WO2023142182A1

WO2023142182A1 - High-throughput cell micromanipulation device based on microfluidic chip, and control method

Info

Publication number: WO2023142182A1
Application number: PCT/CN2022/076519
Authority: WO
Inventors: 周鸣川; 郭祥雨
Original assignee: 浙江大学
Priority date: 2022-01-25
Filing date: 2022-02-16
Publication date: 2023-08-03
Also published as: CN114591827A; CN114591827B

Abstract

A high-throughput cell (2) micromanipulation device based on a microfluidic chip (42), and a control method. The device comprises a digital camera (37), a computer (38), a controller (39), a driver (40), the microfluidic chip (42), an injection device (41), micro plunger pumps (43), and an inverted microscope manipulation table (44); the micro plunger pumps (43) are electrically connected to the computer (38) by means of the driver (40) and the controller (39); five blind holes communicated with five micro plunger pumps (43) are formed in the microfluidic chip (42), a main channel (1) is formed inside the microfluidic chip (42), three channels are formed on one side of the main channel (1), and a conical hole (6) is formed on the other side of the main channel (1); the injection device (41) comprises an injection needle (12), an inner connector (14), an outer connector (15), a piezoelectric element (16), and a retaining rod (17), and the injection needle (12) on the injection device (41) is wrapped by an elastic material (13), embedded in the conical hole (6), and used for injecting a cell (2). According to the device, closed-loop control is formed by means of the digital camera (37) and a stepping motor (36), assembly line-type automatic manipulation for the cell (2) is achieved, the labor of manual manipulation is reduced, the manipulation efficiency and quality are improved, and animal and plant breeding efficiency and scientific research are accelerated.

Description

Microfluidic chip-based high-throughput cell micromanipulation device and control method

technical field

The invention belongs to a microfluidic chip, and in particular relates to a microfluidic chip-based high-throughput cell micromanipulation device and a control method, which realize the adjustment of the two-dimensional posture of the cell.

Background technique

Cell micromanipulation technology is an important means of cell research at the microscopic scale, and has been widely used in animal and plant breeding, biomedical research and other fields. Traditional cell operations require experienced operators to complete the operation. During the operation, the microscope lens must be frequently switched, and the suction needle and the injection needle must be coordinated to complete the cell operation. It is labor-intensive. Long-term operation makes people tired, and the consistency of the results cannot be guaranteed. In addition, manual operation can only realize the operation of a single cell, but cannot realize the batch processing of cells, which restricts the efficiency of cell operation. Although optical tweezers, electric tweezers and other operating methods can also achieve single-cell manipulation, these methods will cause physical damage or potential physiological damage to cells.

Contents of the invention

In order to solve the problems in the background technology, the present invention provides a high-throughput cell micromanipulation device and control method based on a microfluidic chip. Position and posture, realize the continuous pipeline operation of cells, and alleviate the problems of low operation efficiency and cell damage caused by manual adjustment for a long time.

The technical scheme that the present invention adopts is as follows:

1. A high-throughput cell micromanipulation device based on a microfluidic chip

Including inverted microscope operating table, microfluidic chip, six micro plunger pumps, injection device, driver, controller and computer;

The microfluidic chip is placed on the mobile stage on the operating table of the inverted microscope, and the stage and the moving parts are adjusted at the same time, so that the objective lens directly below the mobile stage is aligned with the microfluidic chip; there is a microfluidic chip on the microfluidic chip. A U-shaped main channel arranged horizontally and five blind holes arranged vertically. The side is provided with three passages respectively connected with the bottom of the three blind holes in the middle: the first passage, the second passage and the third passage, and a tapered hole is arranged on the other side in the middle of the main passage; the three passages are connected with the main passage. The communication position forms an arc surface that bends toward the main channel, and the arc surface is arranged opposite to the tapered hole.

A moving part is installed on one side of the moving stage, and the moving part is provided with an injection needle holder for clamping the injection device. The injection needle of the injection device is aimed at the tapered hole of the microfluidic chip, and the injection needle is parallel to the moving carrier. The object table is arranged, and the injection needle is pushed by the piezoelectric block for injection.

Each micropiston pump includes a stepping motor, a ball screw, and a microinjection tube. Both the stepping motor and the microinjection tube are mounted on a fixed plate through a support frame, and the screw of the ball screw is connected to the output shaft of the stepping motor. The nut of the ball screw is connected with the piston rod of the microinjection tube; the stepper motor of each micropiston pump is connected with the driver, and the driver is connected to the computer through the controller.

The water injection hole of the injection device communicates with the nozzle of the micro injection tube of one of the micro plunger pumps, and the nozzles of the micro injection tubes of the other five micro plunger pumps communicate with the tops of the five blind holes respectively.

The injection device is mainly composed of an injection needle, an inner connector, an outer connector, a piezoelectric element, and a support rod; a threaded hole is opened on the front end of the outer connector, and an inner hole that communicates with the threaded hole and does not penetrate the outer connector is opened in the middle. Cavity, above the inner cavity, there is a water injection hole that communicates with the inner cavity and protrudes from the top of the outer connector; the rear end of the outer connector is provided with a boss; the rear end of the inner connector is connected with the threaded hole of the outer connector through threads, and There are front and rear through holes for fixing the injection needle. The injection head at the front of the injection needle protrudes out of the through hole and enters the tapered hole of the microfluidic chip. The gap between the injection head and the tapered hole is filled with elasticity to seal the gap between the two. Material: the end of the injection needle passes through the threaded hole and communicates with the inner cavity, and a tapered rubber ring clamping the injection needle is embedded between the inner cavity and the end of the injection needle, and the injection needle and the inner cavity are kept sealed by the tapered rubber ring , The tapered rubber ring firmly binds the injection needle in the inner cavity of the outer joint under the extrusion of the inner joint.

The piezoelectric element includes a piezoelectric shell, a piezoelectric block, and a rear end cover. The boss of the external connector extends into the card slot opened at the front of the piezoelectric shell, and a connecting boss and a card are embedded between the boss and the card slot. The elastic washer of the groove; the piezoelectric block is installed in the middle of the piezoelectric shell, and the wire connected to the piezoelectric block passes through the piezoelectric shell and is connected to the driver; The rear end cover installed at the end is provided with a bump corresponding to the position of the piezoelectric block, and the piezoelectric block is squeezed by the bump to make the front end of the piezoelectric block fit into the groove; one end of the holding rod is connected to the rear end cover of the piezoelectric element, The other end is fixed on the injection needle holder.

A digital camera connected to the computer is installed on the microscope operator, and the digital camera transmits the captured cell image information to the computer; the microscope operator is equipped with a focusing handwheel for adjusting the clarity of the field of view.

The first channel, the second channel and the third channel are arranged in parallel, the second channel is located in the center of the arc surface and facing the tapered hole, the second channel and the third channel are distributed on both sides of the second channel; the diameter of the arc surface is larger than the cells to be injected diameter, and less than 1.2 times the diameter of the cells to be injected.

The blind hole connected to the input end of the main channel is the fifth blind hole, the blind hole connected to the output end of the main channel is the first blind hole, and the blind holes connected to the first channel, the second channel and the third channel are respectively the second blind hole. Blind hole, third blind hole and fourth blind hole.

The microinjection tube is adapted to the diameter of the first blind hole, and the diameter of the first blind hole is 0.75 mm; the diameter of the main channel is 1.2 to 1.5 times the diameter of the cells to be injected, and the diameter of the second channel is the diameter of the cells to be injected One-fifth of the diameter of the first channel and the third channel are two-fifths of the diameter of the cells to be injected.

The core of the controller is a single-chip microcomputer, which adjusts the flow rate of the micro-piston pump through the PID control method, thereby controlling the flow rate of the internal channel of the microfluidic chip and realizing the adjustment of the cell posture.

The microfluidic chip integrates the functions of cell transport, cell holding, cell rotation, cell injection and cell release.

2. Control method of high-throughput cell micromanipulation device based on microfluidic chip

Include the following steps:

Step 1: Install the device on the micromanipulation bench, and adjust the mobile stage, moving parts, objective lens and focusing handwheel to make the cell imaging through the main channel of the microfluidic chip the clearest;

Step 2) The micro plunger pump connected to the fifth blind hole is loaded with half the capacity of the cell suspension, and an appropriate amount of cell viability dye is added to the cell suspension, which can stain the cells and facilitate the detection and identification of the cells; and the first blind hole The three micro plunger pumps connected to the second blind hole and the fourth blind hole respectively are loaded with half capacity of pure liquid; the micro plunger pump connected to the third blind hole is not loaded with liquid. The plunger pump is connected with the injection needle; the micro plunger pump connected with the syringe is loaded with half the volume of exogenous substances;

Step 3: The micro plunger pump connected to the first blind hole and the fifth blind hole is started at the same time, and the cell suspension is injected into the main channel at a constant speed. The computer processes the image taken by the digital camera. When it is detected that the cells entering the main channel are located in the arc When facing the corresponding position, the computer stops the micro plunger pump connected with the first blind hole and the fifth blind hole through the controller, starts the micro plunger pump connected with the third blind hole, and passes through the The second channel adsorbs the cells to fix the cells; after the cells are fixed, the micro plunger pump connected to the second blind hole and the fourth blind hole starts simultaneously: the micro plunger pump injects fluid through the first channel and absorbs fluid through the third channel , so that the cells rotate forward without moving; on the contrary, the micro plunger pump injects fluid through the third channel and absorbs fluid through the first channel, so that the cells rotate in the reverse direction without moving; the cells are adjusted from the current posture to the target posture;

Step 4: When the computer detects that the cell has adjusted to the target posture, the micro plunger pump connected to the second blind hole and the fourth blind hole stops; the controller controls the piezoelectric element in the injection device to be energized through the driver, and the piezoelectric block becomes The long-driven injection needle pierces the cell through the tapered hole, and the micro plunger pump connected to the injection needle starts to inject the foreign substance into the cell. After the injection time reaches the preset time, the micro plunger pump connected to the injection needle stops. When the piezoelectric element is powered off, the piezoelectric block returns to its original length, the elastic material and the elastic washer reset and drive the injection needle to be pulled out from the inside of the cell; the micro plunger pump connected to the third blind hole resets to release the cell, and at the same time starts the injection with the third blind hole. A micro plunger pump connecting the first blind hole and the fifth blind hole moves the injected cells towards the first blind hole;

Step 5: Repeat steps 3) to 4) to achieve continuous cell injection.

The beneficial effects of the present invention are:

The present invention realizes the pipeline operation of cells in a closed space by designing a microfluidic chip. On the premise of not destroying the cells, the flow rate of the internal channel is controlled by adjusting the flow rate of the micro plunger pump to adjust the cell posture and position, which not only reduces the The labor load of manual operation can be improved, the quality and efficiency of cell operation can be improved, and the whole process of cell operation can be controlled.

Description of drawings

Fig. 1 is the structural representation of device of the present invention;

Fig. 2 is the micropiston pump schematic diagram of device of the present invention;

Fig. 3 is the structural representation of the inverted microscope of device of the present invention;

Fig. 4 is the structural diagram of the injection device of the device of the present invention;

5 is a structural diagram of the microfluidic chip and the main channel of the device of the present invention;

Fig. 6 is a channel layout diagram of the microfluidic chip of the device of the present invention;

Figure 7 is a schematic diagram of the operation strategy of the microfluidic chip, (a) is a schematic diagram of cell transport, (b) is a schematic diagram of cell holding, (c) is a schematic diagram of cell rotation, (d) is a schematic diagram of cell injection, (e) is a schematic diagram of cell release schematic.

Fig. 8 is a control flowchart of the device of the present invention.

In the figure: 1. main channel, 2. cell, 3. first channel, 4. second channel, 5. third channel, 6. tapered hole, 7. first blind hole, 8. second blind hole, 9. Third blind hole, 10. Fourth blind hole, 11. Fifth blind hole, 12. Injection needle, 13. Elastic material, 14. Inner connector, 15. Outer connector, 16. Piezoelectric element, 17 , Reinforcing rod, 18, Card slot, 19, Tapered rubber ring, 20, Inner hole, 21, Water injection hole, 22, Elastic washer, 23, Boss, 24, Electric wire, 25, Piezoelectric block, 26, Rear end Cover, 27, light source regulator, 28, moving parts, 29, injection needle holder, 30, objective lens, 31, moving stage, 32, focusing handwheel, 33, fixed plate, 34, microinjection tube, 35. Ball screw, 36. Stepper motor, 37. Digital camera, 38. Computer, 39. Controller, 40. Driver, 41. Injection device, 42. Microfluidic chip, 43. Micro plunger pump, 44 , Inverted microscope console.

Detailed ways

The present invention will be further described below in conjunction with drawings and embodiments.

As shown in Figure 1, the device of the present invention comprises a digital camera 37, a computer 38, a controller 39, a driver 40, an injection device 41, a microfluidic chip 42, six micropiston pumps 43 and an inverted microscope console 44; The device 41 and the microfluidic chip 42 are installed on an inverted microscope console 44, the microfluidic chip 43 is respectively connected to the injection device 41 and five micropiston pumps 43, and the stepping motors 36 of all micropiston pumps 43 are driven 40, the controller 39, the computer 38 and the digital camera 37 are electrically connected in sequence.

As shown in Figure 2, each micropiston pump 43 comprises a stepper motor 36, a ball screw 35, a microinjection tube 34 and a fixed plate 33, the output shaft of the stepper motor 36 is connected with the input shaft of the ball screw 35, and the ball screw The output shaft of the rod 35 is connected with the microinjection tube 34 and installed on the fixed plate 33; the microfluidic chip 42 is provided with a first blind hole 7, a second blind hole 8, a third blind hole 9, and a fourth blind hole with the same depth. The blind hole 10 and the fifth blind hole 11 are respectively connected with five micro plunger pumps 43, with a tapered hole 6 on the side, and a main channel 1 inside the microfluidic chip 42, and on the side of the main channel 1 There are first channel 3, second channel 4, and third channel 5;

As shown in Figure 3, the inverted microscope console 44 includes a light source adjuster 27, a moving part 28, an injection needle holder 29, an objective lens 30, a movable stage 31, and a focusing handwheel 32, and is located on the movable stage 31. The objective lens 30 directly below faces the cells passing through the main channel of the microfluidic chip 42 , and the digital camera 37 is installed on the inverted micromanipulator 44 .

As shown in Figure 4, the injection device 41 includes an injection needle 12, an inner connector 14, an outer connector 15, a piezoelectric element 16, and a support rod 17, and the outer connector 15 includes an inner cavity 20, a water injection hole 21, an elastic washer 22, a convex Platform 23, piezoelectric element 16 includes card slot 18, electric wire 24, piezoelectric block 25, rear end cover 26, injection needle 12 passes through the inner cavity of inner connector 14, and the end of injection needle 12 is nested with tapered rubber ring 19, and the inner The joint 14 and the outer joint 15 are connected by threads, and the conical rubber ring 19 is squeezed by the inner joint 14 to firmly bind the injection needle in the inner cavity 20 of the outer joint 15, and the outer joint 15 has a water injection hole 21, It is connected with the sixth micro-piston pump 28; the boss 23 at the end of the outer joint 15 is equipped with an elastic washer 22, and the card slot 18 in the piezoelectric element 16 locks the boss 23, and the piezoelectric block 25 is assembled with the recess of the boss 23. In the groove, the center of the piezoelectric block 25 is aligned with the rear end cover 26 and squeezed tightly.

As shown in Figure 5, the main channel 1 in the microfluidic chip 42 communicates with the first channel 3, the second channel 4, the third channel 5, and the tapered hole 6, and the first blind hole 7 and the fifth blind hole 11 communicate with the main channel. Channel 1 communicates, the second blind hole 8 communicates with the first channel 3, the third blind hole 9 communicates with the first channel 4, the fourth blind hole 10 communicates with the first channel 5, and the tapered hole 6 communicates with the first channel 3, the second The channel 4 and the third channel 5 are not on the same side, and the position of the second channel 4 is directly opposite to the position of the tapered hole 6 .

As shown in Figure 6, the communication positions of the first channel 3, the second channel 4, the third channel 5 and the main channel 1 form an arc surface curved toward the main channel 1, the first channel 3, the second channel 4, the third channel 5 are arranged parallel to each other, the second channel 4 is in the center of the arc surface, the first channel 3 and the third channel 5 are distributed on both sides of the second channel 4, and are coplanar with the equator of the arc surface, and the diameter of the arc surface is larger than the cells to be operated The diameter is less than 1.2 times the diameter of the cell to be manipulated.

The core of the controller 25 is a single-chip microcomputer, and adjusts the flow rate of the micro plunger pump 28 through the PID control method, thereby controlling the flow rate of the internal channel of the microfluidic chip 27 and realizing the adjustment of the cell posture.

As shown in Figure 7, the working principle and steps of the present invention are as follows:

1) Install the device of the present invention on a micromanipulation experiment bench, move the stage 31, the objective lens 30 and adjust the focusing handwheel 32 until the image of the cell 2 in the microfluidic chip 42 is the clearest.

2) The micro plunger pump 43 is pre-loaded with a half-capacity specific solution, and the micro plunger pump 43 connected to the fifth blind hole 11 is loaded with a half-capacity cell suspension, and the first blind hole 7, the second blind hole 8, The micropiston pump 43 connected to the fourth blind hole 10 is loaded with half capacity of pure liquid, and the micropiston pump 28 connected to the third blind hole 9 is not loaded with any liquid, and the internal air is completely excluded. The micropiston pump 43 and The water injection hole 21 on the injection device 41 is connected.

3) The injection needle 12 is used in conjunction with the elastic material 13, embedded in the tapered hole 6 to ensure that the tapered hole 6 does not leak, the injection device 41 is installed on the injection holder 29, and the electric wire 24 connected to the piezoelectric block 25 is connected to the driver 40 .

4) The six micropiston pumps 43 are electrically connected to the driver 40, the controller 39, the computer 38 and the digital camera 37 in sequence, and further checks are made to ensure that the device can work normally.

5) Cell delivery: start the micro plunger pump 43 connected to the first blind hole 7 and the fifth blind hole 11, and inject the cell suspension into the internal main channel 1 at a constant speed.

6) Cell holding: the computer 38 processes the image taken by the digital camera 37, and when it is detected that the cell 2 is located at the corresponding position of the second channel 4, the computer 38 makes the first blind hole 7 and the fifth blind hole 11 through the controller 39 The connected micropiston pump 43 stops moving, and the micropiston pump 43 connected to the third blind hole 9 starts to generate a constant negative pressure to adsorb cells and fix the cells.

7) Cell rotation: the micropiston pump 43 communicating with the second blind hole 8 and the fourth blind hole 10 starts simultaneously, the micropiston pump 43 communicating with the second blind hole 8 injects fluid, and communicates with the fourth blind hole 10 When the micro plunger pump 43 sucks the fluid, the cells rotate clockwise without moving, or the micro plunger pump 43 connected to the fourth blind hole 10 ejects the fluid, and the micro plunger pump 43 connected to the second blind hole 8 When the fluid is sucked, the cells only rotate without moving, and the cells adjust from the current posture to the target posture, realizing the adjustment of the two-dimensional posture of the cells.

8) Cell injection: when the computer 38 detects that the cells are adjusted to the target posture, the micro plunger pump 43 connected to the second blind hole 8 and the fourth blind hole 10 stops; the controller 39 controls the injection device 41 through the driver 40 The piezoelectric element 16 is energized, the piezoelectric block 25 becomes longer and drives the injection needle 12 to pierce the cell 2 through the tapered hole 6, and the micro plunger pump 43 connected to the injection needle 12 starts to inject the foreign substance into the cell 2. The injection time After the preset time is reached, the micro plunger pump 43 connected to the injection needle 12 stops, the piezoelectric element 16 is powered off, the piezoelectric block 25 returns to its original length, the elastic material 13 and the elastic washer 22 reset and drive the injection needle 12 from the cell 2 Internally unplugged.

9) Cell release: the micro plunger pump 43 connected to the third blind hole 9 is reset to release the cells, the micro plunger pump 43 connected to the third blind hole 9 is reset to release the cells, and at the same time start the connection with the first blind hole 7 is communicated with the fifth blind hole 11 and the micro plunger pump 43 moves the injected cells towards the first blind hole 7 to complete cell injection.

10) Repeat the above steps 4)-9) to realize continuous cell operation.

Claims

A high-throughput cell micromanipulation device based on a microfluidic chip, characterized in that it includes an inverted microscope console (44), six micro plunger pumps (43), a microfluidic chip (42), and an injection device (41), driver (40), controller (39) and computer (38);

The microfluidic chip (42) is placed on the movable stage (31) in the middle of the microscope operating table (44), and the objective lens (30) directly below the movable stage (31) is aligned with the microfluidic chip (42); The microfluidic chip (42) is provided with a horizontally arranged U-shaped main channel (1) and five blind holes arranged vertically. The bottoms of the two blind holes are connected; the microfluidic chip (42) is provided with three channels on one side in the middle of the main channel (1): the first channel (3), the second channel (4), and the third channel (5), One end of the three channels communicates with the bottoms of the three blind holes in the middle, and the other end communicates with the main channel (1); on the other side in the middle of the main channel (1), there is a tapered hole communicating with the main channel (1) (6); The communication positions of the three passages and the main passage (1) form an arc surface curved toward the main passage (1), and the arc surface is arranged opposite to the tapered hole (6);

One side of the moving stage (31) is equipped with a moving part (28), and the moving part (28) is provided with an injection needle holder (29) for clamping the injection device (41). The injection of the injection device (41) The needle (12) is aligned with the tapered hole (6) of the microfluidic chip (42), and the injection needle (12) is arranged parallel to the moving stage (31), and the injection needle (12) is pushed by the piezoelectric block (25). )injection;

Each micropiston pump (43) comprises a stepper motor (36), a ball screw (35), a microinjection tube (34), and the stepper motor (36) and the microinjection tube (34) are all installed on the On the fixed plate (33), the screw rod of the ball screw (35) is connected with the output shaft of the stepper motor (36), and the nut of the ball screw (35) is connected with the piston rod of the microinjection tube (34); The stepping motor (36) of plunger pump (43) is all connected with driver (40), and driver (40) is connected to computer (38) through controller (39);

The water injection hole (21) of the injection device (41) is communicated with the microinjection tube (34) nozzle of one of the micropiston pumps (43), and the microinjection tubes (34) of the other five micropiston pumps (43) are connected to each other. The ports communicate with the tops of the five blind holes respectively.
A high-throughput cell micromanipulation device based on a microfluidic chip according to claim 1, characterized in that: the injection device (41) is mainly composed of an injection needle (12), an inner connector (14), Composed of an external connector (15), a piezoelectric element (16), and a support rod (17);

The front end of the outer connector (15) is provided with a threaded hole, and the middle is provided with an inner chamber (20) which communicates with the threaded hole and does not pass through the outer connector (15). A water injection hole (21) that communicates and protrudes from the top of the outer connector (15); the rear end of the outer connector (15) is provided with a boss;

The rear end of the inner connector (14) is threadedly connected to the threaded hole of the outer connector (15), and there are front and rear through holes for fixing the injection needle (12) in the middle. Into the tapered hole (6) of the microfluidic chip (42), the elastic material (13) that seals the gap between the two is filled between the injection head and the tapered hole; the end of the injection needle (12) passes through the threaded hole It communicates with the inner cavity (20), and a tapered rubber ring (19) for clamping the injection needle (12) is embedded between the inner cavity (20) and the end of the injection needle (12). Sealing is maintained between the injection needle (12) and the inner chamber (20);

The piezoelectric element (16) comprises a piezoelectric housing, a piezoelectric block (25), a rear end cover (26), and the boss (23) of the external connector (15) stretches into the slot (18) provided at the front of the piezoelectric housing. ), an elastic washer (22) is embedded between the boss (23) and the slot (18); a piezoelectric block (25) is installed in the middle of the piezoelectric shell, and the electric wire (24) connected to the piezoelectric block (25) ) is connected with the driver (40) after passing through the piezoelectric shell; the rear end surface of the boss (23) is provided with a groove for assembling the piezoelectric block (25), and the rear end cover (26) installed at the rear end of the piezoelectric shell is provided with The bump corresponding to the position of the piezoelectric block (25) squeezes the piezoelectric block (25) through the bump so that the front end surface of the piezoelectric block (25) fits into the groove;

One end of the reinforcing rod (17) is connected with the rear end cover (26) of the piezoelectric element (16), and the other end is fixed on the injection needle holder (29).
A high-throughput cell micromanipulation device based on a microfluidic chip according to claim 1, characterized in that: a digital camera (37) connected to a computer (38) is installed on the microscope console (44), The digital camera (37) transmits the captured cell image information to the computer (38); the microscope console (45) is equipped with a focusing handwheel (32) for adjusting the clarity of the field of view.
A high-throughput cell micromanipulation device based on a microfluidic chip according to claim 1, characterized in that: the first channel (3), the second channel (4) and the third channel (5) are arranged in parallel , the second channel (4) is located in the center of the arc surface and facing the tapered hole, the second channel (4) and the third channel (5) are distributed on both sides of the second channel (4); the diameter of the arc surface is larger than the diameter of the cells to be injected , and less than 1.2 times the diameter of the cells to be injected.
A high-throughput cell micromanipulation device based on a microfluidic chip according to claim 1, characterized in that: the blind hole communicated with the input end of the main channel (1) is the fifth blind hole (11), and The blind hole connected to the output end of the main channel (1) is the first blind hole (7), and the blind holes connected to the first channel (3), the second channel (4) and the third channel (5) are respectively the second blind hole. hole (8), the third blind hole (9) and the fourth blind hole (10).
A high-throughput cell micromanipulation device based on a microfluidic chip according to claim 5, characterized in that: the microinjection tube (34) is adapted to the diameter of the first blind hole (7); The diameter of the channel (1) is 1.2 to 1.5 times the diameter of the cells to be injected, the diameter of the second channel (4) is one-fifth of the diameter of the cells to be injected, and the diameter of the first channel (3) and the third channel (5) The diameter is two-fifths the diameter of the cells to be injected.
A high-throughput cell micromanipulation device based on a microfluidic chip according to claim 1, characterized in that: the core of the controller (39) is a single-chip microcomputer, and the micro plunger pump (44) is adjusted by a PID control method flow, so as to control the flow rate of the internal channel of the microfluidic chip (43), and realize the adjustment of cell attitude.
The control method using the device described in any one of claims 1 to 7, characterized in that: comprising the following steps:

Step 1) Install the device on the micromanipulation bench, and image the cells passing through the main channel of the microfluidic chip (42) by adjusting the mobile stage (31), objective lens (30) and focusing handwheel (32) clearest;

Step 2) The micropiston pump (44) that is communicated with the fifth blind hole (11) is loaded with half-capacity cell suspension, and cell viability dye is added in the cell suspension; and the first blind hole (7), the second blind hole Hole (8), the three micropiston pumps (43) that the 4th blind hole (10) communicates respectively all load the pure liquid of half capacity; The micropiston pump (43) that communicates with the 3rd blind hole (9) does not After loading the liquid and completely removing the internal air, connect the micro plunger pump (43) to the water injection hole (21) on the injection device (41); the micro plunger pump (43) connected to the injection device (41) is loaded with half capacity foreign substances;

Step 3) Cell transport: the micro plunger pump (43) connected to the first blind hole (7) and the fifth blind hole (11) is started simultaneously, and the cell suspension is injected into the main channel (1) at a uniform speed;

Step 4) cell holding: the computer (38) processes the image taken by the digital camera (37), and when it is detected that the cell (2) entering the main channel is located at the corresponding position on the arc surface, the computer (38) passes the controller ( 39) Make the micropiston pump (43) communicated with the first blind hole (7) and the fifth blind hole (11) stop moving, and the micropiston pump (43) communicated with the 3rd blind hole (9) start, After generating a constant negative pressure, the cells are adsorbed through the second channel (4) to fix the cells;

Step 5) cell rotation: after the cells are fixed, the micro plunger pump (43) communicated with the second blind hole (8) and the fourth blind hole (10) starts simultaneously: the micro plunger pump (43) passes through the first channel ( 3) Inject fluid, absorb fluid through the third channel (5), make the cells rotate forward without moving; otherwise, the micro plunger pump (43) inject fluid through the third channel (5), and pass through the first channel (3) Suction the fluid to make the cell reverse rotation without moving; make the cell adjust from the current posture to the target posture;

Step 6) cell injection: after the computer (38) detects that the cell is adjusted to the target posture, the micro plunger pump (43) connected to the second blind hole (8) and the fourth blind hole (10) stops; the controller ( 39) Control the piezoelectric block (25) in the injection device (41) to be energized through the driver (40), and the piezoelectric block (25) is extended to push the boss on the outer connector (15) to move forward, thereby driving the injection needle ( 12) Puncture the cell (2) through the tapered hole (6), and the micro plunger pump (43) connected to the injection needle (12) starts to inject the foreign substance into the cell (2), and the injection time reaches the preset time. After the setting time, the micro plunger pump (43) connected to the injection needle (12) stops, the control piezoelectric block (25) is powered off, the piezoelectric block (25) recovers the original length, and the elastic material (13) and the elastic washer ( 22) Reset under the action of elastic force and drive the injection needle (12) to be pulled out from the cell (2);

Step 8) cell release: reset the micro plunger pump (43) communicated with the third blind hole (9), release the cells, and start the micro plunger pump communicated with the first blind hole (7) and the fifth blind hole (11) simultaneously. The plunger pump (43) moves the injected cells towards the first blind hole (7);

Step 9) Repeat steps 3) to 8) to realize continuous cell injection.
[Corrected under Rule 26 16.03.2022]
The control method according to claim 8, characterized in that the microfluidic chip (42) integrates the functions of cell transport, cell holding, cell rotation, cell injection, and cell release.