CN114636744A - Microelectrode array chip based on nano porous membrane and high-flux intracellular electric signal continuous monitoring system - Google Patents
Microelectrode array chip based on nano porous membrane and high-flux intracellular electric signal continuous monitoring system Download PDFInfo
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Abstract
The invention provides a microelectrode array chip based on a nano porous membrane and a high-flux intracellular electric signal continuous monitoring system, wherein the microelectrode array chip comprises a nano porous membrane substrate and a microelectrode array attached to the nano porous membrane substrate. And the micro-channel device is integrated at the bottom of the nano porous membrane substrate. The system comprises a nano porous membrane microelectrode array sensor for detecting cell electric signals. The invention utilizes the porous characteristic of the porous membrane to generate an uneven three-dimensional electrode structure, and enhances the coupling by increasing the contact area between the electrode and the cell, thereby obtaining higher-quality electric signal recording. The nano porous membrane microelectrode array sensor is integrated above the micro-channel device, and the drug test solution is applied to the micro-channels of different channels by utilizing the characteristics of the multi-channel of the micro-channel, so that the high-flux drug experiment test can be carried out, and the high-flux drug screening can be realized.
Description
Technical Field
The invention relates to a micromachining technology of a biosensor, in particular to a high-flux cellular electric signal continuous monitoring system based on nano porous membrane electroporation.
Background
The human body has two vital organs, the brain and the heart. The former is the control center of the human body, and almost all normal physiological activities are directly or indirectly controlled by the brain; the latter provides power for blood flow, transports blood to various parts of the body, and maintains normal physiological activities of the human body. However, cardiovascular disease is currently the number one killer of modern human health.
Most of the cardiovascular diseases such as heart failure, hypertension, coronary heart disease and arrhythmia are related to the physiological activities of the heart, such as mechanical pulsation, and the physiological behaviors of the heart, such as mechanical pulsation, are closely related to the electrophysiological activities of cells. Researchers have conducted extensive research into the mechanisms of cardiac work and cellular electrophysiological activity in the prevention and treatment of cardiovascular disease. The models established for the research of the heart electrophysiological mechanism are mainly divided into an in vivo animal model and an in vitro animal model, wherein the in vitro animal model monitors and analyzes the electrophysiological properties of the cardiac muscle cells or neurons on the level of single cells or cell networks. Currently, there are two main methods for monitoring single cell electrophysiological activity: patch clamp recording techniques and microelectrode array techniques.
The patch clamp technique is a method for accurately measuring transmembrane ion current. The signal in and among cells is transmitted through ion channels on cell membranes, the ion channels are composed of some special proteins produced by the cells, the special proteins are gathered and embedded on the cell membranes, the ion channels have selectivity and switching performance, and the signal transmission is realized through the regulation and control of the concentration of specific ions. In vivo, different kinds of ions pass through ion channels to generate current, and neurons and muscle cells are stimulated; in the sense organ, a channel converts a physical or chemical stimulus into an electrical signal of the nervous system. Even cells not associated with the central nervous system, such as blood cells in the blood, leukocytes of the immune system, cells of the liver or other organs, use ion channels for signaling.
The current patch clamp technology is 'gold standard' for detecting cell electric signals, and is a technology for reflecting various ion transport activities on a membrane by recording the current change of an ion channel on the membrane. In the early days, the accuracy that has limited this technique was mainly the area of the adsorption film and the tightness of the adsorption of the electrode to the film, and although changes in current could be detected, the noise was large. Later, with the continuous improvement and perfection of the technology by researchers, the researchers find that after the surface of the glass microelectrode is cleaned and negative pressure is added to the glass microelectrode, the contact area between the microelectrode and a film can be greatly reduced, the adsorption between the microelectrode and the film is more compact, the tip of the microelectrode and the adsorbed film surface form a G omega-level closed resistance, background noise is greatly reduced, the signal-to-noise ratio is improved, and mechanical isolation and electrical isolation are formed.
The patch clamp detection technology is used as the earliest developed electrophysiological research technology, changes of ion channels on cell membranes are generally detected by voltage or circuit clamping, the space-time resolution of signal detection is high, the signal-to-noise ratio is high, and the signal detection is always used as the 'gold standard' of cell electrical signal detection by researchers, but the technology has high operation difficulty and can be completed by experiential experimenters; moreover, the patch clamp technology generally aims at recording electric signals of single cells, so that the efficiency is low; more importantly, the patch clamp detection technology is an invasive detection method, certain damage is caused to cells, the electrophysiological activity of the cells cannot be detected for a long time, and the application occasions of the technology are greatly limited. With the rapid development of microelectronic technology, the microelectrode array-based cell electrical signal detection technology attracts the attention of researchers with the advantages of non-invasion, long-time signal recording, high throughput, and the like.
Microelectrode Arrays (microelectrodes Arrays) refer to chips formed by processing electrode patterns with cell sizes on certain substrates with good biocompatibility through a micro-nano processing technology, and are used for high-flux synchronous detection of cell electrical signals. Microelectrode array technology has been developed for over 50 years, and its source can trace back to 1972, and Thomas group first combined with microelectronic technology developed microelectrode array and successfully recorded the electrical signals of in vitro cultured chicken embryos. Then, the Gross group successfully detected the electrophysiological activity of the brain neuron cells of the snails by preparing a high-density microelectrode array by using a thin film lithography and electroplating process and an ultraviolet laser microbeam method. The Pine group adopts microelectrode array technology to detect the extracellular electric signals and simultaneously adopts patch clamp technology to detect the intracellular electric signals, which proves that the intracellular action potential detected by the patch clamp is related to the extracellular electric signals detected by the microelectrode. Since then, microelectrode array technology is widely used to detect electrophysiological activity of cardiomyocytes and neuronal cells. In the related research of myocardial cells, the MEAs play more and more important roles in the research fields of heart research, heart related drug screening and the like by virtue of the advantages of simple operation, high flux, long-time detection of cell electric signals and the like; in the related research of neuronal cells, MEAs are often used to research neuroelectrophysiology by virtue of their high-throughput and multi-channel properties, including the change of the electrophysiological activity of neuronal cells under the action of drugs, the change of the electrophysiological activity after stimulation of retinal nerves and olfactory nerves, and the like.
In the study of cellular electrophysiology, researchers are always more interested in intracellular electrical signals. Among the two methods for detecting the electrophysiological activity of the single cell, although the patch clamp recording technology can realize the high-precision and low-noise recording of the electrophysiological signals of the cell, the two methods have the defects of complex operation, large difficulty coefficient, low flux and irreversible damage to the cell, and are always not negligible; the latter microelectrode array technology can realize non-invasive long-time high-flux recording of extracellular electric signals, which are enough to explain problems in some application occasions such as observation of arrhythmia and beating frequency of myocardial cells, but the electric signals are still lack of some key characteristics compared with natural transmembrane potential, and thus the electrophysiological activity of the myocardial cells is prevented from being researched.
In recent years, techniques using microelectrode arrays in combination with electroporation have made optimistic progress in the multi-channel recording of electrical signals in cardiomyocytes. By applying an electric pulse with a low voltage (2V) to the microelectrode array, the cell membrane can be effectively perforated, and thus, the microelectrode array is contacted with the intracellular environment. Thus, the microelectrode records the action potential distribution in the cell. On the other hand, since the microelectrode array has a multi-channel recording performance, the technique of microelectrode electroporation can realize synchronous recording of intracellular electrical signals of a plurality of myocardial cells. However, microelectrode arrays are based on solid substrates and solid electrode structures, and do not have the capability of multi-channel microfluidic devices to integrate drug delivery to cells. Therefore, for the conventional microelectrode array chip, the medicament can only be directly added into the cell culture solution, and has no spatial resolution performance. Although the microelectrodes have multiple pathways, the recorded cellular electrical signals are similar and do not have the capability to test multiple drugs at high throughput on a single microelectrode array chip.
Therefore, the problem to be solved by those skilled in the art is how to design a non-invasive system for stably monitoring electrical signals in cardiomyocytes at high throughput for a longer time (e.g., more than half a hour) so as to help researchers to better study the electrophysiological properties of cardiomyocytes.
Disclosure of Invention
The invention aims to solve the problem that the prior art can not realize non-invasive high-flux and long-time stable monitoring of myocardial cell intracellular electric signals, and develops a microelectrode microfluidic chip based on nanoporous membrane electroporation and a high-flux intracellular electric signal continuous monitoring system to realize high-flux and long-time (> half an hour) stable measurement of myocardial cell electric signals. A porous membrane with nano-pores (the diameter of 400-1000nm) is used as a chip substrate, a metal microelectrode array is prepared on the surface of the porous membrane through micro-processing, and a non-electrode part is insulated by photoresist. And then, a photoetching technology is combined with dimethyl siloxane (PDMS) reverse mould to prepare the multi-micro-channel device. The nanoporous membrane microelectrode may be integrated over a microchannel device. The drug test solution is applied through the micro-channel at the bottom of the microelectrode of the nano porous membrane, and the drug test solution can be diffused to the cells above the microelectrode along the nano holes of the nano porous membrane. The myocardial cells are cultured on the surface of the nano porous membrane microelectrode and are influenced by the drug solution delivered by the micro-channel at the bottom of the nano-pore to change the action potential signals. On the other hand, the nano porous membrane microelectrode can record an extracellular action potential signal of the myocardial cells cultured on the surface of the nano porous membrane microelectrode. And the cell membrane can be punctured instantaneously by applying electric pulse through the microelectrode, so that the microelectrode records the action potential in the cell. As the micro-nano topological structure on the surface of the nano porous membrane has a better coupling effect with cells, the rupture time of the cell membrane after electroporation can be effectively prolonged, and the record of electric signals in the cells can be maintained for more than half an hour from experimental results.
The purpose of the invention is realized by the following technical scheme:
a nanoporous membrane based microelectrode array chip comprising: a nano porous membrane (Nanoporous membrane) substrate and a microelectrode array attached to the nano porous membrane substrate.
Further, the material of the nano porous membrane substrate is polyethylene terephthalate.
Further, still include: and the micro-channel device is integrated at the bottom of the nano porous membrane substrate.
Further, the micro flow channel device is a dimethyl siloxane (PDMS) micro flow channel chip with multiple channels (4-10 channels).
A high-throughput cellular electric signal monitoring system based on nanoporous membrane electroporation, comprising:
the nano porous membrane microelectrode array sensor comprises the microelectrode array chip based on the nano porous membrane and a cell culture cavity fixed above the microelectrode array chip, and is mainly used for detecting cell electrical signals.
And the sensor electric signal conditioning module is mainly used for filtering and amplifying the cell electric signals detected by the nano porous membrane microelectrode array sensor.
And the electroporation circuit module is connected with the electrodes on the nano porous membrane microelectrode array sensor and is mainly used for controlling the opening and closing of electroporation signals.
The signal acquisition module is connected with the sensor electric signal conditioning module and is used for receiving and recording the cell electric signals detected by the sensor; the signal acquisition module is connected with the electroporation circuit module and used as a pulse source to generate pulses under different conditions, and the electroporation circuit module is used as a switch between the acquisition card and the electrodes to achieve the purpose of applying different electroporation signals to the electrodes at any time.
And the upper computer module is connected with the signal acquisition module and is used for displaying the detected cell electric signals and/or controlling to generate different pulse signals.
And the power supply module is mainly used for supplying power to the sensor electric signal conditioning module, the electroporation circuit module and the signal acquisition module.
Furthermore, the sensor electrical signal conditioning module is composed of a primary amplifier module, a high-pass filter module, a low-pass filter module and a secondary amplifier module which are connected in sequence.
Furthermore, the signal acquisition module is a signal acquisition card.
Furthermore, the microelectrode array sensor of the nano porous membrane is provided with a pin header, the sensor electrical signal conditioning circuit board is provided with corresponding jacks, each pin header is connected with each working electrode, and the jacks on the circuit board are connected with the sensor electrical signal conditioning module; meanwhile, the input end of the data acquisition module is connected with the output end of the sensor electric signal conditioning module, the analog output end of the data acquisition module is connected with the output line of the electroporation circuit module, and the output end of the data acquisition module is connected with the input end of the upper computer.
Further, the nano porous membrane microelectrode array sensor is prepared by the following steps:
(1) a porous film with a substrate thickness of 0.5mm was selected, on which an RZJ-390PG-50 positive photoresist was spin-coated, and heated on a hot plate at 120 ℃ for 2 minutes.
(2) And covering a metal mask plate on the porous membrane, exposing, and developing by using an RZX3038 developing solution for 35s to remove the redundant photoresist.
(3) Then, Ti with the thickness of 10nm is subjected to magnetron sputtering on the substrate, Au with the thickness of 100nm is further sputtered, and then the photoresist and the redundant metal are stripped by acetone to form an electrode and a lead.
(4) Then, for covering the sensor with an insulating layer, the sensor was first spin-coated with SU-82002 photoresist to a thickness of 2 μm, heated on a hot plate at 95 ℃ for 1 minute, exposed with a metal mask, heated at 95 ℃ for 1 minute, developed with Propylene Glycol Methyl Ether Acetate (PGMEA) for 1 minute, finally cleaned with isopropyl alcohol, dried with a nitrogen gun and hardened at 150 ℃ for one hour.
Further, the multichannel microchannel device was prepared as follows: polydimethylsiloxane (PDMS) and a curing agent were mixed in a 10: 1, poured onto a prepared microchannel mold and cured at 90 ℃ for one and a half hours. The microfluidic channel has a length of 10mm, a width of 200 μm and a thickness of 100 μm.
Further, after the sensor chip is prepared, the sensor chip needs to be packaged, and the main packaging process is as follows:
firstly, preparing Polydimethylsiloxane (PDMS) and a curing agent, wherein the proportion of the PDMS to the curing agent is 10: 1, then coating a layer of PDMS curing agent on a specific PCB substrate, then pasting and fixing a chip on the PCB substrate, aligning an electrode of the chip with a metal interface of the PCB substrate, connecting and conducting the interface and the electrode by conductive silver adhesive, and then heating the chip in an oven at 80 ℃ for about 8 minutes to finish curing; fixing the glass culture cavity on the chip by using a PDMS curing agent, and heating and curing in an oven; and finally, welding the pin header on the PCB substrate. Then, a multi-channel micro-channel is attached to the bottom of the microelectrode chip and is connected with an external pump for conveying solution by a conduit.
Furthermore, the system is additionally provided with a metal shielding box for shielding power frequency noise and high frequency noise.
Compared with the prior art, the invention has the following advantages:
1. the microelectrode array sensor is prepared by taking the porous membrane as a substrate, the rugged three-dimensional electrode structure is generated by utilizing the porous characteristic of the porous membrane, and the coupling is enhanced by increasing the contact area between the electrode and the cell, so that the higher-quality electric signal record can be obtained.
2. The nano porous membrane microelectrode array sensor is integrated above the micro-channel device, and the drug test solution is applied to the micro-channels of different channels by utilizing the characteristics of the multi-channel of the micro-channel, so that the high-flux drug experiment test can be carried out, and the high-flux drug screening can be realized.
Drawings
FIG. 1 is a schematic diagram of a microelectrode array chip;
FIG. 2 is a schematic view showing a process for fabricating a porous membrane microelectrode array chip;
FIG. 3 is a schematic view showing a micro-electrode array sensor and a micro-channel device including a micro-electrode array chip based on a nanoporous membrane;
FIG. 4 is a system block diagram of a high throughput cardiomyocyte electrical signal recording system;
FIG. 5 is a schematic diagram of an electrical signal conditioning module of the microelectrode array sensor;
FIG. 6 is a schematic diagram of a cell electrical signal conditioning circuit;
FIG. 7 is a schematic diagram of an electroporation circuit module;
FIG. 8 is a schematic diagram of an electroporation circuit;
FIG. 9 is a diagram of the extracellular electrical signals recorded by the system;
FIG. 10 is a graph of the electrical signals recorded by the system;
in the figure, a pin header 1, a cell culture glass cavity 2, a reference platinum wire electrode 3, a PCB substrate 4, a nano porous membrane substrate 5, a microelectrode array 6, a microelectrode array sensor 7 of the porous membrane substrate, a cell electrical signal conditioning circuit 8, a pin header jack 9 for connecting a microelectrode array sensor chip, an electrical signal conditioning circuit PCB substrate 10, an electrical signal conditioning circuit output terminal 11, a data acquisition card input terminal 12, an electrical signal conditioning circuit module power input terminal 13, a pin header jack 14 for connecting a power supply perforating circuit module, an electroporation circuit module pin header 15, an electroporation circuit output terminal 16, a data acquisition card output terminal 17, an electroporation circuit module power input terminal 18, an electroporation circuit PCB substrate 19, an electroporation circuit 20, a primary amplifier 21, a high-pass filter 22, a low-pass filter 23, a secondary amplifier 24, a section of extracellular electrical signals 25, a reference platinum wire electrode 3, a PCB substrate 4, a nano porous membrane substrate 5, a microelectrode array 6, a microelectrode array sensor 7 of the porous membrane substrate, a cell electrical signal conditioning circuit module power input terminal 14, an electroporation circuit module power supply, An extracellular electrical signal 26, an intracellular electrical signal 27, an intracellular electrical signal 28, and a micro-channel device 29.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
A micro-electrode array chip based on a nano porous membrane, as shown in FIG. 1, comprises: a nanoporous membrane substrate 5 and a microelectrode array 6 attached to the nanoporous membrane substrate 5.
The microelectrode array chip based on the porous membrane has a good coupling effect with cells due to the micro-nano topological structure on the surface of the porous membrane, the rupture time of the cell membrane after electroporation can be effectively prolonged, and the record of electric signals in the cells can be maintained for more than half an hour from experimental results. Preferably, the material of the nanoporous membrane substrate is polyethylene terephthalate (PET), and illustratively, the microelectrode array chip based on the nanoporous membrane can be prepared by the following method, and the preparation process is shown in fig. 2:
(1) a nano porous film with a substrate thickness of 0.5mm is selected, an RZJ-390PG-50 positive photoresist is spin-coated on the nano porous film, and the nano porous film is heated on a hot plate at 120 ℃ for 2 minutes.
(2) And covering a metal mask with a microelectrode array pattern on the nano porous membrane, exposing, and developing with an RZX3038 developing solution for 35s to remove the redundant photoresist.
(3) Then, Ti with the thickness of 10nm is subjected to magnetron sputtering on the nano porous membrane substrate, Au with the thickness of 100nm is sputtered, and then the photoresist and the redundant metal are stripped by acetone to form an electrode and a lead.
(4) Then covering an insulating layer on the surface, firstly using SU-82002 photoresist to spin-coat, the thickness is 2 μm, heating for 1 minute on a hot plate at 95 ℃, exposing by using a metal mask, heating for 1 minute at 95 ℃, then using Propylene Glycol Methyl Ether Acetate (PGMEA) to develop for 1 minute, finally using isopropanol to clean, using a nitrogen gun to dry, and hardening for one hour at 150 ℃ to obtain the microelectrode array chip based on the porous membrane.
The microelectrode array 6 is mainly used as a working electrode, and in order to reduce crosstalk between the electrodes as much as possible, the electrodes of the microelectrode array 6 need to have a certain distance.
In order to facilitate circuit connection, as a preferred embodiment, a PCB substrate is further provided, the microelectrode array chip is fixed on the PCB substrate, and the electrodes of the microelectrode array 6 are aligned with the metal interfaces on the PCB substrate and are connected and conducted.
Further, the micro-electrode array chip further includes: and a micro-channel device 29 integrated on the nano porous membrane substrate, wherein the micro-channel device 29 is preferably a dimethyl siloxane micro-channel chip. The micro flow channel device 29 is prepared as follows: polydimethylsiloxane (PDMS) and a curing agent were mixed at a ratio of 10: 1, poured onto a prepared microchannel mold and cured at 90 ℃ for one and a half hours. The microfluidic channel has a length of 10mm, a width of 200 μm and a thickness of 100 μm.
The drug test solution is applied through the micro-channel at the bottom of the nano-porous membrane microelectrode and can diffuse to the cells above the microelectrode along the nano-pores of the nano-porous membrane substrate 5. The myocardial cells are cultured on the surface of the nano porous membrane microelectrode and are influenced by the drug solution delivered by the micro-channel at the bottom of the nano-pore, so that action potential signals of the myocardial cells are changed, and the performance of testing various drugs is realized.
Corresponding to the microelectrode array chip, the invention also provides a nano porous membrane microelectrode array sensor, fig. 3 is a schematic structural diagram of the microelectrode array sensor and a micro-channel device comprising the microelectrode array chip based on the nano porous membrane, and as shown in fig. 3, the microelectrode array sensor based on the nano porous membrane mainly comprises a microelectrode array 6 serving as a working electrode, a reference platinum wire electrode 3, a cell glass culture cavity 2, a nano porous membrane substrate 5 and a PCB substrate 4. The size of the microelectrode array 6 is 20mm multiplied by 20mm, 32 working electrodes are totally arranged, the diameter of each working electrode is 10 μm, the minimum distance between the working electrodes is set to be 150 μm, and the working electrodes are connected with the PCB substrate 4 through conductive silver adhesive. The cell glass culture cavity 2 is fixed on the nano porous membrane substrate 5 by PDMS curing agent, one end of the reference platinum wire electrode 3 is connected with the ground, and the other end is arranged in the cell glass culture cavity 2.
4 groups of pin headers 1 are distributed on the PCB substrate 4 and are connected with pin header jacks 9 for connecting the microelectrode array sensor chip through the pin headers 1.
Corresponding to the nano porous membrane microelectrode array sensor, the invention also provides a high-throughput intracellular electric signal continuous monitoring system, and fig. 4 is a structural schematic diagram of the high-throughput intracellular electric signal continuous monitoring system, as shown in fig. 4, comprising:
the nano porous membrane microelectrode array sensor 7 is mainly used for detecting cell electric signals.
And the sensor electric signal conditioning module is mainly used for filtering and amplifying the cell electric signals detected by the nano porous membrane microelectrode array sensor 7.
The electroporation circuit module is mainly used for controlling the on and off of electroporation signals.
The signal acquisition module is used for receiving and recording cell electric signals detected by the sensor and generating pulse electroporation signals under different conditions, and generally a data acquisition card can be adopted.
And the upper computer module is used for displaying the detected cell electric signals and/or controlling to generate different pulse signals.
And the power supply module is mainly used for supplying power to the sensor electric signal conditioning module, the electroporation circuit module and the signal acquisition module.
Specifically, as shown in fig. 5, the sensor electrical signal conditioning module is composed of a cell electrical signal conditioning circuit 8, a pin header jack 9 for connecting a microelectrode array sensor chip, an electrical signal conditioning circuit PCB substrate 10, an electrical signal conditioning circuit output terminal 11, an electrical signal conditioning circuit power input terminal 13, and a pin header jack 14 for connecting a power supply perforation circuit module. The nano porous membrane microelectrode array sensor 7 is inserted on the pin header jack 9 through the pin header 1, so that the corresponding connection between the working electrode and the cell electrical signal conditioning circuit 8 is realized, and the detected cell electrical signal is input to the cell electrical signal conditioning circuit 8 for filtering and amplifying. The cell electric signal conditioning circuit 8 mainly comprises a primary amplifier 21, a high-pass filter 22, a low-pass filter 23 and a secondary amplifier 24, fig. 6 is an exemplary electric signal conditioning circuit diagram, specific parameters are as shown in fig. 6, each cell electric signal conditioning circuit 8 is dual-channel and corresponds to 32 working electrodes, the whole sensor electric signal conditioning module has 16 same circuits in total and has 32 channels in total, the output of each channel is collected to an electric signal conditioning circuit output terminal 11, meanwhile, the electric signal conditioning circuit output terminal 11 is connected with an input end 12 of a data acquisition card, and a power supply input end 13 of the electric signal conditioning circuit module is externally connected with a +/-5V power supply for supplying power. In addition, the pin header jack 14 to which the electroporation circuit module is connected to the electroporation circuit module.
Further, fig. 7 is a schematic structural diagram of an electroporation circuit module, which is composed of an electroporation circuit module pin header 15, an electroporation circuit output terminal 16, an electroporation circuit module power input terminal 18, an electroporation circuit PCB substrate 19, and an electroporation circuit 20. The electroporation circuit module is inserted on the pin header jacks 14 through the pin header 15 so as to be connected with the working electrode of the nano porous membrane microelectrode array sensor 7. The schematic diagram of the electroporation circuit 20 is shown in fig. 8, the PMOS transistor is controlled to operate through the switch, when the switch control inputs a low level 0V, at this time, Q2 is turned on, the voltage follower U2 operates, and a pulse signal is synchronously output. The operational amplifier in the schematic shown in fig. 8 is a single operational amplifier, controlling the electroporation of a single working electrode, requiring 32 identical circuits corresponding to the 32 working electrodes; preferably, the electroporation circuit 20 may employ four operational amplifiers to control four channels of electroporation, and only 8 identical circuits are required corresponding to 32 working electrodes. The output of each channel is collected at the output terminal 16 of the electroporation circuit, and the output terminal 16 of the electroporation circuit is connected with the input terminal 17 of the data acquisition module, and the pulse signal of electroporation is provided by the data acquisition module.
Further, the cell electric signal collection working process of the invention is as follows: culturing the myocardial cells of the mouse in the cell glass culture cavity, starting a power supply, starting the system to work, collecting and obtaining extracellular electric signals of the myocardial cells by a working electrode of the nano porous membrane microelectrode array sensor 7, transmitting the extracellular electric signals to the sensor electric signal conditioning module for filtering and amplifying, and collecting and transmitting the signals to an upper computer by a signal collecting device for storage and/or display; when intracellular signals are collected, the upper computer controls the data collection card to send out electroporation pulse signals, the electroporation pulse signals are controlled by the electroporation circuit module to be transmitted to the working electrode of the nano porous membrane microelectrode array sensor 7 and applied to the myocardial cells on the working electrode, the working electrode collects the intracellular electric signals of the myocardial cells, the intracellular electric signals are transmitted to the sensor electric signal conditioning module to be filtered and amplified, and then the signals are collected by the signal collection device and transmitted to the upper computer to be stored and/or displayed. FIGS. 9-10 are graphs showing the results of signals collected by the system of the present invention, in which FIG. 9 is a graph showing the recorded electrical signals before electroporation, i.e., extracellular electrical signals, including a segment of extracellular electrical signal 25 and an amplified extracellular electrical signal 26; where FIG. 10 is a graph of the electrical signals recorded after electroporation, including a segment of intracellular electrical signal 27, and an amplified intracellular electrical signal 28.
The portion of the duration of the intracellular electrical signal maintenance across the experimental dish may be up to 1 hour or more, with 12 pathways of intracellular electrical signal maintenance for > half an hour.
Further, six different test solutions were prepared, including sodium ion solution, potassium ion solution, calcium ion solution, amiodarone solution, flecainide solution, diltiazem solution. Wherein standard solutions are purchased on the net for sodium ion solution, potassium ion solution and calcium ion solution, the sodium ion solution is 1mg/mL, the potassium ion solution is 0.1mg/mL, and the calcium ion solution is 1 mg/mL. Amiodarone solution, flecainide solution and diltiazem solution are diluted by Phosphate Buffered Saline (PBS), wherein the amiodarone solution is configured to be 40 mu g/mL, the flecainide solution is configured to be 0.1 mu g/mL, and the diltiazem solution is configured to be 10 ng/mL. These six solutions were then injected into the corresponding microchannels through the catheters. The microelectrode on the corresponding micro-channel can record the extracellular electric signal change of the myocardial cell. The electric signal of the cell recorded by the microelectrode is converted from the electric signal outside the cell into the electric signal inside the cell by applying electroporation through the microelectrode. By utilizing the characteristics of the micro-channel and the multi-channel, the drug test solution is applied to the micro-channels of different channels, and the system disclosed by the invention is utilized to carry out high-throughput drug experiment test, so that high-throughput drug screening is realized.
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.
Claims (7)
1. A microelectrode array chip based on a nano porous membrane is characterized by comprising: a nano porous membrane substrate and a microelectrode array attached on the nano porous membrane substrate.
2. The microelectrode array chip of claim 1, wherein the nanoporous membrane substrate is polyethylene terephthalate.
3. The microelectrode array chip of claim 1, further comprising: and the micro-channel device is integrated at the bottom of the nano porous membrane substrate.
4. The microelectrode array chip of claim 3, wherein the micro-channel device is a dimethylsiloxane micro-channel chip.
5. A high-throughput continuous monitoring system for electrical signals in a cell, comprising:
a nanoporous membrane microelectrode array sensor comprising the nanoporous membrane based microelectrode array chip of any of claims 1 to 4 and a cell culture chamber fixed above the microelectrode array chip, the nanoporous membrane microelectrode array sensor being primarily for detecting electrical signals of cells.
And the sensor electric signal conditioning module is mainly used for filtering and amplifying the cell electric signals detected by the nano porous membrane microelectrode array sensor.
And the electroporation circuit module is connected with the electrodes on the nano porous membrane microelectrode array sensor and is mainly used for controlling the opening and closing of electroporation signals.
The signal acquisition module is connected with the sensor electric signal conditioning module and is used for receiving and recording the cell electric signals detected by the sensor; the signal acquisition module is connected with the electroporation circuit module and is used for generating pulse electroporation signals under different conditions.
And the upper computer module is connected with the signal acquisition module and is used for displaying the detected cell electric signals and/or controlling to generate different pulse signals.
And the power supply module is mainly used for supplying power to the sensor electric signal conditioning module, the electroporation circuit module and the signal acquisition module.
6. The continuous high-throughput intracellular electrical signal monitoring system according to claim 5, wherein the sensor electrical signal conditioning module comprises a primary amplifier module, a high-pass filter module, a low-pass filter module and a secondary amplifier module which are connected in sequence.
7. The system for continuous monitoring of high throughput intracellular electrical signals according to claim 5, wherein the signal acquisition module is a signal acquisition card.
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