CN113528332B - Intracellular and extracellular electrophysiological recording system and method for automatic electroporation regulation screening - Google Patents
Intracellular and extracellular electrophysiological recording system and method for automatic electroporation regulation screening Download PDFInfo
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- 230000002107 myocardial effect Effects 0.000 claims abstract description 18
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- 238000004113 cell culture Methods 0.000 claims description 11
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q3/00—Condition responsive control processes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
Abstract
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 myocardial cell intracellular and extracellular electrical signal recording 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 cells, can realize the comparison of electroporation results under different conditions in one experiment, can detect the change of cell electrophysiological phenomena, and realizes the high-space-time resolution cell electrical signal recording. Meanwhile, high-efficiency automatic analysis is realized through the upper computer, different electroporation conditions are traversed, the optimal conditions of electroporation are screened out by combining with data analysis, and cell electroporation is carried out under the optimal conditions, so that the intracellular electrical signals of myocardial cells are recorded with high quality.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and relates to an extracellular and intracellular electrophysiological recording system and method for automatic electroporation regulation and screening.
Background
Since the intracellular electrical signal can provide a high signal to noise ratio action potential signal, in order to obtain a high quality action potential signal, a pulse is applied to the electrode to crack the surface of the cell membrane, usually based on the electroporation technology of a planar electrode, so that the electrode can record the intracellular electrical signal. Currently, 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 can not be exerted, and in addition, all channels of the existing instrument are subjected to electroporation at the same time, crosstalk can be caused between the two channels, and the electroporation efficiency is low. In the experiment of detecting the intracellular electric signals, the experimenter needs to test different electroporation conditions, the workload is large, the operation is complex, and the method 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, comprising a microelectrode array sensor, an extracellular and extracellular electrical signal conditioning circuit, an electroporation control circuit, a data acquisition card, a metal shielding box, an upper computer and a power module. The system can realize the functions of cell electroporation and myocardial cell intracellular and extracellular electrical signal recording 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 cells, can realize the comparison of electroporation results under different conditions in one experiment, can detect the change of cell electrophysiological phenomena, and realizes the high-space-time resolution cell electrical signal recording. Meanwhile, high-efficiency automatic analysis is realized through the upper computer, different electroporation conditions are traversed, the optimal conditions of electroporation are screened out by combining with data analysis, and cell electroporation is carried out under the optimal conditions, so that the intracellular electrical signals of myocardial cells are recorded with high quality.
The invention is realized by the following technical scheme:
an automated electroporation-mediated screening intracellular and extracellular electrophysiological recording system, 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 microelectrodes and a common reference electrode lead-out.
The intracellular and extracellular electric signal conditioning module comprises multiple paths of extracellular and extracellular electric signal conditioning circuits with the same number as the microelectrodes, and each path of input end is respectively connected with each working electrode leading-out end and is used for filtering noise and amplifying the collected extracellular and extracellular electric signals. And the ground wire of the intracellular and extracellular electric signal conditioning module is connected with the reference electrode leading-out end.
The electroporation control module comprises a plurality of electroporation control circuits with the same number as the microelectrodes, and each output end is respectively connected with each working electrode leading-out end and used for controlling the on-off of electroporation signals of each microelectrode.
The data acquisition card, the analog input of data acquisition card links to each other with the output of extracellular and intracellular electrical signal conditioning module, the analog output of data acquisition card links to each other with electroporation control module's input, produces the electroporation signal of corresponding microelectrode respectively.
And the upper computer is connected with the data acquisition card and used for controlling the data acquisition card.
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 extracellular and extracellular electric signal conditioning module, the electroporation control module and the data acquisition card.
Further, each path of extracellular and intracellular electric signal conditioning circuit is composed of a high-pass filter circuit, a first-stage amplifying circuit, a band-pass filter circuit and a second-stage amplifying 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.
Further, each 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 tube and a PMOS tube and is used for controlling the power supply on-off of the amplifying circuit and the channel control circuit according to a control signal output by a digital output end of the data acquisition card; the amplifying circuit is an in-phase proportional amplifier and is used for amplifying electroporation signals output by an 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.
Further, the power supply 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, wherein the grid electrode of the NMOS tube Q5 is connected with the digital output end of the data acquisition card and is connected with the ground simultaneously, the source electrode of the NMOS tube Q5 is grounded, the drain electrode of the NMOS tube Q5 is connected with the grid electrode of the PMOS tube Q4 and is connected with the output end of the power supply module simultaneously with the resistor R13, the source electrode of the PMOS tube Q4 is connected with the output end of the power supply module, the drain electrode of the PMOS tube Q4 is connected with the 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 is used for outputting VCC after filtering to supply power for the amplifying circuit and the channel control circuit.
Further, the metal shielding box is used for shielding external power frequency interference and high frequency interference.
Further, the metal shielding box is also provided with an openable metal shielding cover.
The invention also provides a high-efficiency automatic electroporation regulation condition screening and measuring method, which specifically comprises the following steps:
and (3) carrying out electroporation test by adopting a microelectrode array sensor, setting electroporation conditions with the same quantity according to the quantity of 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 collecting myocardial cell intracellular electrical signals of the corresponding microelectrode.
And analyzing the intracellular electrical signals of the myocardial cells of all 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.
Further, the electroporation signal has a pulse with an amplitude of 0-20V, a frequency range of 5 Hz-100 kHz, and a pulse width range of 10 μs-0.2 s.
Compared with the prior art, the invention has the following advantages:
1. the functions of cell electroporation and intracellular and extracellular electrical signal recording are realized on one instrument, so that the defect that the existing instrument cannot be integrated and the cell stimulation control and the intracellular and extracellular signal detection recording are required to be respectively carried out on the two instruments is overcome;
2. the method can control each channel to generate a pulse signal with adjustable frequency, amplitude and pulse width, and the channel for generating electroporation is optional, different electroporation conditions are traversed by traversing each channel, so that the optimal condition screening is completed in one experiment, meanwhile, high-efficiency automatic analysis is realized through 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 the recording of the intracellular electric signals of myocardial cells with high quality is realized, and the method is favorable for more intensive study on the cell electrophysiological phenomenon.
Drawings
FIG. 1 is a schematic diagram of a microelectrode array sensor;
FIG. 2 is a diagram of an extracellular and intracellular electrical signal conditioning module;
FIG. 3 is a block diagram of an electroporation control module;
FIG. 4 is a schematic diagram of a high-throughput cell stimulation control and intracellular and extracellular electrical signal detection and analysis system;
FIG. 5 is a block diagram of a high throughput cell stimulation control and intracellular and extracellular electrical signal detection analysis system;
FIG. 6 is a block diagram of a metallic shielding cage;
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 host computer interface of the high throughput cell stimulation control and intracellular and extracellular electrical signal detection and analysis system;
FIG. 11 is a graph of the results of the acquisition of extracellular electrical signals by the high-throughput cell stimulation control and extracellular electrical signal detection analysis system;
FIG. 12 is a graph of the results of the acquisition of intracellular electrical signals by the high throughput cell stimulation control and intracellular and extracellular electrical signal detection and analysis system;
in the figure, a pin 1, a reference electrode 2, a cell culture chamber 3, a glass substrate 4, a PCB 5, a working electrode 6, an extracellular and intracellular electric signal conditioning module output wiring terminal 7, an extracellular and intracellular electric signal conditioning circuit 8, an electrode array sensor pin arrangement slot 9, an electroporation control module pin arrangement slot 10, a microelectrode array sensor 11, an extracellular and intracellular electric signal conditioning module PCB substrate 12, an extracellular and intracellular electric signal conditioning module output wiring terminal 13, an extracellular and intracellular electric signal conditioning module power interface 14, a channel control circuit 15, an electroporation control module pin 16, an amplifying circuit 17, an electroporation control module power interface 18, an electroporation control module signal input end 19, an electroporation control module power control end 20, an electroporation control module power control circuit 21, an electroporation control module PCB substrate 22, an electroporation control module channel control end 23, a metal shielding cover 24, a data acquisition card 25, a power module 26, a metal chassis 27 and hinge 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, thereby forming a double-layer charge at the interface between the electrode and the electrolyte, functioning like a capacitor, and being referred to as an electric double layer structure. The electroporation circuit generates electrons and transfers the charges to the metal electrode immersed in the cell culture solution or the buffer solution through circuit conduction, and the cells are cultured above the microelectrode array and are in direct contact with the metal electrode to form tight coupling, so that the electroporation causes the metal electrode to generate electric field change, and further the action and the physiological activity of the cells are influenced. The cell generates action potential, the concentration of 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 extracellular electric signal of the cell can be obtained by detecting the change of the electric signal of the metal electrode. When electroporation causes the cells to be in a transient high-electric-field environment, the high-electric-field environment causes a plurality of pores to appear on the surface of the cell membrane, so that the metal electrode can record the intracellular electric signals of the cells.
The invention provides an extracellular and intracellular electrophysiological recording system for automatic electroporation regulation screening, which is described in detail below with reference to examples and drawings:
the invention discloses an automatic electroporation, regulation and screening intracellular and extracellular electrophysiological recording system, which comprises a microelectrode array sensor 11, an extracellular and extracellular electrical 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 includes a working electrode 6, a reference electrode 2, a PCB board 5, and a cell culture chamber 3. The working electrode 6 is a microelectrode array, and in this embodiment, the microelectrode used is 10 μm in diameter, 40 μm in pitch, 5 μm in internal lead line width, and 0.3mm in external lead line width, but is not limited thereto. Each microelectrode is connected with the input end of an extracellular and intracellular electric signal conditioning circuit 8 and the output end of an electroporation control module. In this embodiment, but not limited to, a Pt wire electrode with a diameter of 0.5mm as the reference electrode 2 is placed in the cell culture chamber 3 and connected to the ground. Wherein, a glass cylinder body is used for sealing and bonding 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 comprises multiple paths of extracellular and intracellular electrical signal conditioning circuits 8 with the same number as the microelectrodes, the electroporation control module comprises multiple paths of electroporation control circuits with the same number as the microelectrodes, and the microelectrodes, the extracellular and extracellular electrical signal conditioning circuits and the electroporation control circuits are in one-to-one correspondence to form a multichannel system.
The intracellular and extracellular electric signal conditioning circuit 8 is composed of a high-pass filter circuit, a first-stage amplifying circuit, a band-pass filter circuit and a second-stage amplifying circuit which are sequentially connected, specifically, as shown in fig. 7, the high-pass filter circuit is composed of a capacitor C2 and a resistor R5 which are mutually connected, the other end of the capacitor C2 is used as an input end of the extracellular and extracellular electric signal conditioning circuit 8, the extracellular and extracellular electric signals collected by corresponding microelectrodes are received, noise with the frequency of less than 1Hz is filtered, direct-current components are filtered, 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 and is connected with the input end of the first-stage amplifying circuit. The first-stage amplifying circuit consists of three resistors R1, R6 and R9, a feedback capacitor C5 and a precise operational amplifier U1B, wherein the resistor R6 is connected between the reverse input end and the output end of the precise operational amplifier U1B, the feedback capacitor C5 is connected in parallel with the resistor R6, the resistor R9 is connected with the reverse input end and the ground of the precise operational amplifier U1B, the resistor R1 is connected with the output end of the high-pass filter circuit and the forward input end of the precise operational amplifier U1B, and the other end of the resistor R1 is used as the input end of the first-stage amplifying circuit. The feedback capacitor C5 has the function of phase compensation and prevents the precision operational amplifier U1B from generating self-oscillation. The band-pass filter circuit comprises an RC high-pass filter circuit formed by C3 and R7 and a Butterworth low-pass filter circuit formed by resistors R2 and R3, a low-noise operational amplifier U2B and capacitors C1 and C4, wherein the forward input end of the low-noise operational amplifier U2B is connected with one end of the capacitor C4 and one end of the 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, and 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 and R10, a feedback capacitor C6 and a precise operational amplifier U3B, the resistor R8 is connected between the reverse input end and the output end of the precise operational amplifier U3B, the feedback capacitor C6 is connected in parallel with the resistor R8, the resistor R10 is connected with the reverse input end and the ground of the precise operational amplifier U3B, the resistor R4 is connected with the output end of the band-pass filter circuit and the forward input end of the precise operational amplifier U3B, the output end of the precise operational amplifier U3B is connected with the analog input end of the data acquisition card 25, and other channels of the intracellular and extracellular electric signal conditioning module are identical to the circuits.
The electronic components related to the circuits are welded on an intracellular and extracellular electric signal conditioning module, as shown in fig. 2, the extracellular and extracellular electric signal conditioning module PCB substrate 12 of the module further comprises 31 paths of identical extracellular and extracellular electric signal conditioning circuits 8 to form a 32-channel extracellular and extracellular electric signal conditioning module, the extracellular and extracellular electric signal conditioning module is provided with two wiring terminals 7 and 13 corresponding to 32 microelectrodes in fig. 1, the extracellular and extracellular electric signal conditioning module is connected with a data acquisition card 25 by a flat cable, and the extracellular 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 1, the intracellular and extracellular electrical signal conditioning module is provided with a corresponding upward electrode array sensor pin slot 9, the pin 1 is connected with a corresponding microelectrode, and the electrode array sensor pin slot 9 is connected with a corresponding extracellular and extracellular electrical signal conditioning circuit 8.
The electroporation control circuit is composed of a POWER supply control circuit 21, an amplifying circuit 17 and a channel control circuit 15, as shown in fig. 3, the POWER supply control circuit 21 is composed of resistors R13 and R15, a capacitor C8, an inductor L2, an NMOS tube Q5 and a PMOS tube Q4, the grid electrode of the NMOS tube Q5 is connected with the digital output end of the data acquisition card 25 and is connected with the ground of the resistor R15, the source electrode is grounded, the drain electrode is connected with the grid electrode of the PMOS tube Q4 and is connected with a POWER supply input POWER (POWER supply module) with the resistor R13, the source electrode of the PMOS tube Q4 is connected with the POWER supply input POWER (POWER supply module), the drain electrode is connected with the inductor L2, the other end of the inductor L2 is connected with the capacitor C8 to form an LC filter circuit, and the filtered POWER supply VCC is supplied to the amplifying 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 when electroporation is performed.
The amplifying circuit 17 is configured to amplify the electroporation SIGNAL, as shown IN fig. 8b, where the positive input terminal of the op amp U4A is connected to the electroporation SIGNAL input SIGNAL, the negative input terminal is connected to ground with the resistor R14, and is connected to the output terminal of the op amp U4A with the resistor R16, the output terminal is the amplified electroporation SIGNAL output pulsein, and the electroporation SIGNAL input SIGNAL is connected to the analog output terminal of the data acquisition card 25 through the electroporation control module SIGNAL input terminal 19.
The amplifying circuit 17 may be formed by a one-channel or multi-channel op-amp, for example, a four-channel op-amp U4.
The channel control circuit 15 is a control switch, and may be an independent control switch or a multi-channel control switch, in this embodiment, a 1:1 four-channel multiplexer IC1 is used, and meanwhile, a voltage follower is placed in each channel, so that the driving capability of the pulse can be improved, even if some microelectrodes do not have a cell attachment current as large as possible, the perforation current of the pulse on other paths will not be weakened, the perforation efficiency of the cell is remarkably improved by separating electroporation of multiple pulses, and the detection flux of the system is improved.
As shown IN fig. 8c, the channel control circuit 15 is composed of a four-channel op-amp U5, a 1:1 four-channel multiplexer IC1, resistors R18, R19, R20, R21, taking a channel U5A of the op-amp and a channel of the multiplexer as an example, the reverse input end and the output end of the op-amp U5A are connected to form a voltage follower, the forward input end of the op-amp U5A is connected to the electroporation signal output pulsin, 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 lead-out end of the microelectrode array sensor 11, and the control end 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 of the multiplexer is connected to each microelectrode output of the microelectrode sensor 11 via the electroporation control circuit module power control 20.
Therefore, the four-channel operational amplifier comprises a 32-channel power supply control circuit 21, 8 groups of four-channel operational amplifiers U4 and 8 groups of channel control circuits 15, all channels are connected in one-to-one correspondence, 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 an electroporation control module power supply interface 18 is arranged on the electroporation control module and 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 16, the intracellular and extracellular electrical signal conditioning module is provided with a corresponding downward pin arrangement slot 10, the pin arrangement 16 is connected with a corresponding electroporation control circuit, and the pin arrangement slot 10 is connected with a corresponding microelectrode.
The power module 26 is used for providing +/-5V power for the intracellular and extracellular electric signal conditioning module, 20V power for the electroporation control module and 12V power for the data acquisition card 25; the input end of the power module 26 is connected with a 12V power supply, generates +/-5V power supply through a DC/DC chip, and is provided with a control switch for controlling the opening and closing of the +/-5V power supply, and the control switch is connected with a digital output port of the data acquisition card 25; a 20V power supply is generated by a DC/DC chip.
Preferably, the metal shielding box is further included, as shown in fig. 5, and is composed of a metal case 27, hinge 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 of the invention is as follows: when the electric signal is collected, the digital output end of the data collection card 25 outputs a high level, the +/-5V power supply of the control power supply module 26 is started to supply power to the extracellular and extracellular electric signal conditioning module, the extracellular ion concentration is changed due to action potential generated by cells in the cell culture cavity 3, the electric field is changed, the microelectrode transmits the electric field change to the input end of the extracellular and extracellular electric signal conditioning circuit 8, the cellular electric signal passes through the high-pass filter circuit, the primary amplification circuit, the band-pass filter circuit and the secondary amplification circuit, the amplitude of the cellular electric signal is ensured to be large enough, and finally the cellular electric signal is collected by the analog input end of the data collection card 25 and is transmitted to the upper computer for display and storage. When 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, the multiplexer of the electroporation control circuit is turned on, so that the channel can generate an 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 an electroporation signal. After electroporation, the microelectrode records the intracellular electric signals, and the intracellular electric signals are also amplified and filtered by the intracellular and extracellular electric signal conditioning circuit 8 and collected by the data collecting card 25.
An automatic electroporation-regulated screening intracellular and extracellular electrophysiological recording system, the workflow of which is shown in figure 9, specifically comprises the following steps:
firstly, myocardial cells and culture fluid thereof are placed in the microelectrode array sensor 11, the microelectrode array sensor 11 is inserted into an extracellular and intracellular electric signal conditioning module, and the microelectrode array sensor 11 is covered by a metal shielding cover 24.
And step two, starting upper computer software, setting the amplitude, frequency and pulse width range of the electroporation signal which is automatically traversed, controlling the generation of electroporation signals with different conditions channel by channel after electroporation duration, recording the intracellular electrical signals of the myocardial cells, and drawing and displaying an electrical signal diagram.
And thirdly, analyzing the acquired intracellular electric signals to obtain the optimal electroporation condition.
And step four, the upper computer re-controls all channels to generate electroporation signals with optimal conditions, records and stores the myocardial cell intracellular electrical signals, and displays the acquired intracellular electrical signal diagram in real time.
The application case of the present invention is given below.
The automatic electroporation regulation screening intracellular and extracellular electrophysiological recording system and method are mainly used for automatic detection of the myocardial cell intracellular electrical signals. Firstly, an electroporation control module is arranged below an extracellular and intracellular electric signal conditioning module, connecting lines among the modules, adding myocardial cells and culture fluid thereof into a cell culture cavity 3, inserting a microelectrode array sensor 11 into a slot 9, covering the microelectrode array sensor 11 by a metal shielding cover 24, opening an upper computer, entering a main interface, as shown in fig. 10, clicking to start, traversing electroporation conditions with different frequencies, amplitudes and pulse widths by the upper computer, as shown in fig. 11-12, selecting optimal electroporation conditions, and performing electroporation under the optimal electroporation conditions to acquire extracellular electric signals of myocardial cells. When the cell electric signal collection is completed, 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 myocardial intracellular electrophysiological recording high-efficiency automatic electroporation regulation screening system and method, can apply controllable electroporation to cells in different channels according to requirements, and can also detect changes of cell electrophysiological phenomena and specific parameters of electric signals. By controlling each channel to generate electroporation one by one, the efficiency of cell electroporation is improved, high-efficiency automatic analysis is realized through an upper computer, and the optimal conditions of electroporation are found out through traversing different electroporation conditions and combining data analysis, so that the intracellular electrical signals of myocardial cells are recorded with high quality.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary or exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (6)
1. An automated electroporation-mediated screening of extracellular and intracellular electrophysiological recording system, 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 microelectrodes and a common reference electrode lead-out;
the intracellular and extracellular electric signal conditioning module comprises a plurality of paths of extracellular and extracellular electric signal conditioning circuits with the same number as the microelectrodes, and each path of input end is respectively connected with each working electrode leading-out end and is used for filtering noise and amplifying the collected extracellular and extracellular electric signals; the ground wire of the intracellular and extracellular electric signal conditioning module is connected with the reference electrode leading-out end;
the electroporation control module comprises a plurality of electroporation control circuits with the same number as the microelectrodes, and each output end is respectively connected with each working electrode leading-out end and used for controlling the on-off of electroporation signals of each microelectrode;
the data acquisition card is characterized in that an analog input end of the data acquisition card is connected with an output end of the intracellular and extracellular electrical signal conditioning module, and an analog output end of the data acquisition card is connected with an input end of the electroporation control module to respectively generate electroporation signals corresponding to the microelectrodes;
the upper computer is connected with the data acquisition card and used for controlling the data acquisition card;
the power 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 extracellular and extracellular electric signal conditioning module, the electroporation control module and the data acquisition card;
each path of intracellular and extracellular electric signal conditioning circuit consists of a high-pass filter circuit, a first-stage amplifying circuit, a band-pass filter circuit and a second-stage amplifying 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;
each 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 tube and a PMOS tube and is used for controlling the power supply on-off of the amplifying circuit and the channel control circuit according to a control signal output by a digital output end of a data acquisition card; the amplifying circuit is an in-phase proportional amplifier and is used for amplifying electroporation signals output by an 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; the control end of the control switch is connected with the digital output end of the data acquisition card;
the power supply control circuit consists of a resistor R13, a resistor R15, a capacitor C8, an inductor L2, an NMOS tube Q5 and a PMOS tube Q4, wherein the grid electrode of the NMOS tube Q5 is connected with the digital output end of the data acquisition card and is connected with the resistor R15 at the same time, the source electrode of the NMOS tube Q5 is grounded, the drain electrode of the NMOS tube Q5 is connected with the grid electrode of the PMOS tube Q4 and is connected with the resistor R13 at the same time to the output end of the power supply module, the source electrode of the PMOS tube Q4 is connected with the output end of the power supply module, the drain electrode of the PMOS tube Q4 is connected with the inductor L2, and the other end of the inductor L2 is connected with the capacitor C8 to form an LC filter circuit, and the LC filter circuit is used for supplying power for the amplifying circuit and the channel control circuit after filtering.
2. The system of claim 1, further comprising a metallic shielding cage for shielding the external power frequency interference and the high frequency interference.
3. The system of claim 2, wherein the metallic shielding cage is further provided with an openable metallic shielding lid.
4. An efficient and automatic electroporation regulation condition screening measurement method, which is characterized in that the method is realized based on the extracellular electrophysiological recording system for automatic electroporation regulation screening according to any one of claims 1-3, and specifically comprises the following steps:
performing electroporation test by using a microelectrode array sensor, setting electroporation conditions with the same number according to the number of 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 collecting myocardial cell intracellular electrical signals of the corresponding microelectrode;
and analyzing the intracellular electrical signals of the myocardial cells of all microelectrodes to obtain the optimal electroporation condition.
5. The method of claim 4, wherein the electroporation conditions comprise electroporation signal amplitude, frequency and pulse width ranges, and electroporation duration.
6. The method of claim 5, wherein the electroporation signal is pulses having a magnitude of 0 to 20V, a frequency ranging from 5Hz to 100kHz, and a pulse width ranging from 10 μs to 0.2 s.
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