Microelectrode array device, preparation method thereof and detection system

Abstract

The invention discloses a microelectrode array device and a preparation method thereof and a detection system.The microelectrode array device comprises a substrate and a plurality of microelectrodes arranged on the surface of one side of the substrate, and the microelectrodes are distributed around the substrate and form a preset pattern; each microelectrode comprises a first conductive layer and a second conductive layer, the first conductive layer is arranged between the substrate and the second conductive layer, the second conductive layer comprises a three-dimensional nano carbon layer, the preset pattern comprises a recording area and a non-recording area, and the non-recording area is covered with an insulating layer. According to the embodiment of the invention, multi-channel intracellular action potential detection is realized based on electroporation, the difficulty of a preparation process is reduced, and the method can be widely applied to the technical field of signal detection.

Description

Technical Field

[0001] The present invention relates to the technical field of signal detection, and in particular to a microelectrode array device and a preparation method and a detection system thereof. Background Art

[0002] High-content monitoring of intracellular action potentials in cardiomyocytes is of great significance for studying cardiac electrophysiology, heart diseases, and drug development. High-content action potential monitoring can provide high-throughput and high-resolution multidimensional data, such as the depolarization and repolarization time of cardiomyocytes. In recent years, patch clamp technology has become a gold standard for recording intracellular AP (Action Potential), which measures transmembrane potentials by directly entering the cell through a glass pipette. The intracellular AP recorded by patch clamp has a high signal-to-noise ratio (SNR), time resolution, and signal fidelity, and usually up to 10 patch clamps are operated in parallel. Due to its low detection throughput and complex operation, patch clamp technology is difficult to efficiently reveal complex pathophysiology such as heart disease, which limits its application in high-throughput cell electrophysiology research. Although several multi-channel automated patch clamp techniques have been proposed to increase sample processing, these techniques usually require the use of isolated cells in suspension, which damages the correlation of electrical connections between cells.

[0003] The microelectrode array platform is widely used to monitor cellular electrical signals in large cell populations because of its multi-channel detection capabilities. In order to improve the accuracy of electrophysiological detection. However, the current microelectrode array process is very complicated and not conducive to mass production; and the current microelectrodes are mainly limited to recording extracellular field potentials and cannot record intracellular action potentials. In related technologies, ultrafast pulsed lasers are used to generate hot carriers to perforate the cell membrane. The method based on optical perforation can successfully penetrate the cell membrane, but it requires complex optical alignment and it is difficult to achieve synchronous optical perforation. Summary of the invention

[0004] In view of this, in order to solve one of the above problems, an embodiment of the present invention aims to provide a microelectrode array device and a preparation method and a detection system thereof, which realize multi-channel intracellular action potential detection based on electroporation and reduce the difficulty of the preparation process.

[0005] On the one hand, an embodiment of the present invention provides a microelectrode array device, comprising a substrate and a plurality of microelectrodes arranged on one side surface of the substrate, wherein the plurality of microelectrodes are distributed around the substrate and form a preset pattern, each of the microelectrodes comprises a first conductive layer and a second conductive layer, the first conductive layer is arranged between the substrate and the second conductive layer, the second conductive layer comprises a three-dimensional nanocarbon layer, the preset pattern comprises a recording area and a non-recording area, and the non-recording area is covered with an insulating layer.

[0006] Optionally, the three-dimensional nanocarbon layer includes a graphene layer or a carbon nanotube layer.

[0007] Optionally, the graphene layer includes graphene in a petal structure.

[0008] The implementation of the embodiments of the present invention includes the following beneficial effects: a microelectrode array device includes a substrate and a plurality of microelectrodes on the substrate, each microelectrode includes a first conductive layer and a second conductive layer, and the second conductive layer includes a three-dimensional nanocarbon layer; during the test process, the electrode of the three-dimensional nanocarbon layer structure is tightly wrapped by the cell membrane, thereby reducing the gap distance between the cell membrane and the electrode, improving the sealing resistance, and improving the signal recording quality of the microelectrode array; in addition, since the three-dimensional nanocarbon microelectrode is small in size, gravity when the cell falls or the diffusion of the cell on the substrate can cause the microelectrode to penetrate the cell membrane, which makes it possible to detect intracellular action potentials.

[0009] On the other hand, an embodiment of the present invention provides a method for preparing a microelectrode array device, comprising:

[0010] providing a substrate, and cleaning the substrate;

[0011] A first conductive layer and a second conductive layer are sequentially prepared on one side surface of the substrate; the second conductive layer comprises a three-dimensional nanocarbon layer;

[0012] Etching the first conductive layer and the second conductive layer to form a preset pattern; the preset pattern includes a plurality of microelectrodes distributed around the substrate;

[0013] An insulating layer is prepared in the non-recording area of ​​the preset pattern; the preset pattern includes a recording area and a non-recording area.

[0014] Optionally, the first conductive layer includes a metal layer, and the first conductive layer and the second conductive layer are sequentially prepared on a surface of one side of the substrate, including:

[0015] Using magnetron sputtering to prepare the first conductive layer on one side surface of the substrate;

[0016] The second conductive layer is prepared on the surface of the first conductive layer by using a vapor deposition method.

[0017] Optionally, etching the first conductive layer and the second conductive layer to form a preset pattern includes:

[0018] By adopting a laser etching method, on a substrate prepared with a first conductive layer and a second conductive layer, redundant first conductive layer and second conductive layer are removed according to a preset pattern to form the preset pattern.

[0019] The implementation of the embodiment of the present invention includes the following beneficial effects: first, a first conductive layer and a second conductive layer are prepared on a substrate, and then the first conductive layer and the second conductive layer are etched to form a preset pattern, and an insulating layer is prepared in a non-recording area of ​​the preset pattern. The preparation process of the microelectrode array device is simple, the difficulty of the preparation process is reduced, and large-scale production is facilitated.

[0020] On the other hand, an embodiment of the present invention provides a detection system for a microelectrode array device, comprising a circuit board, a cell culture chamber and the above-mentioned microelectrode array device, wherein the cell culture chamber is fixed in the recording area of ​​the microelectrode array device, and several microelectrodes of the microelectrode array device are connected to the circuit board.

[0021] Optionally, the detection system further comprises a signal processing module, an electroporation module and a processor, the circuit board connects the signal processing module and the electroporation module, and the processor connects the signal processing module and the electroporation module.

[0022] Optionally, the signal processing module includes an amplifier, a filter and an analog-to-digital converter.

[0023] Optionally, the electroporation module includes a signal generator and a voltage buffer.

[0024] The implementation of the embodiment of the present invention includes the following beneficial effects: the detection system of the microelectrode array device includes a circuit board, a cell culture chamber and a microelectrode array device, the cell culture chamber is fixed in the recording area of ​​the microelectrode array device, and several microelectrodes of the microelectrode array device are connected to the circuit board. A voltage signal is applied to the microelectrode through the circuit board to form a local electric field on both sides of the cell membrane to generate nanopores in the cell membrane, that is, intracellular action potential detection is achieved through electroporation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic structural diagram of a microelectrode array device provided by an embodiment of the present invention;

[0026] Figure 2 It is a schematic flow chart of the steps of a method for preparing a microelectrode array device provided by an embodiment of the present invention;

[0027] Figure 3 It is a schematic flow chart of the steps of another method for preparing a microelectrode array device provided by an embodiment of the present invention;

[0028] Figure 4 is a SEM image of a microelectrode array provided by an embodiment of the present invention;

[0029] Figure 5 is a schematic diagram of a detection system of a microelectrode array device provided by an embodiment of the present invention;

[0030] Figure 6 It is a structural block diagram of a detection system of a microelectrode array device provided by an embodiment of the present invention;

[0031] Figure 7 It is a structural block diagram of another detection system of a microelectrode array device provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The step numbers in the following embodiments are only provided for the convenience of explanation and description, and the order between the steps is not limited in any way. The execution order of each step in the embodiment can be adaptively adjusted according to the understanding of those skilled in the art.

[0033] See also Figure 1 The embodiment of the present invention provides a microelectrode array device, comprising a substrate (1-1) and a plurality of microelectrodes (1-2) arranged on a side surface of the substrate (1-1), wherein the plurality of microelectrodes (1-2) are distributed around the substrate (1-1) and form a preset pattern, wherein each microelectrode (1-2) comprises a first conductive layer and a second conductive layer, wherein the first conductive layer is arranged between the substrate (1-1) and the second conductive layer, and the second conductive layer comprises a three-dimensional nanocarbon layer, wherein the preset pattern comprises a recording area (1-3) and a non-recording area (1-4), and the non-recording area (1-4) is covered with an insulating layer.

[0034] Microelectrodes are used to detect intracellular action potentials. Microelectrodes can be divided into segments of different thicknesses. The recording area refers to the area where intracellular action potentials can be detected, and the non-recording area refers to the area that is not used to detect intracellular action potentials. For example, see Figure 1 The microelectrode is divided into two sections of different thicknesses. The thinner section is used to detect the signal of intracellular action potential, and the thicker section is used to transmit the signal of intracellular action potential. The area formed by the thinner section of the electrode is the recording area, and the area formed by the thicker section of the electrode is the non-recording area.

[0035] It should be noted that the first conductive layer includes but is not limited to a metal layer, such as a gold layer, a titanium layer, a chromium layer or a mixed metal layer, etc. The number of microelectrodes on the substrate is determined according to actual applications and is not specifically limited in this embodiment. For example, 32 microelectrodes are provided on the substrate.

[0036] Optionally, the three-dimensional nanocarbon layer includes a graphene layer or a carbon nanotube layer.

[0037] Graphene or carbon nanotubes have good electrical conductivity, which can further improve the accuracy of the test results. In addition, the preparation process of graphene or carbon nanotubes is mature, which can reduce the difficulty of the production process.

[0038] Optionally, the graphene layer includes graphene in a petal structure.

[0039] Optimization of microelectrode dimensions and structural morphology: By constructing three-dimensional graphene structures (such as three-dimensional porous networks, three-dimensional micro / nano interconnected scaffolds, etc.), the specific surface area of ​​graphene can be significantly increased, providing more contact sites for electrolyte ions, thereby improving capacitance performance and energy density. For example, by controlling the loading amount of graphene to optimize the three-dimensional structure, ensure uniform current line distribution, enhance the accessibility of electrolyte ions and promote ion dynamics.

[0040] Microelectrode surface functionalization: chemical modification, such as modifying the graphene surface by chemical methods (such as oxidation, reduction, doping, etc.), introducing functional groups or changing the surface charge state, thereby improving the interaction between graphene and the electrolyte, improving capacitance performance and cycle stability. In addition, graphene can also be compounded with other materials (such as metal oxides, conductive polymers, etc.) to form composite electrodes; these composite materials can play their respective advantages, such as the high pseudocapacitance of metal oxides, the high conductivity and stability of conductive polymers, thereby improving the performance of the overall electrode.

[0041] The implementation of the embodiments of the present invention includes the following beneficial effects: a microelectrode array device includes a substrate and a plurality of microelectrodes on the substrate, each microelectrode includes a first conductive layer and a second conductive layer, and the second conductive layer includes a three-dimensional nanocarbon layer; during the test process, the electrode of the three-dimensional nanocarbon layer structure is tightly wrapped by the cell membrane, thereby reducing the gap distance between the cell membrane and the electrode, improving the sealing resistance, and improving the signal recording quality of the microelectrode array; in addition, since the three-dimensional nanocarbon microelectrode is small in size, gravity when the cell falls or the diffusion of the cell on the substrate can cause the microelectrode to penetrate the cell membrane, which makes it possible to detect intracellular action potentials.

[0042] See also Figure 2 and Figure 3 The present invention provides a method for preparing a microelectrode array device, comprising:

[0043] S100, providing a substrate, and cleaning the substrate.

[0044] The substrate is determined according to the actual application, and this embodiment does not make any specific restrictions, such as quartz glass, see Figure 3 The cleaning method of the substrate is determined according to the actual application, and is not specifically limited in this embodiment. For example, the quartz glass substrate is cleaned with water and anhydrous ethanol respectively.

[0045] S200, sequentially preparing a first conductive layer and a second conductive layer on one side surface of the substrate; the second conductive layer includes a three-dimensional nanocarbon layer.

[0046] The first conductive layer and the second conductive layer are determined according to actual applications, and are not specifically limited in this embodiment. The preparation methods of the first conductive layer and the second conductive layer are determined according to their respective materials. For example, if the conductive layer is a metal material, a magnetron sputtering method can be used, and if the conductive layer is a carbon material, a vapor deposition method can be used.

[0047] Optionally, the first conductive layer includes a metal layer, and the first conductive layer and the second conductive layer are sequentially prepared on a surface of one side of the substrate, including:

[0048] S210, preparing a first conductive layer on one surface of the substrate by magnetron sputtering;

[0049] S220, preparing a second conductive layer on the surface of the first conductive layer by using a vapor deposition method.

[0050] In a specific embodiment, a gold film (Au purity 99.999%) with a thickness of 30 nm is sputtered on one surface of the substrate using a magnetron sputtering apparatus. Figure 3 (b). Next, the Au-coated quartz glass substrate was transferred to a radio frequency microwave plasma enhanced chemical vapor deposition (MPECVD) device. The substrate was heated to 950°C in a vacuum chamber (25 Pa) and heated to 20 sccm of hydrogen (H 2 ) flow rate, the substrate was pretreated with 1500W plasma for 15 minutes. Subsequently, H 2 and methane (CH 4 ) was introduced into a vacuum chamber and deposited at a plasma power of 1500 W for 20 min to grow nano-petal structure graphene on the Au surface, such as Figure 3 Middle (c).

[0051] S300, etching the first conductive layer and the second conductive layer to form a preset pattern; the preset pattern includes a plurality of microelectrodes distributed around the substrate.

[0052] It should be noted that the first conductive layer and the second conductive layer have the same shape and area, and the preset pattern and the intervals between the plurality of microelectrodes distributed around the substrate are determined according to actual applications, and this embodiment does not impose any specific restrictions.

[0053] Optionally, etching the first conductive layer and the second conductive layer to form a preset pattern includes:

[0054] S310, using a laser etching method, on a substrate having a first conductive layer and a second conductive layer, removing excess first conductive layer and second conductive layer according to a preset pattern to form a preset pattern.

[0055] In a specific embodiment, after the substrate covered with the first conductive layer and the second conductive layer is cooled, the excess graphene and the gold connecting layer are removed by ultraviolet laser etching technology to obtain an uninsulated MEA (Microelectrode Array) chip, such as Figure 3 Middle (d).

[0056] S400, preparing an insulating layer in a non-recording area of ​​a preset pattern; the preset pattern includes a recording area and a non-recording area.

[0057] Finally, a layer of uncured PDMS (PDMS: curing agent = 10:1) was spin-coated on the non-recording area of ​​the graphene electrode as an insulating layer and thermally cured at 80°C for 2 hours. Finally, a 32-channel carbon petal electrode array chip was obtained, such as Figure 3 Middle(e).

[0058] The SEM characterization process of the nanocarbon petal microelectrode array is as follows: A scanning electron microscope equipped with a field emission gun (FEG) was used to observe the sample morphology. High-resolution images of 1024×768 pixels were collected at an accelerating voltage of 10 kV and a working distance of 9 mm. Before imaging, the sample was gold-sprayed to improve the image quality. Pseudo-colors were added to different parts of the SEM image of the nanocarbon petal electrode array to distinguish different structures. Figure 4 As shown in (a), the wire part of the nanocarbon petals MEA is insulated by PDMS, and only the nanocarbon petals MEA for recording cell electrical signals are exposed. Figure 4 As shown in (b), the surface of the nanocarbon petal electrode presents a typical nanosheet structure with very high roughness at the nanoscale. Figure 4 As shown in (c), there are empty nanogaps between graphene protrusions, which is beneficial for promoting cell adhesion and better electrical contact. In addition, the structure of vertically grown graphene sheets is similar to the nanostructure of vertical silicon materials, which will be beneficial for achieving nano-electroporation and extending the time of intracellular recording, such as Figure 4 Middle (d).

[0059] The implementation of the embodiment of the present invention includes the following beneficial effects: first, a first conductive layer and a second conductive layer are prepared on a substrate, and then the first conductive layer and the second conductive layer are etched to form a preset pattern, and an insulating layer is prepared in a non-recording area of ​​the preset pattern. The preparation process of the microelectrode array device is simple, the difficulty of the preparation process is reduced, and large-scale production is facilitated.

[0060] See also Figure 5An embodiment of the present invention provides a detection system for a microelectrode array device, comprising a circuit board (2-3), a cell culture chamber (2-1) and the above-mentioned microelectrode array device (2-2), wherein the cell culture chamber (2-1) is fixed to a recording area of ​​the microelectrode array device (2-2), and a plurality of microelectrodes of the microelectrode array device (2-2) are connected to the circuit board.

[0061] In a specific embodiment, the assembly process of the detection system of the microelectrode array device is as follows: the nanocarbon petal MEA chip is connected to a PCB circuit board to facilitate the connection of the nanocarbon petal MEA to the signal acquisition circuit system. First, the nanocarbon petal electrode array chip is fixed to a customized PCB using PDMS (PDMS: curing agent = 10:1), and 32 electrodes are connected to the PCB using a conductive silver adhesive. Subsequently, a 1 cm wide and 1.5 cm high glass ring is glued to the center of the device with PDMS as a cell culture chamber. The reference electrode is fixed to the plastic cover and electrically connected to the PCB. Finally, a row of pin interfaces is welded on the PCB to match the interface of the electrophysiological signal acquisition system, and a carbon petal electrode array device is obtained through the above operations. The nanocarbon petal electrode array device uses a universal reference electrode for electrophysiological signal recording and nano-electroporation.

[0062] Optionally, see Figure 6 The detection system also includes a signal processing module, an electroporation module and a processor. The circuit board connects the signal processing module and the electroporation module, and the processor connects the signal processing module and the electroporation module.

[0063] The signal processing module is used to process the intracellular action potential collected by the microelectrode array and send the processed data to the processor; the processor is used for data processing, waveform display, data storage and pulse control of the electroporation module; the electroporation module is used to generate a voltage acting on the microelectrode array according to the pulse control signal of the processor.

[0064] Optionally, the signal processing module includes an amplifier, a filter and an analog-to-digital converter.

[0065] See also Figure 7 The signal processing module includes an amplifier, a filter and an analog-to-digital converter. The amplifier is used to amplify the intracellular action potential signal collected by the microelectrode array, the filter is used to filter the amplified intracellular action potential signal, and the analog-to-digital converter is used to perform data conversion on the filtered intracellular action potential signal.

[0066] Optionally, the electroporation module includes a signal generator and a voltage buffer.

[0067] See also Figure 7The electroporation module includes a signal generator and a voltage buffer. The signal generator is used to generate a voltage signal according to a pulse control signal sent by a controller, and the voltage buffer is used to filter the voltage signal to reduce burrs.

[0068] The implementation of the embodiment of the present invention includes the following beneficial effects: the detection system of the microelectrode array device includes a circuit board, a cell culture chamber and a microelectrode array device, the cell culture chamber is fixed in the recording area of ​​the microelectrode array device, and several microelectrodes of the microelectrode array device are connected to the circuit board. A voltage signal is applied to the microelectrode through the circuit board to form a local electric field on both sides of the cell membrane to generate nanopores in the cell membrane, that is, intracellular action potential detection is achieved through electroporation.

[0069] In a specific embodiment, the detection process of intracellular action potential detection is as follows. Neonatal rat ventricular myocytes are isolated from the hearts of rats 1-3 days after birth. We incubate the myocytes at 37°C and 5% CO 2 The cells are cultured in an incubator, and after the cells beat synchronously, the electrophysiological signal collection begins. The 32-channel microelectrode array device detection system in the embodiment of the present invention is used. Since the amplitude of the cell electrophysiological signal obtained by the microelectrode is low, and noise is introduced during the transmission process, it is easy to cause the electrophysiological signal to attenuate and distort. The digital pulse signal is converted into a voltage pulse and applied to the nanocarbon petal microelectrode through the electroporation module. The electroporation parameters are 3V, 20Hz, 200μs square wave electric pulse, and the total time is 1s. All circuits are installed in a metal box to shield the interference of power frequency noise and high-frequency noise. The processor module is responsible for electrophysiological signal display, electroporation parameter control and data storage. When intracellular AP recording is performed, the nanocarbon petal electrode array device is connected.

[0070] In a specific embodiment, the intracellular AP of the nanocarbon petal electrode array is monitored in real time. By connecting the primary cardiomyocytes of mice to nanocarbon petal microelectrodes of different sizes, the cardiomyocytes acquire spontaneous and regular electrical activity and mechanical contraction after at least 3 days of culture. Before and after 3V nano-electroporation, the 32-channel nanocarbon petal electrode array records the extracellular and intracellular electrophysiological signals with consistent beating. Before the nano-electroporation operation, the recorded extracellular FP shows a biphasic spike morphology, which provides information about the Na on the cell membrane. + Current (upstroke), Ca 2+ Current (Platform Phase) and K +The field potential amplitude recorded by the nanocarbon petal electrode array is 30-150μV, which is consistent with the measurement results of traditional planar microelectrodes. The results show that under physiological conditions, that is, in the absence of any external stimulation to perforate the cell membrane, intracellular APs recorded using nanocarbon petal electrode array electrodes do not occur. This shows that despite the tight adhesion between the cell and the nanosheet material, the nanocarbon petal electrode array is not spontaneously internalized by the cardiomyocytes. After applying a 3V pulse voltage to induce the formation of transient nanopores in the cell membrane, the nanocarbon petal electrode array is able to contact the intracellular environment. The electrical signal recorded by the nanocarbon petal electrode array changes from an extracellular FP with an extracellular biphasic spike morphology to an intracellular AP with a single pulse waveform. The amplitude of the action potential is 50-400μV. The intracellular AP waveform shows the depolarization and repolarization process, which is consistent with the measurement results of the traditional patch clamp technique.

[0071] In order to explore the effect of the size of nanocarbon petal microelectrodes on the electrophysiological parameters of cardiomyocytes, this study used nanocarbon petal microelectrodes with an electrode width of 50μm and a length range of 115μm to 496μm to continuously record the extracellular FP and intracellular AP of cardiomyocytes. Then, the effect of the size of nanocarbon petal microelectrodes on the peak-to-peak voltage of FP (FP Vpp) and the peak-to-peak voltage of AP (AP Vpp), the ratio of intracellular AP to extracellular FP Vpp, the SNR of AP and FP, the normalized 50% of action potential duration (APD50), the duration of AP, and the beating frequency of cardiomyocytes before and after NE were quantitatively analyzed by extracting eigenvalues. The results showed that with the increase of the microelectrode size, the peak-to-peak value of the extracellular field potential (FP Vpp) showed a gradual increase. FP Vpp increased from 30.39±0.62μV at 115μm to 146.18±6.07μV at 496μm, an increase of about 380%. This is because as the electrode size increases, the nanocarbon petal microelectrode can detect the electrophysiological activities of more cells around the electrode. As the electrode size increases in the range of 115μm to 350μm, the AP Vpp also increases. However, when the electrode size continues to increase, the change in intracellular AP Vpp is not obvious, mainly concentrated in the range of 161.08±2.11μV to 366.27±7.73μV. When the size of the nanocarbon petal microelectrode is smaller than the size of the cardiomyocyte, as the size of the nanocarbon petal microelectrode increases, the electrode electroporation area of ​​a single cardiomyocyte gradually increases, and the impedance of the cell membrane gradually decreases. Therefore, as the electrode size increases, the intracellular AP amplitude recorded by the nanocarbon petal microelectrode gradually increases. When the size of the nanocarbon petal microelectrode is larger than the size of a single cardiomyocyte, the nanocarbon petal microelectrode still performs nano-electroporation on the entire bottom area of ​​a single cardiomyocyte. However, the cultured cardiomyocytes also include myocardial fibroblasts that cannot discharge. Therefore, as the electrode size increases, the intracellular AP amplitude recorded by the nanocarbon petal microelectrode is the intracellular AP of a single cell. In the range of 115μm to 496μm, the ratio of AP Vpp to FP Vpp recorded by the nanocarbon petal MEA gradually decreased with the increase of size, from 8.29±0.39 at 115μm to 2.27±0.10 at 496μm. In addition, the effects of different electrode sizes on FP SNR and AP SNR were analyzed. The results showed that with the increase of electrode size, FP SNR showed an increasing trend, while AP SNR only increased in the range of 115μm to 300μm. This trend of change is consistent with the trend of extracellular FP and intracellular AP changing with electrode size. FP SNR increased from 10.42±1.29dB at 115μm to 24.07±1.66dB at 496μm, an increase of about 130%.The AP SNR change trend is not obvious. When the electrode size is less than 350μm, the AP SNR is concentrated around 14.92±2.27dB. When the electrode size is greater than 350μm, the AP SNR is concentrated around 33.24±1.20dB.

[0072] In order to explore the effect of electrode size on the autonomy of cardiomyocytes, APD50, action potential duration, and fluctuation frequency before and after electroporation were analyzed. The results showed that after the same voltage perforation treatment, the duration of APD50 did not change significantly with the electrode size, mainly concentrated in the range of 97.01±0.009ms to 157.80±0.008ms. This is because the nano-electroporation activity mainly produces aqueous pores in the phospholipid bilayer of the cell membrane, without significantly affecting the ion channel proteins on the cell membrane surface. Since the aqueous pores in the cell membrane will gradually close over time during the reversible aqueous pore formation process, we define the period from the beginning of the formation of aqueous pores to the beginning of the waveform characteristics of the field potential as the action potential duration. As the electrode size increases, the action potential duration gradually decreases, from 494ms at 115μm to 207ms at 496μm, a decrease of about 138%. The electrode size of the nanocarbon petal MEA had no effect on the beating frequency of the extracellular FP and intracellular AP, which was 81.64 bmp and 100.56 bmp, respectively. The nanoelectroporation process slightly increased the beating frequency of cardiomyocytes by about 23%.

[0073] In order to reduce the difference between intracellular action potentials caused by electrode size, the intracellular action potentials measured by electrodes of different sizes were divided by the corresponding electrode area to obtain the effective action potential. The intracellular action potential waveforms of a single contraction-relaxation cycle of cardiomyocytes recorded by 32 channels were superimposed. The results showed that electrodes of different sizes recorded typical intracellular action potential waveforms, and the single contraction-relaxation cycle was similar. In order to more finely analyze the amplitude of intracellular action potentials of cardiomyocytes at different time points, the intracellular action potentials recorded by the 32-channel nanocarbon petal microelectrode were segmented, thereby achieving analysis with higher time resolution.

[0074] The above is a specific description of the preferred implementation of the present invention, but the invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A microelectrode array device, characterized in that: It includes a substrate and a plurality of microelectrodes arranged on one side surface of the substrate, wherein the plurality of microelectrodes are distributed around the substrate and form a preset pattern, each of the microelectrodes includes a first conductive layer and a second conductive layer, the first conductive layer is arranged between the substrate and the second conductive layer, the second conductive layer includes a three-dimensional nanocarbon layer, the preset pattern includes a recording area and a non-recording area, and the non-recording area is covered with an insulating layer.

2. The microelectrode array device according to claim 1, characterized in that: The three-dimensional nanocarbon layer includes a graphene layer or a carbon nanotube layer.

3. The microelectrode array device according to claim 2, characterized in that: The graphene layer includes graphene in a petal structure.

4. A method for preparing a microelectrode array device, characterized in that: include: providing a substrate, and cleaning the substrate; Sequentially preparing a first conductive layer and a second conductive layer on a surface of one side of the substrate; The second conductive layer includes a three-dimensional nanocarbon layer; Etching the first conductive layer and the second conductive layer to form a preset pattern; The preset pattern includes a plurality of microelectrodes distributed around the substrate; An insulating layer is prepared in the non-recording area of ​​the preset pattern; the preset pattern includes a recording area and a non-recording area.

5. The preparation method according to claim 4, characterized in that: The first conductive layer includes a metal layer, and the first conductive layer and the second conductive layer are sequentially prepared on a surface of one side of the substrate, including: Using magnetron sputtering to prepare the first conductive layer on one side surface of the substrate; The second conductive layer is prepared on the surface of the first conductive layer by using a vapor deposition method.

6. The preparation method according to claim 4, characterized in that: The etching of the first conductive layer and the second conductive layer to form a preset pattern includes: By adopting a laser etching method, on a substrate prepared with a first conductive layer and a second conductive layer, redundant first conductive layer and second conductive layer are removed according to a preset pattern to form the preset pattern.

7. A detection system for a microelectrode array device, characterized in that: It comprises a circuit board, a cell culture chamber and a microelectrode array device as described in any one of claims 1 to 3, wherein the cell culture chamber is fixed in the recording area of ​​the microelectrode array device, and a plurality of microelectrodes of the microelectrode array device are connected to the circuit board.

8. The detection system according to claim 7, characterized in that: The detection system further comprises a signal processing module, an electroporation module and a processor. The circuit board connects the signal processing module and the electroporation module, and the processor connects the signal processing module and the electroporation module.

9. The detection system according to claim 8, characterized in that: The signal processing module includes an amplifier, a filter and an analog-to-digital converter.

10. The detection system according to claim 8, characterized in that: The electroporation module includes a signal generator and a voltage buffer.

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