Multi-channel flexible nanoneedle electrode array and preparation method thereof

Abstract

The invention discloses a multi-channel flexible nanoneedle electrode array and a preparation method thereof, the multi-channel flexible nanoneedle electrode array comprises a flexible substrate, nanoneedle electrodes, a flexible conductive layer, an insulating layer and a flexible external lead, the flexible substrate comprises a plurality of nanopores, the nanoneedle electrodes penetrate through nanoholes of the flexible substrate to form nanoneedle electrode parts to be exposed, the nanoneedle electrodes share one electrode disc, the surface of the electrode disc is covered with the flexible conductive layer, the electrode disc and the flexible conductive layer are wrapped by the insulating layer, and the insulating layer is arranged on the flexible substrate. The electrode disc is connected with the flexible external lead, and the exposed nanoneedle electrode is modified with a positive ionic polymer. The embodiment of the invention can accurately monitor electric signals in cells for a long time, and can be widely applied to the technical field of sensors.

Description

Technical Field

[0001] The present invention relates to the technical field of sensors, and particularly to a multi-channel flexible nano-needle electrode array and a preparation method thereof. Background Art

[0002] The advantages of micro-nano sensors lie in the high-sensitivity electrical properties endowed by micro-nano precision materials and the unique potential of inserting into cells to record electrical signals. Currently, the micro-nano sensors applied to record intracellular electrical signals mainly include field-effect transistor sensors and conductive nano-electrode array sensors. The methods based on field-effect transistor sensors and conductive nano-electrode array sensors require cells to be cultured above the nanowires in an in vitro form. Since cells are cultured on micro-nano structured materials for a long time, there is a possibility that the behavior of cells is interfered. And by using perforation techniques such as electroporation and optoporation, the cell membrane usually heals within a few minutes after rupture. Therefore, intracellular recording can only be carried out within a very short time (shorter than half an hour), and it is difficult to record for a long time. Moreover, the electrical stimulation or photothermal effect of electroporation and optoporation has a greater impact on cells, easily changing the discharge behavior of neuron cells, not suitable for multiple uses, and there is a risk of changing the functional behavior of cells.

[0003] Currently, most of the flexible electrode technologies for recording brain physiological electrical signals are based on planar electrode structures, which can only record electrical signals from the outside of cells and do not have the function of penetrating the living cell membrane, so the intracellular electrical signals cannot be recorded. In addition, when the electrode is attached to the surface of the soft brain tissue, large deformations will occur, so the conductive path is easily damaged. Summary of the Invention

[0004] In view of this, to solve one of the above problems, an object of an embodiment of the present invention is to provide a multi-channel flexible nano-needle electrode array and a preparation method thereof, which can accurately monitor intracellular electrical signals for a long time.

[0005] On the one hand, an embodiment of the present invention provides a multi-channel flexible nano-needle electrode array, including a flexible substrate, nano-needle electrodes, a flexible conductive layer, an insulating layer, and a flexible external lead. The flexible substrate includes a plurality of nano-holes. The nano-needle electrodes pass through the nano-holes of the flexible substrate to form partial exposure of the nano-needle electrodes. A plurality of the nano-needle electrodes share an electrode disk. The surface of the electrode disk is covered with the flexible conductive layer. The electrode disk and the flexible conductive layer are wrapped by the insulating layer. The electrode disk is connected to the flexible external lead. The exposed nano-needle electrodes are modified with a cationic polymer.

[0006] Optionally, the diameter range of the nano-holes of the flexible substrate is 90 - 110 nm, and the density range of the nano-holes of the flexible substrate is 0.05 - 0.15 holes / μm 2 .

[0007] Optionally, the material of the nano-needle electrode includes any one of metal, graphene or carbon nanotubes.

[0008] Optionally, the flexible conductive layer includes a liquid metal layer doped with carbon nanomaterials.

[0009] On the other hand, an embodiment of the present invention provides a method for preparing a multi-channel flexible nano-needle electrode array, which is applied to the above multi-channel flexible nano-needle electrode array, and includes:

[0010] Construct an electrode pad pattern on one side surface of the flexible substrate and prepare the electrode pad;

[0011] Prepare a flexible conductive layer on the surface of the electrode pad and prepare the insulating layer on one side surface of the flexible substrate;

[0012] Prepare nano-needle electrodes in the holes on the other side surface of the flexible substrate, etch part of the flexible substrate to expose part of the nano-needle electrodes, and modify the surface of the exposed nano-needle electrodes with a cationic polymer.

[0013] Optionally, the constructing an electrode pad pattern on one side surface of the flexible substrate and preparing the electrode pad includes:

[0014] Prepare a mask for the electrode pad pattern on one side surface of the flexible substrate;

[0015] Prepare an electrode layer with a preset thickness by magnetron sputtering and remove the mask to obtain the electrode pad.

[0016] Optionally, the constructing an electrode pad pattern on one side surface of the flexible substrate and preparing the electrode pad includes:

[0017] Prepare the electrode pad pattern on one side surface of the flexible substrate by photolithography;

[0018] Prepare an electrode layer with a preset thickness by magnetron sputtering and remove the photoresist to obtain the electrode pad.

[0019] Optionally, the preparing a flexible conductive layer on the surface of the electrode pad includes:

[0020] Add carbon nanomaterials to liquid metal to obtain liquid metal doped with carbon nanomaterials;

[0021] Roll the liquid metal doped with carbon nanomaterials on the flexible substrate immersed in a dilute hydrochloric acid bath, so as to selectively coat a flexible conductive layer on the electrode pad of the flexible substrate.

[0022] Optionally, the preparing nano-needle electrodes in the holes on the other side surface of the flexible substrate includes:

[0023] Prepare an electroplating solution and perform electrodeposition in the electroplating solution to form solid nanoneedle electrodes in the holes on the other surface of the flexible substrate.

[0024] Optionally, modifying the surface of the exposed nanoneedle electrodes with a cationic polymer includes:

[0025] Clean the nanoneedle electrodes once to remove surface contaminants;

[0026] Clean the nanoneedle electrodes twice to activate the electrode surface to form a hydroxyl or oxide layer;

[0027] Immerse the nanoneedle electrodes in a preset solution for reaction to form a stable amino group layer;

[0028] Immerse the nanoneedle electrodes in a cationic polymer solution so that the cationic polymer is adsorbed electrostatically or bonded covalently to the electrode surface.

[0029] Implementing the embodiments of the present invention includes the following beneficial effects: The multi-channel flexible nanoneedle electrode array in this embodiment includes a flexible substrate, nanoneedle electrodes, a flexible conductive layer, an insulating layer, and a flexible external lead. The flexible substrate includes a number of nanopores. The nanoneedle electrodes pass through the nanopores of the flexible substrate to form a part of the nanoneedle electrodes exposed. A number of nanoneedle electrodes share one electrode disk. The surface of the electrode disk is covered with a flexible conductive layer. The flexible conductive layer has the properties of flexibility, bendability, and stretchability, reducing the damage to the conductivity of the circuit during the bending process. The electrode disk and the flexible conductive layer are wrapped by the insulating layer. The electrode disk is connected to the flexible external lead. The exposed nanoneedle electrodes are modified with a cationic polymer, carrying a large number of positive charges, which can interact with the negatively charged cell membrane phospholipid molecules, increasing the affinity between the nanoneedles and the cell membrane. Moreover, the cationic polymer can provide a certain degree of flexibility, reducing the mechanical damage of the nanoneedle surface to the cell membrane, optimizing the conditions for the long-term penetration of the nanoneedle electrodes into the cell membrane, and thus enabling the long-term and accurate monitoring of the electrical signals inside the cells. Description of the Drawings

[0030] Figure 1 is a cross-sectional view of a multi-channel flexible nanoneedle electrode array provided by an embodiment of the present invention;

[0031] Figure 2 is a top view of a multi-channel flexible nanoneedle electrode array provided by an embodiment of the present invention;

[0032] Figure 3 is a schematic flow chart of the steps of a preparation method of a multi-channel flexible nanoneedle electrode array provided by an embodiment of the present invention;

[0033] Figure 4It is a schematic diagram of the step flow of another preparation method of the multi-channel flexible nanoneedle electrode array provided by the embodiments of the present invention;

[0034] Figure 5 It is a schematic diagram of the step flow of modifying a multi-channel flexible nanoneedle electrode array with a positive ion polymer provided by the embodiments of the present invention;

[0035] Figure 6 It is a test diagram of intracellular electrical signals provided by the embodiments of the present invention. Detailed implementation manners

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. For the step numbers in the following embodiments, they are only set for the convenience of explanation and illustration, and no limitation is imposed on the order between steps. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0037] As Figure 1 shown, the embodiments of the present invention provide a multi-channel flexible nanoneedle electrode array, including a flexible substrate P1, nanoneedle electrodes P5, a flexible conductive layer P2, an insulating layer P4, and flexible external leads. The flexible substrate P1 includes a plurality of nanopores. The nanoneedle electrodes P5 pass through the nanopores of the flexible substrate P1 to form a part of the nanoneedle electrodes P5 exposed. A plurality of nanoneedle electrodes P5 share one electrode disk. The surface of the electrode disk is covered with a flexible conductive layer P2. The electrode disk and the flexible conductive layer P2 are wrapped by the insulating layer P4. The electrode disk is connected to the flexible external lead. The exposed nanoneedle electrodes are modified with a positive ion polymer P6.

[0038] It should be noted that conductive substances P3 can be doped in the flexible conductive layer P2 to improve the conductivity of the flexible conductive layer P2, such as doping carbon nanomaterials. The multi-channel flexible nanoneedle electrode array is used for testing intracellular electrical signals of neuron cells P7.

[0039] It should be noted that the material of the flexible substrate is determined according to actual applications. For example, using biodegradable materials such as natural materials (such as silk fibroin or gelatin) can reduce the long-term impact of the implant on the organism; or using polycarbonate nanoporous membranes, etc. The size of each electrode disk and the number of nanoneedle electrodes connected are determined according to actual applications.

[0040] Optionally, the diameter range of the nanopores of the flexible substrate is 90 - 110 nm, and the density range of the nanopores of the flexible substrate is 0.05 - 0.15 pores / μm 2 .

[0041] Refer to Figure 2 Refer to Figure 2Shows the top view and partial enlarged view of a multi-channel flexible nano-needle electrode array. The diameter and density of the nanopores on the flexible substrate are determined according to actual applications and are not specifically limited in this embodiment. For example, the flexible substrate is a polycarbonate nanoporous membrane with nanopores having a pore diameter of 100 nm and a pore density of 0.1 pore / μm 2 . Use photolithography or masking process to deposit a gold nano-needle array and a flexible circuit adhesion layer on the substrate surface to form electrode pads. The side length of the electrode pads is a square with a side length of 10 μm. According to the spacing of the nanopores on the porous membrane, each prepared electrode pad array will have an average of 10 nano-needles; and the diameter of each neuron cell > 10 μm, so each cell will contact an average of 10 nano-needles, improving the signal monitoring accuracy.

[0042] It should be noted that the material of the insulating layer is determined according to actual applications. For example, the material of the insulating layer is selected as polydimethylsiloxane (PDMS).

[0043] Optionally, the material of the nano-needle electrode includes any one of metal, graphene or carbon nanotubes.

[0044] Specifically, the metal includes but is not limited to materials with good electrical conductivity and ductility such as gold and silver; using graphene or carbon nanotubes can further improve electrical conductivity and mechanical flexibility, while reducing the rigid damage of the nano-needles.

[0045] Optionally, the flexible conductive layer includes a liquid metal layer doped with carbon nanomaterials.

[0046] The intrinsically stretchable electronic device reduces mechanical mismatch and immune response with biological tissues, constructs liquid metal or conductive polymer wires to ensure electrical interconnection, and increases substrate flexibility and electrode durability.

[0047] Refer to Figure 3 , this embodiment of the present invention provides a preparation method for a multi-channel flexible nano-needle electrode array, which is applied to the above multi-channel flexible nano-needle electrode array, including:

[0048] S100. Construct an electrode pad pattern on one side surface of the flexible substrate and prepare the electrode pads;

[0049] S200. Prepare a flexible conductive layer on the surface of the electrode pads and prepare an insulating layer on one side surface of the flexible substrate;

[0050] S300. Prepare nano-needle electrodes in the holes on the other side surface of the flexible substrate, etch part of the flexible substrate to expose the nano-needle electrode part, and modify a positive ion polymer on the surface of the exposed nano-needle electrode.

[0051] Specifically, first, a flexible substrate is selected, and an electrode pad pattern is constructed on one side surface of the flexible substrate. An electrode pad is prepared according to the electrode pad pattern, and the electrode pad provides an adhesion layer for the subsequent preparation of microneedles. Then, a flexible conductive layer is prepared on the surface of the electrode pad, and the flexible conductive layer covers the electrode pad. An insulating layer is prepared on one side surface of the flexible substrate, and the insulating layer covers one side surface of the flexible substrate. Finally, nano-needle electrodes are prepared in the holes on the other side surface of the flexible substrate until the nano-needle electrodes reach the height of the flexible substrate. Then, a part of the flexible substrate is etched to expose a part of the nano-needle electrodes, and a positive ion polymer is modified on the surface of the exposed nano-needle electrodes. In a specific embodiment, in order to expose the microneedle structure, first, the excess metal on the back of the polycarbonate nanoporous membrane is polished off mechanically, and then the excess polycarbonate nanoporous membrane is etched to half of its thickness using an oxygen plasma etching process to expose the gold nano-needles.

[0052] Optionally, constructing an electrode pad pattern on one side surface of the flexible substrate and preparing an electrode pad includes:

[0053] Preparing a mask for the electrode pad pattern on one side surface of the flexible substrate;

[0054] Using magnetron sputtering to prepare an electrode layer with a preset thickness and removing the mask to obtain the electrode pad.

[0055] Determine the electrode pad pattern according to factors such as the distribution of the electrode pad and the nano-needle electrodes, and prepare a mask for the electrode pad pattern on one side surface of the flexible substrate based on the electrode pad pattern; based on the mask of the electrode pad pattern, use magnetron sputtering to prepare an electrode layer with a preset thickness. After the electrode layer is prepared, remove the mask to obtain the electrode pad.

[0056] Optionally, constructing an electrode pad pattern on one side surface of the flexible substrate and preparing an electrode pad includes:

[0057] Preparing the electrode pad pattern on one side surface of the flexible substrate by photolithography;

[0058] Using magnetron sputtering to prepare an electrode layer with a preset thickness and removing the photoresist to obtain the electrode pad.

[0059] Determine the electrode pad pattern according to factors such as the distribution of the electrode pad and the nano-needle electrodes, and prepare the photoresist for the electrode pad pattern on one side surface of the flexible substrate based on the electrode pad pattern; based on the photoresist of the electrode pad pattern, use magnetron sputtering to prepare an electrode layer with a preset thickness. After the electrode layer is prepared, remove the mask to obtain the electrode pad.

[0060] Optionally, preparing a flexible conductive layer on the surface of the electrode pad includes:

[0061] Adding carbon nanomaterials to liquid metal to obtain liquid metal doped with carbon nanomaterials;

[0062] Roll the liquid metal doped with carbon nanomaterials on a flexible substrate immersed in a dilute hydrochloric acid bath, so as to selectively coat a flexible conductive layer on the electrode pads of the flexible substrate.

[0063] First, add carbon nanomaterials to the liquid metal, and determine the dissolution temperature and time according to the actual application to fully dissolve the carbon nanomaterials into the liquid metal, obtaining the liquid metal doped with carbon nanomaterials; then, roll the liquid metal doped with carbon nanomaterials on a flexible substrate immersed in a dilute hydrochloric acid bath, so as to selectively coat a flexible conductive layer on the electrode pads of the flexible substrate.

[0064] Optionally, prepare nano-needle electrodes in the holes on the other surface of the flexible substrate, including:

[0065] Prepare an electroplating solution and perform electroplating in the electroplating solution to form solid nano-needle electrodes in the holes on the other surface of the flexible substrate.

[0066] The electroplating solution contains ions of the nano-needle electrode material, and electroplating is performed in the electroplating solution to form solid nano-needle electrodes in the holes on the other surface of the flexible substrate.

[0067] The following takes a specific embodiment to illustrate the preparation process of the multi-channel flexible nano-needle electrode array.

[0068] Refer to Figure 4, a gold nanoneedle electrode array and a flexible circuit pattern are constructed on a flexible substrate of a polycarbonate nanoporous membrane. The entire gold nanoneedle electrode array mainly consists of two parts: a 6×6 electrode array and a flexible circuit pattern for electrical signal recording. To achieve a patterned arrangement of the gold nanoneedle array, first, a mask plate with a 6×6 square (size 10μm×10μm) array pattern is designed, or a photoresist pattern of a 6×6 square (size 10μm×10μm) array is exposed on the back side of the porous membrane through ultraviolet lithography technology. Then, a layer of Au about 40nm thick is deposited by magnetron sputtering, and then the mask plate is directly removed, or the photoresist at the unexposed area is removed by developing, thus obtaining a 6×6 arranged Au layer square (size 10μm×10μm) pattern. A flexible circuit is also designed, which is composed of liquid metal doped with carbon nanomaterials bonded to the sputtered gold surface. The liquid metal doped with carbon nanomaterials is prepared by adding liquid Ga into a Teflon beaker through a plastic syringe or dropper and heating it to 40°C. Next, a small amount of carbon nanotube powder is gradually added under vigorous mechanical stirring. Then, a layer of liquid metal is selectively coated on the patterned gold film on the polycarbonate nanoporous membrane substrate by simply rolling a large piece of liquid metal doped with carbon nanomaterials on the entire substrate immersed in a dilute hydrochloric acid bath. The flexible circuit pattern serves as a lead to connect the nanoneedle sensing module and the external circuit contact module. The flexible circuit has flexible and bendable properties, and at the same time, the liquid metal doped with carbon nanomaterials has stretchable characteristics, avoiding the destruction of conductivity in the bending process of a rigid circuit design. Then, a PDMS insulating layer is spin-coated on the back side of the liquid metal to encapsulate the device.

[0069] These square block electrodes can provide good conductive contact for the electrodeposition of metal nanoneedles, enabling the deposition of nanoneedle structures only in the pores where the gold pattern is located. Then, using Ag / AgCl, Pt, and the gold layer at the bottom of the porous membrane as the reference electrode, counter electrode, and working electrode respectively, an electroplating solution containing HAuCl4 (0.1 mM / L), potassium sulfate (10 mM / L), sulfuric acid (1 mM / L), and potassium chloride (1 mM / L) is used for electroplating. In the constant voltage working mode, gold is deposited on the inner wall of the porous membrane to form a solid columnar structure. After the electroplating of gold is completed, a serpentine flexible structure circuit (line width 1μm) is fabricated by photolithography to connect the square electrodes externally, and a counter electrode is prepared to form a loop. Finally, a small amount of excess gold film on the front side is removed by mechanical polishing, and the upper surface of the polycarbonate membrane is etched with O2 plasma to expose the gold nanoneedle structure with a diameter of 100nm and a length of 2μm.

[0070] Optionally, a cationic polymer is modified on the surface of the exposed nanoneedle electrode, including:

[0071] The nanoneedle electrode is cleaned once to remove surface contaminants;

[0072] The nanoneedle electrode is cleaned twice to activate the electrode surface to form a hydroxyl or oxide layer;

[0073] The nanoneedle electrode is immersed in a preset solution for reaction to form a stable amino group layer;

[0074] The nanoneedle electrode is immersed in a positive ion polymer solution so that the positive ion polymer is adsorbed electrostatically or bonded covalently to the electrode surface.

[0075] Specifically, refer to Figure 5 , clean the nanoneedle electrode, and ultrasonically clean it with deionized water, ethanol or acetone to remove surface organic and inorganic pollutants. After cleaning, the electrode surface is further activated by ultraviolet-ozone (UV-Ozone) treatment or oxygen plasma cleaning to generate a rich hydroxyl or oxide layer. Immerse the nanoneedles in a mixed solution of APTES and absolute ethanol (usually at a concentration of 1-5%), and react at room temperature or 50 °C for 1-2 hours. Then rinse with ethanol and dry to form a stable amino group layer. Prepare a PEI solution (usually 10 mg / mL, pH adjusted to 7.4). Immerse the pretreated nanoneedle electrode in the PEI solution and let it stand for 2-6 hours so that the PEI molecules are adsorbed electrostatically or bonded covalently to the electrode surface. To enhance the modification stability, a crosslinking agent can be introduced, such as glutaraldehyde (GA), to fix the PEI on the electrode surface by forming covalent bonds. Immerse the PEI-modified electrode in a GA solution (0.1%-0.5%) for 30 minutes to 1 hour, and then wash it thoroughly with PBS to remove the unreacted crosslinking agent. Rinse the electrode with deionized water to remove the unbound PEI and other residual substances.

[0076] The preparation process of the multi-channel flexible nanoneedle electrode array also includes integration, encapsulation and characterization: lead out the serpentine circuit through metal leads for easy connection to an external power supply. Encapsulate the device to prevent leakage of electricity or liquid. Observe the distribution of the serpentine circuit through an optical microscope and a scanning electron microscope to ensure the integrity and reliability of the flexible electrode. For in vitro cell culture and electroporation exploration, in order to make the experimental conditions simpler and easier to operate, after fabricating the nanoneedle sensing module and the flexible circuit on the back side of the polycarbonate film flexible substrate, build a cell culture chamber on the front side of the flexible substrate, and assemble the PDMS module containing the cell culture chamber on one side of the front side of the substrate film. For experiments on ex vivo or in vivo tissues, there is no need to construct a PDMS culture chamber, and the device can be directly buckled on the surface of the tissue for experiments. An Ag / AgCl electrode is used as a reference electrode for electrical signal recording. Evaluation of the electrical detection performance of the multi-channel flexible nanoneedle electrode array:

[0077] The nano-needle sensor will be used for bioelectric signal recording. Therefore, it is necessary to test its basic electrical properties to evaluate its ability to record high-quality signals. Based on the fact that the nano-needle array we fabricated and the electrode leads on the back are two technological steps, in order to ensure the reliability of the electrical signal recording function of the nano-needle sensor, it is first necessary to conduct electrochemical tests on it. Using the cyclic voltammetry method (CV), in a K3Fe(CN)6 solution, scan the CV curve of the nano-needle sensor. By judging from the experimental current, determine whether the connection between the nano-needle and the back electrode of the substrate is good. At the same time, use the electrochemical impedance spectroscopy test method to scan the impedance spectrum of the nano-needle sensor in a phosphate buffer solution (PBS) between 100 Hz and 1 MHz, and compare it with the impedance spectra of typical microelectrode arrays and nano-sensors to further determine whether the basic performance of the developed nano-needle sensor is good. After the basic electrical property tests, it is necessary to evaluate the blank background noise level of the nano-needle sensor using the standard instrument for microelectrode arrays. Use the nano-needle sensor to record the blank background noise of electrically excitable cells cultured in vitro to evaluate the electrical signal noise level under in vitro conditions. At the same time, use the nano-needle sensor to record the blank background noise of the brain and heart organ tissues of animals to evaluate the electrical signal noise level under in vivo conditions. By analyzing the impedance spectrum and noise level, calculate the coefficient of variation (CV) to evaluate the consistency between multi-channel nano-needle sensors. Through the above various performance tests, this study will further optimize and improve the processing technology of the nano-needle sensor to develop a nano-needle sensor with good electrical signal recording performance.

[0078] Flexibility test of the multi-channel flexible nano-needle electrode array: In addition to being used for recording the electrical signals of electrically excitable cells in vitro, the nano-needle electrode array will also be used for recording the electrical signals of the brain and heart in vivo. In order to achieve a good bio-device interface coupling, it is necessary to use a flexible material as the sensor substrate. Different from the micro-nano sensors with a rigid substrate, it is necessary to conduct a flexibility test on the micro-nano sensors based on a flexible material substrate to evaluate the stability and robustness of their electrical properties under the tensile and torsional deformation states generated during brain and heart applications. Through impedance spectrum scanning, study whether there are significant changes in the impedance performance of the nano-needle sensor under different curvature conditions to determine the stability of the electrical properties of the sensor with a flexible substrate. At the same time, conduct a fatigue resistance test on the flexible sensor with repeated tensile and torsional deformations. Determine its robustness through the impedance spectrum characteristics before and after the test. Finally, determine the optimal working conditions and service life of the flexible sensor through the above two aspects of tests.

[0079] The following uses a specific embodiment to illustrate the process of testing using the multi-channel flexible nano-needle electrode array.

[0080] Culture of hippocampal neurons in vitro: Before cell culture, the sensor surface was modified overnight with polylysine containing 5 mg / mL to facilitate cell adhesion. Primary hippocampal neurons were isolated from neonatal C57 mice, regardless of gender. First, several neonatal C57 mice at 24 h old were selected, disinfected with 75% alcohol throughout the body, decapitated, and the scalp and skull were incised layer by layer under sterile conditions. The brain region was exposed with curved forceps, and the whole brain was carefully removed and washed several times with D-hanks solution. Second, starting from the midline of the brain, the temporal cortex of the brain was carefully separated to expose the crescent-shaped hippocampal gyrus. The hippocampal tissue was carefully clamped out and placed in ice-cold D-hanks solution. Microvessels were carefully removed and the tissue was sufficiently minced. Third, 10 - 20 U / mL of papain and 400 U / mL of DNAse were used. The bottle mouth was covered with tin foil and digested in an incubator at 37°C for about 30 min. Several drops of fetal bovine serum were added to terminate digestion. The cells were gently pipetted for several minutes until the liquid became a rice paste, and then the pipetting was stopped. The operation should be gentle. The cell suspension was filtered through a 40-mesh sieve. Fourth, the cells were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM / F12 medium and gently pipetted. Trypan blue staining was used for rapid cell counting. According to the counting results, the final cell concentration was adjusted to 10,000 cells / ml. After adding fetal bovine serum at a final concentration of 10%, the cells were seeded on the sensor surface and cultured in an incubator at 37°C with 5% CO2. Shaking was prohibited within 12 hours. Finally, 24 hours after seeding, the culture medium was completely replaced with serum-free DMEM / F12 medium. At 48 hours, cytosine arabinoside at a final concentration of 10 μmol / L was added to inhibit the overgrowth of non-neuronal cells. Subsequently, the medium was changed by half every 3 days. After 72 hours of culture, the electrical signals of the neuronal cells could be recorded.

[0081] In vivo brain tissue: The in vivo brain tissue also used a rat brain model. First, the rats were anesthetized by inhaling 5% isoflurane. After the rats lost consciousness and had no pain response, they were fixed on a stereotaxic apparatus for the brain, and then the anesthesia was maintained by inhaling 1 - 1.5% isoflurane. A special heating pad was used to maintain the body temperature of the rats, and the heating pad was set at a constant temperature of 37 ± 1°C. Second, a hair clipper and depilatory cream were used to remove the hair on the rat's head, the skin was exposed, and 0.1 ml of 0.25% bupivacaine solution was injected and waited for 1 minute. Third, the scalp was incised to expose the skull, and a circular area of 3 mm in diameter was removed with a drill bit to further expose the in vivo brain tissue. The sensor was gently placed on the exposed brain tissue surface. Fourth, a glass slide with a diameter of 4 mm was covered above the circular opening area of the skull, fixed with dental cement after sealing, and the sensor wire was led out to record the electrical signals of the in vivo brain tissue.

[0082] Evaluation of the sensitivity of intracellular electrical signal recording: To detect intracellular electrical signals, excitable neuron cells in vivo or in vitro are used as the objects for research, testing, and application. The cells are cultured in a multi-well plate. During the experiment, through a micromanipulator, the height of the nano-needle electrode device modified with a cationic polymer is adjusted, and it is gradually pressed above the cell. If the nano-needle penetrates the cell membrane and the electrode contacts the intracellular environment, the intracellular electrical signal can be recorded and participate in Figure 6 . Usually, the intracellular electrical signal is similar in shape to the standard action potential. Through data analysis, the amplitude of the electrical signal recorded by the nano-needle sensor can be calculated. To evaluate the sensitivity of the sensor's electrical signal recording, the gold-standard patch clamp technique is used to synchronously record the action potential of the same cell, and then the ratio of the intracellular potential amplitude to the standard action potential amplitude is calculated to determine the sensitivity of the nano-needle sensor in recording intracellular signals. At the same time, the signal-to-noise ratio can be calculated based on the noise levels of the two recording methods, so as to comprehensively evaluate the basic performance of the nano-needle sensor combined with the optoelectronic permeable membrane technique in recording intracellular electrical signals.

[0083] Implementing the embodiments of the present invention includes the following beneficial effects: The multi-channel flexible nano-needle electrode array of this embodiment includes a flexible substrate, nano-needle electrodes, a flexible conductive layer, an insulating layer, and a flexible external lead. The flexible substrate includes several nano-holes, and the nano-needle electrodes pass through the nano-holes of the flexible substrate to form a part of the nano-needle electrodes exposed. Several nano-needle electrodes share an electrode disk, and the surface of the electrode disk is covered with a flexible conductive layer. The flexible conductive layer has the properties of flexibility, bendability, and stretchability, reducing the damage to conductivity during the bending process of the circuit. The electrode disk and the flexible conductive layer are wrapped by an insulating layer. The electrode disk is connected to the flexible external lead. The exposed nano-needle electrodes are modified with a positive ion polymer, which carries a large amount of positive charges and can interact with the negatively charged cell membrane phospholipid molecules, increasing the affinity between the nano-needle and the cell membrane. Moreover, the positive ion polymer can provide a certain degree of flexibility, reducing the mechanical damage of the nano-needle surface to the cell membrane, optimizing the conditions for the nano-micro needle electrode to penetrate the cell membrane for a long time, and thus being able to accurately monitor the intracellular electrical signal for a long time.

[0084] The above is a specific description of the preferred embodiments of the present invention, but the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent deformations or substitutions without departing from the spirit of the present invention, and these equivalent deformations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A multi-channel flexible nano-needle electrode array, characterized in that It includes a flexible substrate, nano-needle electrodes, a flexible conductive layer, an insulating layer, and a flexible external lead. The flexible substrate includes a number of nanopores. The nano-needle electrodes pass through the nanopores of the flexible substrate to form a part of the nano-needle electrodes exposed. A number of the nano-needle electrodes share one electrode disk. The surface of the electrode disk is covered with the flexible conductive layer. The electrode disk and the flexible conductive layer are wrapped by the insulating layer. The electrode disk is connected to the flexible external lead. The exposed nano-needle electrodes are modified with a cationic polymer.

2. The multi-channel flexible nano-needle electrode array according to claim 1, wherein The diameter range of the nanopores of the flexible substrate is 90-110 nm, and the nanopore density range of the flexible substrate is 0.05-0.15 pores / μm 2 .

3. The multi-channel flexible nano-needle electrode array according to claim 1, wherein The material of the nano-needle electrodes includes any one of metal, graphene, or carbon nanotubes.

4. The multi-channel flexible nano-needle electrode array according to claim 1, wherein, The flexible conductive layer includes a liquid metal layer doped with carbon nanomaterials.

5. A preparation method of a multi-channel flexible nano-needle electrode array, characterized in that, Applied to the multi-channel flexible nano-needle electrode array according to any one of claims 1-4, it includes: Construct an electrode disk pattern on one side surface of the flexible substrate and prepare the electrode disk. Prepare a flexible conductive layer on the surface of the electrode disk and prepare the insulating layer on one side surface of the flexible substrate. Prepare nano-needle electrodes in the holes on the other side surface of the flexible substrate, etch part of the flexible substrate to make a part of the nano-needle electrodes exposed, and modify the surface of the exposed nano-needle electrodes with a cationic polymer.

6. The preparation method according to claim 5, wherein The constructing an electrode disk pattern on one side surface of the flexible substrate and preparing the electrode disk includes: Prepare a mask plate of the electrode disk pattern on one side surface of the flexible substrate. Use magnetron sputtering to prepare an electrode layer with a preset thickness and remove the mask plate to obtain the electrode disk.

7. The preparation method according to claim 5, characterized in that, The constructing an electrode disk pattern on one side surface of the flexible substrate and preparing the electrode disk includes: Prepare the electrode disk pattern on one side surface of the flexible substrate by photolithography. Use magnetron sputtering to prepare an electrode layer with a preset thickness and remove the photoresist to obtain the electrode disk.

8. The preparation method according to claim 5, characterized in that, The preparing a flexible conductive layer on the surface of the electrode disk includes: Add carbon nanomaterials to liquid metal to obtain liquid metal doped with carbon nanomaterials. Roll the liquid metal doped with carbon nanomaterials on the flexible substrate immersed in a dilute hydrochloric acid bath, so as to selectively coat a flexible conductive layer on the electrode disk of the flexible substrate.

9. The preparation method according to claim 5, characterized in that, The preparing nano-needle electrodes in the holes on the other side surface of the flexible substrate includes: Prepare an electroplating solution and perform electro-deposition in the electroplating solution to form solid nano-needle electrodes in the holes on the other side surface of the flexible substrate.

10. The preparation method according to claim 5, characterized in that, The modifying the surface of the exposed nano-needle electrodes with a cationic polymer includes: Perform a first cleaning on the nano-needle electrodes to remove surface contaminants. Perform a second cleaning on the nano-needle electrodes to activate the electrode surface to form a hydroxyl or oxide layer. Immerse the nano-needle electrodes in a preset solution for reaction to form a stable ammonia-based layer. Immerse the nano-needle electrodes in a cationic polymer solution so that the cationic polymer is adsorbed by electrostatic force or bonded by covalent bonds to the electrode surface.

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