Three-dimensional integrated cell electrophysiology platform and preparation method and application thereof

Abstract

The invention belongs to the technical field of signal detection, and relates to a three-dimensional integrated cell electrophysiology platform and a preparation method and application thereof.The three-dimensional integrated cell electrophysiology platform is composed of microelectrodes and a three-dimensional micro-scaffold, and the microelectrodes comprise a two-dimensional plane microelectrode and a three-dimensional microelectrode; wherein the microelectrode is used for applying stimulation to cells in a two-dimensional plane and a three-dimensional space and collecting electrophysiological signals; the three-dimensional micro-scaffold is used for simulating a three-dimensional microenvironment of normal physiological activities of cells; the average diameter of the two-dimensional planar microelectrode and the average diameter of the three-dimensional microelectrode are each independently 1 [mu] m or less. The three-dimensional integrated cell electrophysiology platform provided by the invention has the functions of high-precision processing, high structural design flexibility and high integration, and is suitable for various scenes such as cell culture, cell detection, cell excitation and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of signal detection, and relates to a three-dimensional integrated cell electrophysiological platform, in particular to a three-dimensional integrated cell electrophysiological platform and a preparation method and application thereof. BACKGROUND

[0002] Accurate detection of cell electrophysiological signals is a core technical support for analyzing single cell functional characteristics, revealing cell group synergistic mechanism, and promoting early diagnosis and pathological research of diseases. At present, the mainstream cell electrophysiological signal detection technologies mainly include patch clamp technology and microelectrode array technology, which have their own characteristics in principle and application scenarios, and also have obvious technical limitations.

[0003] Among them, the patch clamp technology realizes high-resolution recording of single cell electrical signals by forming a tight seal between a glass microelectrode and a cell. However, this technology has significant shortcomings: first, the overall cost of the equipment is high, and the operation threshold is high; second, the equipment requires strict environmental stability (such as temperature, vibration, and humidity) during operation, which is difficult to apply on a large scale in a conventional laboratory environment; third, due to the sealing method of the electrode and the cell, it is difficult to realize long-term and dynamic electrical signal monitoring of the same cell or cell group, and the application is limited in long-term physiological process research.

[0004] In comparison, the microelectrode array technology has a broader application prospect in the field of cell electrophysiological monitoring due to its unique advantages. This technology can realize non-invasive synchronous recording of electrical signals of multiple cells by integrating a microelectrode array on a cell culture substrate, and can also continuously stimulate cells, so it is suitable for single cell electrical activity analysis and long-term dynamic monitoring of cell group function. However, the current commercial microelectrode array is based on a two-dimensional planar substrate, and the electrode can only form a local contact with the bottom of the cell, which directly leads to three technical problems: first, the cell electrical signal acquisition sensitivity is low, which makes it difficult to capture weak electrophysiological changes; second, the signal signal-to-noise ratio is poor, which is easily disturbed by background noise, affecting data accuracy; third, the recording stability is weak, and the signal is easy to drift during long-term monitoring. At the same time, the two-dimensional structure cannot realize accurate extraction and targeted electrical stimulation of electrical signals at different sites of the cell in three-dimensional space, and it is difficult to fully reflect the three-dimensional electrophysiological characteristics of the cell.

[0005] From the perspective of physiological environment simulation, all physiological activities of cells in a living organism occur within a complex three-dimensional physical / biochemical microenvironment. Two-dimensional planar microelectrode arrays cannot replicate this realistic physiological environment, leading to discrepancies between the detected cellular electrical signals and actual in vivo signals. In severe cases, this can even mislead experimental conclusions, hindering the translation of cellular electrophysiological research results into clinical applications. Therefore, developing an integrated electrophysiological platform that combines three-dimensional cell culture capabilities with three-dimensional electrical signal detection has become an urgent need in the fields of cell biology and biomedical engineering.

[0006] In the fabrication of three-dimensional electrophysiological platforms, the mainstream technology still employs traditional semiconductor processes, constructing three-dimensional microelectrode structures through a series of complex processes such as photolithography, etching, and thin film deposition. These methods have inherent drawbacks: the process flow is cumbersome and involves many steps, with stringent requirements for the processing environment (such as cleanliness and vacuum); the investment cost of processing equipment is high, making low-cost, large-scale production difficult; furthermore, limited by photolithographic resolution and the flexibility of etching processes, the design freedom of the three-dimensional structure is low, making it impossible to quickly adjust structural parameters according to different cell types or experimental needs, thus failing to meet personalized research requirements.

[0007] In recent years, 3D printing technology has provided a new technical path for the fabrication of three-dimensional electrophysiological platforms due to its superior three-dimensional structure forming capabilities and flexible customization advantages. However, commonly used 3D printing technologies (such as inkjet printing and extrusion printing) still have significant shortcomings: on the one hand, the conductivity of the printing materials is poor, making it difficult to meet the high conductivity requirements of microelectrodes, requiring additional conductive layer modification, which increases the complexity of the process; on the other hand, the printing accuracy is limited, failing to meet the processing requirements of subcellular scale (<10μm) microelectrodes, thus making it difficult to achieve accurate detection of electrical signals in tiny areas of single cells.

[0008] It is evident that existing processing technologies cannot simultaneously meet the core requirements of a three-dimensional integrated cell electrophysiology platform for high-precision processing, high structural design flexibility, and high integration functionality. Overcoming these technological bottlenecks has become the key to driving cell electrophysiology research towards higher dimensions and higher precision. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a three-dimensional integrated cell electrophysiology platform, its preparation method, and its application, which combines high-precision processing, high structural design flexibility, and high integration functionality, and is suitable for various scenarios such as cell culture, cell detection, and cell stimulation.

[0010] To achieve this objective, the present invention employs the following technical solution:

[0011] In a first aspect, the present invention provides a three-dimensional integrated cell electrophysiology platform, which consists of microelectrodes and a three-dimensional microscaffold, wherein the microelectrodes include two-dimensional planar microelectrodes and three-dimensional microelectrodes.

[0012] The microelectrodes are used to stimulate cells and collect electrophysiological signals in two-dimensional plane and three-dimensional space; the three-dimensional microscaffold is used to simulate the three-dimensional microenvironment of normal cell physiological activities.

[0013] The average diameter of the two-dimensional planar microelectrode and the three-dimensional microelectrode is independently less than 1 μm.

[0014] The three-dimensional integrated cell electrophysiology platform provided by this invention simulates the three-dimensional microenvironment of normal cellular physiological activities through a three-dimensional microscaffold. At the same time, it uses microelectrodes to stimulate cells and collect electrophysiological signals in two-dimensional plane and three-dimensional space, which truly restores the cellular physiological environment, reduces the deviation between the detected signal and the real signal in vivo, and realizes the integrated function of "culture-stimulation-recording". Moreover, the microelectrode size of less than 1μm reaches the subcellular scale, meets the high precision requirements, and well balances high precision processing, high structural design flexibility and high integration function. It is suitable for various scenarios such as cell culture, cell detection and cell stimulation.

[0015] Preferably, the three-dimensional microscaffold includes a hollow load-bearing portion and an edge support portion for supporting and culturing cells.

[0016] Preferably, the material of the three-dimensional microscaffold includes a polymer, and the polymer monomer includes pentaerythritol triacrylate (PETA).

[0017] Preferably, the two-dimensional planar microelectrode is disposed through the bottom of the three-dimensional micro-support, and the microelectrode contacts are exposed in the hollow area of ​​the hollow support portion.

[0018] Preferably, the two-dimensional planar microelectrode refers to a microelectrode having a two-dimensional shape on a plane.

[0019] Preferably, the material of the two-dimensional planar microelectrode includes platinum.

[0020] Preferably, the three-dimensional microelectrode is attached to the surface of the three-dimensional microscaffold, and the three-dimensional microelectrode includes a microelectrode with a three-dimensional spatial location.

[0021] Preferably, the microelectrode with a three-dimensional spatial location includes a two-dimensional shaped microelectrode with a three-dimensional spatial location and / or a three-dimensional shaped microelectrode with a three-dimensional spatial location.

[0022] Preferably, the three-dimensional microelectrode further includes a microelectrode having a three-dimensional shape on a plane.

[0023] Preferably, the material of the three-dimensional microelectrode includes platinum.

[0024] Preferably, the number and shape of the microelectrodes and the three-dimensional microscaffolds are designed in combination according to the cell type to be detected, and the cell type to be detected includes any one or a combination of at least two of single cells, cell populations or organoids.

[0025] In a second aspect, the present invention provides a method for preparing a three-dimensional integrated cell electrophysiological platform as described in the first aspect, comprising the following steps:

[0026] (1) The substrate is cleaned and silanized sequentially;

[0027] (2) A two-dimensional planar microelectrode is fabricated on the surface of the substrate;

[0028] (3) A three-dimensional microscaffold is printed on the surface of the substrate, the three-dimensional microscaffold covering the two-dimensional planar microelectrode;

[0029] (4) Three-dimensional microelectrodes are prepared on the surface of the substrate and the three-dimensional microscaffold to obtain the three-dimensional integrated cell electrophysiological platform.

[0030] Preferably, the fabrication methods of the two-dimensional planar microelectrode and the three-dimensional microelectrode each independently include laser direct writing and / or photolithographic deposition, and more preferably laser direct writing.

[0031] Preferably, the laser direct writing includes: coating a platinum precursor ink, focusing a laser on the surface to be processed, and generating metallic platinum through a photoreduction reaction at the laser focal point.

[0032] Preferably, the method for printing the three-dimensional microscaffold includes: coating the surface of the substrate with negative photoresist, and performing three-dimensional scanning by controlling the focus through a 3D printing program to obtain the three-dimensional microscaffold.

[0033] Thirdly, the present invention provides a macro-micro integrated electrophysiological signal acquisition device, comprising an acquisition device, a printed circuit board, and a three-dimensional integrated cell electrophysiological platform as described in the first aspect, which are connected in sequence.

[0034] Preferably, a conversion device is electrically connected between the acquisition device and the printed circuit board.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The three-dimensional integrated cell electrophysiology platform provided by this invention simulates the three-dimensional microenvironment of normal cellular physiological activities through a three-dimensional microscaffold. At the same time, it uses microelectrodes to stimulate cells and collect electrophysiological signals in two-dimensional plane and three-dimensional space, which truly restores the cellular physiological environment, reduces the deviation between the detected signal and the real signal in vivo, and realizes the integrated function of "culture-stimulation-recording". Moreover, the microelectrode size of less than 1μm reaches the subcellular scale, meets the high precision requirements, and well balances high precision processing, high structural design flexibility and high integration function. It is suitable for various scenarios such as cell culture, cell detection and cell stimulation. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of the three-dimensional integrated cell electrophysiology platform provided by the present invention;

[0038] Figure 2 These are micrographs of the three-dimensional integrated cell electrophysiology platform provided in Example 1;

[0039] Figure 3 These are microscopic images of the three-dimensional microelectrode-microscaffold structure of the laser-written platinum wire and the platinum wire passing through the pre-reserved groove at the bottom of the microscaffold in the three-dimensional integrated cell electrophysiology platform provided in Example 1.

[0040] Figure 4 These are micrographs of the three-dimensional integrated cell electrophysiology platform provided in Example 2;

[0041] Figure 5 This is a schematic diagram of the electrophysiological signal acquisition device provided in Example 1-2.

[0042] Wherein: 10-microelectrode; 11-two-dimensional planar microelectrode; 12-three-dimensional microelectrode; 20-three-dimensional microscaffold. Detailed Implementation

[0043] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0044] One embodiment of this invention provides a three-dimensional integrated cell electrophysiology platform, such as... Figure 1 As shown, the three-dimensional integrated cell electrophysiology platform consists of microelectrodes 10 and three-dimensional microscaffolds 20, and the microelectrodes 10 include two-dimensional planar microelectrodes 11 and three-dimensional microelectrodes 12.

[0045] The microelectrode 10 is used to stimulate cells and collect electrophysiological signals in a two-dimensional plane and a three-dimensional space; the three-dimensional microscaffold 20 is used to simulate the three-dimensional microenvironment of normal cell physiological activities.

[0046] The average diameter of the two-dimensional planar microelectrode 11 and the three-dimensional microelectrode 12 is independently less than 1 μm, for example, it can be 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm or 1 μm, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0047] The three-dimensional integrated cell electrophysiology platform provided by this invention simulates the three-dimensional microenvironment of normal cellular physiological activities through a three-dimensional microscaffold 20. At the same time, microelectrodes 10 are used to stimulate cells and collect electrophysiological signals in two-dimensional plane and three-dimensional space, which truly restores the cellular physiological environment, reduces the deviation between the detected signal and the real signal in vivo, and realizes the integrated function of "culture-stimulation-recording". Moreover, the size of the microelectrodes 10 below 1μm reaches the subcellular scale, meets the high precision requirements, and well balances high precision processing, high structural design flexibility and high integration function. It is suitable for various scenarios such as cell culture, cell detection and cell stimulation.

[0048] In some embodiments, the three-dimensional microscaffold 20 includes a hollow support portion and an edge support portion for supporting and culturing cells.

[0049] The hollow support portion includes a central hollow area and a sloping area surrounding the edge of the hollow area. However, it is not limited to whether or not it has a sloping area. As long as it can fulfill the function of supporting and culturing cells, other shapes are within the protection scope of this invention.

[0050] In some embodiments, the material of the three-dimensional microscaffold 20 includes a polymer, and the polymer monomer includes pentaerythritol triacrylate (PETA).

[0051] The present invention selects PETA as the polymer monomer for the three-dimensional microscaffold 20 because this material has high processing resolution, fast polymerization speed, and excellent biocompatibility, effectively balancing processing precision, processing efficiency, and detection accuracy.

[0052] In some embodiments, the two-dimensional planar microelectrode 11 is disposed through the bottom of the three-dimensional micro-support 20, and the microelectrode contacts are exposed in the hollow area of ​​the hollow support portion.

[0053] The two-dimensional planar microelectrode 11 refers to a microelectrode with a two-dimensional shape on a plane, that is, a two-dimensional microelectrode without a clear three-dimensional shape or three-dimensional height on a plane. For example, it can be a microelectrode line or microelectrode sheet with a two-dimensional shape on a plane.

[0054] In this invention, the two-dimensional planar microelectrode 11 is provided with a certain length at the end of the hollowed-out area so that the microelectrode and the cell can contact to form a conductive path.

[0055] In some embodiments, the material of the two-dimensional planar microelectrode 11 includes platinum.

[0056] In some embodiments, the three-dimensional microelectrode 12 is attached to the surface of the three-dimensional microscaffold 20, and the three-dimensional microelectrode 12 includes a microelectrode with a three-dimensional spatial location.

[0057] In some embodiments, the microelectrode with a three-dimensional spatial location includes a two-dimensional shaped microelectrode with a three-dimensional spatial location and / or a three-dimensional shaped microelectrode with a three-dimensional spatial location.

[0058] The two-dimensional microelectrode with a three-dimensional spatial location can be a two-dimensional microelectrode line or a two-dimensional microelectrode sheet with a three-dimensional shape or a three-dimensional height, and the three-dimensional microelectrode with a three-dimensional spatial location can be a microneedle or a microcolumn with a three-dimensional shape or a three-dimensional height.

[0059] In some embodiments, the three-dimensional microelectrode 12 further includes a microelectrode having a three-dimensional shape on a plane, such as a microneedle or micropillar having a three-dimensional shape on a plane.

[0060] In some embodiments, the material of the three-dimensional microelectrode 12 includes platinum.

[0061] In this invention, both the two-dimensional planar microelectrode 11 and the three-dimensional microelectrode 12 are made of platinum metal because platinum metal is not easily oxidized, has low resistance, and excellent biocompatibility.

[0062] In some embodiments, the number and shape of the microelectrodes 10 and the three-dimensional microscaffold 20 are designed in combination according to the cell type to be detected, and the cell type to be detected includes any one or a combination of at least two of single cells, cell populations or organoids.

[0063] One embodiment of the present invention also provides a method for preparing the three-dimensional integrated cell electrophysiology platform described in any of the above embodiments, comprising the following steps:

[0064] (1) The substrate is cleaned and silanized sequentially;

[0065] (2) A two-dimensional planar microelectrode 11 is fabricated on the surface of the substrate;

[0066] (3) A three-dimensional microscaffold 20 is printed on the surface of the substrate, the three-dimensional microscaffold 20 covering the two-dimensional planar microelectrode 11;

[0067] (4) A three-dimensional microelectrode 12 is prepared on the surface of the substrate and the three-dimensional microscaffold 20 to obtain the three-dimensional integrated cell electrophysiological platform.

[0068] In some embodiments, the fabrication methods of the two-dimensional planar microelectrode 11 and the three-dimensional microelectrode 12 independently include laser direct writing and / or photolithographic deposition, with laser direct writing being more preferred.

[0069] Compared to photolithography deposition, the laser direct writing method preferred in this invention can directly process microelectrode structures of specific shapes in a two-dimensional plane or three-dimensional space by controlling the laser power and the laser focus position. Moreover, processing different materials only requires cleaning and replacing the precursor ink, which is convenient and quick.

[0070] In some embodiments, the laser direct writing includes: coating a platinum precursor ink, focusing a laser on the surface to be processed, and generating metallic platinum through a photoreduction reaction at the laser focal point.

[0071] Optionally, the photolithography deposition includes: coating a positive photoresist onto the surface to be processed, selectively patterning the photoresist using a laser, exposing the patterned area after development, and depositing conductive platinum metal in the exposed area to finally complete the fabrication of the three-dimensional electrophysiological platform.

[0072] In some embodiments, the printing method of the three-dimensional microscaffold 20 includes: coating the surface of the substrate with negative photoresist, and performing three-dimensional scanning by controlling the focus through a 3D printing program to obtain the three-dimensional microscaffold 20.

[0073] This invention uses laser direct writing and 3D printing to prepare microelectrodes and microscaffolds respectively, which can share a single processing system, have a short preparation cycle, and do not require cleanroom equipment, thus significantly reducing preparation costs.

[0074] One embodiment of the present invention also provides a macro-micro integrated electrophysiological signal acquisition device, comprising an acquisition device, a printed circuit board, and the three-dimensional integrated cell electrophysiological platform described in any of the above embodiments, which are connected in sequence.

[0075] In some embodiments, a conversion device is also electrically connected between the acquisition device and the printed circuit board.

[0076] Example 1

[0077] This embodiment provides a three-dimensional integrated cell electrophysiology platform and its preparation method, specifically including the following steps:

[0078] (1) Material preparation

[0079] PETA negative photoresist: Slowly add 0.25wt% of 7-diethylamino-3-(2-thienyl)coumarin (DETC) to PETA, stir continuously for 2 hours, and store at room temperature away from light;

[0080] Platinum precursor ink: Prepare a 70mM ammonium chloroplatinate solution and a 500mM ferric ammonium oxalate solution, seal and protect from light, and mix them evenly in a 1:1 volume ratio before use.

[0081] (2) Substrate pretreatment

[0082] Using a standard coverslip as the substrate, the substrate was ultrasonically cleaned in deionized water, acetone, and isopropanol for 5 minutes in sequence. The surface residual liquid was gently dried with nitrogen and then placed in a 150°C oven for 30 minutes. After the substrate cooled, it was treated with oxygen plasma at 100W power for 10 minutes. The treated substrate was then immersed in a toluene solution containing 0.2% by volume of (3-aminopropyl)triethoxysilane for 120 minutes. Finally, the substrate was removed and cleaned in sequence with toluene, acetone, and isopropanol, and then gently dried with nitrogen.

[0083] (3) Fabrication of two-dimensional planar microelectrodes

[0084] Platinum precursor ink is dropped onto a substrate, and the substrate is placed into a laser processing system. A femtosecond laser with a wavelength of 780 nm is focused on the substrate to process platinum wires. The processing power is controlled at 3.5±1.5mW, and the processing speed is controlled at 12.5±7.5μm / s. Platinum wires with a linewidth of less than 1μm are processed. After processing, the substrate is removed, gently rinsed with ethanol, and the surface moisture is gently dried with nitrogen to obtain a two-dimensional planar microelectrode.

[0085] (4) 3D micro-scaffold printing

[0086] PETA negative photoresist was dropped onto a substrate with conductive contacts, and a clean cover glass was placed on top to cover it. The air was gently pressed out, and the substrate was tightly sealed around the edges and placed into a laser processing system. A femtosecond laser with a wavelength of 780nm was focused on the substrate, and the focus was controlled by a 3D printing program to perform three-dimensional scanning to process the polymer three-dimensional microscaffold. The laser power was controlled at 15±10mW, and the processing speed was controlled at 150±50μm / s. After processing, the substrate was removed, immersed in isopropanol for 20 minutes to remove excess unpolymerized photoresist, and finally dried with nitrogen to obtain the three-dimensional microscaffold.

[0087] (5) Fabrication of three-dimensional microelectrodes

[0088] Platinum precursor ink was dropped onto a substrate, which was then placed into a laser processing system. A femtosecond laser with a wavelength of 780 nm was focused onto the substrate to process platinum wires. The processing power was controlled at 3.5 ± 1.5 mW, and the processing speed was controlled at 12.5 ± 7.5 μm / s. Platinum wires with a linewidth of less than 1 μm were processed. After processing, the substrate was removed, gently rinsed with ethanol, and the surface moisture was gently dried with nitrogen gas to obtain a three-dimensional integrated cell electrophysiology platform.

[0089] Micrographs of the three-dimensional integrated cell electrophysiology platform obtained in this embodiment are shown below. Figure 2 .

[0090] Among them, the three-dimensional microelectrode-microscaffold structure of the laser-written platinum wire and the micromorphology of the platinum wire passing through the pre-reserved groove at the bottom of the microscaffold are shown in [the figure]. Figure 3 .

[0091] Example 2

[0092] This embodiment provides a three-dimensional integrated cell electrophysiology platform and its preparation method, specifically including the following steps:

[0093] (1) Material preparation

[0094] Metal deposition targets: Cr targets, Pt targets;

[0095] Positive photoresist and developer: AZ4620 photoresist, AZ400K developer, store at low temperature and protected from light;

[0096] PETA negative photoresist: Slowly add 0.25wt% of 7-diethylamino-3-(2-thienyl)coumarin (DETC) to PETA, stir continuously for 2 hours, and store at room temperature away from light.

[0097] (2) Substrate pretreatment

[0098] This step is the same as in Example 1, and will not be repeated here.

[0099] (3) Fabrication of two-dimensional planar microelectrodes

[0100] Positive photoresist AZ4620 was diluted with PGMEA at a mass ratio of 1:3 and stirred thoroughly to reduce the viscosity of the photoresist, thereby reducing the film thickness after spin coating. The photoresist was spin-coated onto the substrate surface at 2000 rpm for 30 seconds and then heated in a 100℃ oven for 2 minutes. Subsequently, the substrate was placed in a laser processing system, and a 780nm femtosecond laser was focused onto the substrate. The laser was controlled by a printing program to pattern the photoresist, with the processing power controlled at 15±5mW and the processing speed controlled at 75±25. μm / s; After processing, the substrate is removed and immersed in AZ400K developer for 2 min; After development, the substrate is rinsed with distilled water, then the surface moisture is gently blown dry with nitrogen, and placed in a 100℃ oven for 3 min; Cr / Pt conductive layer is sputtered on the substrate surface by magnetron sputtering, with the thickness of Cr controlled at 5 nm and the thickness of Pt at 100 nm; Finally, the sputtered substrate is immersed in acetone and gently shaken until the residual photoresist is completely removed, exposing the processed microelectrode structure, thus obtaining the two-dimensional planar microelectrode.

[0101] (4) 3D micro-scaffold printing

[0102] This step is the same as in Example 1, and will not be repeated here.

[0103] (5) Fabrication of three-dimensional microelectrodes

[0104] Positive photoresist AZ4620 was diluted with PGMEA at a mass ratio of 1:3 and stirred thoroughly to reduce the viscosity of the photoresist, thereby reducing the film thickness after spin coating. The photoresist was spin-coated onto the substrate surface at 2000 rpm for 30 seconds and then heated in a 100℃ oven for 2 minutes. Subsequently, the substrate was placed in a laser processing system, and a femtosecond laser with a wavelength of 780 nm was focused onto the substrate. The laser was controlled by a printing program to pattern the photoresist, with the processing power controlled at 15±5 mW and the processing speed controlled at 75±25 μm / s. After processing, the substrate is removed and immersed in AZ400K developer for 2 minutes. After development, the substrate is rinsed with distilled water, then the surface moisture is gently dried with nitrogen gas and placed in a 100°C oven for 3 minutes. A Cr / Pt conductive layer is sputtered on the substrate surface by magnetron sputtering, with the thickness of Cr controlled at 5 nm and the thickness of Pt at 100 nm. Finally, the sputtered substrate is immersed in acetone and gently shaken until the residual photoresist is completely removed, exposing the processed microelectrode structure, and finally a three-dimensional integrated cell electrophysiology platform is obtained.

[0105] Micrographs of the three-dimensional integrated cell electrophysiology platform obtained in this embodiment are shown below. Figure 4 .

[0106] Application Example 1-2

[0107] In this set of application examples, macro- and micro-integrated electrophysiological signal acquisition devices were assembled using the three-dimensional integrated cell electrophysiological platforms obtained in Examples 1 and 2, respectively. Figure 5 As shown, the electrophysiological signal acquisition device includes an acquisition device, a conversion device, a printed circuit board (PCB), and a three-dimensional integrated cell electrophysiological platform that are connected in sequence.

[0108] Before assembly, conductive contacts need to be fabricated on the substrate where the electrophysiology platform is located using photomask ultraviolet lithography and metal deposition. This allows the microelectrodes of the electrophysiology platform to connect with the contacts on the substrate during laser processing. The specific method is as follows:

[0109] (1) Material preparation

[0110] Metal deposition targets: Cr targets, Pt targets;

[0111] Positive photoresist and developer: AZ4620 photoresist, AZ400K developer, store at low temperature and away from light.

[0112] (2) Fabrication of peripheral contacts for macro-micro integrated electrophysiological platforms

[0113] Positive photoresist AZ4620 was spin-coated onto a silanized substrate at 4000 rpm for 30 seconds. The substrate was then placed in a 100°C oven for 2 minutes and stored away from light. The substrate was then subjected to UV lithography, with the photomask determining the distribution and shape of the peripheral contacts. The exposure time was 20 seconds. After exposure, the substrate was immersed in AZ400K developer for 2 minutes. After development, the substrate surface was rinsed with distilled water to remove residual developer, and the surface moisture was gently dried with nitrogen. The substrate was then placed in a 100°C oven for 3 minutes. Subsequently, a Cr / Pt conductive layer was sputtered onto the substrate surface using magnetron sputtering, with the Cr thickness controlled at 5 nm and the Pt thickness at 100 nm. Finally, the sputtered substrate was immersed in acetone and gently agitated until the residual photoresist was completely removed, exposing the fabricated microelectrode structure and forming conductive contacts.

[0114] like Figure 5As shown, this application example designs a PCB board for connection. The PCB board has pads corresponding to the conductive contacts on the substrate. The conductive contacts on the substrate can be connected to the pads on the PCB board via soldering or curing with conductive materials. All pads on the PCB board are led out and connected to the male / female connectors of the corresponding interfaces on the acquisition device. Since the interface of the acquisition device is not a commonly used interface on the PCB board, an adapter is set up to convert the commonly used interface on the PCB board into the interface corresponding to the acquisition device, thereby realizing a closed loop for signal transmission from the device to the PCB board to the electrophysiological platform on the substrate.

[0115] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A three-dimensional integrated cell electrophysiology platform, characterized in that, The three-dimensional integrated cell electrophysiology platform consists of microelectrodes and a three-dimensional microscaffold, and the microelectrodes include two-dimensional planar microelectrodes and three-dimensional microelectrodes. The microelectrodes are used to stimulate cells and collect electrophysiological signals in two-dimensional plane and three-dimensional space; the three-dimensional microscaffold is used to simulate the three-dimensional microenvironment of normal cellular physiological activities. The average diameter of the two-dimensional planar microelectrode and the three-dimensional microelectrode is independently less than 1 μm.

2. The three-dimensional integrated cell electrophysiology platform according to claim 1, characterized in that, The three-dimensional microscaffold includes a hollow load-bearing part and an edge support part, which are used to support and culture cells; And / or, the material of the three-dimensional microscaffold includes a polymer, and the polymer monomer includes pentaerythritol triacrylate.

3. The three-dimensional integrated cell electrophysiology platform according to claim 2, characterized in that, The two-dimensional planar microelectrode is disposed through the bottom of the three-dimensional micro-support, and the microelectrode contacts are exposed in the hollow area of ​​the hollow support part; Wherein, the two-dimensional planar microelectrode refers to a microelectrode having a two-dimensional shape on a plane; And / or, the material of the two-dimensional planar microelectrode includes platinum.

4. The three-dimensional integrated cell electrophysiology platform according to claim 2, characterized in that, The three-dimensional microelectrode is attached to the surface of the three-dimensional microscaffold, and the three-dimensional microelectrode includes a microelectrode with a three-dimensional spatial position. The microelectrode with a three-dimensional spatial location includes a two-dimensional microelectrode with a three-dimensional spatial location and / or a three-dimensional microelectrode with a three-dimensional spatial location. And / or, the three-dimensional microelectrode further includes a microelectrode having a three-dimensional shape on a plane; And / or, the material of the three-dimensional microelectrode includes platinum.

5. The three-dimensional integrated cell electrophysiology platform according to any one of claims 1-4, characterized in that, The number and shape of the microelectrodes and three-dimensional microscaffolds are designed in combination according to the cell type to be detected, and the cell type to be detected includes any one or a combination of at least two of single cells, cell populations or organoids.

6. A method for preparing a three-dimensional integrated cell electrophysiological platform as described in any one of claims 1-5, characterized in that, The preparation method includes the following steps: (1) The substrate is cleaned and silanized sequentially; (2) A two-dimensional planar microelectrode is fabricated on the surface of the substrate; (3) A three-dimensional microscaffold is printed on the surface of the substrate, the three-dimensional microscaffold covering the two-dimensional planar microelectrode; (4) Three-dimensional microelectrodes are prepared on the surface of the substrate and the three-dimensional microscaffold to obtain the three-dimensional integrated cell electrophysiological platform.

7. The method for preparing the three-dimensional integrated cell electrophysiological platform according to claim 6, characterized in that, The fabrication methods for the two-dimensional planar microelectrode and the three-dimensional microelectrode each independently include laser direct writing and / or photolithographic deposition; The laser direct writing process includes: coating a platinum precursor ink, focusing a laser on the surface to be processed, and generating metallic platinum through a photoreduction reaction at the laser focal point.

8. The method for preparing the three-dimensional integrated cell electrophysiological platform according to claim 6 or 7, characterized in that, The method for printing the three-dimensional microscaffold includes: coating the surface of the substrate with negative photoresist, and performing three-dimensional scanning by controlling the focus through a 3D printing program to obtain the three-dimensional microscaffold.

9. A macro-micro combined electrophysiological signal acquisition device, characterized in that, The electrophysiological signal acquisition device includes an acquisition device, a printed circuit board, and a three-dimensional integrated cell electrophysiological platform as described in any one of claims 1-5, which are connected in sequence.

10. The macro-micro combined electrophysiological signal acquisition device according to claim 9, characterized in that, A conversion device is also electrically connected between the acquisition device and the printed circuit board.

Images