and the movement of the particles follows the direction of the flow rate, smaller-sized particles will enter the first sorting inlet 52, and the flow rate of the first sorting inlet 52 is not sufficient to suck in larger-sized particles, so the larger-sized particles will continue to flow; until reaching the second sorting inlet 52, since the flow rates of the sorting inlets 52 increase sequentially from front to back, the flow rate of the second sorting inlet 52 can absorb particles of a larger size range. In this way, particles of each interval range of particle sizes can be absorbed in sequence, and thus a classification is carried out. It should be noted that the particles here are also cells, and the flow rate of the sorting inlet 52 can be adjusted by the diameter of the sorting inlet 52;

[0032] Next, the sorting tube has a plurality of straight portions 53 connected end to end and a winding portion 54. At the place where the straight portion 53 is close to the winding portion 54, there is a receiving structure. The particle flow follows a rule that it first moves in the direction with less flow resistance. Since the winding portion 54 is curved, its flow resistance is greater than that of the receiving structure at the straight portion 53. Therefore, the particles will preferentially move towards the receiving structure. At this time, the acoustic wave transducer assembly 60 acts on the straight portion 53. Since the larger the particle diameter, the greater the acoustic wave force, the acoustic wave emitted by the acoustic wave transducer assembly 60 is first set to a smaller value, so that the larger particles in the particles sorted once at the straight portion 53 are driven closer to the receiving structure by the acoustic wave, and the smaller particles do not have a large offset distance. After the receiving structure is blocked by a particle (i.e., a single cell), the flow resistance at the receiving structure will be greater than that 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 single cells in descending order; the reason why the curvatures of the plurality of winding portions 54 gradually increase 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 received by the sample flow, preventing the sample flow from not having enough kinetic energy to continue moving forward in the later section.

[0033] The present invention can perform secondary sorting on a cell population, improving the sorting efficiency of cells.

[0034] Specifically, in this embodiment, the sheath flow channel assembly includes a first sheath flow pipeline 41 and a second sheath flow pipeline 42 respectively 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 rate of the second sheath flow pipeline 42 to that of the first sheath flow pipeline 41 is 2 - 5.

[0035] It can be understood that, please refer to Figure 2 , through the shearing action of the first sheath flow pipeline 41 and the second sheath flow pipeline 42, the cell population 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 that of the first sheath flow pipeline 41 is 1:1, and the ratio of the flow rate of the main channel 20 to that of the second sheath flow pipeline 42 is 1:5. By changing the ratio of the outlet flow rate of the second sheath flow pipeline 42 to that of the first sheath flow pipeline 41, it can be found that the greater the flow rate of the second sheath flow pipeline 42, the closer the cell population in the sample flow will be to the sorting channel 50, so as to make the cell population arranged in a straight line more likely to enter the sorting inlet 52.

[0036] Furthermore, the ratio of the flow rate of the sorting inlet 52 to that of the main channel 20 is 0.3 - 0.6; the pressure of the optical fiber pressure device 30 is 10PN - 12PN.

[0037] It can be understood that, please refer to Figure 3, the present invention is described with two sorting inlets 52. By setting the flow rate of the sorting inlets 52 to increase sequentially from the front to the back, it can be seen that the particle group with a smaller size range will enter the first sorting inlet 52 first, and the particle group with a larger size range will continue to move forward and enter the second sorting inlet 52. With the cooperation of the optical fiber pressure device 30, the adjustment of the particle group can be made more precise. Preferably, the pressure of the optical fiber pressure device 30 gradually increases from the front to the back, so as to further improve the accuracy of the primary sorting. It should be noted that the ratio of the flow rate of the sorting inlet 52 to the flow rate of the main channel 20 can be adjusted by the size of the sorting inlet 52.

[0038] Furthermore, the accommodating structure includes an accommodating portion 55 that is connected to one side of the straight portion 53 and is semicircularly arranged, and a blocking portion 56 that is respectively connected to the other side of the accommodating portion 55 and the straight portion 53. The accommodating portion 55 is used to accommodate single cells.

[0039] It can be understood that the size of the part where the accommodating portion 55 is connected to the blocking portion 56 needs to be at least larger than the smallest single cell, so that the accommodating portion 55 can accommodate single cells. When a single cell enters the accommodating portion 55, the blocking portion 56 will be blocked, so that the flow resistance of the accommodating structure is greater than that of the meandering portion 54, and the remaining cell groups will continue to flow towards the meandering portion 54 and wait to reach the accommodating structure again for the sorting of the next single cell.

[0040] Furthermore, the acoustic wave transducer assembly 60 includes a first acoustic wave transducer 61 and a second acoustic wave transducer 62 that are respectively arranged at both ends of the straight portion 53. The sine signals of the first acoustic wave transducer 61 and the second acoustic wave transducer 62 are the same, and the amplitude is 10V - 20V; the inclination angles of the first acoustic wave transducer 61 and the second acoustic wave transducer 62 are 15° - 30°.

[0041] It can be understood, please refer to Figure 4, due to the inverse piezoelectric effect, under the action of the first acoustic wave transducer 61 and the second acoustic wave transducer 62, surface acoustic waves will be generated, which will further cause the internal pressure of the sample flow to change, forming a periodically distributed pressure, and further causing the cell population in the sample flow to shift. The tilt angle in this embodiment can further enhance the degree of shift, enabling the cell population to move further towards the receiving structure. Since single cells with larger sizes have a greater degree of shift, the amplitudes of the sine signals of the first acoustic wave transducer 61 and the second acoustic wave transducer 62 are set to allow single cells with larger sizes to pass through. While single cells with smaller sizes have a smaller offset distance, enabling single cells with larger sizes to enter the receiving structure and the smaller-sized single cell population to enter the next receiving structure. By sequentially increasing the amplitudes of the sine signals of the first acoustic wave transducer 61 and the second acoustic wave transducer 62, all single cells can be sorted in descending order of size.

[0042] Further, the ratio of the flow rate of the receiving portion 55 to the flow rate of the nearest meandering portion 54 is 1.43 to 1.51.

[0043] It can be understood that by making the ratio of the flow rate of the receiving portion 55 to the flow rate of the nearest meandering portion 54 an optimal value, single cells can enter the receiving portion 55 better, avoiding the situation where the ratio is too small and single cells fail to enter the receiving portion 55, and also avoiding the situation where the ratio is too large and excessive single cells enter the receiving portion 55, resulting in sorting errors.

[0044] Further, 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 communicated with the sheath flow channel assembly, the sample flow inlet 21 is communicated with the starting point of the main channel 20, the waste liquid port 22 is communicated with the end point of the main channel 20, and the microfluidic chip is also provided with an air outlet 51 communicated with the sorting tube.

[0045] It can be understood that the above structures are all conventional setting structures in the microfluidic chip, used for introducing sheath flow, introducing sample flow, and collecting excess sample flow, etc., and will not be elaborated here.

[0046] In this embodiment, by selecting the values of the parameters in each component, the capture rate of the present invention for cell sorting is verified.

[0047]

[0048] It should be noted that the cell capture rate at each sorting inlet 52 and the cell capture rate at each accommodating structure are both based on more than 50 simulation experiments, and there are 10 accommodating structures in the simulation model in the first sorting channel 50 (only 5 are shown in the schematic diagram in the figure); the signal amplitudes of the first acoustic transducer and the second acoustic transducer increase successively in an arithmetic progression.

[0049] As can be seen from the above table, through the structure in the present invention, whether it is the first sorting at the sorting inlet 52 or the second sorting at the accommodating structure, the cell sorting accuracy rate can reach more than 90%, providing a good idea for those skilled in the art in the research and development of microfluidic chips.

[0050] As for the preparation method of the microfluidic chip, a conventional photolithography and development method can be used for preparation.

[0051] In summary, the cell sorting device in the above embodiments of the present invention can perform a first sorting on the cell population through the setting of the fiber optic pressure device and the sorting inlet flow rate, and can perform a second sorting on the cell population through the setting of the flow rate ratio of the accommodating part to the meandering part and the setting of the amplitude of the acoustic transducer, improving the cell sorting efficiency.

[0052] The second embodiment of the present invention also provides a flow cytometer, which has the cell sorting device as described in the first embodiment. It should be noted that basic components such as microscopes and oscilloscopes in the flow cytometer are all prior arts, and the present invention has not made improvements, so no description will be given here.

[0053] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0054] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended 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, 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 inclined toward the containing structure and arranged at both ends of the straight section, 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 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; The containing structure includes a containing part which is connected to one side of the straight part and is arranged in a semicircular shape, and a blocking part which is respectively connected to the containing part and the other side of the straight part, and the containing part is used to contain single cells; the sheath flow channel assembly includes a first sheath flow pipeline and a second sheath flow pipeline which are 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; the acoustic wave transducer assembly includes a first acoustic wave transducer and a second acoustic wave transducer which are respectively arranged at both ends of the straight part, the sinusoidal signals of the first acoustic wave transducer and the second acoustic wave transducer are consistent, and the amplitude is 10V~20V; the ratio of the flow of the containing part to the flow of the nearest winding part is 1.43~1.51; the ratio of the flow of the sorting inlet to the flow of the main channel is 0.3~0.6, and the ratio of the flow of the sorting inlet to the flow of the main channel is adjusted by the size of the sorting inlet.

2. The cell sorting device according to claim 1, characterized in that: The inclination angles of the first acoustic wave transducer and the second acoustic wave transducer are 15° to 30°.

3. 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.

4. 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.

5. A flow cytometer, characterized in that: A cell sorting device comprising the cell sorting device according to any one of claims 1 to 4.

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