Coupled microelectroporation multi-size microelectrode array detection device and method of manufacture

Abstract

The application relates to the field of cell signal detection, in particular to a multi-size microelectrode array detection device coupled with microelectroporation and a preparation method thereof. The multi-size microelectrode array detection device comprises a first microelectrode array device, a second microelectrode array device, and an electrophysiological signal acquisition module. The first microelectrode array device comprises a first chip, a first printed circuit board and a first cell culture chamber, and the first chip comprises a plurality of microelectrodes with mixed sizes. The second microelectrode array device comprises a second chip, a second printed circuit board and a second cell culture chamber, and the second chip comprises a plurality of microelectrodes with the same size. The microelectrodes on the second chip of different second microelectrode array devices are not the same in size. The electrophysiological signal acquisition module collects the electrophysiological signals of cells collected by the first microelectrode array device and the second microelectrode array device. An upper computer stores and displays the electrophysiological signal data collected by the electrophysiological signal acquisition module. The application improves the adaptability of the cell detection device and the accuracy of the cell electrophysiological signal detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell signal detection, in particular to a multi-size microelectrode array detection device coupled with microelectroporation and a preparation method. BACKGROUND

[0002] The intracellular action potential detection method of cardiomyocytes has important physiological value for in-depth understanding of the pathogenesis of cardiovascular diseases and heart drug screening. Generally, the intracellular action potential detection method of cardiomyocytes includes patch clamp technology, which directly contacts the inside of the cell by destroying part of the cell membrane. The patch clamp technology can sensitively record the intracellular action potential, but its high invasiveness and difficulty in operation limit its feasibility in long-term and large-scale applications. The microelectrode array with customizable patterns can perform non-invasive long-term and multi-channel extracellular field potential measurement. However, the quality and spatial resolution of the measured electrical signals are low.

[0003] In related technologies, in order to meet the time and spatial resolution requirements of extracellular measurement, nanoscale electrodes are developed, and the small geometry is crucial for enhancing signal detection. Because the cell membrane is tightly wrapped around the vertical electrode, this reduces the gap between the cell membrane and the electrode, improving the accuracy of extracellular action potential detection results. However, due to the small size of the nanoelectrode, when the cell lands, the gravity-induced puncture or the penetration caused by the cell spreading on the substrate, this spontaneous penetration efficiency is usually low, resulting in that the nanoelectrode cannot be large-scale captured for research on the basic characteristics of cardiomyocytes. The nanoscale electrode can only detect the action potential signal record of a single heart cell, and cannot detect the action potential of multiple cardiomyocytes at the same time; in addition, the sizes of cardiomyocytes of different organisms are different, and the fixed size of the nanoscale electrode detection device cannot meet the detection requirements of cardiomyocytes of different sizes, the adaptability of the fixed size of the nanoscale electrode detection device is low, resulting in that the intracellular action potential detection result of the cardiomyocyte is not accurate. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the embodiments of the present application provide a multi-size microelectrode array detection device coupled with microelectroporation and a preparation method, which is beneficial to improve the adaptability of the cell detection device, and at the same time improve the accuracy of the electrical physiological signal detection result corresponding to the intracellular action potential of the cell.

[0005] In a first aspect, the embodiments of the present application provide a multi-size microelectrode array detection device coupled with microelectroporation, comprising:

[0006] At least one first microelectrode array device includes a first chip, a first printed circuit board, and a first cell culture chamber. The first chip is fixed on the first printed circuit board. The first chip includes a plurality of microelectrodes of mixed sizes. The plurality of microelectrode pins on the first chip are connected to the first printed circuit board through conductive silver paste. The first cell culture chamber and the first chip abut against each other.

[0007] Multiple second microelectrode array devices include a second chip, a second printed circuit board, and a second cell culture chamber. The second chip is fixed on the second printed circuit board and includes multiple microelectrodes of the same size. The microelectrodes on the second chips of different second microelectrode array devices are of different sizes. The multiple microelectrode pins on the second chip are connected to the second printed circuit board through conductive silver paste. The second cell culture chamber and the second chip are in contact.

[0008] An electrophysiological signal acquisition module is connected to the first microelectrode array device and the second microelectrode array device. The electrophysiological signal acquisition module is used to acquire the electrophysiological signals of cells acquired by the first microelectrode array device and the electrophysiological signals of cells acquired by the second microelectrode array device.

[0009] The host computer is communicatively connected to the electrophysiological signal acquisition module. The host computer is used to store the electrophysiological signal data acquired by the electrophysiological signal acquisition module and to display the electrophysiological signal data.

[0010] An electroporation module is connected to both the first chip and the second chip. The host computer controls the electroporation module to apply a preset voltage pulse. The preset voltage pulse is applied to the cells to be tested in the first cell culture chamber through multiple microelectrodes on the first chip, thereby achieving electroporation of the cells in the first cell culture chamber. Similarly, the preset voltage pulse is applied to the cells to be tested in the second cell culture chamber through multiple microelectrodes on the second chip, thereby achieving electroporation of the cells in the second cell culture chamber.

[0011] The technical solution of the first aspect of this application has at least one of the following advantages or beneficial effects: the first chip includes multiple microelectrodes of mixed sizes, which can detect intracellular action potentials of cells of different sizes, thus improving the adaptability of the first microelectrode array device for cell detection; the second chip includes multiple microelectrodes of the same size, and the microelectrodes on the second chips of different second microelectrode array devices are of different sizes, which can simultaneously detect intracellular action potentials of cells of different sizes, thus improving the adaptability of the second microelectrode array device for cell detection. By setting the multi-size microelectrode array detection device coupled with micro-electroporation to include at least one first microelectrode array device and multiple second microelectrode array devices, the multi-size microelectrode array detection device coupled with micro-electroporation can simultaneously detect multiple test cells of different sizes, further improving the adaptability of the multi-size microelectrode array detection device coupled with micro-electroporation, thereby improving the accuracy of the electrophysiological signal detection results corresponding to intracellular action potentials. Because the electrophysiological signal acquisition module is connected to the first and second microelectrode array devices, it can acquire the electrophysiological signals of cells collected by the first and second microelectrode array devices in real time. During the acquisition process, the host computer controls the electroporation module to apply a preset voltage pulse. The electroporation module is connected to the first and second chips. The preset voltage pulse is applied to the test cells in the first cell culture chamber through multiple microelectrodes of the first chip, and to the test cells in the second cell culture chamber through multiple microelectrodes of the second chip, thus achieving cell electroporation. Because the electrophysiological signal acquisition module is connected to the host computer, it can transmit the acquired cell electrophysiological signals to the host computer. The host computer stores the electrophysiological signal data acquired by the first and second microelectrode array devices and displays the electrophysiological signal data of the cell, improving the visualization of the electrophysiological signal detection results corresponding to the intracellular action potential.

[0012] According to some embodiments of this application, the first microelectrode array device includes a first microelectrode array mask, one side of which is printed with a mixed-size electrode pattern, and the other side of which is printed with an encapsulation layer pattern.

[0013] According to some embodiments of this application, the mixed-size microelectrodes on the first chip include an 8-channel microelectrode of a first preset size, an 8-channel microelectrode of a second preset size, an 8-channel microelectrode of a third preset size, a 4-channel microelectrode of a fourth preset size, and a 4-channel microelectrode of a fifth preset size.

[0014] According to some embodiments of this application, the minimum distance between the edges of adjacent microelectrodes of the first microelectrode array device and the second microelectrode array device is 250 μm.

[0015] According to some embodiments of this application, the second microelectrode array device includes a second microelectrode array mask. One side of the second microelectrode array mask is printed with an electrode pattern of a preset size, and the other side of the second microelectrode array mask is printed with an encapsulation layer pattern. The size of the electrode pattern printed on different second microelectrode array masks is different.

[0016] According to some embodiments of this application, the dimensions of the plurality of microelectrodes on the second chip include a 32-channel sixth preset size microelectrode, a 32-channel seventh preset size microelectrode, a 32-channel eighth preset size microelectrode, a 32-channel ninth preset size microelectrode, and a 32-channel tenth preset size microelectrode.

[0017] According to some embodiments of this application, a first reference electrode and a second reference electrode are also included. The first reference electrode is electrically connected to a first printed circuit board of the first microelectrode array device. The first reference electrode is used for microelectroperfusion of cells in the first cell culture chamber and for recording electrophysiological signals acquired by the first microelectrode array device. The second reference electrode is electrically connected to a second printed circuit board of the second microelectrode array device. The second reference electrode is used for microelectroperfusion of cells in the second cell culture chamber and for recording electrophysiological signals acquired by the second microelectrode array device.

[0018] Secondly, embodiments of this application provide a method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, comprising:

[0019] Fabrication of a first microelectrode array mask and second microelectrode array masks of different sizes;

[0020] Positive photoresist is spin-coated onto a glass substrate at a first preset speed, and the glass substrate is placed in an oven at a first preset temperature and baked for a first preset time.

[0021] The first microelectrode array mask is placed in the mechanical structure of the photolithography machine, and the distance between the first microelectrode array mask and the glass substrate is controlled by the mechanical structure. The electrode pattern layer of the first microelectrode array mask is irradiated with 365 nm ultraviolet light for a second preset time, and then developed with developer for a third preset time, so that the first electrode pattern is exposed on the glass substrate.

[0022] A titanium or gold layer is deposited on a glass substrate with a first electrode pattern exposed by magnetron sputtering, and the positive photoresist is stripped with acetone to form a first microelectrode array.

[0023] A predetermined number of baked glass substrates are taken, and each glass substrate corresponds to a second microelectrode array mask of a certain size. The second microelectrode array masks of different sizes are placed sequentially on the mechanical structure of the photolithography machine. The distance between the second microelectrode array mask and the glass substrate is controlled by the mechanical structure. The electrode pattern layer of the first microelectrode array mask is irradiated with 365 nm ultraviolet light for a second predetermined time, and then developed with developer for a third predetermined time, so that the exposed second electrode pattern is formed on the glass substrate.

[0024] Titanium or gold layers are deposited on a glass substrate with a second electrode pattern exposed by magnetron sputtering, and the positive photoresist is stripped with acetone to form a second microelectrode array.

[0025] The technical solution of the second aspect of this application has at least one of the following advantages or beneficial effects: By printing electrode patterns of mixed sizes on one side of the first microelectrode array mask and irradiating the electrode pattern layer of the first microelectrode array mask with 365 nm ultraviolet light to form exposed first electrode patterns on the glass substrate, the first microelectrode array includes multiple microelectrodes of mixed sizes. The multiple microelectrodes of mixed sizes can detect intracellular action potentials of cells of different sizes, thereby improving the adaptability of the first microelectrode array device for cell detection; by printing electrode patterns of preset sizes on one side of the second microelectrode array mask, and the electrode patterns of different second microelectrode array masks having different sizes, and irradiating the electrode pattern layers of different second microelectrode array masks sequentially with 365 nm ultraviolet light to form multiple exposed second electrode patterns on multiple glass substrates, multiple different second microelectrode array devices can simultaneously detect intracellular action potentials of cells of different sizes, thereby improving the adaptability of the second microelectrode array device for cell detection.

[0026] According to some embodiments of this application, negative photoresist is spin-coated onto the surface of the first microelectrode array at a second preset speed;

[0027] The first microelectrode array mask placed on the mechanical structure of the lithography machine is flipped, and the distance between the first microelectrode array mask and the glass substrate is controlled by the mechanical structure. The encapsulation layer pattern of the first microelectrode array mask is irradiated with 365 nm ultraviolet light for a fourth preset time, developed in propylene glycol methyl ether acetate for a fifth preset time, and the first microelectrode array is cleaned with isopropanol to form a first insulating layer.

[0028] The first microelectrode array is placed at a second preset temperature and baked for a sixth preset time to obtain the first chip;

[0029] The negative photoresist is spin-coated onto the surface of the second microelectrode array at a second preset speed;

[0030] The second microelectrode array mask placed on the mechanical structure of the lithography machine is flipped, and the distance between the second microelectrode array mask and the glass substrate is controlled by the mechanical structure. The encapsulation layer pattern of the second microelectrode array mask is irradiated with 365 nm ultraviolet light for a fourth preset time, developed in propylene glycol methyl ether acetate for a fifth preset time, and the second microelectrode array is cleaned with isopropanol to form a second insulating layer.

[0031] The second microelectrode array is placed at a second preset temperature and baked for a sixth preset time to obtain the second chip.

[0032] According to some embodiments of this application, a first chip is fixed on a first printed circuit board using PDMS, and conductive silver paste is used to connect the microelectrode pins of the first chip to the first printed circuit board.

[0033] The first cell culture chamber was adhered to the top of the first chip using PDMS;

[0034] Electrically connect the first reference electrode and the first printed circuit board; solder the first pin interface to the first printed circuit board to match the interface of the multi-size microelectrode array detection device to obtain the first microelectrode array device;

[0035] The second chip is fixed on the second printed circuit board using PDMS, and the microelectrode pins of the second chip are connected to the second printed circuit board using conductive silver paste.

[0036] The second cell culture chamber was adhered to the top of the second chip using PDMS;

[0037] Electrically connect the second reference electrode and the second printed circuit board; solder the second pin interface to the second printed circuit board to match the interface of the multi-size microelectrode array detection device to obtain the second microelectrode array device.

[0038] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0039] Figure 1This is a schematic diagram of the structure of a multi-size microelectrode array detection device coupled with microelectroporation provided in an embodiment of this application;

[0040] Figure 2 This is a schematic diagram of the structure of a first microelectrode array device provided in an embodiment of this application;

[0041] Figure 3 This is a schematic diagram of the structure of a second microelectrode array device provided in an embodiment of this application;

[0042] Figure 4 This is a schematic diagram of the electrode pattern of a first microelectrode array mask provided in an embodiment of this application;

[0043] Figure 5 This is a schematic diagram of the encapsulation layer pattern of a first microelectrode array mask provided in an embodiment of this application;

[0044] Figure 6 This is a schematic diagram of the electrode pattern of a second microelectrode array mask provided in an embodiment of this application;

[0045] Figure 7 This is a schematic diagram of the electrode patterns of various second microelectrode array masks provided in the embodiments of this application;

[0046] Figure 8 This is a schematic diagram of the encapsulation layer pattern of a second microelectrode array mask provided in an embodiment of this application;

[0047] Figure 9 This is a schematic diagram of multiple microelectrodes on a first chip provided in an embodiment of this application;

[0048] Figure 10 This is a schematic diagram of multiple microelectrodes on a second chip provided in an embodiment of this application;

[0049] Figure 11 This is a flowchart illustrating a method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, as provided in an embodiment of this application.

[0050] Figure 12 This is a flowchart of another method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, provided in an embodiment of this application;

[0051] Figure 13 This is a flowchart of another method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, provided in an embodiment of this application. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various implementations. Simultaneously, the steps or actions described in the method description can be rearranged or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various orders in the specification and drawings are merely for the clear description of a particular embodiment and do not imply a mandatory order, unless otherwise stated that a particular order must be followed.

[0053] In the description of this application, "several" means one or more, "more than" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0054] The serial numbers assigned to components in this document, such as "first" and "second," are used only to distinguish the described objects and have no sequential or technical meaning. The terms "connection" and "linkage" used in this application, unless otherwise specified, include both direct and indirect connections (linkages).

[0055] Methods for detecting intracellular action potentials in cardiomyocytes are of significant physiological value for understanding the pathogenesis of cardiovascular diseases and screening cardiac drugs. Typically, methods for detecting intracellular action potentials in cardiomyocytes include patch-clamp techniques, which directly access the cell interior by disrupting a portion of the cell membrane. While patch-clamp techniques can sensitively record intracellular action potentials, their high invasiveness and operational complexity limit their feasibility for long-term and large-scale applications. Customizable patterned microelectrode arrays can perform non-invasive, long-term, and multi-channel measurements of extracellular field potentials. However, the quality and spatial resolution of the measured electrical signals are relatively low.

[0056] In related technologies, nanoscale electrodes have been developed to meet the temporal and spatial resolution requirements of extracellular measurements. Their tiny geometry is crucial for enhancing signal detection. Because the cell membrane tightly surrounds the vertical electrode, the gap between the cell membrane and the electrode is reduced, improving the accuracy of extracellular action potential detection results. However, due to the small size of nanoelectrodes, spontaneous permeation efficiency is typically low, resulting from gravity-induced puncture during cell descent or cell diffusion on the substrate. This limits the large-scale capture of fundamental characteristics of cardiomyocytes for research. Nanoscale electrodes can only detect action potential signals from single cardiomyocytes, unable to simultaneously detect the action potentials of multiple cardiomyocytes. Furthermore, cardiomyocytes vary in size across different organisms; fixed-size nanoscale electrode detection devices cannot meet the detection needs of cardiomyocytes of different sizes, resulting in low adaptability and inaccurate intracellular action potential detection results for cardiomyocytes.

[0057] Based on this, the present application provides a detection device and preparation method for a multi-size microelectrode array coupled with microelectroporation, which is beneficial to improve the adaptability of cell detection devices and improve the accuracy of the detection results of electrophysiological signals corresponding to intracellular action potentials.

[0058] The present application will now be further described with reference to the accompanying drawings.

[0059] Reference Figures 1 to 3 As shown, the multi-size microelectrode array detection device 1000 coupled with microelectropertomy includes at least one first microelectrode array device 100, multiple second microelectrode array devices 200, an electrophysiological signal acquisition module 300, and a host computer 400. The first microelectrode array device 100 includes a first chip 110, a first printed circuit board 120, and a first cell culture chamber 130. The first chip 110 is fixed on the first printed circuit board 120 and includes multiple microelectrodes of mixed sizes. The multiple microelectrode pins on the first chip 110 are connected to the first printed circuit board 120 through conductive silver paste. The first cell culture chamber 130 and the first chip 110 abut against each other. The second microelectrode array device 200 includes a second chip, a second printed circuit board 220, and a second cell culture chamber 230. The second chip is fixed on the second printed circuit board 220 and includes multiple microelectrodes of the same size. The microelectrodes on the second chips of different second microelectrode array devices 200 have different sizes. The multiple microelectrode pins on the second chip are connected to the second printed circuit board 220 through conductive silver paste. The second cell culture chamber 230 and the second chip are in contact.

[0060] The electrophysiological signal acquisition module 300 is connected to the first microelectrode array device 100 and the second microelectrode array device 200. The electrophysiological signal acquisition module 300 is used to acquire the electrophysiological signals of cells acquired by the first microelectrode array device 100 and the second microelectrode array device 200. The host computer 400 is communicatively connected to the electrophysiological signal acquisition module 300. The host computer 400 is used to store the electrophysiological signal data acquired by the electrophysiological signal acquisition module 300 and to display the electrophysiological signal data.

[0061] An electroporation module 500 is connected to a first chip 110 and a second chip 210. A host computer 400 controls the electroporation module 500 to apply a preset voltage pulse. The preset voltage pulse is applied to the cells to be tested in the first cell culture chamber 130 through multiple microelectrodes on the first chip 110, thereby achieving electroporation of the cells in the first cell culture chamber 130. Similarly, the preset voltage pulse is applied to the cells to be tested in the second cell culture chamber 230 through multiple microelectrodes on the second chip 210, thereby achieving electroporation of the cells in the second cell culture chamber 230.

[0062] In this application, the first chip 110 includes multiple microelectrodes of mixed sizes, which can detect intracellular action potentials of cells of different sizes, thus improving the adaptability of the first microelectrode array device 100 for cell detection; the second chip includes multiple microelectrodes of the same size, and the microelectrodes on the second chips of different second microelectrode array devices 200 are of different sizes, so that multiple different second microelectrode array devices 200 can simultaneously detect intracellular action potentials of cells of different sizes, thus improving the adaptability of the second microelectrode array device 200 for cell detection. A first cell culture chamber 130 and a first chip 110 are connected. The first cell culture chamber 130 is used to culture cells to be tested. The first chip 110 is fixed on a first printed circuit board 120, and multiple microelectrode pins on the first chip 110 are connected to the first printed circuit board 120 through conductive silver paste. Multiple microelectrodes of mixed sizes on the first chip 110 perform intracellular action potential detection on the cells to be tested in the first cell culture chamber 130. A second cell culture chamber 230 and a second chip are connected. The second cell culture chamber 230 is used to culture cells to be tested. The second chip is fixed on a second printed circuit board 220, and multiple microelectrode pins on the second chip are connected to the first printed circuit board 120 through conductive silver paste. Multiple microelectrodes on the second chip perform intracellular action potential detection on the cells to be tested in the second cell culture chamber 230. Intracellular action potential detection: Since the electrophysiological signal acquisition module 300 is connected to the first microelectrode array device 100 and the second microelectrode array device 200, the electrophysiological signals corresponding to the intracellular action potentials of cells acquired by multiple microelectrodes of mixed sizes on the first chip 110 and multiple microelectrodes on the second chip can be transmitted to the electrophysiological signal acquisition module 300. During the acquisition process, the host computer 400 controls the electroporation module 500 to apply a preset voltage pulse. The electroporation module 500 is connected to the first chip 110 and the second chip 210. The preset voltage pulse is applied to the test cells in the first cell culture chamber 130 through multiple microelectrodes of the first chip 110, and to the test cells in the second cell culture chamber 230 through multiple microelectrodes of the second chip 210, thereby realizing cell electroporation. Because the electrophysiological signal acquisition module 300 and the host computer 400 are connected, the electrophysiological signal data acquired by the electrophysiological signal acquisition module 300 can be transmitted to the host computer 400. The host computer 400 stores and displays the electrophysiological signal data, which improves the visualization of the electrophysiological signal detection results corresponding to the intracellular action potential.

[0063] This application provides a multi-size microelectrode array detection device 1000 coupled with microelectroporation, which includes at least one first microelectrode array device 100 and multiple second microelectrode array devices 200. This enables the multi-size microelectrode array coupled with microelectroporation to simultaneously detect multiple test cells of different sizes, improving the adaptability of the multi-size microelectrode array detection device 1000 coupled with microelectroporation and thus improving the accuracy of the electrophysiological signal detection results corresponding to intracellular action potentials.

[0064] In some embodiments of this application, the surfaces of multiple microelectrodes on the first chip 110 and the surfaces of multiple microelectrodes on the second chip are modified with one of hydrogel, polycarbazole, and polypyrrole. By modifying the surfaces of multiple microelectrodes on the first chip 110 and the surfaces of multiple microelectrodes on the second chip with one of hydrogel, polycarbazole, and polypyrrole, the quality of electrophysiological signal recording acquired by the first chip 110 and the second chip can be improved, thereby improving the accuracy of the electrophysiological signal detection results characterizing intracellular action potentials acquired by the first microelectrode array device 100 and the second microelectrode array device 200.

[0065] In some embodiments of this application, multiple microelectrodes on the first chip 110 and multiple microelectrodes on the second chip are micrometer-sized electrodes. Microelectrode arrays based on micrometer-sized electrodes can achieve stable electroporation, enabling low-cost and high-quality recording of intracellular action potentials. Cell culture and intracellular action potential recording were performed using the first microelectrode array device 100 and the second microelectrode array device 200, respectively. Different microelectrode sizes have different effects on the amplitude, amplitude ratio, signal-to-noise ratio, duration, automaticity, single-cell signal ratio, and action potential yield of intracellular action potentials before and after microelectroporation. In this application, intracellular action potentials were recorded using a mixed-size microelectrode array on the first chip 110 in the first microelectrode array device 100 and microelectrode arrays of the same size on the second chips in multiple second microelectrode array devices 200, respectively. The obtained results showed the same pattern of influence on the recording of electrophysiological signals corresponding to intracellular action potentials. This multi-size microelectrode array detection device 1000 coupled with microelectroporation paves the way for accurate recording of intracellular action potentials using micrometer-sized electrodes.

[0066] In some embodiments of this application, the minimum edge distance between adjacent microelectrodes of the first microelectrode array device 100 and the second microelectrode array device 200 is 250 μm. That is, the minimum edge distance between any two adjacent microelectrodes between multiple microelectrodes on the first chip 110 and multiple microelectrodes on the second chip is 250 μm. By setting the minimum edge distance between any two adjacent microelectrodes between multiple microelectrodes on the first chip 110 and multiple microelectrodes on the second chip to 250 μm, it is possible to effectively avoid the microelectrodes on the first chip 110 and the microelectrodes on the second chip from collecting the same electrophysiological signal corresponding to the intracellular action potential of cells. This avoids the mutual interference between the microelectrodes of the first microelectrode array device 100 and the microelectrode array device 200 in the acquisition sensitivity, improves the detection accuracy of the multi-size microelectrode array detection device 1000 coupled with micro-electroporation, and at the same time improves the accuracy of the detection results of the electrophysiological signal corresponding to the intracellular action potential of cells.

[0067] Reference Figure 4 As shown, Figure 4 This is a schematic diagram of the electrode pattern of a first microelectrode array mask provided in an embodiment of this application. In some embodiments of this application, the first microelectrode array device 100 includes a first microelectrode array mask, on which a mixed-size electrode pattern is printed. It is understood that the mixed-size electrode pattern on the first microelectrode array mask is used for exposure under 365 nm ultraviolet light and then developed using a developer to form an exposed electrode pattern on a glass substrate. The electrode pattern formed by the first microelectrode array mask is a mixed-size electrode pattern.

[0068] In one embodiment, the mixed-size electrode patterns include electrode patterns of 20 μm, 50 μm, 100 μm, 200 μm and 500 μm. Those skilled in the art can set the size of the mixed size of the electrode patterns according to the actual situation. The embodiments of this application do not limit the size of the mixed size.

[0069] Reference Figure 5 As shown, Figure 5 This is a schematic diagram of the encapsulation layer pattern of a first microelectrode array mask provided in an embodiment of this application. In some embodiments of this application, the first microelectrode array mask is also printed with an encapsulation layer pattern. It is understood that the encapsulation layer pattern is used to expose under 365 nm ultraviolet light and then develop in propylene glycol methyl ether acetate to form a first insulating layer.

[0070] In some embodiments of this application, the mixed-size multiple microelectrodes on the first chip 110 include an 8-channel microelectrode of a first preset size, an 8-channel microelectrode of a second preset size, an 8-channel microelectrode of a third preset size, a 4-channel microelectrode of a fourth preset size, and a 4-channel microelectrode of a fifth preset size.

[0071] It should be noted that in some embodiments of this application, the first preset size is 20 μm, the second preset size is 50 μm, the third preset size is 100 μm, the fourth preset size is 200 μm, and the fifth preset size is 500 μm. Those skilled in the art can set the mixed size of multiple microelectrodes on the first chip 110 according to the size of the cells to be tested. The embodiments of this application do not limit the size of the first preset size, the second preset size, the third preset size, the fourth preset size, and the fifth preset size.

[0072] Reference Figure 9 , Figure 9 This is a schematic diagram of multiple microelectrodes on a first chip 110 provided in an embodiment of this application. In one embodiment, the multiple microelectrodes of mixed sizes on the first chip 110 are 32 microelectrodes, including 8-channel 20 μm microelectrodes, 8-channel 50 μm microelectrodes, 8-channel 100 μm microelectrodes, 4-channel 200 μm microelectrodes, and 4-channel 500 μm microelectrodes. By setting the first chip 110 to include microelectrodes of various mixed sizes, microelectrodes of different sizes can detect intracellular action potentials of cells of different sizes, improving the adaptability of the first microelectrode array device 100. Different microelectrode sizes have different effects on the amplitude, amplitude ratio, signal-to-noise ratio, duration, automaticity, single-cell signal ratio, and action potential yield of intracellular action potentials before and after microelectroporation, paving the way for precise recording of intracellular action potentials by micron-sized electrodes.

[0073] Reference Figure 6 As shown, Figure 6 This is a schematic diagram of the electrode pattern of a second microelectrode array mask provided in an embodiment of this application. In some embodiments of this application, the second microelectrode array device 200 includes a second microelectrode array mask on which electrode patterns of a preset size are printed. The size of the electrode patterns printed on different second microelectrode array masks is different. It is understood that the electrode patterns of the preset size on the second microelectrode array mask are used for exposure under 365 nm ultraviolet light, followed by development with a developer, thereby forming exposed electrode patterns on a glass substrate. The electrode patterns formed by the second microelectrode array mask are electrode patterns of the preset size.

[0074] Reference Figure 7 As shown, Figure 7This is a schematic diagram of electrode patterns on various second microelectrode array masks provided in embodiments of this application. In one embodiment, the preset electrode patterns are 20 μm, 50 μm, 100 μm, 200 μm, and 500 μm electrode patterns. The sizes of the electrode patterns printed on different second microelectrode array masks are different. Those skilled in the art can set the preset size of the electrode patterns according to actual conditions. This application does not limit the size of the preset size.

[0075] Reference Figure 8 As shown, Figure 8 This is a schematic diagram of the encapsulation layer pattern of a second microelectrode array mask provided in an embodiment of this application. In some embodiments of this application, the second microelectrode array mask is also printed with an encapsulation layer pattern. It is understood that the encapsulation layer pattern is used to expose under 365 nm ultraviolet light and then develop in propylene glycol methyl ether acetate to form a second insulating layer.

[0076] In some embodiments of this application, the dimensions of the plurality of microelectrodes on the second chip 210 include those of a 32-channel sixth preset size microelectrode, a 32-channel seventh preset size microelectrode, a 32-channel eighth preset size microelectrode, a 32-channel ninth preset size microelectrode, and a 32-channel tenth preset size microelectrode.

[0077] It should be noted that in some embodiments of this application, the sixth preset size is 20 μm, the seventh preset size is 50 μm, the eighth preset size is 100 μm, the ninth preset size is 200 μm, and the tenth preset size is 400 μm, as shown in the reference. Figure 10 , Figure 10 This is a schematic diagram of multiple microelectrodes on a second chip 210 provided in an embodiment of this application. Figure 10 The size of the multiple microelectrodes on the second chip 210 is 800 μm. Those skilled in the art can set the size of the multiple microelectrodes on the second chip 210 according to the size of the cells to be tested. In this embodiment, the size of the sixth, seventh, eighth, ninth and tenth preset sizes is not limited.

[0078] In one embodiment, the second chip 210 has 32 microelectrodes, and the sizes of these microelectrodes include 32-channel 20 μm microelectrodes, 32-channel 50 μm microelectrodes, 32-channel 100 μm microelectrodes, 32-channel 200 μm microelectrodes, and 32-channel 400 μm microelectrodes. By setting the second chip 210 to include 32 channels of microelectrodes of preset sizes, the sizes of the microelectrodes on the second chip 210 of different second microelectrode array devices 200 are different. Microelectrodes of different sizes can detect intracellular action potentials of cells of different sizes, improving the adaptability of the second microelectrode array device 200. Different microelectrode sizes have different effects on the amplitude, amplitude ratio, signal-to-noise ratio, duration, automaticity, single-cell signal ratio, and action potential yield of intracellular action potentials before and after microelectroporation, paving the way for precise recording of intracellular action potentials by micron-sized electrodes.

[0079] In some embodiments of this application, the multi-size microelectrode array detection device 1000 coupled with microelectroperfusion further includes a first reference electrode. The first reference electrode is electrically connected to the first printed circuit board 120 of the first microelectrode array device 100, and a row of pin interfaces is soldered to the first printed circuit board 120 to match the interface of the multi-size microelectrode array detection device 1000 coupled with microelectroperfusion. The first microelectrode array device 100 uses a common first reference electrode for electrical signal recording and microelectroperfusion. It is understood that the first reference electrode is used for microelectroperfusion of cells in the first cell culture chamber 130, and the first reference electrode is also used for recording electrophysiological signals acquired by the first microelectrode array device 100.

[0080] The multi-size microelectrode array detection device 1000 coupled with microelectroporation also includes a second reference electrode. The second reference electrode is electrically connected to the second printed circuit board 220 of the second microelectrode array device 200, and a row of pin interfaces is soldered to the second printed circuit board 220 to match the interface of the multi-size microelectrode array detection device 1000 coupled with microelectroporation. It is understood that the second reference electrode is used for microelectroporation of cells in the second cell culture chamber 230, and also for recording electrophysiological signals acquired by the second microelectrode array device 200.

[0081] In some embodiments of this application, the electrochemical performance of the multi-size microelectrode array detection device 1000 coupled with microelectroporation is characterized using an electrochemical workstation in a three-electrode device via cyclic voltammetry and electrochemical impedance spectroscopy. An electrolyte is added to the first cell culture chamber 130 to connect the microelectrodes of the first microelectrode array device 100 to the electrolyte, with a platinum wire as the control electrode and an Ag / AgCl electrode as the reference electrode. The potential range of the cyclic voltammetry is controlled from -0.4 V to 0.4 V, and the scan rate is 100 mV / s. The electrochemical impedance spectroscopy experiment is controlled at a frequency range of 1 to 1 MHz to characterize the electrochemical performance of the first microelectrode array device 100. Similarly, an electrolyte is added to the second cell culture chamber 230 to connect the microelectrodes of the second microelectrode array device 200 to the electrolyte, with a platinum wire as the control electrode and an Ag / AgCl electrode as the reference electrode. Cyclic voltammetry was performed with a potential range of -0.4 V to 0.4 V and a scan rate of 100 mV / s. Electrochemical impedance spectroscopy was conducted with a frequency range of 1 to 1 MHz to characterize the electrochemical performance of the second microelectrode array device 200.

[0082] In some embodiments of this application, the cell electroporation model simulation was performed using the current module in COMSOL Multiphysics 6.0 software. Since the cells are distributed in different locations, three-dimensional cell-Au electrode models were constructed for different cell distribution locations. First, a 2 mm thick glass substrate, a 100 nm thick Au electrode, and a 2 μm thick SU-8 encapsulation layer were designed sequentially from bottom to top. Then, cells with a diameter of 80 μm were coupled to the surface of the Au electrode or encapsulation layer, with a cell membrane thickness of 10 nm. A 100 nm gap was set at the cell-growth interface, and the remaining part of the model was defined as the cell culture chamber, with a reference electrode placed on top of the culture medium. During the simulation, different perforation voltages of 1 to 5 V were applied through the Au electrode, and the transmembrane voltage at different cell locations was analyzed after reaching steady state. To facilitate the study of the transmembrane potential of the cell membrane, five three-dimensional cutoff lines with a length of 80 μm and a spacing of 10 μm were constructed using a two-point method. The potential along the three-dimensional cut-off line was extracted from the results, and the potential difference when the three-dimensional cut-off line passed through the cell membrane was statistically analyzed, thereby realizing the simulation of the cell electroporation model.

[0083] In some embodiments of this application, the electrophysiological signals corresponding to intracellular action potentials are recorded by maintaining the cells at a temperature of 37 °C and 5% CO2, with electrophysiological signals being collected starting on the third day of cell synchronous beating. The multi-size microelectrode array detection device 1000 coupled with micro-electroporation includes at least one first microelectrode array device 100, multiple second microelectrode array devices 200, an electrophysiological signal acquisition module 300, and a host computer 400. The electrophysiological signal acquisition module 300 is used to acquire the cell electrophysiological signals acquired by the first microelectrode array device 100 and the cell electrophysiological signals acquired by the second microelectrode array devices 200. The host computer 400 is communicatively connected to the electrophysiological signal acquisition module 300 and is used to store the electrophysiological signal data acquired by the electrophysiological signal acquisition module 300 and to display the electrophysiological signal data, thereby improving the visualization of the electrophysiological signal detection results corresponding to intracellular action potentials.

[0084] It should be noted that the electrophysiological signals need to be bandpass recorded at a sampling rate of 15 kHz from 1 Hz to 7.5 kHz. During microelectroporation, a 2500-cycle, 3V, 200 μs square wave electrical pulse is applied to the first chip 110 of the first microelectrode array device 100 and / or the second chip 210 of the second microelectrode array device 200, for a total duration of 1 s.

[0085] Reference Figure 11 As shown, Figure 11 This is a flowchart illustrating a method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, as provided in an embodiment of this application. The method includes, but is not limited to, steps S100 to S150. Specifically,

[0086] Step S100: Fabricate a first microelectrode array mask and second microelectrode array masks of different sizes;

[0087] Step S110: Spin-coat positive photoresist onto a glass substrate at a first preset speed, and place the glass substrate in an oven at a first preset temperature for a first preset time;

[0088] Step S120: Place the first microelectrode array mask on the mechanical structure of the photolithography machine, and control the distance between the first microelectrode array mask and the glass substrate through the mechanical structure. Irradiate the electrode pattern layer of the first microelectrode array mask with 365 nm ultraviolet light for a second preset time, and then develop it with developer for a third preset time, so that the exposed first electrode pattern is formed on the glass substrate.

[0089] Step S130: Deposit a titanium or gold layer on a glass substrate with the first electrode pattern exposed by magnetron sputtering, and use acetone to remove the positive photoresist to form a first microelectrode array.

[0090] Step S140: Take a preset number of baked glass substrates, one glass substrate corresponds to a second microelectrode array mask of a certain size, and place the second microelectrode array masks of different sizes in sequence on the mechanical structure of the photolithography machine. Control the distance between the second microelectrode array mask and the glass substrate by the mechanical structure. Irradiate the electrode pattern layer of the first microelectrode array mask with 365 nm ultraviolet light for a second preset time, and then develop it with developer for a third preset time to form an exposed second electrode pattern on the glass substrate.

[0091] Step S150: Deposit a titanium or gold layer on a glass substrate with the second electrode pattern exposed by magnetron sputtering, and use acetone to remove the positive photoresist to form a second microelectrode array.

[0092] In some embodiments of this application, the fabrication method of the multi-size microelectrode array detection device coupled with microelectroporation includes: First, fabricating a first microelectrode array mask and second microelectrode array masks of different sizes. Specifically, a mixed-size electrode pattern is printed on one side of the first microelectrode array mask, and an encapsulation layer pattern is printed on the other side. A preset-size electrode pattern is printed on one side of the second microelectrode array mask, and an encapsulation layer pattern is printed on the other side. The electrode patterns of different second microelectrode array masks have different sizes. Then, the glass substrate is baked, and the first microelectrode array mask is placed in the mechanical structure of a photolithography machine. The electrode pattern layer of the first microelectrode array mask is irradiated with 365 nm ultraviolet light and then developed with a developer to form an exposed first electrode pattern on the glass substrate. Finally, a titanium or gold layer is deposited on the glass substrate with the exposed first electrode pattern using magnetron sputtering technology to form the first microelectrode array. Different sizes of second microelectrode array masks are placed sequentially on the mechanical structure of the photolithography machine. The electrode pattern layer of the second microelectrode array mask is irradiated with 365 nm ultraviolet light and then developed with a developer to expose the second electrode pattern on the glass substrate. Finally, titanium or gold layers are deposited on the glass substrate with the exposed second electrode pattern by magnetron sputtering technology to form the second microelectrode array.

[0093] In this application, by printing electrode patterns of mixed sizes on one side of a first microelectrode array mask and irradiating the electrode pattern layer of the first microelectrode array mask with 365 nm ultraviolet light to form exposed first electrode patterns on a glass substrate, the first microelectrode array includes multiple microelectrodes of mixed sizes. These multiple microelectrodes of mixed sizes can detect intracellular action potentials of cells of different sizes, thus improving the cell detection adaptability of the first microelectrode array device. Furthermore, by printing electrode patterns of a preset size on one side of a second microelectrode array mask, and by using 365 nm ultraviolet light to sequentially irradiate the electrode pattern layers of different second microelectrode array masks to form multiple exposed second electrode patterns on multiple glass substrates, multiple different second microelectrode array devices can simultaneously detect intracellular action potentials of cells of different sizes, further improving the cell detection adaptability of the second microelectrode array device.

[0094] It should be noted that in some embodiments of this application, the first preset speed is 3000 rpm / min, the first preset temperature is 120 degrees Celsius, and the first preset time is 2 minutes. Those skilled in the art can set the magnitude of the first preset speed, the first preset temperature, and the first preset time according to the actual situation. The embodiments of this application do not limit the magnitude of the first preset speed, the first preset temperature, and the first preset time.

[0095] It should be noted that in some embodiments of this application, the first preset distance is 0.1 micrometers, the first microelectrode array mask is 0.1 micrometers away from the glass substrate, and the electrode pattern layer of the first microelectrode array mask is exposed under vacuum by 365 nm ultraviolet light to form an exposed first electrode pattern on the glass substrate. Those skilled in the art can set the size of the first preset distance according to the actual situation. The embodiments of this application do not limit the size of the first preset distance.

[0096] It should be noted that in some embodiments of this application, the second preset time is 2 seconds and the third preset time is 35 seconds. Those skilled in the art can set the size of the second preset time and the third preset time according to the actual situation. The embodiments of this application do not limit the size of the second preset time and the third preset time.

[0097] In one embodiment, a first microelectrode array and a second microelectrode array are fabricated using standard photolithography. Using a 4-inch square glass substrate as the base, a 2.5 μm thick layer of positive photoresist RZJ-390PG-30 is spin-coated onto the glass substrate at 3000 rpm / min. After baking at 120 °C for 2 minutes, the first microelectrode array mask is placed on the mechanical structure of the photolithography machine, and the distance between the first microelectrode array mask and the glass substrate is controlled by the mechanical structure. The electrode pattern layer of the first microelectrode array mask is irradiated with 365 nm ultraviolet light for 2 seconds, followed by development with developer RZX3038 for 35 seconds, thereby forming an exposed first electrode pattern on the glass substrate. Then, a 10 nm titanium / 100 nm gold layer is deposited by magnetron sputtering, and the photoresist is stripped with acetone to form electrodes. Finally, a 2 μm thick layer of negative photoresist SU-8 2002 is spin-coated onto the electrode surface at 1000 rpm / min to obtain the first microelectrode array. According to the above method, the first microelectrode array mask is replaced with the second microelectrode array mask to complete the fabrication of the second microelectrode array.

[0098] Reference Figure 12 As shown, Figure 12 This is a flowchart of another method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, provided in the embodiments of this application, including but not limited to steps S200 to S250. Specifically,

[0099] Step S200: Spin-coat the negative photoresist onto the surface of the first microelectrode array at a second preset speed;

[0100] Step S210: Flip the first microelectrode array mask placed on the mechanical structure of the photolithography machine, control the distance between the first microelectrode array mask and the glass substrate through the mechanical structure, irradiate the encapsulation layer pattern of the first microelectrode array mask with 365 nm ultraviolet light for a fourth preset time, develop it in propylene glycol methyl ether acetate for a fifth preset time, and clean the first microelectrode array with isopropanol to form a first insulating layer;

[0101] Step S220: Place the first microelectrode array at a second preset temperature and bake for a sixth preset time to obtain the first chip;

[0102] Step S230: Spin-coat the negative photoresist onto the surface of the second microelectrode array at a second preset speed;

[0103] Step S240: Flip the second microelectrode array mask placed on the mechanical structure of the photolithography machine, control the distance between the second microelectrode array mask and the glass substrate by the mechanical structure, irradiate the encapsulation layer pattern of the second microelectrode array mask with 365 nm ultraviolet light for a fourth preset time, develop in propylene glycol methyl ether acetate for a fifth preset time, and clean the second microelectrode array with isopropanol to form a second insulating layer;

[0104] Step S250: Place the second microelectrode array at a second preset temperature and bake for a sixth preset time to obtain the second chip.

[0105] In some embodiments of this application, the method for fabricating a multi-size microelectrode array detection device coupled with micro-electroperving further includes: flipping a first microelectrode array mask placed on the mechanical structure of a lithography machine, such that the encapsulation layer pattern of the first microelectrode array mask is irradiated with 365 nm ultraviolet light to form a first insulating layer; similarly, flipping a second microelectrode array mask placed on the mechanical structure of a lithography machine, such that the encapsulation layer pattern of the second microelectrode array mask is irradiated with 365 nm ultraviolet light to form an insulating layer.

[0106] It should be noted that in some embodiments of this application, the second preset speed is 1000 rpm / min. Those skilled in the art can set the size of the second preset speed according to the actual situation. The embodiments of this application do not limit the size of the second preset speed.

[0107] It should be noted that in some embodiments of this application, the fourth preset time is 2 seconds, the fifth preset time is 1 minute, the second preset temperature is 150 degrees Celsius, and the sixth preset time is 30 minutes. Those skilled in the art can set the values ​​of the fourth preset time, the fifth preset time, the sixth preset time, and the second preset temperature according to actual conditions. This application does not limit the values ​​of the fourth preset time, the fifth preset time, the sixth preset time, and the second preset temperature.

[0108] In one embodiment, the encapsulation layer pattern of the first microelectrode array mask is exposed to 365 nm ultraviolet light for 2 seconds, then developed in propylene glycol methyl ether acetate for 1 minute, and then cleaned with isopropanol to form a second insulating layer. Next, the device is baked at 150°C for 30 minutes. Finally, a 4-inch square glass substrate is cut into 25 2×2 cm² first microelectrode array chips, completing the fabrication of the first microelectrode array chip. Similarly, using the above method, the encapsulation layer pattern of the first microelectrode array mask is replaced with the encapsulation layer pattern of the second microelectrode array mask to fabricate the second microelectrode array chip.

[0109] Reference Figure 13 As shown, Figure 13This is a flowchart of another method for fabricating a multi-size microelectrode array detection device coupled with microelectroporation, provided in the embodiments of this application, including but not limited to steps S300 to S350. Specifically,

[0110] Step S300: Use PDMS to fix the first chip onto the first printed circuit board, and use conductive silver paste to connect the microelectrode pins of the first chip to the first printed circuit board;

[0111] Step S310: Use PDMS to adhere the first cell culture chamber to the top of the first chip;

[0112] Step S320: Electrically connect the first reference electrode and the first printed circuit board; solder the first pin interface to the first printed circuit board to match the interface of the multi-size microelectrode array detection device to obtain the first microelectrode array device;

[0113] Step S330: Use PDMS to fix the second chip onto the second printed circuit board, and use conductive silver paste to connect the microelectrode pins of the second chip to the second printed circuit board.

[0114] Step S340: Use PDMS to adhere the second cell culture chamber to the top of the second chip;

[0115] Step S350: Electrically connect the second reference electrode and the second printed circuit board; solder the second pin interface to the second printed circuit board to match the interface of the multi-size microelectrode array detection device to obtain the second microelectrode array device.

[0116] In some embodiments of this application, the fabrication method of the multi-size microelectrode array detection device coupled with microelectroporation further includes: fixing a first chip to a first printed circuit board using PDMS Sylgard 184, and connecting 32 microelectrodes to the first printed circuit board using conductive silver paste. Subsequently, a glass ring 1 cm wide and 1.5 cm high is bonded to the top of the first chip using PDMS to serve as a first cell culture chamber. A first reference electrode is electrically connected to the first printed circuit board. Finally, a row of pin interfaces is soldered to the first printed circuit board to match the interface of the multi-size microelectrode array detection device, and the first microelectrode array device performs electrophysiological signal recording and microelectroporation through the first reference electrode. Similarly, a second chip is fixed to a second printed circuit board using PDMS Sylgard 184, and 32 microelectrodes are connected to the second printed circuit board using conductive silver paste. Subsequently, a glass ring 1 cm wide and 1.5 cm high is bonded to the top of the second chip using PDMS to serve as a second cell culture chamber. A second reference electrode is electrically connected to the second printed circuit board. Finally, a row of pin interfaces is soldered onto the second printed circuit board to match the interface of the multi-size microelectrode array detection device. The second microelectrode array device records electrophysiological signals and performs microelectroperforation through the second reference electrode.

[0117] The above is a detailed description of the preferred embodiments of this application. However, this application is not limited to the above embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of this application. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A coupled microelectroporation multi-size microelectrode array detection device, characterized by, include: At least one first microelectrode array device includes a first chip, a first printed circuit board, and a first cell culture chamber. The first chip is fixed on the first printed circuit board. The first chip includes a plurality of microelectrodes of mixed sizes. The plurality of microelectrode pins on the first chip are connected to the first printed circuit board through conductive silver paste. The first cell culture chamber and the first chip abut against each other. Multiple second microelectrode array devices include a second chip, a second printed circuit board, and a second cell culture chamber. The second chip is fixed on the second printed circuit board and includes multiple microelectrodes of the same size. The microelectrodes on the second chips of different second microelectrode array devices are of different sizes. The multiple microelectrode pins on the second chip are connected to the second printed circuit board through conductive silver paste. The second cell culture chamber and the second chip are in contact. An electrophysiological signal acquisition module is connected to the first microelectrode array device and the second microelectrode array device. The electrophysiological signal acquisition module is used to acquire the electrophysiological signals of cells acquired by the first microelectrode array device and the electrophysiological signals of cells acquired by the second microelectrode array device. The host computer is communicatively connected to the electrophysiological signal acquisition module. The host computer is used to store the electrophysiological signal data acquired by the electrophysiological signal acquisition module and to display the electrophysiological signal data. An electroporation module is connected to the first chip and the second chip respectively. The host computer controls the electroporation module to apply a preset voltage pulse. The preset voltage pulse is applied to the cells to be tested in the first cell culture chamber through multiple microelectrodes on the first chip, thereby realizing electroporation of the cells in the first cell culture chamber. The preset voltage pulse is applied to the cells to be tested in the second cell culture chamber by multiple microelectrodes on the second chip, thereby achieving cell electroporation in the second cell culture chamber.

2. The coupled microelectroporation multi-size microelectrode array detection device of claim 1, wherein, The first microelectrode array device includes a first microelectrode array mask, one side of which is printed with a mixed-size electrode pattern, and the other side of which is printed with an encapsulation layer pattern.

3. The coupled microelectroporation multi-size microelectrode array detection device of claim 2, wherein, The first chip has a plurality of microelectrodes of mixed sizes, including an 8-channel microelectrode of a first preset size, an 8-channel microelectrode of a second preset size, an 8-channel microelectrode of a third preset size, a 4-channel microelectrode of a fourth preset size, and a 4-channel microelectrode of a fifth preset size.

4. The coupled microelectroporation multi-size microelectrode array detection device of claim 1, wherein, The minimum distance between the edges of adjacent microelectrodes in the first microelectrode array device and the second microelectrode array device is 250 μm.

5. The coupled microelectroporation multi-size microelectrode array detection device of claim 1, wherein, The second microelectrode array device includes a second microelectrode array mask. One side of the second microelectrode array mask is printed with an electrode pattern of a preset size, and the other side of the second microelectrode array mask is printed with an encapsulation layer pattern. The size of the electrode pattern printed on different second microelectrode array masks is different.

6. The coupled microelectroporation multi-size microelectrode array detection device of claim 5, wherein, The size of the plurality of microelectrodes on the second chip includes 32-channel sixth preset size microelectrodes, 32-channel seventh preset size microelectrodes, 32-channel eighth preset size microelectrodes, 32-channel ninth preset size microelectrodes and 32-channel tenth preset size microelectrodes.

7. The coupled microelectroporation multi-size microelectrode array detection device of claim 1, wherein, Further comprising a first reference electrode and a second reference electrode, the first reference electrode and the first printed circuit board of the first microelectrode array device are electrically connected, the first reference electrode is used for micro-electroporation of cells in the first cell culture chamber and for recording the electrophysiological signals collected by the first microelectrode array device, and the second reference electrode and the second printed circuit board of the second microelectrode array device are electrically connected, the second reference electrode is used for micro-electroporation of cells in the second cell culture chamber and for recording the electrophysiological signals collected by the second microelectrode array device.

8. A method of fabricating a coupled microelectroporation multi-size microelectrode array detection device as claimed in claim 1, wherein, It comprises: Preparation of first microelectrode array mask and second microelectrode array mask with different sizes; The positive photoresist is spin-coated on the glass substrate at a first preset speed, and the glass substrate is placed in an oven at a first preset temperature for baking for a first preset time; The first microelectrode array mask is placed in the mechanical structure of the photoetching machine, and the first microelectrode array mask is controlled to be at a first preset distance from the glass substrate by the mechanical structure, and the electrode pattern layer of the first microelectrode array mask is irradiated with 365 nm ultraviolet light for a second preset time, then developed with a developing agent for a third preset time, so that the exposed first electrode pattern is formed on the glass substrate; A titanium or gold layer is deposited on the glass substrate with the exposed first electrode pattern by magnetron sputtering technology, and the positive photoresist is stripped using acetone to form a first microelectrode array; A preset number of baked glass substrates are taken, one of which corresponds to a second microelectrode array mask with a certain size, and different sizes of second microelectrode array masks are placed in the mechanical structure of the photoetching machine in turn, and the second microelectrode array mask is controlled to be at a first preset distance from the glass substrate by the mechanical structure, and the electrode pattern layer of the first microelectrode array mask is irradiated with 365 nm ultraviolet light for a second preset time, then developed with a developing agent for a third preset time, so that the exposed second electrode pattern is formed on the glass substrate; A titanium or gold layer is deposited on the glass substrate with the exposed second electrode pattern by magnetron sputtering technology, and the positive photoresist is stripped using acetone to form a second microelectrode array.

9. The method of claim 8, wherein the microelectrode array is a microelectrode array for a microelectroporation coupled multi-size microelectrode array detection device. Further comprising: The negative photoresist is spin-coated on the surface of the first microelectrode array at a second preset speed; The first microelectrode array mask placed in the mechanical structure of the photoetching machine is turned over, and the first microelectrode array mask is controlled to be at a first preset distance from the glass substrate by the mechanical structure; 365 nm ultraviolet light is used to irradiate the packaging layer pattern of the first microelectrode array mask for a fourth preset time, and the first microelectrode array is developed in propylene glycol methyl ether acetate for a fifth preset time and washed with isopropyl alcohol to form a first insulating layer; placing the first microelectrode array at a second preset temperature and baking for a sixth preset time to obtain a first chip; spinning the negative photoresist on the surface of the second microelectrode array at a second preset speed; turning over the second microelectrode array mask placed on the mechanical structure of the photoetching machine, controlling the distance between the second microelectrode array mask and the glass substrate by the mechanical structure, irradiating the encapsulation layer pattern of the second microelectrode array mask with 365 nm ultraviolet light for a fourth preset time, developing in propylene glycol methyl ether acetate for a fifth preset time, and cleaning the second microelectrode array with isopropyl alcohol to form a second insulating layer; placing the second microelectrode array at a second preset temperature and baking for a sixth preset time to obtain a second chip.

10. The method of claim 8, wherein the microelectrode array is a microelectroporation- coupled multi-size microelectrode array detection device. Further comprising: fixing the first chip on a first printed circuit board by using PDMS and connecting the microelectrode pins of the first chip and the first printed circuit board by using conductive silver glue; adhering the first cell culture chamber above the first chip by using PDMS; electrically connecting the first reference electrode and the first printed circuit board, and welding the first pin interface to the first printed circuit board to match the interface of the multi-size microelectrode array detection device to obtain a first microelectrode array device; fixing the second chip on a second printed circuit board by using PDMS and connecting the microelectrode pins of the second chip and the second printed circuit board by using conductive silver glue; adhering the second cell culture chamber above the second chip by using PDMS; electrically connecting the second reference electrode and the second printed circuit board, and welding the second pin interface to the second printed circuit board to match the interface of the multi-size microelectrode array detection device to obtain a second microelectrode array device.

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