阅读说明：本技术 自动化电穿孔调控筛选的胞内外电生理记录系统与方法 (Intracellular and extracellular electrophysiological recording system and method for automatic electroporation regulation and screening ) 是由 胡宁 刘铮杰 谢曦 张明悦 夏其坚 陈惠琄 于 2021-07-19 设计创作，主要内容包括：本发明提供了一种自动化电穿孔调控筛选的胞内外电生理记录系统与方法,该系统包括微电极阵列传感器、胞内外电信号调理模块、电穿孔控制模块、数据采集卡、上位机和电源模块。通过该系统可以在一台仪器上实现细胞电穿孔和心肌细胞胞内外电信号记录的功能,能控制各通道逐一产生不同频率、幅值、脉宽的脉冲信号施加到细胞上,可实现在一次实验内不同条件电穿孔结果的对比,又能检测细胞电生理现象的变化,实现高时空分辨率的细胞电信号记录。同时,通过上位机能实现高效自动化分析,通过遍历不同的电穿孔条件,结合数据分析筛选出电穿孔的最优条件,以最优条件进行细胞电穿孔,从而实现高质量地对心肌细胞的胞内电信号进行记录。(The invention provides an intracellular and extracellular electrophysiological recording system and method for automatic electroporation regulation and screening. The system can realize the functions of cell electroporation and the recording of the intracellular and extracellular electric signals of the myocardial cells on one instrument, can control each channel to generate pulse signals with different frequencies, amplitudes and pulse widths one by one to be applied to the cells, can realize the comparison of the electroporation results under different conditions in one experiment, can detect the change of the cell electrophysiological phenomenon, and realizes the recording of the cell electric signals with high space-time resolution. Meanwhile, high-efficiency automatic analysis can be realized through the upper computer, the optimal conditions of electroporation can be screened out by traversing different electroporation conditions and combining data analysis, and cell electroporation can be carried out under the optimal conditions, so that the intracellular electric signals of the cardiac muscle cells can be recorded with high quality.)

1. An intracellular and extracellular electrophysiological recording system with automatic electroporation regulation and screening function, comprising:

the microelectrode array sensor comprises a substrate, a cell culture cavity positioned on the substrate and a microelectrode array formed on the substrate, wherein the microelectrode array comprises a plurality of microelectrodes and is positioned in the cell culture cavity; an electrode lead-out including a working electrode lead-out corresponding to each of the plurality of micro-electrodes and a common reference electrode lead-out.

The intracellular and extracellular electric signal conditioning module comprises a plurality of paths of intracellular and extracellular electric signal conditioning circuits with the same number as the microelectrodes, and each path of input end is respectively connected with the leading-out end of each working electrode and is used for filtering noise and amplifying the collected intracellular and extracellular electric signals. And the ground wire of the intracellular and extracellular electric signal conditioning modules is connected with the leading-out end of the reference electrode.

And the electroporation control module comprises a plurality of electroporation control circuits with the same number as the microelectrodes, and each output end of each circuit is respectively connected with the leading-out end of each working electrode and used for controlling the on-off of the electroporation signal of each microelectrode.

And the analog input end of the data acquisition card is connected with the output end of the intracellular and extracellular electric signal conditioning module, and the analog output end of the data acquisition card is connected with the input end of the electroporation control module to respectively generate electroporation signals corresponding to the microelectrodes.

And the upper computer is connected with the data acquisition card and is used for controlling the data acquisition card.

And the power supply module is connected with the intracellular and extracellular electric signal conditioning module, the electroporation control module and the data acquisition card and is used for providing a stable power supply for the intracellular and extracellular electric signal conditioning module, the electroporation control module and the data acquisition card.

2. The system of claim 1, wherein each of the intracellular and extracellular electrical signal conditioning circuits comprises a high-pass filter circuit, a first stage amplifier circuit, a band-pass filter circuit and a second stage amplifier circuit connected in series, wherein the band-pass filter circuit comprises an RC high-pass filter circuit and a butterworth low-pass filter circuit.

3. The system according to claim 1, wherein each of the electroporation control circuits comprises a power control circuit, an amplifying circuit and a channel control circuit, wherein the power control circuit comprises an NMOS transistor and a PMOS transistor, and is used for controlling the power on/off of the amplifying circuit and the channel control circuit according to a control signal output by a digital output terminal of the data acquisition card; the amplifying circuit is an in-phase proportional amplifier and is used for amplifying the electroporation signal output by the analog output end of the data acquisition card; the channel control circuit comprises a voltage follower and a control switch, wherein the input end of the voltage follower is connected with the output end of the amplifying circuit, the output end of the voltage follower is connected with one end of the control switch, and the other end of the control switch is connected with a microelectrode leading-out end of the microelectrode array sensor. And the control end of the control switch is connected with the digital output end of the data acquisition card.

4. The system of claim 3, wherein the power control circuit is composed of a resistor R13, a resistor R15, a capacitor C8, an inductor L2, an NMOS transistor Q5, and a PMOS transistor Q4, a gate of the NMOS transistor Q5 is connected to the digital output terminal of the data acquisition card and to the ground of the resistor R15, a source of the NMOS transistor Q5 is connected to the ground, a drain of the NMOS transistor Q5 is connected to the gate of the PMOS transistor Q4 and to the output terminal of the power module of the resistor R13, a source of the PMOS transistor Q4 is connected to the output terminal of the power module, a drain of the PMOS transistor Q4 is connected to the inductor L2, and the other end of the inductor L2 is connected to the capacitor C8 to form an LC filter circuit for filtering to provide a power output VCC for the amplification circuit and the channel control circuit.

5. The system of claim 1, further comprising a metal shielding box for shielding external power frequency interference and high frequency interference.

6. The system of claim 1, wherein the metallic shield can is further provided with an openable metallic shield cover.

7. A high-efficiency automatic electroporation regulation and control condition screening and measuring method is characterized by comprising the following steps:

the method comprises the steps of carrying out electroporation test by adopting a microelectrode array sensor, setting the electroporation conditions with the same quantity according to the quantity of the microelectrodes in the microelectrode array sensor, controlling each microelectrode to generate electroporation signals under different conditions one by one according to the electroporation conditions, and simultaneously collecting the myocardial cell intracellular electric signals of the corresponding microelectrode.

And analyzing the myocardial cell intracellular electric signals of all the microelectrodes to obtain the optimal electroporation condition.

8. The method of claim 7, wherein the electroporation conditions include electroporation signal amplitude, frequency and pulse width range, electroporation duration, and the like.

9. The method of claim 8, wherein the electroporation signal is a pulse having an amplitude of 0 to 20V, a frequency in the range of 5Hz to 100kHz, and a pulse width in the range of 10 μ s to 0.2 s.

Technical Field

The invention belongs to the field of biomedical engineering, and relates to an intracellular and extracellular electrophysiological recording system and method for automatic electroporation regulation and screening.

Background

Because the intracellular electric signal can provide a high signal-to-noise ratio action potential signal, in order to obtain a high-quality action potential signal, the electroporation technology based on a planar electrode is generally adopted, and a pulse is applied to the electrode to enable the surface of a cell membrane to be cracked, so that the electrode can record the intracellular electric signal. At present, a signal generator is generally adopted as electroporation equipment, the number of channels is limited, the advantage that an electric field can be generated by using two or more microelectrodes in a microelectrode array cannot be exerted, all channels of an existing instrument are subjected to electroporation simultaneously, crosstalk can be caused between the two channels, and the electroporation efficiency is low. In the experiment for detecting the intracellular electric signals, experimenters need to test different electroporation conditions, the workload is large, the operation is complex, and the experiment becomes an obstacle for deeply researching the electrophysiological phenomenon of the myocardial cells.

Disclosure of Invention

The invention aims to provide an intracellular and extracellular electrophysiological recording system and method for automatic electroporation regulation and screening. The system can realize the functions of cell electroporation and the recording of the intracellular and extracellular electric signals of the myocardial cells on one instrument, can control each channel to generate pulse signals with different frequencies, amplitudes and pulse widths one by one to be applied to the cells, can realize the comparison of the electroporation results under different conditions in one experiment, can detect the change of the cell electrophysiological phenomenon, and realizes the recording of the cell electric signals with high space-time resolution. Meanwhile, high-efficiency automatic analysis can be realized through the upper computer, the optimal conditions of electroporation can be screened out by traversing different electroporation conditions and combining data analysis, and cell electroporation can be carried out under the optimal conditions, so that the intracellular electric signals of the cardiac muscle cells can be recorded with high quality.

The invention is realized by the following technical scheme:

an intracellular and extracellular electrophysiological recording system for automated electroporation regulatory screening, comprising:

the microelectrode array sensor comprises a substrate, a cell culture cavity positioned on the substrate and a microelectrode array formed on the substrate, wherein the microelectrode array comprises a plurality of microelectrodes and is positioned in the cell culture cavity; an electrode lead-out including a working electrode lead-out corresponding to each of the plurality of micro-electrodes and a common reference electrode lead-out.

The intracellular and extracellular electric signal conditioning module comprises a plurality of paths of intracellular and extracellular electric signal conditioning circuits with the same number as the microelectrodes, and each path of input end is respectively connected with the leading-out end of each working electrode and is used for filtering noise and amplifying the collected intracellular and extracellular electric signals. And the ground wire of the intracellular and extracellular electric signal conditioning modules is connected with the leading-out end of the reference electrode.

And the electroporation control module comprises a plurality of electroporation control circuits with the same number as the microelectrodes, and each output end of each circuit is respectively connected with the leading-out end of each working electrode and used for controlling the on-off of the electroporation signal of each microelectrode.

And the analog input end of the data acquisition card is connected with the output end of the intracellular and extracellular electric signal conditioning module, and the analog output end of the data acquisition card is connected with the input end of the electroporation control module to respectively generate electroporation signals corresponding to the microelectrodes.

And the upper computer is connected with the data acquisition card and is used for controlling the data acquisition card.

And the power supply module is connected with the intracellular and extracellular electric signal conditioning module, the electroporation control module and the data acquisition card and is used for providing a stable power supply for the intracellular and extracellular electric signal conditioning module, the electroporation control module and the data acquisition card.

Furthermore, each path of the intracellular and extracellular electric signal conditioning circuit consists of a high-pass filter circuit, a first-stage amplification circuit, a band-pass filter circuit and a second-stage amplification circuit which are sequentially connected, wherein the band-pass filter circuit comprises an RC high-pass filter circuit and a Butterworth low-pass filter circuit.

Furthermore, each circuit of the electroporation control circuit consists of a power supply control circuit, an amplifying circuit and a channel control circuit, wherein the power supply control circuit consists of an NMOS (N-channel metal oxide semiconductor) tube and a PMOS (P-channel metal oxide semiconductor) tube and is used for controlling the power supply of the amplifying circuit and the channel control circuit to be switched on and off according to a control signal output by the digital output end of the data acquisition card; the amplifying circuit is an in-phase proportional amplifier and is used for amplifying the electroporation signal output by the analog output end of the data acquisition card; the channel control circuit comprises a voltage follower and a control switch, wherein the input end of the voltage follower is connected with the output end of the amplifying circuit, the output end of the voltage follower is connected with one end of the control switch, and the other end of the control switch is connected with a microelectrode leading-out end of the microelectrode array sensor. And the control end of the control switch is connected with the digital output end of the data acquisition card.

Furthermore, the power control circuit is composed of a resistor R13, a resistor R15, a capacitor C8, an inductor L2, an NMOS tube Q5 and a PMOS tube Q4, a grid electrode of the NMOS tube Q5 is connected with a digital output end of the data acquisition card, the grid electrode of the NMOS tube Q15 is connected to the ground, a source electrode of the NMOS tube Q5 is grounded, a drain electrode of the NMOS tube Q5 is connected with a grid electrode of the PMOS tube Q4, the drain electrode of the NMOS tube Q13 is connected to an output end of the power module, a source electrode of the PMOS tube Q4 is connected with an output end of the power module, a drain electrode of the PMOS tube Q4 is connected with an inductor L2, the other end of the inductor L2 is connected with the capacitor C8 to form an LC filter circuit, and the LC filter circuit provides power for a power output VCC after filtering and supplies power for the amplifying circuit and the channel control circuit.

Furthermore, the device also comprises a metal shielding box for shielding external power frequency interference and high frequency interference.

Furthermore, the metal shielding box is also provided with a metal shielding cover which can be opened and closed.

The invention also provides a high-efficiency automatic electroporation regulation and control condition screening and measuring method, which specifically comprises the following steps:

the method comprises the steps of carrying out electroporation test by adopting a microelectrode array sensor, setting the electroporation conditions with the same quantity according to the quantity of the microelectrodes in the microelectrode array sensor, controlling each microelectrode to generate electroporation signals under different conditions one by one according to the electroporation conditions, and simultaneously collecting the myocardial cell intracellular electric signals of the corresponding microelectrode.

And analyzing the myocardial cell intracellular electric signals of all the microelectrodes to obtain the optimal electroporation condition.

Further, the electroporation conditions include electroporation signal amplitude, frequency and pulse width ranges, electroporation duration, and the like.

Furthermore, the amplitude of the electroporation signal is 0-20V, the frequency range is 5 Hz-100 kHz, and the pulse width range is 10 mus-0.2 s.

Compared with the prior art, the invention has the following advantages:

1. the functions of cell electroporation and recording of electric signals inside and outside the cell are realized on one instrument, and the defects that the existing instrument cannot be integrated, cell stimulation control needs to be respectively carried out on the two instruments, and the detection and recording of the signals inside and outside the cell are avoided;

2. the system can control each channel to generate pulse signals with adjustable frequency, amplitude and pulse width, the channel for generating electroporation is selectable, different electroporation conditions are traversed by traversing each channel, so that optimal condition screening is completed in one experiment, high-efficiency automatic analysis is realized by the upper computer, the optimal condition of electroporation is screened out by combining data analysis, and cell electroporation is performed under the optimal condition, so that intracellular electric signals of cardiac myocytes are recorded in high quality, and the system is favorable for deeply researching the cell electrophysiological phenomenon.

Drawings

FIG. 1 is a schematic view of a microelectrode array sensor;

FIG. 2 is a diagram of an intracellular and extracellular electrical signal conditioning module;

FIG. 3 is a block diagram of an electroporation control module;

FIG. 4 is a schematic diagram of the structure of the system for controlling the stimulation of high-throughput cells and detecting and analyzing the intracellular and extracellular electric signals;

FIG. 5 is a block diagram of a system for high throughput cell stimulation control and detection and analysis of intracellular and extracellular electrical signals;

fig. 6 is a structural view of a metal shield case;

FIG. 7 is a schematic diagram of an intracellular and extracellular electrical signal conditioning circuit;

FIG. 8 is a schematic diagram of an electroporation control circuit;

FIG. 9 is a program flow diagram of the upper computer control software;

FIG. 10 is a top-level computer main interface of the high-throughput cell stimulation control and intracellular and extracellular electrical signal detection and analysis system;

FIG. 11 is a diagram showing the results of the extracellular electrical signal collection in the high-throughput cell stimulation control and intracellular and extracellular electrical signal detection and analysis system;

FIG. 12 is a graph showing the results of intracellular signal collection in the high-throughput cell stimulation control and intracellular and extracellular signal detection and analysis system;

in the figure, a pin header 1, a reference electrode 2, a cell culture chamber 3, a glass substrate 4, a PCB 5, a working electrode 6, an output connection terminal 7 of an intracellular and extracellular electric signal conditioning module, an intracellular and extracellular electric signal conditioning circuit 8, a pin header slot 9 of an electrode array sensor, a pin header slot 10 of an electroporation control module, a microelectrode array sensor 11, a PCB substrate 12 of the intracellular and extracellular electric signal conditioning module, an output connection terminal 13 of the intracellular and extracellular electric signal conditioning module, a power interface 14 of the intracellular and extracellular electric signal conditioning module, a channel control circuit 15, a pin header 16 of the electroporation control module, an amplification circuit 17, a power interface 18 of the electroporation control module, a signal input terminal 19 of the electroporation control module, a power control terminal 20 of the electroporation control module, a power control circuit 21 of the electroporation control module, a PCB substrate 22 of the electroporation control module, and a channel control terminal 23 of each electroporation control module, The device comprises a metal shielding cover 24, a data acquisition card 25, a power module 26, a metal case 27 and folding hardware 28.

Detailed Description

When the metal electrode is immersed in the electrolyte, free electrons on the metal electrode and ions in the electrolyte will move, so that an electric opposite double-layer charge is formed at the interface between the electrode and the electrolyte, and the structure is similar to a capacitor and is called an electric double-layer structure. The electroporation circuit generates electrons and transfers the charges to a metal electrode immersed in cell culture solution or buffer solution through circuit conduction, the cells are cultured above the microelectrode array and directly contact with the metal electrode to form tight coupling, and the electroporation causes the metal electrode to generate electric field change, thereby further influencing the behavior and physiological activities of the cells. The cell generates action potential, the concentration of the extracellular ions changes, the electric field changes to polarize the surface of the metal electrode, the double-layer capacitor charges and discharges, and the change of the electric signal of the metal electrode is detected to obtain the change of the extracellular electric signal of the cell. When the electroporation makes the cell in a transient high electric field environment, the high electric field environment can make the surface of the cell membrane have a plurality of small holes, so that the metal electrode can record the intracellular electric signal of the cell.

The invention provides an intracellular and extracellular electrophysiological recording system for automatic electroporation regulation and screening, which is described in detail by combining an example and the attached drawings as follows:

the invention relates to an intracellular and extracellular electrophysiological recording system for automatic electroporation regulation and screening, which comprises a microelectrode array sensor 11, an intracellular and extracellular electric signal conditioning module, an electroporation control module, a data acquisition card 25, a metal shielding box, an upper computer and a power supply module 26.

As shown in FIG. 1, the microelectrode array sensor 11 comprises a working electrode 6, a reference electrode 2, a PCB 5 and a cell culture chamber 3. The working electrode 6 is a micro-electrode array, and in this embodiment, micro-electrodes having a diameter of 10 μm, a pitch of 40 μm, a line width of an internal lead of 5 μm, and a line width of an external lead of 0.3mm are used, but not limited thereto. Each microelectrode is connected with the input end of the intracellular and extracellular electric signal conditioning circuit 8 and the output end of the electroporation control module. In this embodiment, a Pt wire electrode having a diameter of 0.5mm as the reference electrode 2 is disposed in the cell culture chamber 3 and connected to the ground, but not limited thereto. Wherein, a glass cylinder is adopted to be sealed and adhered to the top of the microelectrode array to be used as a cell culture cavity 3.

As shown in fig. 2-6, the intracellular and extracellular electrical signal conditioning module includes a plurality of intracellular and extracellular electrical signal conditioning circuits 8 with the same number as that of the microelectrodes, the electroporation control module includes a plurality of electroporation control circuits with the same number as that of the microelectrodes, and the microelectrodes, the intracellular and extracellular electrical signal conditioning circuits, and the electroporation control circuits correspond to one another to form a multi-channel system.

The intracellular and extracellular electric signal conditioning circuit 8 comprises a high-pass filter circuit, a first-stage amplification circuit, a band-pass filter circuit and a second-stage amplification circuit which are connected in sequence, specifically, as shown in fig. 7, the high-pass filter circuit comprises a capacitor C2 and a resistor R5 which are connected with each other, the other end of the capacitor C2 is used as the input end of the intracellular and extracellular electric signal conditioning circuit 8, receives intracellular and extracellular electric signals collected by corresponding microelectrodes, filters noise with the frequency of below 1Hz and simultaneously filters direct current components, and the other end of the resistor R5 is grounded; the junction of the capacitor C2 and the resistor R5 is used as the output end of the high-pass filter circuit to be connected with the input end of the first-stage amplifying circuit. The first-stage amplification circuit is composed of three resistors R1, R6, R9, a feedback capacitor C5 and a precision operational amplifier U1B, a resistor R6 is connected between the reverse input end and the output end of the precision operational amplifier U1B, the feedback capacitor C5 is connected with the resistor R6 in parallel, a resistor R9 is connected with the reverse input end and the ground of the precision operational amplifier U1B, a resistor R1 is connected with the output end of the high-pass filter circuit and the forward input end of the precision operational amplifier U1B, and the other end of the resistor R1 is used as the input end of the first-stage amplification circuit. The feedback capacitor C5 is used for phase compensation and preventing the precision operational amplifier U1B from self-oscillation. The band-pass filter circuit comprises an RC high-pass filter circuit consisting of C3 and R7 and a Butterworth low-pass filter circuit consisting of resistors R2, R3, a low-noise operational amplifier U2B, capacitors C1 and C4, wherein the forward input end of the low-noise operational amplifier U2B is connected with one ends of a capacitor C4 and a resistor R3, the other end of the capacitor C4 is grounded, the other end of the resistor R3 is connected with the capacitor C1 and the resistor R2, the other end of the capacitor C1 is connected with the output end of the low-noise operational amplifier U2B, the other end of the resistor R2 is connected with the output end of the RC high-pass filter circuit, and the output end of the low-noise operational amplifier U2B is connected with the reverse input end. The second-stage amplifying circuit is composed of three resistors R4, R8, R10, a feedback capacitor C6 and a precision operational amplifier U3B, a resistor R8 is connected between a reverse input end and an output end of the precision operational amplifier U3B, the feedback capacitor C6 is connected with the resistor R8 in parallel, a resistor R10 is connected with the reverse input end of the precision operational amplifier U3B and the ground, a resistor R4 is connected with the output end of the band-pass filter circuit and the forward input end of the precision operational amplifier U3B, the output end of the precision operational amplifier U3B is connected with the analog input end of the data acquisition card 25, and other channels of the indoor and outdoor electric signal conditioning module are the same as the circuits.

The electronic components related to the circuit are welded on the intracellular and extracellular electric signal conditioning module, as shown in figure 2, the substrate 12 of the intracellular and extracellular electric signal conditioning module PCB of the module also comprises 31 other same intracellular and extracellular electric signal conditioning circuits 8 to form a 32-channel intracellular and extracellular electric signal conditioning module, which corresponds to 32 microelectrodes in figure 1, the intracellular and extracellular electric signal conditioning module is provided with two connecting terminals 7 and 13, the connecting terminals are connected with a data acquisition card 25 by a flat cable, and the intracellular and extracellular electric signal conditioning module is provided with a power interface 14 and is connected with +/-5V voltage supplied by a power module 26.

Preferably, the microelectrode array sensor 11 is provided with a pin header 1, the intracellular and extracellular electric signal conditioning module is provided with a corresponding upward electrode array sensor pin header slot 9, the pin header 1 is connected with a corresponding microelectrode, and the electrode array sensor pin header slot 9 is connected with a corresponding intracellular and extracellular electric signal conditioning circuit 8.

The electroporation control circuit is composed of a POWER control circuit 21, an amplification circuit 17 and a channel control circuit 15, as shown in fig. 3, the POWER control circuit 21 is composed of resistors R13 and R15, a capacitor C8, an inductor L2, an NMOS Q5 and a PMOS Q4 as shown in fig. 8a, a gate of the NMOS Q5 is connected to a digital output terminal of the data acquisition card 25 and is connected to the ground with the resistor R15, a source of the NMOS Q5 is connected to the ground, a drain of the NMOS Q4 is connected to the gate of the PMOS Q4 and is connected to a POWER input POWER module with the resistor R13, a source of the PMOS Q4 is connected to the POWER input POWER module, a drain of the PMOS Q4 is connected to the inductor L2, and the other end of the inductor L2 is connected to the capacitor C8 to form an LC filter circuit which is a POWER output VCC after filtering and supplies POWER to the amplification circuit 17 and the channel control circuit 15. The heat generated by the operational amplifier can be reduced by controlling the power supply to be turned off, and the power supply is turned on again when the electroporation is carried out.

The amplifier circuit 17 is used to amplify the electroporation SIGNAL, as shown IN fig. 8b, the operational amplifier U4A has a positive input terminal connected to the electroporation SIGNAL input SIGNAL, a negative input terminal connected to the resistor R14 and connected to ground via the resistor R16 to the output terminal of the operational amplifier U4A, the output terminal is the amplified electroporation SIGNAL output PULSE _ IN, and the electroporation SIGNAL input SIGNAL is connected to the analog output terminal of the data acquisition card 25 via the SIGNAL input terminal 19 of the electroporation control module.

The amplifying circuit 17 may be formed by a one-channel or multi-channel operational amplifier, for example, a four-channel operational amplifier U4.

The channel control circuit 15 is a control switch, and can adopt a single control switch or a multi-channel control switch, in the embodiment, a 1:1 four-channel multiplexer IC1 is adopted, and meanwhile, a voltage follower is arranged in each channel, so that the driving capability of the pulse can be improved, even if some microelectrodes have no cell attached branch current and are large, the perforation current of the pulse on other paths cannot be weakened, the electroporation separating multi-channel pulses obviously improves the perforation efficiency of the cells, and the detection flux of the system is improved.

As shown IN FIG. 8c, the channel control circuit 15 comprises a four-channel operational amplifier U5, a 1:1 four-channel multiplexer IC1, resistors R18, R19, R20 and R21, taking a channel U5A of the operational amplifier and a channel of the multiplexer as an example, the reverse input end and the output end of the operational amplifier U5A are connected to form a voltage follower, the forward input end of the operational amplifier U5A is connected to the PULSE _ IN, the output end is connected to a resistor R18, the other end of the resistor R18 is connected to a channel input end S1 of the multiplexer, a channel output end D1 of the multiplexer is connected to a microelectrode leading-out end of the microelectrode array sensor 11, and the control terminal SEL1 is connected to the digital output end of the data acquisition card 25. Similarly, the other 3 channels are connected in the same manner.

The output end of the multiplexer is connected with each microelectrode leading-out end of the microelectrode sensor 11 through the electroporation control circuit module power supply control end 20.

Therefore, the circuit comprises 32 power supply control circuits 21, 8 groups of four-channel operational amplifiers U4 and 8 groups of channel control circuits 15, wherein the channels are connected in a one-to-one correspondence manner, electronic components related to the circuits are welded on a PCB substrate 22, and finally a 32-channel electroporation control circuit module is formed, and the electroporation control module is also provided with an electroporation control module power supply interface 18 which is connected with 20V voltage supplied by a power supply module 26.

Preferably, the electroporation control module is provided with an electroporation control module pin header 16, the intracellular and extracellular electrical signal conditioning modules are provided with corresponding downward pin header slots 10, the pin header 16 is connected with corresponding electroporation control circuits, and the pin header slots 10 are connected with corresponding microelectrodes.

The power supply module 26 is used for providing a +/-5V power supply for the intracellular and extracellular electric signal conditioning modules, providing a 20V power supply for the electroporation control module and providing a 12V power supply for the data acquisition card 25; the input end of the power module 26 is connected with a 12V power supply, a +/-5V power supply is generated through a DC/DC chip, a control switch is arranged to control the +/-5V power supply to be turned on and off, and the control switch is connected with a digital output port of the data acquisition card 25; the 20V power supply is generated by the DC/DC chip.

Preferably, the shielding box further comprises a metal shielding box, as shown in fig. 5, the metal shielding box is composed of a metal chassis 27, a folding hardware 28 and a metal shielding cover 24, and the metal shielding box is connected with a ground wire and is used for shielding external power frequency interference and high frequency interference.

The working process of the intracellular and extracellular electric signal conditioning circuit and the electroporation control circuit is as follows: when electric signals are collected, the digital output end of the data acquisition card 25 outputs high level, the +/-5V power supply of the power supply module 26 is controlled to be turned on to supply power to the intracellular and extracellular electric signal conditioning modules, the cells in the cell culture cavity 3 generate action potential to cause the concentration of extracellular ions to change, so that the electric field changes, the microelectrode transmits the electric field changes to the input end of the intracellular and extracellular electric signal conditioning circuit 8, the cell electric signals pass through the high-pass filter circuit, the primary amplification circuit, the band-pass filter circuit and the secondary amplification circuit to ensure that the amplitude of the cell electric signals is large enough, and finally the cell electric signals are collected by the analog input end of the data acquisition card 25 and transmitted to an upper computer to be displayed and stored. When the electroporation is performed, the analog output end of the data acquisition card 25 generates an electroporation pulse signal, the digital output end of the data acquisition card 25 outputs a high level, and the multiplexer of the electroporation control circuit is turned on, so that the channel can generate the electroporation signal, and if the digital output end of the data acquisition card 25 outputs a low level, the multiplexer of the electroporation control circuit is turned off, and the channel cannot generate the electroporation signal. After the cell is electroporated, the microelectrode records an intracellular electric signal, and the intracellular electric signal is amplified and filtered by an intracellular and extracellular electric signal conditioning circuit 8 and is acquired by a data acquisition card 25.

The work flow of the system for automatically regulating and screening the intracellular and extracellular electrophysiological recording by electroporation is shown in fig. 9, and the system specifically comprises the following steps:

step one, placing cardiac muscle cells and culture solution in the microelectrode array sensor 11, inserting the microelectrode array sensor 11 into the intracellular and extracellular electric signal conditioning module, and covering the microelectrode array sensor 11 with a metal shielding cover 24.

And step two, starting the upper computer software, setting the amplitude, frequency and pulse width range of the electroporation signal traversed automatically and the electroporation duration, controlling channel-by-channel generation of the electroporation signal under different conditions, recording the intracellular electric signal of the myocardial cell, and drawing and displaying an electric signal graph.

And step three, analyzing the collected intracellular electric signals to obtain the optimal electroporation condition.

And step four, the upper computer controls all the channels again to generate electroporation signals with optimal conditions, records and stores the intracellular electric signals of the cardiac muscle cells, and displays the acquired intracellular electric signal graph in real time.

The following gives examples of applications of the present invention.

The system and the method for the intracellular and extracellular electrophysiological recording of the automatic electroporation regulation and screening are mainly used for the automatic detection of the intracellular electric signals of the myocardial cells. Firstly, an electroporation control module is arranged below an intracellular and extracellular electric signal conditioning module, connecting lines among the modules, adding myocardial cells and a culture solution thereof into a cell culture cavity 3, inserting a microelectrode array sensor 11 into a slot 9, covering the microelectrode array sensor 11 with a metal shielding cover 24, opening an upper computer, entering a main interface as shown in figure 10, starting clicking, traversing electroporation conditions with different frequencies, amplitudes and pulse widths by the upper computer, as shown in figures 11-12, selecting the optimal conditions of electroporation, then performing electroporation under the optimal conditions of electroporation, and acquiring the intracellular electric signals of the myocardial cells. When the cell electric signal collection is finished, the metal shielding cover 24 is opened, the microelectrode array sensor 11 is taken out, and the experiment is finished.

The invention adopts microelectrode array to develop high-quality high-efficiency automatic electroporation regulation and screening system and method for myocardial intracellular electrophysiological recording, can apply controllable electroporation to cells in different channels according to requirements, and can detect the change of cell electrophysiological phenomena and specific parameters of electric signals. The efficiency of cell electroporation is improved by controlling each channel to generate electroporation one by one, efficient automatic analysis can be realized by the upper computer, and the optimal conditions of electroporation can be found out by traversing different electroporation conditions and combining data analysis, thereby realizing the high-quality recording of the intracellular electric signals of the myocardial cells.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should all embodiments be exhaustive. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.