阅读说明：本技术 一种复合脉冲细胞电融仪和控制方法 (Composite pulse cell electrofusion instrument and control method ) 是由 柯强 沈婷 李新皓 于 2021-09-03 设计创作，主要内容包括：本发明公开了一种复合脉冲细胞电融仪和控制方法,包括保护盒、电路控制组件和定位组件,电路控制组件包括电源盒、高频高压正弦模块、微秒脉冲模块和纳秒脉冲模块；定位组件设置在固定仓的底部,定位组件包括两个第一固定块和两个第二固定块,两个第一固定块和两个第二固定块正面的中部穿插连接有双向螺杆,两个第一固定块的两侧均设置有控制机构,两个第二固定块的两侧均设置有夹定机构。本发明的有益效果是：本发明利用了纳秒脉冲能够对不同尺寸的细胞均能进行穿孔的特性,能很好的融合不同尺寸的细胞；利用了微秒脉冲使细胞在纳秒脉冲作用下产生的穿孔继续进一步扩大,便于细胞的融合,有效地提高了细胞融合的成功率。(The invention discloses a composite pulse cell electrofusion instrument and a control method, wherein the composite pulse cell electrofusion instrument comprises a protection box, a circuit control assembly and a positioning assembly, wherein the circuit control assembly comprises a power supply box, a high-frequency high-voltage sine module, a microsecond pulse module and a nanosecond pulse module; the positioning assembly is arranged at the bottom of the fixed bin and comprises two first fixed blocks and two second fixed blocks, the two positive middle parts of the two first fixed blocks and the two second fixed blocks are alternately connected with a bidirectional screw rod, the two sides of the two first fixed blocks are provided with control mechanisms, and the two sides of the two second fixed blocks are provided with clamping mechanisms. The invention has the beneficial effects that: the invention utilizes the characteristic that nanosecond pulse can perforate cells with different sizes, and can well fuse the cells with different sizes; the microsecond pulse is utilized to ensure that the perforation generated by the cells under the action of the nanosecond pulse is continuously and further expanded, thereby facilitating the fusion of the cells and effectively improving the success rate of the cell fusion.)

1. A composite pulse cell electrofusion apparatus, comprising:

the protective box comprises a protective box (1), wherein a packaging cover (101) is arranged at the top end of the protective box (1), and a fixed bin is arranged inside the protective box (1);

the circuit control assembly comprises a power box (10), a high-frequency high-voltage sine module (9), a microsecond pulse module (8) and a nanosecond pulse module (7), wherein the power box (10), the high-frequency high-voltage sine module (9), the microsecond pulse module (8) and the nanosecond pulse module (7) are electrically connected in sequence;

locating component, locating component sets up the bottom in fixed storehouse, locating component includes two first fixed blocks (2) and two second fixed blocks (4), two second fixed block (4) set up between two first fixed blocks (2), two first fixed block (2) and two positive middle parts of second fixed block (4) alternate and are connected with two-way screw rod (6), two the both sides of first fixed block (2) all are provided with control mechanism, two the both sides of second fixed block (4) all are provided with presss from both sides fixed establishment.

2. The composite pulse cell electrofusion apparatus according to claim 1, wherein: the combined groove has been seted up on the top of protection box (1) both sides wall, both sides fixedly connected with compoboard (102) of encapsulation lid (101) bottom, two compoboard (102) and two combined groove joints, two direction spout (103) have been seted up to the inner wall of fixed storehouse bottom both sides, wherein one side of protection box (1), and run through the fixed storehouse and seted up viewing aperture and a plurality of control hole, the opposite side of protection box (1), and run through the fixed storehouse and seted up a plurality of wiring mouths, the front of protection box (1), and run through the fixed storehouse and seted up the regulation hole.

3. The composite pulse cell electrofusion apparatus according to claim 1, wherein: two equal fixedly connected with cardboard (201) in one side that first fixed block (2) carried on the back mutually, two the regulation screw has all been seted up at the positive middle part of first fixed block (2) and two cardboard (201), two-way screw rod (6) and four regulation screw thread alternate connection, two the equal fixedly connected with in top of cardboard (201) barrier plate (202).

4. The composite pulse cell electrofusion apparatus according to claim 1, wherein: two the rotation mouth has all been seted up to the both sides of second fixed block (4), two the interlude hole has all been seted up at the positive middle part of second fixed block (4), two-way screw rod (6) and two interlude hole sliding connection, one of them one end of two-way screw rod (6) is provided with turning block (601), just turning block (601) run through the regulation hole and set up in the front of protection box (1).

5. The composite pulse cell electrofusion apparatus according to claim 1, wherein: clamping mechanism includes two backup pads (401), two backup pad (401) and one of them direction spout (103) slip interlude are connected, two bullport (402) have all been seted up in the front of backup pad (401), two be provided with swinging arms (5) between backup pad (401), two it has the rotary rod to rotate to alternate between the top of backup pad (401) and swinging arms (5), one side fixedly connected with clamping plate (501) at swinging arms (5) top.

6. The composite pulse cell electrofusion apparatus according to claim 1, wherein: the control mechanism comprises a control block (3), the control block (3) is connected with one of the guide sliding grooves (103) in a sliding and penetrating mode, a control insertion rod (301) is fixedly connected to the back face of the control block (3), and the control insertion rod (301) is connected with a guide hole (402) in a sliding and penetrating mode.

7. The composite pulse cell electrofusion apparatus according to claim 1, wherein: the back surfaces of the high-frequency high-voltage sine module (9), the microsecond pulse module (8) and the nanosecond pulse module (7) are respectively provided with a connecting pole column (11), the front surfaces of the power box (10), the high-frequency high-voltage sine module (9) and the microsecond pulse module (8) are respectively provided with two connecting holes (12), six connecting pole columns (11) and six connecting holes (12) are correspondingly inserted, and one side of the power box (10) is provided with a display screen (1001);

a third switch relay is arranged on one side of the high-frequency high-voltage sine module (9) and is set to be K3;

a second switching relay and a fifth switching relay are arranged on one side of the microsecond pulse module (8) and are respectively set to be K2 and K5;

one side of the nanosecond pulse module (7) is provided with a first switch relay and a fourth switch relay which are respectively set to K1 and K4.

8. A control method of a composite pulse cell electrofusion instrument is characterized in that: comprises one of the following working modes;

the first working mode comprises the following steps:

s1: setting the number of output pulses of the microsecond pulse module (8), the number of output pulses of the nanosecond pulse module (7) and the output time of the high-frequency high-voltage sine module (9);

s2: firstly, the controller controls the third switch relay K3 to be switched on, then controls the first switch relay K1, the second switch relay K2, the fourth switch relay K4 and the fifth switch relay K5 to be switched off, starts the high-frequency high-voltage sine module (9), applies sine alternating-current voltage to a culture dish containing cells and fusion liquid, and promotes the cells to form a queue and be tightly attached through the sine alternating-current voltage;

s3: when the output time of the sine alternating voltage reaches a set time value, the controller closes the third switch relay K3 to stop applying the sine alternating voltage to the culture dish;

s4: the first switch relay K1 is conducted through the controller, the nanosecond pulse module (7) is electrified to start to operate, and nanosecond square pulse waves are applied to the culture dish;

s5: after the pulse number of the nanosecond pulse module (7) reaches the set number, the controller controls to disconnect the first switch relay K1, and the nanosecond pulse module (7) stops applying nanosecond square pulse waves to the culture dish;

s6: controlling a second switch relay K2 to be conducted, electrifying a microsecond pulse module (8) to start to operate, and applying microsecond square pulse waves to the culture dish;

s7: after the pulse number of the microsecond pulse module (8) reaches the set number, the controller controls to turn off the second switch relay K2, and the microsecond pulse module (8) stops applying nanosecond square pulse waves to the culture dish;

s8: controlling the fourth switching relay K4 to be switched on, electrifying a discharging loop connected with the nanosecond pulse module (7), and starting discharging the nanosecond pulse module (7) through the first discharging resistor;

s9: the fifth switch relay K5 is controlled to be switched on, a discharging loop connected with the microsecond pulse module (8) is electrified, and the microsecond pulse module (8) starts to discharge through a second discharging resistor R2;

the second working mode comprises the following steps:

the first step is as follows: setting the number of output pulses of the microsecond pulse module (8), the number of output pulses of the nanosecond pulse module (7) and the output time of the high-frequency high-voltage sine module (9);

the second step is that: firstly, the controller controls the third switch relay K3 to be switched on, then controls the first switch relay K1, the second switch relay K2, the fourth switch relay K4 and the fifth switch relay K5 to be switched off, starts the high-frequency high-voltage sine module (9), applies sine alternating-current voltage to a culture dish containing cells and fusion liquid, and promotes the cells to form a queue and be tightly attached through the sine alternating-current voltage;

the third step: when the output time of the sine alternating voltage reaches a set time value, the controller closes the third switch relay K3 to stop applying the sine alternating voltage to the culture dish;

the fourth step: controlling a second switch relay K2 to be conducted, electrifying a microsecond pulse module (8) to start to operate, and applying microsecond square pulse waves to the culture dish;

the fifth step: after the pulse number of the microsecond pulse module (8) reaches the set number, the controller controls to turn off the second switch relay K2, and the microsecond pulse module (8) stops applying nanosecond square pulse waves to the culture dish;

and a sixth step: the first switch relay K1 is conducted through the controller, the nanosecond pulse module (7) is electrified to start to operate, and nanosecond square pulse waves are applied to the culture dish;

the seventh step: after the pulse number of the nanosecond pulse module (7) reaches the set number, the controller controls to disconnect the first switch relay K1, and the nanosecond pulse module (7) stops applying nanosecond square pulse waves to the culture dish;

eighth step: controlling the fourth switching relay K4 to be switched on, electrifying a discharging loop connected with the nanosecond pulse module (7), and starting discharging the nanosecond pulse module (7) through the first discharging resistor R1;

the ninth step: and controlling the fifth switch relay K5 to be switched on, electrifying a discharging loop connected with the microsecond pulse module (8), and starting discharging the microsecond pulse module (8) through a second discharging resistor R2.

9. The method for controlling a composite pulse cell thawing apparatus according to claim 8, wherein: the sine voltage output by the high-frequency high-voltage sine module (9) ranges from 0V to 500V, the frequency ranges from 0 MHz to 3MHz, and the duration time ranges from 0s to 100 s.

10. The method for controlling a composite pulse cell thawing apparatus according to claim 8, wherein: the nanosecond pulse module (7) outputs pulses with the amplitude of 0-10kV, the pulse width of 50-1000 ns, the pulse frequency of 0.1-1000 Hz, the number of the pulses is adjustable, and the waveform is a pulse square wave;

the microsecond pulse module (8) outputs pulses with the amplitude of 0-5kV, the pulse width of 1us-1000us, the pulse frequency of 0.1Hz-1000Hz, the number of the pulses is adjustable, and the waveform is pulse square waves.

Technical Field

The invention relates to a cell capacitance instrument, in particular to a composite pulse cell electric fusion instrument and a control method, and belongs to the technical field of cell fusion instruments.

Background

Cell fusion, also known as cell hybridization, refers to the process of forming a cell with binuclear or polynuclear nuclei from two or more cells by means of mediated and cultured, artificial fusion in an ex vivo condition. The sexual reproduction of nature ensures the stability of genetic materials, various barriers are arranged among species, and the artificial induction cell fusion technology can fuse single cells from different kinds of organisms into a heterokaryocyte, and the new cell contains genetic materials of a plurality of parent cells, has new genetic or biological characteristics, and can be cultured into new species, strains or new cell engineering products.

Cell fusion, a rapidly developing new cell engineering method, has achieved pioneering research results in the fields of agriculture, medicine, and the like, and the application field is expanding, becoming a conventional means for studying somatic reprogramming and gene localization. The following four methods are mainly used for cell fusion: viral fusion, cytochemical fusion, cytoelectrofusion and laser fusion of cells.

Compared with other cell fusion technologies, the cell electrofusion technology has the advantages of strong controllability, strong repeatability, high heterologous cell fusion efficiency, no toxicity to cells, wide application objects, low sample loss and the like, and gradually becomes the most important means for realizing cell fusion. The microsecond pulse electrofusion is adopted in the traditional cell electrofusion, but the microsecond pulse has certain defects, when cells with different sizes are fused, large cells can generate perforation earlier than small cells, so that under the condition of acting on an electric field with the same strength, when the small cells are perforated, the large cells can be in a death state of excessive perforation, and the problems of low success rate, high cost, low efficiency, poor repeatability and the like exist in the experimental process.

Disclosure of Invention

The present invention is directed to solving the above problems and providing a composite pulse cell thawing apparatus and a control method.

The invention achieves the above objects by the following technical scheme, a composite pulse cell electric melting instrument, comprising:

the top end of the protection box is provided with a packaging cover, and a fixed bin is arranged inside the protection box;

the circuit control assembly comprises a power box, a high-frequency high-voltage sine module, a microsecond pulse module and a nanosecond pulse module, wherein the power box, the high-frequency high-voltage sine module, the microsecond pulse module and the nanosecond pulse module are electrically connected in sequence;

locating component, locating component sets up the bottom in fixed storehouse, locating component includes two first fixed blocks and two second fixed blocks, two the second fixed block sets up between two first fixed blocks, two the positive middle part interlude of first fixed block and two second fixed blocks is connected with two-way screw rod, two the both sides of first fixed block all are provided with control mechanism, two the both sides of second fixed block all are provided with presss from both sides fixed establishment.

Preferably, the combination groove has been seted up on the top of protection box both sides wall, the both sides fixedly connected with compoboard, two of encapsulation lid bottom the combination board and two combination groove joints, two direction spouts have been seted up to the inner wall of fixed storehouse bottom both sides, one of them side of protection box, and run through fixed storehouse and seted up viewing aperture and a plurality of control hole, the opposite side of protection box just runs through fixed storehouse and has seted up a plurality of wiring mouths, the front of protection box just runs through fixed storehouse and has seted up the regulation hole.

Preferably, two equal fixedly connected with cardboard in one side that first fixed block carried on the back mutually, two the regulation screw has all been seted up at the positive middle part of first fixed block and two cardboards, two-way screw rod and four regulation screw thread interlude are connected, two the equal fixedly connected with barrier plate in top of cardboard.

Preferably, the two sides of the two second fixing blocks are provided with rotating ports, the two right middle parts of the second fixing blocks are provided with penetrating holes, the two-way screw rod and the two penetrating holes are connected in a sliding penetrating mode, one end of the two-way screw rod is provided with a rotating block, and the rotating block penetrates through the adjusting hole and is arranged on the right side of the protection box.

Preferably, the clamping mechanism comprises two supporting plates, two supporting plates and one of the guide sliding grooves are connected in a sliding and penetrating mode, guide holes are formed in the front faces of the supporting plates, swing rods are arranged between the supporting plates, the top of each supporting plate and the swing rods rotate to be connected with rotating rods in a penetrating mode, and one side of the top of each swing rod is fixedly connected with the clamping plate.

Preferably, the control mechanism comprises a control block, the control block is connected with one of the guide chutes in a sliding and penetrating manner, a control inserted rod is fixedly connected to the back of the control block, and the control inserted rod is connected with the guide hole in a sliding and penetrating manner.

Preferably, the back surfaces of the high-frequency high-voltage sine module, the microsecond pulse module and the nanosecond pulse module are provided with connecting poles, the front surfaces of the power box, the high-frequency high-voltage sine module and the microsecond pulse module are provided with two connecting holes, six connecting poles are correspondingly inserted into the six connecting holes, and one side of the power box is provided with a display screen;

a third switch relay is arranged on one side of the high-frequency high-voltage sine module and is set to be K3;

a second switching relay and a fifth switching relay are arranged on one side of the microsecond pulse module and are respectively set to be K2 and K5;

one side of nanosecond pulse module is provided with first switching relay and fourth switching relay, and sets up to K1 and K4 respectively.

A control method of a composite pulse cell electrofusion instrument comprises one of the following working modes;

the first working mode comprises the following steps:

s1: setting the number of output pulses of the microsecond pulse module, the number of output pulses of the nanosecond pulse module and the output time of the high-frequency high-voltage sine module;

s2: firstly, the controller controls the third switch relay K3 to be switched on, then controls the first switch relay K1, the second switch relay K2, the fourth switch relay K4 and the fifth switch relay K5 to be switched off, starts the high-frequency high-voltage sine module, applies sine alternating-current voltage to a culture dish containing cells and fusion liquid, and promotes the cells to form a queue and be tightly attached through the sine alternating-current voltage;

s3: when the output time of the sine alternating voltage reaches a set time value, the controller closes the third switch relay K3 to stop applying the sine alternating voltage to the culture dish;

s4: the first switch relay K1 is conducted through the controller, the nanosecond pulse module is electrified to start to operate, and nanosecond square pulse waves are applied to the culture dish;

s5: after the pulse number of the nanosecond pulse module reaches the set number, the controller controls to disconnect the first switch relay K1, and the nanosecond pulse module stops applying nanosecond square pulse waves to the culture dish;

s6: controlling a second switch relay K2 to be conducted, electrifying the microsecond pulse module to start to operate, and applying microsecond square pulse waves to the culture dish;

s: after the pulse number of the microsecond pulse module reaches the set number, the second switch relay K2 is controlled to be switched off by the controller, and the microsecond pulse module stops applying nanosecond square pulse waves to the culture dish;

s: controlling the fourth switching relay K4 to be switched on, electrifying a discharging loop connected with the nanosecond pulse module, and starting discharging the nanosecond pulse module through the first discharging resistor;

s: the fifth switch relay K5 is controlled to be switched on, a discharging loop connected with the microsecond pulse module is powered on, and the microsecond pulse module starts to discharge through a second discharging resistor R2;

the second working mode comprises the following steps:

the first step is as follows: setting the number of output pulses of the microsecond pulse module, the number of output pulses of the nanosecond pulse module 3 and the output time of the high-frequency high-voltage sine module 1;

the second step is that: firstly, the controller controls the third switch relay K3 to be switched on, then controls the first switch relay K1, the second switch relay K2, the fourth switch relay K4 and the fifth switch relay K5 to be switched off, starts the high-frequency high-voltage sine module, applies sine alternating-current voltage to a culture dish containing cells and fusion liquid, and promotes the cells to form a queue and be tightly attached through the sine alternating-current voltage;

the third step: when the output time of the sine alternating voltage reaches a set time value, the controller closes the third switch relay K3 to stop applying the sine alternating voltage to the culture dish;

the fourth step: controlling a second switch relay K2 to be conducted, electrifying the microsecond pulse module to start to operate, and applying microsecond square pulse waves to the culture dish;

the fifth step: after the pulse number of the microsecond pulse module reaches the set number, the second switch relay K2 is controlled to be switched off by the controller, and the microsecond pulse module stops applying nanosecond square pulse waves to the culture dish;

and a sixth step: the first switch relay K1 is conducted through the controller, the nanosecond pulse module is electrified to start to operate, and nanosecond square pulse waves are applied to the culture dish;

the seventh step: after the pulse number of the nanosecond pulse module reaches the set number, the controller controls to disconnect the first switch relay K1, and the nanosecond pulse module stops applying nanosecond square pulse waves to the culture dish;

eighth step: controlling the fourth switching relay K4 to be switched on, switching on a discharging loop connected with the nanosecond pulse module, and starting discharging the nanosecond pulse module through the first discharging resistor R1;

the ninth step: the fifth switch relay K5 is controlled to be turned on, the discharging loop connected with the microsecond pulse module is powered on, and the microsecond pulse module starts to discharge through the second discharging resistor R2

Preferably, the sinusoidal voltage output by the high-frequency high-voltage sinusoidal module ranges from 0V to 500V, the frequency ranges from 0 MHz to 3MHz, and the duration time ranges from 0s to 100 s.

Preferably, the nanosecond pulse module outputs pulses with the amplitude of 0-10kV, the pulse width of 50-1000 ns, the pulse frequency of 0.1-1000 Hz, the number of the pulses is adjustable, and the waveform is a pulse square wave;

the microsecond pulse module outputs pulses with the amplitude of 0-5kV, the pulse width of 1us-1000us, the pulse frequency of 0.1Hz-1000Hz, the number of the pulses is adjustable, and the waveform is pulse square waves.

The invention has the beneficial effects that:

firstly, the invention utilizes the characteristic that nanosecond pulse can perforate cells with different sizes, and can well fuse the cells with different sizes; the microsecond pulse is utilized to ensure that the perforation generated by the cells under the action of the nanosecond pulse is continuously and further expanded, thereby facilitating the fusion of the cells and effectively improving the success rate of the cell fusion.

Secondly, the clamping assembly is arranged in the protection box, and the circuit control assembly is clamped through the clamping assembly, so that the power supply box, the high-frequency high-voltage sine module, the microsecond pulse module and the nanosecond pulse module can be clamped step by step, if any one of the modules is damaged, disassembly, maintenance and replacement can be conveniently carried out, and the detection time and the production cost are saved.

The clamping assembly is controlled in two aspects, the two first fixing blocks are controlled by the bidirectional screw rod to horizontally and longitudinally clamp the two second fixing blocks, and then the control structure is matched with the two clamping mechanisms to horizontally and transversely clamp the two second fixing blocks, so that the whole clamping process is uniformly carried out, and the clamping is very firm.

Drawings

FIG. 1 is a schematic view of the overall structure of the present invention;

FIG. 2 is a schematic sectional view and an internal structure of the protection case according to the present invention;

FIG. 3 is a schematic diagram of the connection between the circuit control assembly and the positioning assembly of the present invention;

FIG. 4 is a schematic structural view of a positioning assembly of the present invention;

FIG. 5 is an enlarged view of the structure at A in FIG. 4 according to the present invention;

FIG. 6 is a schematic view of the construction of the sway bar of the present invention;

FIG. 7 is a circuit diagram of the circuit control assembly of the present invention;

fig. 8 is a circuit diagram of the present invention.

In the figure: 1. a protection box; 101. a package cover; 102. a composition board; 103. a guide chute; 2. a first fixed block; 201. clamping a plate; 202. a blocking plate; 3. a control block; 301. a control inserted link; 4. a second fixed block; 401. a support plate; 402. a guide hole; 5. a swing lever; 501. clamping a plate; 6. a bidirectional screw; 601. rotating the block; 7. a nanosecond pulse module; 8. a microsecond pulse module; 9. a high-frequency high-voltage sine module; 10. a power supply box; 1001. a display screen; 11. connecting the pole; 12. and connecting the holes.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-8, a composite pulse cell electrofusion apparatus includes:

the protection box comprises a protection box 1, wherein a packaging cover 101 is arranged at the top end of the protection box 1, and a fixed bin is arranged inside the protection box 1;

the combination groove has been seted up on the top of protection box 1 both sides wall, the both sides fixedly connected with compoboard 102 of encapsulation lid 101 bottom, two compoboards 102 and two combination groove joints, two direction spouts 103 have been seted up to the inner wall of fixed storehouse bottom both sides, one of them side of protection box 1, and run through the fixed storehouse and seted up viewing aperture and a plurality of control hole, protection box 1's opposite side, and run through the fixed storehouse and seted up a plurality of wiring mouths, protection box 1's front, and run through the fixed storehouse and seted up the regulation hole.

The circuit control assembly comprises a power box 10, a high-frequency high-voltage sine module 9, a microsecond pulse module 8 and a nanosecond pulse module 7, wherein the power box 10, the high-frequency high-voltage sine module 9, the microsecond pulse module 8 and the nanosecond pulse module 7 are electrically connected in sequence;

the back sides of the high-frequency high-voltage sine module 9, the microsecond pulse module 8 and the nanosecond pulse module 7 are respectively provided with a connecting pole post 11, the front sides of the power box 10, the high-frequency high-voltage sine module 9 and the microsecond pulse module 8 are respectively provided with two connecting holes 12, the six connecting pole posts 11 and the six connecting holes 12 are correspondingly inserted, and one side of the power box 10 is provided with a display screen 1001;

a third switch relay is arranged on one side of the high-frequency high-voltage sine module 9 and is set to be K3;

a second switching relay and a fifth switching relay are arranged on one side of the microsecond pulse module 8 and are respectively set to be K2 and K5;

one side of the nanosecond pulse module 7 is provided with a first switching relay and a fourth switching relay, which are respectively set to K1 and K4

The positioning assembly is arranged at the bottom of the fixed bin and comprises two first fixed blocks 2 and two second fixed blocks 4, the two second fixed blocks 4 are arranged between the two first fixed blocks 2, the middle parts of the front surfaces of the two first fixed blocks 2 and the two second fixed blocks 4 are connected with two-way screws 6 in an inserting mode, two sides of each of the two first fixed blocks 2 are provided with control mechanisms, and two sides of each of the two second fixed blocks 4 are provided with clamping mechanisms;

the clamping mechanism comprises two supporting plates 401, the two supporting plates 401 are connected with one of the guide sliding grooves 103 in a sliding and penetrating manner, guide holes 402 are formed in the front faces of the two supporting plates 401, a swing rod 5 is arranged between the two supporting plates 401, a rotating rod is connected between the tops of the two supporting plates 401 and the swing rod 5 in a rotating and penetrating manner, and one side of the top of the swing rod 5 is fixedly connected with a clamping plate 501;

the control mechanism comprises a control block 3, the control block 3 is connected with one of the guide chutes 103 in a sliding and penetrating way, the back of the control block 3 is fixedly connected with a control inserted rod 301, and the control inserted rod 301 is connected with a guide hole 402 in a sliding and penetrating way

Clamping plates 201 are fixedly connected to the opposite sides of the two first fixing blocks 2, adjusting screw holes are formed in the middles of the front sides of the two first fixing blocks 2 and the front sides of the two clamping plates 201, the two-way screw 6 and the four adjusting screw holes are connected in a threaded penetrating mode, and blocking plates 202 are fixedly connected to the top ends of the two clamping plates 201;

the rotation mouth has all been seted up to the both sides of two second fixed blocks 4, and the interlude hole has all been seted up at the positive middle part of two second fixed blocks 4, and two-way screw rod 6 and two interlude hole slip are connected, and wherein one end of two-way screw rod 6 is provided with turning block 601, and turning block 601 runs through the regulation hole and sets up in the front of protection box 1.

A control method of a composite pulse cell electrofusion instrument comprises one of the following working modes;

the first working mode comprises the following steps:

s1: setting the number of output pulses of the microsecond pulse module 8, the number of output pulses of the nanosecond pulse module 7 and the output time of the high-frequency high-voltage sine module 9;

s2: firstly, the controller controls the third switch relay K3 to be switched on, then controls the first switch relay K1, the second switch relay K2, the fourth switch relay K4 and the fifth switch relay K5 to be switched off, starts the high-frequency high-voltage sine module 9, applies sine alternating-current voltage to a culture dish containing cells and fusion liquid, and promotes the cells to form a queue and be tightly attached through the sine alternating-current voltage;

s3: when the output time of the sine alternating voltage reaches a set time value, the controller closes the third switch relay K3 to stop applying the sine alternating voltage to the culture dish;

s4: the first switch relay K1 is conducted through the controller, the nanosecond pulse module 7 is electrified to start to operate, and nanosecond square pulse waves are applied to the culture dish;

s5: after the number of pulses of the nanosecond pulse module 7 reaches the set number, the controller controls to turn off the first switch relay K1, and the nanosecond pulse module 7 stops applying nanosecond square pulse waves to the culture dish;

s6: controlling a second switch relay K2 to be conducted, electrifying the microsecond pulse module 8 to start to operate, and applying microsecond square pulse waves to the culture dish;

s7: after the number of pulses of the microsecond pulse module 8 reaches the set number, the controller controls to turn off the second switch relay K2, and the microsecond pulse module 8 stops applying nanosecond square pulse waves to the culture dish;

s8: controlling the fourth switching relay K4 to be switched on, electrifying a discharging loop connected with the nanosecond pulse module 7, and starting discharging through the first discharging resistor by the nanosecond pulse module 7;

s9: the fifth switch relay K5 is controlled to be turned on, a discharging loop connected with the microsecond pulse module 8 is powered on, and the microsecond pulse module 8 starts to discharge through a second discharging resistor R2;

the second working mode comprises the following steps:

the first step is as follows: setting the number of output pulses of the microsecond pulse module 8, the number of output pulses of the nanosecond pulse module 7 and the output time of the high-frequency high-voltage sine module 9;

the second step is that: firstly, the controller controls the third switch relay K3 to be switched on, then controls the first switch relay K1, the second switch relay K2, the fourth switch relay K4 and the fifth switch relay K5 to be switched off, starts the high-frequency high-voltage sine module 9, applies sine alternating-current voltage to a culture dish containing cells and fusion liquid, and promotes the cells to form a queue and be tightly attached through the sine alternating-current voltage;

the third step: when the output time of the sine alternating voltage reaches a set time value, the controller closes the third switch relay K3 to stop applying the sine alternating voltage to the culture dish;

the fourth step: controlling a second switch relay K2 to be conducted, electrifying the microsecond pulse module 8 to start to operate, and applying microsecond square pulse waves to the culture dish;

the fifth step: after the number of pulses of the microsecond pulse module 8 reaches the set number, the controller controls to turn off the second switch relay K2, and the microsecond pulse module 8 stops applying nanosecond square pulse waves to the culture dish;

and a sixth step: the first switch relay K1 is conducted through the controller, the nanosecond pulse module 7 is electrified to start to operate, and nanosecond square pulse waves are applied to the culture dish;

the seventh step: after the number of pulses of the nanosecond pulse module 7 reaches the set number, the controller controls to turn off the first switch relay K1, and the nanosecond pulse module 7 stops applying nanosecond square pulse waves to the culture dish;

eighth step: controlling the fourth switching relay K4 to be switched on, electrifying a discharging loop connected with the nanosecond pulse module 7, and starting discharging the nanosecond pulse module 7 through the first discharging resistor R1;

the ninth step: and the fifth switching relay K5 is controlled to be switched on, the discharging loop connected with the microsecond pulse module 8 is switched on, and the microsecond pulse module 8 starts to discharge through the second discharging resistor R2.

The sine voltage output by the high-frequency high-voltage sine module 9 ranges from 0V to 500V, the frequency ranges from 0 MHz to 3MHz, and the duration ranges from 0s to 100 s.

The nanosecond pulse module 7 outputs pulses with the amplitude of 0-10kV, the pulse width of 50-1000 ns, the pulse frequency of 0.1-1000 Hz, the number of the pulses is adjustable, and the waveform is a pulse square wave;

the microsecond pulse module 8 outputs pulses with the amplitude of 0-5kV, the pulse width of 1us-1000us, the pulse frequency of 0.1Hz-1000Hz, the number of the pulses is adjustable, and the waveform is pulse square waves.

When the invention is used, all components are firstly installed, and refer to fig. 1-6;

the first step of operation is implemented, the power supply box 10 and the nanosecond pulse module 7 are respectively clamped at the top ends of the two first fixed blocks 2, and the two side walls of the top of the first fixed block 2 and the blocking plate 202 are used for attaching and clamping the power supply box 10 and the nanosecond pulse module 7.

And implementing the second step of operation, and respectively clamping the microsecond pulse module 8 and the high-frequency high-voltage sine module 9 on the tops of the two second fixed blocks 4.

Implementing the third step of operation, pinching the rotating block 601 to rotate the bidirectional screw 6 clockwise, and performing the following processes;

the first process is as follows: when the bidirectional screw 6 rotates clockwise, the bidirectional screw is matched with the adjusting screw holes of the two first fixing blocks 2, and the two first fixing blocks 2 respectively drive the two power supply boxes 10 and the nanosecond pulse module 7 to move towards opposite directions under the guiding support of the control block 3 and the guiding chute 103;

and a second process: the two first fixed blocks 2 move and simultaneously extrude the two second fixed blocks 4 to move oppositely;

the third process: the two first fixing blocks 2 drive the connected control blocks 3 to move synchronously, the control blocks 3 drive the connected control insertion rods 301 to move synchronously, and meanwhile, the power supply box 10, the high-frequency high-voltage sine module 9, the microsecond pulse module 8 and the connecting pole 11 and the connecting hole 12 arranged in the nanosecond pulse module 7 are electrically inserted;

the process four is as follows: when the two control insertion rods 301 are close to the support plate 401, the two control insertion rods 301 are slowly inserted into the guide holes 402, under the interaction of the structures of the control insertion rods 301 and the swinging rods 5, the swinging rods 5 are extruded by the control insertion rods 301, the tops of the swinging rods swing towards the direction close to the center of the second fixed block 4, at the moment, the clamping plate 501 swings along with the swinging rods 5, and the microsecond pulse module 8 and the high-frequency high-voltage sine module 9 are clamped from two sides through the clamping plates 501;

and a fifth process: when the two first fixing blocks 2 move oppositely to surround and clamp the two second fixing blocks 4, the power supply box 10, the high-frequency high-voltage sine module, the microsecond pulse module 8 and the nanosecond pulse module 7 are inserted into the connecting pole 11 and the connecting hole 12 to complete clamping of the electric conduction circuit control assembly.

And performing the fourth step, namely clamping the packaging cover 101 on the top end of the protection box 1 to complete the assembly of the whole device, and then completing the fusion operation of the cells according to the using method in the specification.

If one of them module of circuit control assembly damages in the use of whole device, then can anticlockwise rotation turning block 601 for two-way screw rod 6 follows turning block 601 and carries out anticlockwise rotation, can overhaul and change the module that damages afterwards.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.