Multiscale micro / nano electrode array chip for neural signal detection, fabrication method and application

Abstract

This invention provides a multi-scale micro / nano electrode array chip for neural signal detection, its fabrication method, and its applications, belonging to sensor technology. The chip consists of a substrate, a micro / nano electrode array, a counter electrode, a reference electrode, leads, and external contact points. The micro-electrode array features an equally spaced array of nanoscale cylindrical units on its micrometer-sized electrode points. It is fabricated using a combination of traditional microelectromechanical systems (MEMS) processes such as sputtering, photolithography, dry etching, and wet etching, and innovative processes including self-assembled microsphere masks and temperature-controlled phase-change melting biotechnology. The nano-cylinder array units of the micro / nano electrode array promote cell-electrode coupling, reduce leakage current, and increase the surface area of ​​the electrodes without changing their geometric diameter, thus reducing impedance. This chip exhibits high signal amplitude, low noise, and comprehensively improves the signal-to-noise ratio for neural signal detection. The chip fabrication method is economical, rapid, and convenient, making it suitable for research in neuroscience.

Description

Technical Field

[0001] This invention belongs to the field of microfabrication technology of biosensors, and relates to a multi-scale micro / nano electrode array chip for neural signal detection and its fabrication method. The chip fabricated by this method is used for high signal-to-noise ratio detection of neural signals. Background Technology

[0002] The nervous system is one of the most complex and core life systems in the human body. As the most basic structural and functional unit of the nervous system, neurons integrate and transmit various complex signals through neurotransmitter chemical conduction and action potential transmission. Real-time, accurate, comprehensive, and synchronous detection of neuronal signals is an important approach and means to promote the development of neuroscience research, diagnosis of neurological diseases, drug development, and rehabilitation treatment.

[0003] Microelectrode arrays based on microelectromechanical systems technology integrate dozens or even hundreds of neural information detection channels into one unit, which can be used for implantation research in animals or for in vitro cell culture, and are developing towards high integration, high signal-to-noise ratio and good biocompatibility.

[0004] Compared to the complexity and multifactorial nature of implantable neural electrode arrays, in vitro microelectrode array technology, which involves culturing neurons on the electrode surface to receive signals from individual neurons or neural networks, offers advantages such as being non-invasive, multi-site, and easy to operate. Existing in vitro microelectrode arrays are mostly planar, which suffers from poor cell-electrode coupling, small signal detection amplitude, and low signal-to-noise ratio. Fabricating three-dimensional nanopillar arrays on the surface of planar microelectrode arrays can promote cell-electrode adhesion by regulating the expression of cell adhesion proteins. However, existing fabrication methods for three-dimensional micro / nanoelectrode arrays based on electron beam lithography and focused ion beam etching suffer from long production cycles and high costs, and these expensive instruments are generally unavailable in most laboratories. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a low-cost, rapid, and multi-scale micro / nano electrode array chip fabrication method for neural signal detection that does not require large, expensive instruments. This method combines self-assembled microsphere masks, temperature-controlled phase change melting technology, and traditional microelectromechanical systems (MEMS) processes such as sputtering, photolithography, dry etching, and wet etching. The process is simple and convenient. The fabricated multi-scale micro / nano electrode array promotes cell adhesion and coupling, increases the surface area of ​​the microelectrode array without changing the electrode's geometric diameter, thus significantly reducing electrode impedance, increasing the detection signal amplitude, and improving the signal-to-noise ratio. This allows for the effective detection of weak signals from neuronal cells.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A neural signal detection multiscale micro / nano electrode array chip, the chip comprising a support substrate 1 for supporting the electrode array, a micro / nano electrode array 2, micron electrode points 3, a nanoscale cylindrical array 4, an electrochemical reference electrode 5, an electrochemical counter electrode 6, an electrophysiological counter electrode 7, a spare reference electrode 8, leads 9, external pins 10, and an insulating layer 11 for insulating the leads from tissue fluid.

[0008] The supporting substrate 1 has a cross-scale micro-nano electrode array 2 at its center. The cross-scale micro-nano electrode array 2 is composed of multiple micro-nano electrodes with the same structure. Each micro-nano electrode includes a circular micron electrode point 3 and a nanoscale cylindrical array 4 on the upper surface of the micron electrode point 3 to promote cell-electrode coupling. Specifically, the multiple circular micron electrode points 3 are arranged in a matrix at the center of the upper surface of the supporting substrate 1, and the upper surface of each micron electrode point 3 is distributed with equally spaced nanoscale cylindrical arrays 4.

[0009] The multi-scale micro / nano electrode array 2 is surrounded by an electrochemical reference electrode 5, an electrochemical counter electrode 6, an electrophysiological counter electrode 7, and a spare reference electrode 8. Each of these micro / nano electrodes, electrochemical reference electrodes 5, electrochemical counter electrodes 6, electrophysiological counter electrodes 7, and spare reference electrodes 8 is connected to an external pin contact 10 via a lead 9 for connection to external devices. The multi-scale micro / nano electrode array 2, electrochemical reference electrodes 5, electrochemical counter electrodes 6, lead 9, and external pin contact 10 are all made of conductive materials. The conductive layer of the electrochemical reference electrode 5 has an Ag / AgCl composite film, and the surface of all lead 9 is covered with an insulating layer 11.

[0010] Furthermore, the material of the supporting substrate 1 is silicon, which can be processed using reactive ion etching. The substrate has a side length of 40mm to 70mm and a thickness of 0.5mm to 2mm.

[0011] Furthermore, the shape of the micron electrode point 3 is circular, with a diameter of 10μm to 30μm and a spacing of 210μm to 300μm;

[0012] Furthermore, the surface area of ​​the electrochemical reference electrode 5 and the electrochemical counter electrode 6 is 3000 μm. 2 ~5000μm 2 ;

[0013] Furthermore, the nanopillars in the nanopillar array 4 have a diameter of 100nm to 200nm, a center-to-center spacing of 1000nm to 1500nm, and a height of 500nm to 1000nm.

[0014] A method for fabricating a multi-scale micro / nano electrode array chip for neural signal detection is disclosed. The chip is fabricated using processes including self-assembled microsphere masking, temperature-controlled phase change melting, sputtering, photolithography, dry etching, and wet etching. First, a metal film is sputtered onto a silicon support substrate 1. Then, a layer of positive photoresist is spin-coated onto the metal film, followed by drying, exposure, and development to obtain a micron-sized electrode point array pattern 3. A microsphere array is then self-assembled on the patterned substrate using a gas-liquid interface method, with the ordered and tightly packed microspheres covering the entire patterned substrate. Finally, the microspheres are peeled off using a lift-off process, leaving only the microsphere array at the microelectrode points. Oxygen plasma etching reduces the diameter of the microspheres; heating melts the microspheres, changing their contact with the substrate from point contact to surface contact; a metal etching solution is used to etch the substrate, removing the metal film in areas not masked by the microspheres, leaving metal nanodots in the masked areas; after removing the microsphere array with an organic solvent, dry etching is performed on the silicon substrate with the metal nanodots as a masking layer to obtain electrode sites with an array of nanopillars; then, using gold as the conductive layer material and photosensitive polyimide as the insulating layer material, photolithography, wet etching, and other processes are used to complete the fabrication of the entire micro / nano electrode array for neural signals across scales. Specifically, the following steps are included:

[0015] (1) Sputter a metal film with a thickness of 50 nm to 80 nm onto a silicon substrate;

[0016] (2) A layer of positive photoresist with a thickness of 1μm to 1.5μm is spin-coated on the metal film. The area without photoresist after photolithography and development is a micron electrode array pattern, which is used to obtain micron electrode 3 later.

[0017] (3) A microsphere array is self-assembled on the surface of the photoresist pattern using the gas-liquid interface method, and the orderly and closely arranged microspheres cover the entire support substrate 1.

[0018] (4) Use a peeling process to remove excess microspheres, leaving only a microsphere mask on the micron electrode array;

[0019] (5) The diameter of the microspheres is reduced by oxygen plasma etching, and then the microspheres are heated and melted, so that the microspheres and the metal substrate change from point contact to surface contact. The metal film in the area without microspheres is removed by immersion in metal etchant, leaving the metal film in the area with microspheres. The microsphere array is removed by immersion in organic solvent.

[0020] (6) The silicon substrate with nano-dot metal film is etched by dry etching to obtain a micro electrode dot array with nano-pillar array 4, and the substrate is immersed in metal etching solution to etch away the metal film used as a masking layer for dry etching.

[0021] (7) Sputter an insulating layer with a thickness of 10 nm to 50 nm and a microelectrode conductive film layer with a thickness of 100 nm to 250 nm onto the silicon substrate after dry etching.

[0022] (8) Conductive micro-nano electrode array 2, electrochemical reference electrode 5, electrochemical counter electrode 6, electrophysiological counter electrode 7, spare reference electrode 8, lead wire 9 and external pin contact 10 are formed by spin coating, photolithography, development, wet etching of metal, and resist removal.

[0023] (9) Spin-coating polyimide, covering the substrate surface with an insulating layer 11 after the conductive thin film layer is prepared, and exposing the micro-nano electrode array 2, electrochemical counter electrode 6, electrochemical reference electrode 5 and external pin contact 10 by photolithography, while retaining the insulating layer 11 covering the surface of all leads 9.

[0024] (10) Coat the surface of the electrochemical reference electrode 5 with Ag / AgCl slurry and dry it to form an Ag / AgCl composite thin film reference electrode.

[0025] Furthermore, the metal thin film in step (1) is prepared using magnetron sputtering (PVD) technology, and the material is one of chromium, gold, or titanium, with a thickness of 50 nm to 100 nm.

[0026] Furthermore, in step (3), the material for self-assembling microspheres on the substrate using the gas-liquid interface method is one of polystyrene, epoxy methacrylate, or polymethyl methacrylate, with a diameter of 1000 nm to 1500 nm. It is prepared as a monodisperse aqueous solution with a mass concentration of 2% w / v to 2.5% w / v. This solution is mixed with an ethanol solution in a volume ratio of 3:2, 1:1, or 2:3. The volume ratio is selected based on the solubility of the microsphere solution and the diameter of the microspheres. The purpose of mixing with the ethanol solution is to reduce the surface tension of the solution so that the Marangoni effect formed when the microspheres come into contact with water at the gas-liquid interface can cause the microspheres to self-assemble at the gas-liquid interface.

[0027] Furthermore, in step (5), the temperature at which the microspheres are heated to melt is above the glass transition temperature of the plastic, which is 90℃~110℃. The heat from the hot plate is transferred to the bottom of the microspheres through the silicon substrate and the metal film. The initial contact between the microspheres and the substrate is a point contact. After heating, the microspheres undergo a phase change and melt, and the point contact becomes a surface contact. The shape of the surface contact is circular. The heating time is 5s~20s. This time must ensure that the bottom point contact of the lower half circle becomes a surface contact, and the upper half circle has not yet undergone a phase change. After heating, the diameter of the circular surface contact is 100nm~200nm, which is the diameter of the pillar in the nanopillar array.

[0028] Furthermore, in step (6), the etching of the silicon substrate is carried out by reactive ion etching, the etching gas is sulfur hexafluoride (SF6), the radio frequency power is 90W to 110W, the working pressure is 2Pa to 4Pa, the gas flow rate is 15sccm to 25sccm, and the etching depth is 500nm to 1000nm. This depth value is the height of the pillars in the nanopillar array.

[0029] A multi-scale micro / nano electrode array chip for neural signal detection, prepared using the above method, is applied to the detection of neural signals with a high signal-to-noise ratio. Specifically, the prepared electrode array chip is sterilized by immersion in alcohol, irradiation with ultraviolet light, and rinsing with deionized water, and then treated with oxygen plasma to make its surface hydrophilic, thereby promoting the adhesion of neuronal cells. Neuronal cells are cultured on the prepared electrode array chip, and after the neuronal cells adhere and grow, the chip is connected to external electrophysiological and electrochemical detection instruments to collect and analyze the neuronal signals.

[0030] The beneficial effects of this invention are as follows:

[0031] The method for fabricating a multi-scale micro / nano electrode array chip for neural signal detection provided by this invention can rapidly and extensively fabricate nanopillar arrays on microelectrode sites, promoting cell-electrode coupling, reducing electrode impedance, increasing detection amplitude, reducing noise, and increasing biocompatibility. It overcomes the limitations of high cost, small area, and long cycle of electron beam lithography and focused ion beam etching, and provides a new approach for manufacturing multi-scale micro / nano neural electrode arrays for studying neural information transmission, pathogenesis, and drug development. Attached Figure Description

[0032] Figure 1 This is a structural diagram of the multi-scale micro / nano electrode array chip for neural signal detection in this invention.

[0033] Figure 2 This is a partially enlarged schematic diagram of the microelectrode array in this invention.

[0034] Figure 3 This is a partially enlarged schematic diagram of the nanopillar array on the microelectrode points in this invention.

[0035] Figure 4a This is a schematic diagram of the apparatus for fabricating microsphere masks using the gas-liquid interface method in this invention.

[0036] Figure 4b This is a schematic diagram of the process of transferring the microsphere mask onto the substrate in this invention.

[0037] Figure 5 is a process flow diagram of the method for fabricating a multi-scale micro / nano electrode array chip for neural signal detection in this invention;

[0038] Figure 5a An array of microelectrode points after sputtering and patterning a thin metal film;

[0039] Figure 5b A microsphere mask for self-assembly on the entire substrate using the gas-liquid interface method;

[0040] Figure 5cTo remove excess microspheres, only a microsphere mask is left on the microelectrode point array;

[0041] Figure 5d To reduce the diameter of microspheres through oxygen plasma etching;

[0042] Figure 5e To heat the microspheres to slightly melt them, the contact between the microspheres and the metal substrate changes from point contact to surface contact;

[0043] Figure 5f To etch away the metal that was not masked by the microspheres, an organic solvent was then used to dissolve and remove the microsphere mask.

[0044] Figure 5g To utilize dry etching of silicon substrates with nano-dot metal masking layers;

[0045] Figure 5h To sputter an insulating layer and a conductive metal layer on a substrate with a nanopillar array;

[0046] Figure 5i To form patterns of microelectrode arrays, reference electrodes, counter electrodes, leads, and pin contacts on a substrate using processes such as spin coating, photolithography, development, wet etching of metal, and resist removal;

[0047] Figure 5j To prepare a photosensitive polyimide insulating layer by photolithography, the insulating layer covers all lead surfaces, exposing the microelectrode, reference electrode, counter electrode and pin contacts;

[0048] Figure 5k To coat the reference electrode with Ag / AgCl slurry to form an Ag / AgCl composite thin film reference electrode.

[0049] Figure 5l for Figures 5a-5k A schematic diagram of each part.

[0050] In the figure: 1 Supporting substrate; 2 Micro / nano electrode array; 3 Micrometer electrode points; 4 Nanoscale cylindrical array; 5 Electrochemical reference electrode; 6 Electrochemical counter electrode; 7 Electrophysiological counter electrode; 8 Spare reference electrode; 9 Lead wire; 10 External pin contact; 11 Insulating layer that insulates the lead wire from the tissue fluid. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0052] The following detailed description of a specific embodiment of the present invention is provided in conjunction with the accompanying drawings.

[0053] A neural signal detection multi-scale micro / nano electrode array chip includes a support substrate 1 for supporting the electrode array, a micro / nano electrode array 2, micron electrode points 3, a nanoscale cylindrical array 4, an electrochemical reference electrode 5, an electrochemical counter electrode 6, an electrophysiological counter electrode 7, a spare reference electrode 8, leads 9, external pins 10, and an insulating layer 11 to insulate the leads from tissue fluid. The multi-scale micro / nano electrode array 2 is located at the center of the support substrate 1. The multi-scale micro / nano electrode array 2 consists of multiple micro / nano electrodes with identical structures. Each micro / nano electrode includes a circular micron electrode point 3 and a nanoscale cylindrical array 4 disposed on the upper surface of the micron electrode point 3. Specifically, the multiple circular micron electrode points 3 are arranged in a matrix at the center of the upper surface of the support substrate 1, and the upper surface of each micron electrode point 3 is distributed with equally spaced nanoscale cylindrical arrays 4. The multi-scale micro / nano electrode array 2 is surrounded by an electrochemical reference electrode 5, an electrochemical counter electrode 6, an electrophysiological counter electrode 7, and a spare reference electrode 8. Each micro / nano electrode, electrochemical reference electrode 5, electrochemical counter electrode 6, electrophysiological counter electrode 7, and spare reference electrode 8 is connected to an external pin contact 10 via a lead 9 for connection to external devices. The multi-scale micro / nano electrode array 2, electrochemical reference electrode 5, electrochemical counter electrode 6, lead 9, and external pin contact 10 are all made of conductive materials. The conductive layer of the electrochemical reference electrode 5 has an Ag / AgCl composite film, and the surface of all lead 9 is covered with an insulating layer 11. Specifically, the structure of the neural signal detection multi-scale micro / nano electrode array chip provided by this invention is as follows: Figure 1 As shown. The entire chip consists of an electrode array supporting substrate 1, a micro / nano electrode array 2, micron electrode points 3, a nanopillar array 4, an electrochemical reference electrode 5, an electrochemical counter electrode 6, an electrophysiological counter electrode 7, a spare reference electrode 8, leads 9, external pins 10, and an insulating layer 11 that insulates the leads from the tissue fluid.

[0054] The substrate is p-type. <100> A silicon wafer with a thickness of 525 μm serves as the substrate supporting the electrode array. The silicon wafer is cut into rectangles 60 mm long and 50 mm wide. At the center of the substrate surface, 36 circular, multi-scale micro / nano electrode points are distributed, forming the micro / nano electrode array 2. Figure 2 As shown, the microelectrode points are arranged in a 6×6 square array with a diameter of 30 μm and a spacing of 210 μm.

[0055] The microspheres were made of polystyrene microspheres with a diameter of 1000 nm, and were prepared into a monodisperse solution with a mass concentration of 2.5% w / v by adding water. The diameter of the microspheres was the spacing between the nanopillars in the nanopillar array. Before assembling the nanosphere mask using the gas-liquid interface method, the polystyrene microsphere solution and the ethanol solution were mixed at a volume ratio of 2:3 to reduce the surface tension of the mixture. Figure 4aTo allow the microsphere mixture solution to be dropped onto the water surface along an inclined glass slide, the Marangoni effect formed when it comes into contact with water causes the microspheres to self-assemble at the gas-liquid interface; Figure 4b To lower the water level after self-assembly, an ordered and tightly packed array of microspheres was transferred to a substrate patterned with electrode sites and dried at room temperature for 24 hours to increase the adhesion between the microspheres and the substrate.

[0056] The silicon substrate is etched using reactive ion etching, with sulfur hexafluoride (SF6) as the etching gas, 100W of radio frequency power, 2.5Pa of working pressure, 20sccm of gas flow rate, and an etching depth of 1000nm. This depth value is the height of the pillars in the nanopillar array.

[0057] The microelectrode points have an array of nanopillars. Figure 3 This is a magnified schematic diagram of a single multi-scale micro / nano electrode point. The nanopillars have a diameter of 100 nm, a spacing of 1000 nm, and a height of 1000 nm. This size allows nerve cells to be completely wrapped around the pillars, increasing coupling with the electrodes. When the electrodes detect neuronal cell signals, there is a gap at the contact point between the electrode and the neuronal cell. The larger the gap, the lower the amplitude of the detected signal; the smaller the gap, the higher the amplitude of the detected signal. The nanopillar array can promote cell adhesion on the electrodes, reduce the aforementioned gap, increase the signal output amplitude, and increase the surface area of ​​the microelectrode point, reducing impedance, reducing noise, and improving the signal-to-noise ratio of the detection.

[0058] The specific preparation process of this invention is detailed below with reference to Figure 5:

[0059] 1. Sputter a 50nm thick titanium metal thin film onto a silicon wafer that has undergone surface cleaning;

[0060] 2. A layer of positive photoresist AZ703, 1.5 μm thick, is spin-coated onto the above surface. After drying at 85°C, photolithography, and development, the area left without photoresist coverage is the microelectrode array. Figure 5a );

[0061] 3. The microsphere monodisperse solution was ultrasonically mixed with ethanol, and a microsphere array was self-assembled on the photoresist pattern surface using a gas-liquid interface method. Figure 5b );

[0062] 4. Immerse in acetone solution and use a peeling process to remove excess microspheres, leaving a microelectrode array masked by microspheres. Figure 5c );

[0063] 5. The diameter of the microspheres was reduced using oxygen plasma etching. The etching time was 45 min, the power was 100 W, and the oxygen flow rate was 100 sccm. After etching, the diameter of the microspheres was 600 nm. Figure 5d );

[0064] 6. Heat the molten microspheres to 110℃ for 10 seconds, changing the point contact between the microspheres and the metal substrate to a circular surface contact. The diameter of the microspheres is 100 nm. Figure 5e );

[0065] The titanium film in areas not masked by microspheres was removed by immersion in a 7.1:10 (H2O:HF) hydrofluoric acid etching solution for 10 seconds. Then, the microsphere mask was removed by immersion in a xylene solution in an organic solvent for 30 minutes. Figure 5f );

[0066] 8. A silicon substrate with a nanodot metal mask was etched using reactive ion dry etching to obtain a microelectrode array with a nanopillar array. The nanopillars had a diameter of 100 nm, a spacing of 1000 nm, and a height of 1000 nm. Figure 5g );

[0067] After immersing in a 9.1:10 (H2O:HF) hydrofluoric acid etching solution for 10 seconds, the titanium metal mask is removed. Then, a 30 nm thick silicon dioxide insulating layer is first magnetron sputtered onto a silicon substrate with a nanopillar array, followed by a 30 nm thick chromium layer to increase the adhesion between the conductive layer and the silicon substrate. Finally, a 200 nm thick gold conductive layer is sputtered. Figure 5h );

[0068] 10. Conductive electrode points, reference electrodes, counter electrodes, leads, and external pin contacts are formed through processes including spin coating, photolithography, development, wet etching of the conductive layer metal, and resist removal. The lead width is 10 μm, and the reference and counter electrodes have an area of ​​3000 μm. 2 rectangle ( Figure 5i );

[0069] 11. Spin-coating polyimide onto the substrate surface where the conductive thin film layer has been prepared, covering it with an insulating layer. The microelectrode array, counter electrode, reference electrode, and lead contacts are then exposed via photolithography. The insulating layer covering all lead surfaces is retained. The thickness of the polyimide insulating layer is 200 nm. Figure 5j );

[0070] 12. Coat the reference electrode surface with Ag / AgCl slurry and dry it to form an Ag / AgCl composite thin film reference electrode with a thickness of 200 nm. Figure 5k ).

[0071] The above embodiments are for illustrative purposes only and are not intended to limit the present invention. Based on the above description, many improvements and changes can be made to the present invention, and all such improvements and changes, as well as the selection of other functional materials, should be within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a neural signal detection cross-scale micro-nano electrode array chip, characterized in that, The preparation method comprises the following steps: 1) sputtering a metal layer with a thickness of 50 nm-80 nm on a silicon substrate (1) supporting an electrode array; 2) spin coating photoresist with a thickness of 1-1.5 microns, and the photoresist-free area after photoetching is a micrometer electrode point (3) array; 3) using a gas-liquid interface method to self-assemble a microsphere array on the surface of the photoresist pattern; 4) using a stripping process to remove the excess microspheres, leaving only the microsphere mask on the micrometer electrode point array; 5) reducing the diameter of the microspheres by oxygen plasma etching, heating and melting the microspheres, changing the contact between the microspheres and the metal substrate from point contact to surface contact, immersing in a metal etching solution to remove the metal in the area not covered by the microsphere mask, and immersing in an organic solvent to remove the microsphere mask; 6) etching the silicon substrate with the nanometer point metal mask using dry etching to obtain a micrometer electrode point array with a nanometer column array (4), and immersing the substrate in a metal etching solution to etch off the metal film; 7) sputtering an insulating layer with a thickness of 10 nm-50 nm and a microelectrode conductive thin film layer with a thickness of 100 nm-250 nm on the silicon substrate after dry etching; 8) forming a conductive micro-nano electrode point array (2), an electrochemical reference electrode (5), an electrochemical counter electrode (6), an electrophysiological counter electrode (7), a standby reference electrode (8), a lead (9), and an external pin contact (10) through the processes of spin coating, photoetching, developing, wet etching of metal, and removing photoresist; 9) spin coating polyimide to cover the insulating layer (11) on the surface of the substrate with the prepared conductive thin film layer, exposing the microelectrode array, the counter electrode, the reference electrode, and the pin contact through photoetching, and retaining the insulating layer covering the surface of all the leads; 10) coating Ag / AgCl slurry on the surface of the reference electrode and drying to form an Ag / AgCl composite thin film reference electrode.

2. The preparation method of the neural signal detection cross-scale micro-nano electrode array chip according to claim 1, characterized in that: the material of the metal layer sputtered in step 1) is one of chromium, titanium, or gold, and the thickness is 50 nm-100 nm.

3. The method of claim 1, wherein the method further comprises: the material of the microspheres self-assembled on the substrate in step 3) is one of polystyrene, glycidyl methacrylate, or polymethyl methacrylate, the diameter is 1000 nm-1500 nm, a single dispersion solution with a mass concentration of 2% w / v-2.5% w / v is prepared by adding water, and the solution is mixed with an ethanol solution in a volume ratio of 3:2, 1:1, or 2:

3.

4. The method of claim 1, wherein the method further comprises: the temperature for heating and melting the microspheres in step 5) is 90-110°C, which is the glass transition temperature of the polymer material, so that the contact between the microspheres and the substrate changes from point contact to a circular surface contact, the heating time is controlled to make the lower half of the microspheres melt, while the upper half of the microspheres does not melt, and the diameter of the circular surface contact after heating is 100-200 nm, which is the diameter of the column in the nanometer column array.

5. The method of claim 1, wherein the method further comprises: an insulating layer is pre-sputtered before forming the electrode conductive layer in step 7), and the material is one of silicon dioxide, silicon nitride, or silicon oxynitride.

6. A neural signal detection cross-scale micro-nano electrode array chip, characterized in that: The nerve signal detection cross-scale micro-nano electrode array chip is prepared by the preparation method in any one of claims 1-5, and comprises an electrode array support substrate (1), a micro-nano electrode array (2), a micro electrode point (3), a nano-scale cylinder array (4), an electrochemical reference electrode (5), an electrochemical counter electrode (6), an electrophysiological counter electrode (7), a standby reference electrode (8), a lead wire (9), an external lead (10), and an insulation layer (11) for insulating the lead wire from tissue fluid. The cross-scale micro-nano electrode array (2) is arranged at the center of the support substrate, and is composed of a plurality of micro-nano electrodes with the same structure, each of which comprises a circular micro electrode point (3) and a nano-scale cylinder array (4) arranged on the surface of the micro electrode point (3). The micro-nano electrode array (2) is surrounded by the electrochemical reference electrode (5), the electrochemical counter electrode (6), the electrophysiological counter electrode (7), and the standby reference electrode (8). The micro-nano electrode, the reference electrode, and the counter electrode are connected to the external lead pin (10) through the lead wire (9) for connection with external equipment. The electrode array, the reference electrode, the counter electrode, the lead wire, and the pin are all made of conductive materials, and the conductive layer of the reference electrode has an Ag / AgCl composite film. The surface of all the lead wires (9) is covered with an insulation layer (11).

7. The nerve signal detection cross-scale micro-nano electrode array chip according to claim 6, wherein the plurality of circular micro electrode points (3) are arranged in a matrix form on the upper surface of the support substrate (1) at the center, and each micro electrode point (3) has an equal-interval nano-scale cylinder array (4) arranged on the upper surface.

8. The nerve signal detection cross-scale micro-nano electrode array chip according to claim 6, wherein the support substrate (1) is made of silicon, and is processed by reactive ion etching. The substrate has a side length of 40 mm-70 mm and a thickness of 0.5 mm-2 mm.

9. The nerve signal detection cross-scale micro-nano electrode array chip according to claim 6, wherein the micro electrode point (3) is circular in shape, has a diameter of 10 μm-30 μm, and a spacing of 210 μm-300 μm; and the nano column in the nano column array (4) has a diameter of 100 nm-200 nm, a center spacing between columns of 1000 nm-1500 nm, and a column height of 500 nm-1000 nm.

10. The use of a neural signal detection cross-scale micro-nano electrode array chip according to any one of claims 6-9, characterized in that, The cross-scale micro-nano electrode array chip is used for high signal-to-noise ratio detection of nerve signals.

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