2.5 D electrophysiological sensor as well as preparation method and test system thereof

Abstract

The invention discloses a 2.5 D electrophysiological sensor, a preparation method thereof and a test system.The 2.5 D electrophysiological sensor comprises a nano suction tube array and a multi-size integrated electrode array, the nano suction tube array comprises a substrate and an insulating nano suction tube array, the insulating nano suction tube array penetrates through the substrate, and the insulating nano suction tube array is arranged on the substrate. The insulating nano straw array comprises insulating nano straw structures with a plurality of diameters and a plurality of heights, the multi-size integrated electrode array comprises a plurality of electrode layers with different shapes and different thicknesses, and the electrode layers are in cascade connection with the insulating nano straw structures. The embodiment of the invention can measure the electrophysiological signal in the tissue, and can be widely applied to the technical field of sensors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sensors, in particular to a 2.5D electrophysiological sensor and a preparation method and test system thereof. BACKGROUND

[0002] Traditional MEA (Micro-Electrode Array) sensor chips are in a planar (2D) structure, which cannot be used to measure key signals in the internal electrophysiological network of brain slices, three-dimensional organoids and other tissues. SUMMARY

[0003] Therefore, to solve one of the above problems, the purpose of the embodiments of the present application is to provide a 2.5D electrophysiological sensor and a preparation method and test system thereof, which can measure the electrophysiological signals inside the tissue.

[0004] In one aspect, the embodiments of the present application provide a 2.5D electrophysiological sensor, comprising a nanostraw array and a multi-size integrated electrode array, the nanostraw array comprising a substrate and an insulating nanostraw array, the insulating nanostraw array penetrating the substrate, the insulating nanostraw array comprising insulating nanostraw structures of several diameters and several heights, the multi-size integrated electrode array comprising electrode layers of several different shapes and several different thicknesses, the electrode layers cascading several insulating nanostraw structures.

[0005] Optionally, the insulating nanostraw structure comprises a hollow structure, and the sensor further comprises a microfluidic base, the microfluidic base being bonded to the substrate of the nanostraw array, and the microfluidic base comprising several microfluidic channels.

[0006] Optionally, the electrode layer comprises an adhesion layer, a conductive layer and an insulating layer, the adhesion layer being arranged between the conductive layer and the nanostraw array, and the insulating layer covering a non-recording area of the conductive layer.

[0007] In another aspect, the embodiments of the present application provide a preparation method of a 2.5D electrophysiological sensor, comprising:

[0008] providing a substrate of a porous membrane, preparing insulating layers of several thicknesses in the porous membrane of the substrate, etching the insulating layer on the top surface of the porous membrane, and etching the porous membrane of different heights to obtain insulating nanostraw structures of several diameters and several heights;

[0009] preparing electrode layers of several different shapes and several different thicknesses at predetermined positions of the nanostraw array, so that the electrode layers cascade several insulating nanostraw structures to obtain a multi-size integrated electrode array.

[0010] Optionally, the method further comprises:

[0011] Optionally, the method further comprises:

[0012] Optionally, the method further comprises:

[0013] Optionally, the method further comprises:

[0014] Optionally, the method further comprises:

[0015] Optionally, the method further comprises:

[0016] Optionally, the method further comprises:

[0017] Optionally, the method further comprises:

[0018] Optionally, the method further comprises:

[0019] In another aspect, the embodiments of the present application provide a test system of a 2.5D electrophysiological sensor, comprising the 2.5D electrophysiological sensor, an amplification and filtering circuit, a signal conversion circuit and a processor.

[0020] The amplification and filtering circuit is configured to amplify and filter the original signal collected by the 2.5D electrophysiological sensor to obtain an analog signal.

[0021] The signal conversion circuit is configured to convert the analog signal into a digital signal.

[0022] The processor is configured to calculate measurement information according to the digital signal.

[0023] Optionally, the processor is further configured to determine feedback information according to the measurement information, the feedback information including electrical stimulation information and substance delivery control information of the microfluidic base.

[0024] Optionally, the test system further comprises a cell culture dish fixed at a center position of the 2.5D electrophysiological sensor.

[0025] The implementation of the embodiment of the present application has the following beneficial effects: the 2.5D electrophysiological sensor in the embodiment comprises a nanostraw array and a multi-size integrated electrode array, the nanostraw array comprises a substrate and an insulating nanostraw array, the insulating nanostraw array penetrates through the substrate, the insulating nanostraw array comprises insulating nanostraw structures of several diameters and several heights, and the multi-size integrated electrode array comprises electrode layers of several different shapes and several different thicknesses, the electrode layers are cascaded with the several insulating nanostraw structures, the heights and diameters of the insulating nanostraw structures at different positions are different, the nanostraw array for measurement can be flexibly selected according to application requirements, and thus the electrical physiological signals inside the tissue can be measured. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a structural schematic diagram of a 2.5D electrophysiological sensor provided by the embodiment of the present application;

[0027] Figure 2 is a scanning electron microscope image of an electrode array and an enlarged image thereof provided by the embodiment of the present application;

[0028] Figure 3 is a flowchart of a preparation method of a 2.5D electrophysiological sensor provided by the embodiment of the present application;

[0029] Figure 4 is a preparation flowchart of a nanostraw array provided by the embodiment of the present application;

[0030] Figure 5 is a preparation flowchart of a multi-size electrode array provided by the embodiment of the present application;

[0031] Figure 6 is another preparation flowchart of a nanostraw array provided by the embodiment of the present application;

[0032] Figure 7 is another preparation flowchart of a multi-size electrode array provided by the embodiment of the present application;

[0033] Figure 8 is a structural schematic diagram of a microfluidic base provided by the embodiment of the present application;

[0034] Figure 9Fig. 1 is a structural schematic diagram of a 2.5D electrophysiological sensor test system according to an embodiment of the present application. DETAILED DESCRIPTION

[0035] The application will be further described below in conjunction with the drawings and specific embodiments. For the step numbers in the following embodiments, they are only set for the convenience of description, and the order between the steps is not limited. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0036] It should be noted that although the functional modules are divided in the device schematic diagram, and the logical order is shown in the flowchart, in some cases, the steps shown or described can be executed in a different order than the module division in the device or the order in the flowchart. The terms "first", "second", and the like in the specification and claims and the above-described drawings are used to distinguish similar objects, and do not necessarily describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of the application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device that includes a series of steps or units does not necessarily limit to those steps or units clearly listed, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the specification is for the purpose of describing embodiments of the application only and is not intended to be limiting of the application.

[0038] In one aspect, the embodiments of the present application provide a 2.5D electrophysiological sensor, comprising a nanostraw array and a multi-size integrated electrode array, the nanostraw array comprising a substrate and an insulating nanostraw array, the insulating nanostraw array penetrating the substrate, the insulating nanostraw array comprising a plurality of insulating nanostraw structures of a plurality of diameters and a plurality of heights, the multi-size integrated electrode array comprising a plurality of electrode layers of a plurality of shapes and a plurality of thicknesses, the electrode layers cascading a plurality of insulating nanostraw structures.

[0039] It should be noted that the material of the substrate and the insulating nanoscale pipette array is determined according to actual application, and the embodiment is not specifically limited. The number of layers and the material of the electrode layer are determined according to actual application, and the embodiment is not specifically limited. In a specific embodiment, the nanoscale pipette array is an insulating nanoscale pipette array structure of aluminum oxide (Al2O3) material with a PET (polyethylene terephthalate) porous membrane as a substrate, which provides physical support for a 2.5D electrode array. In a specific embodiment, the multi-size integrated electrode array is a multi-size electrode array manufactured by successively depositing a titanium (Ti) adhesion layer and a gold (Au) conductive layer on the nanoscale pipette array framework using a mask and a magnetron sputtering method, and the top layer is an insulating layer made of polydimethylsiloxane (PDMS).

[0040] Referring to Figure 1 , Figure 1 (a) in FIG. indicates a small-size electrode, Figure 1 (c) in FIG. indicates a large-size electrode, Figure 1 (b) in FIG. indicates a 2.5D electrophysiological sensor including a multi-electrode array. Referring to Figure 2 , Figure 2 (a) in FIG. indicates a scanning electron microscope image of a multi-size electrode array, Figure 2 (b) in FIG. indicates a magnified image of the scanning electron microscope image of the multi-size electrode array.

[0041] Implementing the embodiment of the present application includes the following beneficial effects: the 2.5D electrophysiological sensor in the embodiment includes a nanoscale pipette array and a multi-size integrated electrode array, the nanoscale pipette array includes a substrate and an insulating nanoscale pipette array, the insulating nanoscale pipette array penetrates the substrate, the insulating nanoscale pipette array includes a plurality of insulating nanoscale pipette structures with a plurality of diameters and a plurality of heights, the multi-size integrated electrode array includes a plurality of electrode layers with a plurality of different shapes and a plurality of different thicknesses, the electrode layer cascades a plurality of insulating nanoscale pipette structures, the heights of the insulating nanoscale pipette structures at different positions are different, the nanoscale pipette array is determined according to application requirements, and thus the electrical physiological signals inside the tissue are measured.

[0042] Optionally, the insulating nanoscale pipette structure includes a hollow structure, and the sensor further includes a microfluid channel base, the microfluid channel base is bonded to the substrate of the nanoscale pipette array, and the microfluid channel base includes a plurality of microfluid channels.

[0043] The insulating nanoscale pipette structure includes a hollow structure, and is hollow below, and can be combined with a microfluid channel base of a corresponding size as a drug delivery channel. It should be noted that the material of the microfluid channel base is determined according to actual application, and the embodiment is not specifically limited.

[0044] In a specific embodiment, the microfluidic channel base is a microfluidic chip formed by molding PDMS (polydimethylsiloxane) negative mold. Since the conventional microelectrode array lacks the geometric structure to realize the microfluidic channel, it is difficult to realize the efficient delivery of nutrients, oxygen, drugs and other substances, which is not conducive to the long-term culture of cells and the analysis of drug efficacy and other research. Therefore, the microfluidic channel base is selectively added to the 2.5D multi-size integrated nanoscale pipette array sensor according to the needs to meet the different needs of drug, nutrient delivery and the like.

[0045] Optionally, the electrode layer comprises an adhesion layer, a conductive layer and an insulating layer, the adhesion layer is arranged between the conductive layer and the nanoscale pipette array, and the insulating layer covers the non-recording area of the conductive layer.

[0046] The adhesion layer is used to improve the adsorption capacity between the conductive layer and the nanoscale pipette array, and the insulating layer is used to isolate the non-recording area of the conductive layer. The non-recording area refers to an area that does not need to measure the electrical signal.

[0047] Referring to Figure 3 The embodiment of the present application provides a preparation method of a 2.5D electrophysiological sensor, which comprises the following steps:

[0048] S100, providing a substrate of a porous membrane, preparing insulating layers with different thicknesses in the porous membrane of the substrate, etching the insulating layers on the top surface of the porous membrane, and etching the porous membrane with different heights to obtain insulating nanoscale pipette structures with different diameters and different heights;

[0049] S200, preparing electrode layers with different shapes and different thicknesses at the preset positions of the nanoscale pipette array, so that the electrode layers cascade the insulating nanoscale pipette structures, and obtaining a multi-size integrated electrode array.

[0050] It should be noted that the diameter and height of the insulating nanoscale pipette structure are determined according to actual application, and the embodiment does not make specific limitation, and the diameter includes the inner diameter and the outer diameter. The shape and thickness of the electrode layer are determined according to actual application, and the embodiment does not make specific limitation.

[0051] In a specific embodiment, referring to Figure 4, the nanostraw array is an array of hollow nanostraw structures of aluminum oxide (Al2O3), and the preparation process is as follows: on a PET porous membrane substrate, aluminum oxide (Al2O3) is deposited by an atomic layer deposition (ALD) system to form a nanostraw array prototype; and then, the top layer of excess aluminum oxide (Al2O3) and the PET porous membrane are etched away by chlorine gas (Cl2) and oxygen (O2) respectively to form a 2.5D nanostraw array. According to the pore diameter of the selected PET porous membrane and the deposition time of aluminum oxide (Al2O3), the inner and outer diameters of the nanostraw can be flexibly controlled; and by controlling the oxygen (O2) etching time, the height of the nanostraw can be flexibly controlled.

[0052] In a specific embodiment, referring to Figure 5 , the multi-size integrated electrode array is an electrode array composed of titanium (Ti) as an adhesion layer, gold (Au) as a conductive layer, and polydimethylsiloxane (PDMS) as an insulating layer. Each electrode in the array is cascaded with multiple nanostraw structures for subsequent control of the flow rate of sensing points and drug delivery. On the nanostraw array skeleton, titanium (Ti) is sputtered as an adhesion layer, and gold (Au) is sputtered as a conductive layer through a mask and a magnetron sputtering process, and finally polydimethylsiloxane (PDMS) is used as an insulating layer to expose the conductive layer only at the predetermined position at the front end of the electrode. By changing the pattern of the mask, the size of the electrode and the form of the electrode array can be flexibly controlled. In addition, by controlling the magnetron sputtering time, the impedance of the conductive layer (negatively related to the thickness) and the inner diameter of each nanostraw can be flexibly controlled. Finally, the sensing chip is electrically connected to the PCB circuit through conductive silver paste, and the glass ring is fixed by polydimethylsiloxane (PDMS) to form a culture dish structure. In addition, a reference electrode is separately introduced from the top and inserted into the culture solution in the glass ring to provide a reference voltage for recording electrophysiological signals of the sensing chip.

[0053] Optionally, several thicknesses of insulating layers are prepared in the porous membrane of the substrate, the insulating layer on the top surface of the porous membrane is etched, and the porous membrane of different heights is etched to obtain several diameters and several heights of insulating nanostraw structures, including:

[0054] S110, cleaning the porous membrane of the substrate, and preparing several thicknesses of insulating layers in the porous membrane of the substrate by atomic deposition method according to the deposition time;

[0055] S120, etching the insulating layer on the top surface of the porous membrane by inductively coupled plasma reaction, and etching the porous membrane of different heights by plasma, to obtain several diameters and several heights of insulating nanostraw structures.

[0056] In a specific embodiment, referring to Figure 6, first using isopropanol and deionized water to clean the selected aperture PET porous membrane to remove surface contaminants and impurities, and after drying, it is placed in the vacuum reaction chamber of the atomic layer deposition (ALD) system, and a 25 nm thick Al2O3 layer is deposited using trimethylaluminum and water as precursors. Among them, trimethylaluminum and water are used as precursors, and 250 cycles of 0.02 s chemical adsorption reaction time and 40 s purge time are alternately set. After the Al2O3 layer is deposited, the Al2O3 layer on the top surface of the porous PET membrane is selectively etched by an inductively coupled plasma reactive ion etching system with 30 sccm Cl2, 20 sccm SiCl4 and 5 sccm Ar at ICP 300W and RF 60W power. After that, the exposed porous PET membrane is removed by oxygen plasma etching with 40 sccm O2 at RF 100W for 30 minutes to reveal the Al2O3 nanoneedle array.

[0057] Optionally, the electrode layer includes an adhesion layer, a conductive layer and an insulating layer, and the electrode layer with different shapes and different thicknesses is prepared at the preset position of the nanoscale straw array, so that the electrode layer cascades several insulating nanoscale straw structures to obtain a multi-size integrated electrode array, including:

[0058] S210, providing a mask, covering the mask on the surface of the substrate of the nanoscale straw array, and depositing an adhesion layer and a conductive layer;

[0059] S220, removing the mask, preparing an insulating layer in the non-recording area of the conductive layer, so that the electrode layer cascades several insulating nanoscale straw structures to obtain a multi-size integrated electrode array.

[0060] In one specific embodiment, referring to Figure 7 The PET porous membrane is mechanically cut into 15x15mm 2The square of the nanoneedle substrate is used as a nanoneedle substrate. A multi-size nanoneedle MEA mask plate pattern is designed for magnetron sputtering, and the pattern is transferred to a 20 μm thick nickel plate by a laser cutter to make a mask plate. Subsequently, the mask plate is covered on the surface of the multi-size nanoneedle substrate, and a Ti / Au (10 nm / 100 nm) layer is deposited on the substrate by magnetron sputtering. After removing the mask plate, the non-recording area of the microelectrode on the multi-size nanoneedle MEA is coated with a layer of PDMS as an insulating layer, and is heat cured at 80℃ for 2 hours to obtain the final 20-channel multi-size nanoneedle MEA device. The multi-size nanoneedle MEA device is fixed on a customized PCB using PDMS, then the MEA chip pins are electrically connected to the PCB pads using conductive silver paste, and the PDMS and conductive silver paste are cured in an oven at 80℃. Then, a glass ring with a diameter of 10 mm and a height of 15 mm is fixed on the center of the MEA as a cell culture well using PDMS. Then, the pin header is soldered to the PCB using high-temperature solder wire to be electrically connected to the subsequent acquisition circuit. A spiral titanium wire is used as an external reference electrode, which is combined with the centrifuge tube cover.

[0061] Optionally, the preparation method further comprises:

[0062] S310, a substrate material is provided, and a microfluid channel pattern is prepared on the surface of the substrate material;

[0063] S320, a microfluid channel base containing a plurality of microfluid channels is prepared according to the microfluid channel pattern, and the microfluid channel base is bonded to the multi-size integrated electrode array.

[0064] In a specific embodiment, referring to Figure 8 If closed-loop feedback is required for substance delivery, photoresist is coated on a single crystal silicon substrate, a microfluid channel pattern is left through exposure and development, a microfluidic chip is cast by molding, and the surface to be bonded is cleaned for about 10 s at a power of 60 w using plasma, and then the nanosyringe chip above is aligned and pressed, so as to be tightly bonded.

[0065] The embodiment of the present application provides a test system of a 2.5D electrophysiological sensor, which comprises the 2.5D electrophysiological sensor, an amplification and filtering circuit, a signal conversion circuit and a processor.

[0066] The amplification and filtering circuit is used for amplifying and filtering original signals collected by the 2.5D electrophysiological sensor to obtain analog signals.

[0067] The signal conversion circuit is used for converting the analog signals into digital signals.

[0068] The processor is used for calculating measurement information according to the digital signals.

[0069] In one specific embodiment, referring to Figure 9 The 2.5D multi-size integrated nanotube array sensor records the electrophysiological signals of excitable cells or living tissues (extracellular signals collected without electrical pulse perforation, and intracellular signals collected after electrical perforation), and then amplifies and filters the signals by a weak analog signal conditioning circuit with amplification and filtering functions, converts the analog signals into digital signals recognizable by a processor by an analog-to-digital converter (ADC), and transmits the signals into the processor, which automatically extracts electrophysiological signal features, i.e., obtains measurement information.

[0070] Optionally, the processor is further configured to determine feedback information according to the measurement information, the feedback information including electrical stimulation information and substance delivery control information of the microfluidic base.

[0071] The processor automatically determines whether feedback is needed according to the processing result, and the feedback information includes electrical stimulation information and substance delivery control information of the microfluidic base.

[0072] Optionally, the test system further includes a cell culture dish fixed at the center of the 2.5D electrophysiological sensor.

[0073] Referring to Figure 5 , the cell culture dish is fixed at the center of the 2.5D electrophysiological sensor, facilitating cell culture and signal measurement.

[0074] It should be noted that the 2.5D electrophysiological sensor can also perform electrical stimulation, electroporation, drug delivery, and the like, the amplification and filtering circuit includes but is not limited to an analog bandpass filter, a voltage / current gain, a signal conversion circuit includes but is not limited to an analog-to-digital conversion and a digital filter circuit, and the processor includes but is not limited to a computer, a microprocessor, an FPGA, and the like, which are devices with computing functions. The functions of the processor include signal storage, extracellular / intracellular signal prediction, electrophysiological feature parameter extraction, feedback control, and the like.

[0075] The 2.5D multi-size integrated nanotube array sensor chip proposed in this embodiment can flexibly adjust the physical sizes of the hollow nanotube, such as the height and diameter, and can flexibly adjust the number of nanotubes integrated on each electrode (for increasing the point position of the detection signal, and can control the flow of drug delivery), so as to customize the measurement of the electrophysiological information of the living tissues at different depths (z-axis direction) at different positions on the xoy plane. The hollow nanotube structure, if combined with a microfluidic chip as a substrate, can realize the function of drug delivery (drug delivery), thereby realizing a monitoring and feedback closed-loop integrated diagnosis and treatment system.

[0076] It should be understood that, in the present application, "at least one" refers to one or more, and "multiple" refers to two or more. "And / or" is used to describe the association relationship of the associated objects, which means that there can be three relationships, for example, "A and / or B" can represent three cases of only A, only B, and A and B existing at the same time, wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it. "At least one of the following" or similar expressions means any combination of these items, including any combination of single or multiple items. For example, at least one of a, b or c can represent a, b, c, "a and b", "a and c", "b and c", or "a and b and c", wherein a, b, and c can be single or multiple.

[0077] In several embodiments provided in the present application, it should be understood that the disclosed system, device and method can be implemented in other ways. For example, the above-described device embodiments are only schematic, for example, the division of the units is only a logical function division, and actual implementation can have another division manner, for example, a plurality of units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the coupling or direct coupling or communication connection between the units or components shown or discussed can be indirect coupling or communication connection through some interface, device or unit, and can be electrical, mechanical or other forms.

[0078] The units described as separate components can or can not be physically separated, and the components shown as units can or can not be physical units, that is, they can be located in one place, or can be distributed on multiple network units. Part or all of the units can be selected according to actual needs to achieve the purpose of the present embodiment.

[0079] The above is a specific description of the preferred embodiment of the present application, but the present application is not limited to the above-mentioned embodiments. Those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application, and these equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A 2.5D electrophysiological sensor, characterized in that, The 2.5D electrophysiological sensor comprises a nanosipper array and a multi-size integrated electrode array, the nanosipper array comprises a substrate and an insulating nanosipper array penetrating through the substrate, the insulating nanosipper array comprises insulating nanosipper structures of several diameters and several heights, and the multi-size integrated electrode array comprises electrode layers of several different shapes and several different thicknesses, and the electrode layers are cascaded with the insulating nanosipper structures.

2. The sensor of claim 1, wherein, The insulating nanosipper structure comprises a hollow structure, and the sensor further comprises a microfluid channel base bonded with the substrate of the nanosipper array, and the microfluid channel base comprises several microfluid channels.

3. The sensor of claim 1, wherein, The electrode layer comprises an adhesion layer, a conductive layer and an insulating layer, the adhesion layer is arranged between the conductive layer and the nanosipper array, and the insulating layer covers the non-recording area of the conductive layer.

4. A method of making a 2.5D electrophysiological sensor, the method comprising: The method comprises the following steps: A substrate is provided, and insulating layers of several thicknesses are prepared in the porous membrane of the substrate, the insulating layer on the top surface of the porous membrane is etched, and the porous membrane of different heights is etched to obtain insulating nanosipper structures of several diameters and several heights; Electrode layers of several different shapes and several different thicknesses are prepared at the preset positions of the nanosipper array, so that the electrode layers are cascaded with the insulating nanosipper structures to obtain a multi-size integrated electrode array.

5. The preparation method according to claim 4, characterized in that, The step of preparing insulating layers of several thicknesses in the porous membrane of the substrate, etching the insulating layer on the top surface of the porous membrane, and etching the porous membrane of different heights to obtain insulating nanosipper structures of several diameters and several heights comprises the following steps: The porous membrane of the substrate is cleaned, and insulating layers of several thicknesses are prepared in the porous membrane of the substrate by using atomic deposition method according to the deposition time; The insulating layer on the top surface of the porous membrane is etched by inductively coupled plasma reaction, and the porous membrane of different heights is etched by plasma etching to obtain insulating nanosipper structures of several diameters and several heights.

6. The preparation method according to claim 4, characterized in that, The electrode layer comprises an adhesion layer, a conductive layer and an insulating layer, and electrode layers of several different shapes and several different thicknesses are prepared at the preset positions of the nanosipper array, so that the electrode layers are cascaded with the insulating nanosipper structures to obtain a multi-size integrated electrode array, which comprises the following steps: A mask is provided, the mask is covered on the surface of the substrate of the nanosipper array, and an adhesion layer and a conductive layer are deposited; The mask is removed, the non-recording area of the conductive layer is prepared with an insulating layer, so that the electrode layers are cascaded with the insulating nanosipper structures to obtain a multi-size integrated electrode array.

7. The preparation method according to claim 4, characterized in that, The preparation method further comprises the following steps: A substrate material is provided, and a microfluid channel pattern is prepared on the surface of the substrate material; A microfluid channel base containing several microfluid channels is prepared according to the microfluid channel pattern, and the microfluid channel base is bonded with the multi-size integrated electrode array.

8. A test system for a 2.5D electrophysiological sensor, the test system comprising: The 2.5D electrophysiological sensor, the amplification and filtering circuit, the signal conversion circuit and the processor are provided according to any one of claims 1-3, wherein The amplification and filtering circuit is configured to amplify and filter the original signal collected by the 2.5D electrophysiological sensor to obtain an analog signal. The signal conversion circuit is configured to convert the analog signal into a digital signal. The processor is configured to calculate measurement information according to the digital signal.

9. The test system of claim 8, wherein, The processor is further configured to determine feedback information according to the measurement information, wherein the feedback information comprises electrical stimulation information and substance delivery control information of the microfluidic channel base.

10. The test system of claim 8, wherein, The test system further comprises a cell culture dish fixed at a central position of the 2.5D electrophysiological sensor.

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