Intracellular implantable multifunctional carbon coating nanoelectrode and preparation method thereof

Abstract

The invention provides an intracellular implantable multifunctional carbon coating nanoelectrode and a preparation method thereof, and the preparation method comprises the following steps: drawing a glass tube to form a nano pipette, sequentially sputtering metal nanoparticles and a metal oxide sensing coating on the surface of the nano pipette, and leading a wire; after the precursor is treated by low-temperature plasma jet, a carbon-based sensing coating is deposited on the surface of the metal oxide sensing coating; carrying out surface modification treatment on the carbon-based sensing coating by using plasma jet; depositing an insulating coating on the coarsened and modified surface of the carbon-based sensing coating by using plasma jet; the tip of the electrode is selectively etched by using branches of plasma jet, so that the exposure degree of the metal oxide sensing coating, the carbon-based sensing coating and the nano pipette outside the insulating coating is controllable. The electrode prepared by the invention can be implanted into an organism and used for detecting markers such as electric signals, pH value and oxygen in cells, and has excellent electrochemical performance, biological pollution resistance, long-term stability and biocompatibility.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to an intracellular implantable multifunctional carbon-coated nanoelectrode and a preparation method thereof. Background Art

[0002] The development of implantable brain-machine interface devices is becoming increasingly important for the study of neurological diseases. In order to obtain more accurate information from neurons, implantable brain-machine interfaces with ultra-micrometer scales facing the intracellular environment have been developed. Reliable detection and analysis of intracellular chemical and biomarker signals at the single-cell level are essential for early disease diagnosis, pathological assessment, and a deeper understanding of basic biology.

[0003] Compared with microelectrodes, nanoelectrodes with a tip diameter of less than one micron have many advantages, such as high spatiotemporal resolution, targeted and precise regulation of neurons, and good biocompatibility. Nanopipettes, nanotubes, nanowires, and sharp nanoelectrodes have received great attention in minimally invasive intracellular electrophysiological detection. Among them, sharp nanoelectrodes show higher detection sensitivity for intracellular signals due to the modification of nanometal particles. They can not only detect monolayers and submonolayers adsorbed on rough metal surfaces, but also detect specific biological or chemical signals in subcellular regions of single cells, such as pH, oxygen, dopamine, and reactive oxygen species.

[0004] The in-situ synchronous acquisition of multiple types of intracellular signals is of great significance for the accurate diagnosis and real-time monitoring of diseases. Due to the limitations of processing technology, traditional nanoelectrodes can usually only record one of the electrical signals, particle signals and biomolecular signals, and cannot simultaneously achieve accurate detection of multiple intracellular electrochemical signals.

[0005] After searching the prior art, we found:

[0006] James G. Roberts et al. from North Carolina State University wrote an article titled "Carbon-fiber nanoelectrodes for real-time discrimination of vesicle cargo in the native cellular environment" in ACS Nano, 2020, 14. They developed a carbon nanoelectrode that can detect dopamine in vesicles. The electrode is insulated by electropolymerization in a phenolic resin solution and direct wax heating and attachment. However, the exposed length of the electrode tip is 5 μm, which cannot be controlled to a length as low as 10 nm, and it does not have the ability to simultaneously detect multiple intracellular signals.

[0007] Yu-Ting Jiao et al. from Wuhan University wrote an article titled "Dual-channel nanoelectrochemical sensor for monitoring intracellular ROSand NADH kinetic variations of their concentrations" in Biosensors and Bioelectronics, 2023, 222, and developed a dual-channel nanowire electrode that can simultaneously monitor ROS and NADH in single cells. However, the electrode is insulated by inserting the internal sensing material into a glass tube and then sealing it with wax. However, the exposed length of the electrode tip is 5μm, and it is also impossible to controllably achieve an exposure length as low as 10nm, and it does not have the ability to detect intracellular pH and electrical signals.

[0008] David L. Hunt et al. of the Howard Hughes Medical Institute wrote an article titled "Multimodal in vivo brain electrophysiology with integrated glass microelectrodes" in Nature Biomedical Engineering, 2019, 3, developing a glass pipette with integrated microelectrodes for simultaneous acquisition of multimodal intracellular and extracellular information, electrochemical evaluation, and optogenetic perturbation of neural activity in vivo. However, the electrode is insulated by directly inserting the modified nanopipette into molten wax. However, the exposed length of the electrode tip is 20 μm, and it is also impossible to controllably achieve an exposure length as low as 10 nm, and it does not have the ability to detect intracellular pH and multiple biomolecular signals.

[0009] Therefore, there is an urgent need to propose an implantable multifunctional nanoelectrode that has excellent electrochemical properties, anti-biological contamination performance, long-term stability and biocompatibility, and can simultaneously realize in situ detection of intracellular electrical signals, pH and multiple biological molecules. Summary of the invention

[0010] In view of the defects in the prior art, the object of the present invention is to provide an intracellular implantable multifunctional carbon-coated nanoelectrode and a preparation method thereof.

[0011] According to one aspect of the present invention, a method for preparing an intracellular implantable multifunctional carbon-coated nanoelectrode is provided, comprising:

[0012] Drawing a glass tube to form a nanopipette, wherein the nanopipette has a nanochannel inside, sequentially sputtering metal nanoparticles and a metal oxide sensing coating on the surface of the nanopipette, and wiring from the metal nanoparticles;

[0013] After treating the precursor with a low-temperature plasma jet, a carbon-based sensing coating is deposited on the surface of the metal oxide sensing coating;

[0014] Using a plasma jet to perform surface modification treatment on the carbon-based sensing coating to obtain a roughened and modified carbon-based sensing coating and expose the nanochannel;

[0015] Depositing an insulating coating on the surface of the roughened and modified carbon-based sensing coating using a plasma jet;

[0016] The electrode tip is selectively etched by using a branch of a plasma jet, so that the extent to which the metal oxide sensing coating, the carbon-based sensing coating and the nanopipette are exposed outside the insulating coating can be controlled.

[0017] Furthermore, the drawing of the glass tube to form the nanopipette includes: drawing the glass tube using a heating drawing method to obtain the nanopipette.

[0018] Furthermore, the precursor is treated with a low-temperature plasma jet, wherein: the precursor is any one or more of methane, hydrogen, argon and boron oxide;

[0019] A carbon-based sensing coating is deposited on the surface of the metal oxide sensing coating, wherein the carbon-based sensing coating is any one of graphene, carbon nanotubes, diamond-like carbon and boron-doped diamond.

[0020] Furthermore, the carbon-based sensing coating is subjected to surface modification treatment by using a plasma jet, wherein the exciting gas of the plasma jet is a mixed gas of oxygen and helium.

[0021] Furthermore, the carbon-based sensing coating is surface modified by using a plasma jet, wherein the angle between the axis of the electrode tip and the axis of the plasma jet is 0-360°.

[0022] Furthermore, the use of plasma jet to deposit an insulating coating on the surface of the roughened and modified carbon-based sensing coating includes: under the action of a sinusoidal high voltage of ±8-±15kV, a mixed gas of methane, hydrogen and argon excites a plasma jet to deposit a diamond insulating coating with a thickness of 0.05-10μm on the electrode surface.

[0023] Furthermore, the method of using a branch of a plasma jet to selectively etch the electrode tip includes: first exciting a plasma jet on a conductive substrate, then controlling the tip of the nanoelectrode to gradually approach the excited jet, and when a predetermined distance is reached, a small branch of the plasma jet is focused on the tip of the nanoelectrode to selectively remove the surface insulating coating, so that the exposure degree of other internal coatings can be controlled.

[0024] Further, the plasma jet is excited on a conductive substrate, wherein: the voltage waveform for exciting the plasma jet is any one of an AC pulse wave, a square wave, a triangle wave, a sound wave, a sine wave, a sawtooth wave and a trapezoidal wave;

[0025] A thin branch of the plasma jet is focused on the tip of the nanoelectrode, wherein the diameter of the thinnest part of the branch of the plasma jet is 0.01-1000 μm.

[0026] Further, the total exposed length of the electrode tip is 0-100 μm.

[0027] According to another aspect of the present invention, there is provided an intracellularly implantable multifunctional carbon-coated nanoelectrode, which is prepared by using the above-mentioned method for preparing the intracellularly implantable multifunctional carbon-coated nanoelectrode.

[0028] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0029] The present invention is based on layer-by-layer deposition and low-temperature plasma jet processing technology. First, metal nanoparticles and metal oxide sensing coatings are sputtered on the surface of the drawn nanopipette; then, a low-temperature plasma jet is used to convert the precursor into a carbon-based sensing coating, and the surface of the carbon-based sensing coating is modified, roughened and oxygen-containing functional groups are introduced; finally, a plasma jet is used to deposit a nanodiamond insulating coating, and a jet branch is used to selectively etch the tip of the electrode so that the sensing coating, the carbon-based sensing coating and the nanopipette are exposed at the same time. The electrode can realize the simultaneous detection of markers such as electrical signals, pH, and biomolecules such as oxygen in cells implanted in a living body. In addition, the electrode has excellent electrochemical properties, anti-biological contamination performance, long-term stability and biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0031] Figure 1 It is a schematic diagram of a process for preparing a method for intracellular implantable multifunctional carbon-coated nanoelectrode in one embodiment of the present invention;

[0032] Figure 2 A schematic diagram of the cross-sectional structure of an intracellularly implantable multifunctional carbon-coated nanoelectrode in one embodiment of the present invention;

[0033] Figure 3 It is a schematic structural diagram of a low-temperature plasma jet processing platform for preparing intracellular implantable multifunctional carbon-coated nanoelectrodes in one embodiment of the present invention;

[0034] The reference numerals in the figure correspond to: 1-nanopipette, 2-nanochannel, 3-metal nanoparticles, 4-metal oxide sensing coating, 5-carbon-based sensing coating, 6-roughened and modified carbon-based sensing coating, 7-insulating coating, 8-plasma jet. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0036] Reference Figure 1 As shown, a method for preparing an intracellular implantable multifunctional carbon-coated nanoelectrode provided by an embodiment of the present invention comprises the following steps:

[0037] S1, drawing a glass tube to form a nanopipette 1, the nanopipette 1 has a nanochannel 2 inside, and after the surface is dried and cleaned, metal nanoparticles 3 and a metal oxide sensing coating 4 are sequentially sputtered on the surface of the nanopipette 1, and wires are formed from the metal nanoparticles 3, the metal nanoparticles 3 are used for conducting electricity and enhancing the bonding force between the substrate and the sensing coating;

[0038] S2, after treating the precursor with a low-temperature plasma jet, a carbon-based sensing coating 5 is deposited on the surface of the metal oxide sensing coating 4, Figure 1 8 in the middle represents the plasma jet;

[0039] S3, using a plasma jet to perform surface modification treatment on the carbon-based sensing coating 5 to obtain a roughened and modified carbon-based sensing coating 6, and exposing the nanochannel 2;

[0040] S4, using a plasma jet to deposit an insulating coating 7 on the surface of the roughened and modified carbon-based sensing coating 6;

[0041] S5. Use the branch of the plasma jet to selectively etch the electrode tip, so that the degree (i.e., exposure length) of the metal oxide sensing coating 4, the carbon-based sensing coating 5, and the nanopipette 1 exposed outside the insulating coating 7 can be controlled.

[0042] In some embodiments, in step S1, a glass tube is drawn using a heating drawing method to obtain a nanopipette 1.

[0043] For example, the glass tube is made of quartz glass or borosilicate glass, and the heating method is laser-assisted heating or butane torch heating. The material of the metal nanoparticles 3 is gold or platinum; the material of the metal oxide sensing coating 4 is iridium oxide. The copper wire is connected to the metal nanoparticles 3 using conductive silver paste.

[0044] In some embodiments, the structural schematic diagram of the low temperature plasma jet processing platform is as follows Figure 3 As shown, it includes a five-axis motion platform, a spectrometer, a waveform generator, a high-voltage amplifier, an oscilloscope, a precursor gas cylinder, a host computer, etc. In step S2, the precursor is any one or more of methane, hydrogen, argon and boron oxide, etc., which is specifically determined by the type of biological molecules to be detected, so as to form different carbon-based sensing coatings to detect different biological molecules. For example, the precursors of methane and hydrogen can be used to form diamond-like coatings to sense oxygen, etc., and the precursors of methane, hydrogen, argon and boron oxide can be used to form boron-doped diamond coatings to sense dopamine, etc. The carbon-based sensing coating 5 is any one of graphene, carbon nanotubes, diamond-like and boron-doped diamond to sense different biological molecules.

[0045] Exemplarily, a low-temperature plasma jet processing platform is used to process precursors of methane, hydrogen and argon, to excite a plasma jet 8, and to deposit a carbon-based sensing coating 5 on the electrode surface. The carbon-based sensing coating 5 has excellent conductive properties and a thickness of 100 nm.

[0046] In some embodiments, in step S3, a plasma jet is used to perform surface modification treatment on the carbon-based sensing coating 5, wherein: the excitation gas of the plasma jet is a mixed gas of oxygen and helium. The reason for selecting helium is that compared with another commonly used inert gas argon, helium has a low molecular weight, stable reaction and low excitation voltage, which is helpful for controlling the surface morphology of the processing area. Oxygen can promote the oxidation reaction on the surface of the carbon material to generate carbon dioxide with less residue, which helps to roughen the surface and increase the specific surface area. The angle between the axis of the electrode tip and the axis of the plasma jet is 0-360°, the specific surface area of ​​the carbon-based sensing coating 5 is increased, the electrochemical performance is improved, and the introduction of oxygen-containing functional groups also improves the anti-biological contamination performance and biocompatibility of the nanoelectrode. At the same time, the nanochannel 2 inside the electrode is exposed.

[0047] In some embodiments, in step S4, under the action of a sinusoidal high voltage of ±8-±15 kV, a mixture of methane, hydrogen and argon excites a low-temperature plasma jet to deposit a diamond or diamond-like insulating coating dominated by a diamond phase with a thickness of 0.05-10 μm on the electrode surface to improve long-term recording stability and anti-electromagnetic interference capabilities. Here, methane serves as a carbon source to provide the carbon atoms required for diamond growth to form diamond crystals. Hydrogen can partially dissociate into atomic hydrogen to saturate the dangling bonds of carbon atoms on the surface of the growing diamond, making the diamond surface tend to be stable. Argon can adjust the reaction atmosphere and protect the diamond coating to prevent the diamond coating from being oxidized or contaminated at high temperatures, thereby ensuring the quality and purity of the diamond coating. At this time, refer to Figure 2 The cross-sectional structure of the electrode is, from the inside out, a nanochannel 2, a nanopipette 1, metal nanoparticles 3, a metal oxide sensing coating 4, a roughened and modified carbon-based sensing coating 6, and an insulating coating 7.

[0048] In some embodiments, in step S5, a plasma jet is first excited on a conductive substrate, and then the tip of the nanoelectrode is controlled to gradually approach the excited jet. When a predetermined distance is reached, a small branch of the plasma jet is focused on the tip of the nanoelectrode, and the surface insulating coating 7 is selectively removed, so that the exposure degree of other internal coatings can be controlled. Thanks to the site selectivity of the plasma jet, the jet will focus on the tip of the nanoelectrode due to the movement of electrons. This is because the high-voltage amplifier forms a high voltage between the ring electrode and the grounded nanoelectrode in the plasma generator, and the tip of the nanoelectrode is closest to the ring electrode. Because it is easier to attract electrons here to form a plasma jet, the carbon-based insulating coating at the tip of the nanoelectrode will be etched first, just exposing the unetched internal sensing coating, etc., thereby achieving selective etching of the insulating coating 7.

[0049] Preferably, the voltage waveform for exciting the plasma jet is any one of an AC pulse wave, a square wave, a triangle wave, a sound wave, a sine wave, a sawtooth wave and a trapezoidal wave, which is specifically related to the density and hardness of the coating to be processed. For example, an AC pulse wave with a larger energy can be selected when processing diamonds, and a relatively stable sine wave high voltage can be selected when processing carbon nanotubes. A small branch of the plasma jet is focused on the tip of the nanoelectrode, wherein the diameter of the thinnest branch of the plasma jet is 0.01-1000μm. When the diameter of the nanoelectrode is smaller, the coating is thinner, the area to be removed is smaller, and the area to be exposed is smaller, it is necessary to select a plasma generating nozzle with a smaller inner diameter, and the diameter of the jet branch is thinner. The total exposed length of the electrode tip is 0-100μm, which is related to the plasma jet processing time. The choice of exposure length is related to the size of the object to be detected. For example, to detect overall marker changes in cells, an exposure length of 10-3000nm can be selected. To detect marker changes at a certain position in the cell, an exposure length of 10-500nm can be selected, which is determined by the size of the area to be detected. To detect marker changes outside the cell or even outside the body, a longer exposure length can be selected.

[0050] For example, the plasma jet is first excited on a stainless steel substrate, and then the tip of the nanoelectrode is controlled to gradually approach the excited jet. When the two are 3 mm apart, a thin branch of the plasma jet with a diameter of about 50 nm at the thinnest part will focus on the tip of the nanoelectrode and stay for 5 seconds. The diamond insulating coating is selectively removed, and the iridium oxide coating is exposed by 1000 nm, the boron-doped diamond coating is exposed by 500 nm, and the nanochannel is exposed by 100 nm, thereby obtaining an intracellular implantable multifunctional carbon-coated nanoelectrode. The electrode has the ability to detect targeted sites in specific intracellular regions, the nanochannel can detect intracellular electrical signals, the iridium oxide coating can detect intracellular pH, the roughened and modified graphene coating can detect reactive oxygen and reactive nitrogen, and the diamond insulating coating can ensure the long-term stability of the detection signal, thereby realizing the simultaneous detection of intracellular electrical signals, pH and biomolecules.

[0051] Another embodiment of the present invention provides an intracellular implantable multifunctional carbon-coated nanoelectrode, which is prepared using the above-mentioned preparation method of the intracellular implantable multifunctional carbon-coated nanoelectrode. The carbon-coated nanoelectrode has the ability to detect targeted sites in specific intracellular regions, the nanochannel can detect intracellular electrical signals, the metal oxide sensing coating can detect intracellular pH, the roughened and modified carbon-based sensing coating can detect oxygen and hydrogen peroxide, and the insulating coating can ensure the long-term stability of the detection signal. Under a microscope, the electrode in the embodiment of the present invention is clamped by a micromanipulator and inserted into the nerve cells of the mouse brain, and current is applied. The signal is amplified by a patch clamp amplifier, and an intracellular continuous spike electrical signal with an amplitude of 10-100mV and a response time of 1-100ms can be detected, based on which diseases can be diagnosed and monitored. The pH detection sensitivity of the electrode is about 40-60mV / pH, so during the course of mouse lesions, the potential difference detected in the cell and the corresponding pH value will fluctuate up and down, based on which diseases can be diagnosed and monitored. In addition, the carbon material on the surface of the nanoelectrode is sensitive to biological molecules such as oxygen. In electrochemical tests, when a voltage of -650mV is applied, different concentrations of dissolved oxygen and other biological molecules will correspond to different current values. Based on this, diseases can be diagnosed and monitored based on changes in the concentrations of biological molecules such as oxygen, thereby achieving simultaneous detection of intracellular electrical signals, pH and biological molecules.

[0052] The scheme of the present application will be explained below in conjunction with specific embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the documents in this area or the product specifications are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained through commercial channels.

[0053] Example 1

[0054] This embodiment provides a method for preparing an intracellular implantable multifunctional carbon-coated nanoelectrode, which is performed according to the following steps:

[0055] S1, refer to Figure 1 As shown, a quartz glass tube is drawn by laser-assisted heating to obtain a nanopipette 1, in which a nanochannel 2 is formed. After the surface is dried and cleaned, metal nanoparticles 3 and a metal oxide sensing coating 4 are sputtered, and the enameled copper wires polished at both ends are connected to the metal nanoparticles 3 with conductive silver paste. The material of the metal nanoparticles 3 here is gold, and the raw material of the metal oxide sensing coating 4 is iridium oxide.

[0056] S2, reference Figure 1 and Figure 3As shown, a low-temperature plasma jet processing platform is used to process precursors methane and hydrogen, stimulate a plasma jet 8, and deposit a carbon-based sensing coating 5 on the electrode surface. The carbon-based sensing coating here is a diamond-like coating containing a certain proportion of graphite phase with excellent conductive properties and a thickness of 200 nm.

[0057] S3, refer to Figure 1 and Figure 3 As shown, the diamond-like coating on the surface of the nanoelectrode is modified by a plasma jet excited by a mixed gas of oxygen and helium to obtain a roughened and modified diamond-like coating. The angle between the axis of the nanoelectrode tip and the axis of the plasma jet is 60°. At this time, the specific surface area of ​​the sensing coating is increased, the electrochemical performance is improved, and the introduction of oxygen-containing functional groups also improves the anti-biological contamination performance and biocompatibility of the nanoelectrode. At the same time, the nanochannel 1 inside the electrode is exposed.

[0058] S4, ref. Figure 1 and Figure 3 As shown in the figure, under the action of ±11kV sinusoidal high voltage, the mixed gas of methane, hydrogen and argon stimulates a plasma jet, depositing a diamond insulating coating with a thickness of 100nm on the electrode surface to improve the long-term recording stability and anti-electromagnetic interference ability. Figure 2 The cross-sectional structure of the electrode is, from the inside out, a nanochannel 2, a nanopipette 1, metal nanoparticles 3 (gold), a metal oxide sensing coating 4 (iridium oxide), a roughened and modified carbon-based sensing coating 6 (diamond-like coating) and an insulating coating 7 (diamond insulating coating).

[0059] S5, refer to Figure 1 and Figure 3 As shown, the plasma jet is first excited on the single crystal silicon substrate, and then the tip of the nanoelectrode is controlled to gradually approach the excited jet. When the two are 2 mm apart, a thin branch of the plasma jet with a diameter of about 100 nm at the thinnest part will focus on the tip of the nanoelectrode and stay for 3 seconds. The diamond insulating coating is selectively removed, and the iridium oxide coating is exposed by 2000 nm, the diamond-like coating is exposed by 1000 nm, and the nanochannel is exposed by 200 nm, thereby obtaining an intracellular implantable multifunctional carbon-coated nanoelectrode. The electrode has the ability to detect targeted sites in specific intracellular regions. The nanochannel can detect intracellular electrical signals, the iridium oxide coating can detect intracellular pH, the roughened and modified diamond-like coating can detect oxygen and hydrogen peroxide, and the diamond insulating coating can ensure the long-term stability of the detection signal, thereby realizing the simultaneous detection of intracellular electrical signals, pH and biomolecules.

[0060] Example 2

[0061] This embodiment provides a method for preparing an intracellular implantable multifunctional carbon-coated nanoelectrode, which is performed according to the following steps:

[0062] S1, refer to Figure 1 As shown, after heating with a butane torch, a quartz glass tube is drawn to obtain a nanopipette 1, in which a nanochannel 2 is formed. After its surface is dried and cleaned, metal nanoparticles 3 and a metal oxide sensing coating 4 are sputtered, and a copper wire is connected to the metal nanoparticles 3 with a conductive silver paste. Here, the material of the metal nanoparticles 3 is platinum, and the raw material of the metal oxide sensing coating 4 is iridium oxide.

[0063] S2, reference Figure 1 and Figure 3 As shown, a low-temperature plasma jet processing platform is used to process precursors such as methane, hydrogen, argon and boron oxide, to excite a plasma jet 8, and to deposit a carbon-based sensing coating 5 on the electrode surface. The carbon-based sensing coating here is a boron-doped diamond coating with excellent electrical conductivity and a thickness of 150 nm.

[0064] S3, refer to Figure 1 and Figure 3 As shown, the boron-doped diamond coating on the surface of the nanoelectrode is modified by a plasma jet excited by a mixed gas of oxygen and helium to obtain a roughened and modified boron-doped diamond coating. The angle between the axis of the nanoelectrode tip and the axis of the plasma jet is 180°. At this time, the specific surface area of ​​the sensing coating is increased, the electrochemical performance is improved, and the introduction of oxygen-containing functional groups also improves the anti-biological contamination performance and biocompatibility of the nanoelectrode. At the same time, the nanochannel 1 inside the electrode is exposed.

[0065] S4, ref. Figure 1 and Figure 3 As shown in the figure, under the action of the ±12kV pulse wave high voltage, the mixed gas of methane, hydrogen and argon stimulates a plasma jet, and a diamond insulating coating with a thickness of 150nm is deposited on the electrode surface to improve the long-term recording stability and anti-electromagnetic interference ability. Figure 2 The cross-sectional structure of the electrode is, from the inside to the outside, a nanochannel 2, a nanopipette 1, metal nanoparticles 3 (platinum), a metal oxide sensing coating 4 (iridium oxide), a roughened and modified carbon-based sensing coating 6 (boron-doped diamond coating) and an insulating coating 7 (diamond insulating coating).

[0066] S5, refer to Figure 1 and Figure 3As shown, the plasma jet is first excited on the aluminum substrate, and then the tip of the nanoelectrode is controlled to gradually approach the excited jet. When the two are 1 mm apart, a thin branch of the plasma jet with a diameter of about 200 nm at the thinnest part will focus on the tip of the nanoelectrode and stay for 1.5 seconds. The diamond insulating coating is selectively removed, and the iridium oxide coating is exposed by 3000 nm, the boron-doped diamond coating is exposed by 1500 nm, and the nanochannel 2 is exposed by 1000 nm, thereby obtaining an intracellular implantable multifunctional carbon-coated nanoelectrode. The electrode has the ability to detect targeted sites in specific intracellular regions. The nanochannel can detect intracellular electrical signals, the iridium oxide coating can detect intracellular pH, the roughened and modified boron-doped diamond coating can detect dopamine and enzymes, and the diamond insulating coating can ensure the long-term stability of the detection signal, thereby realizing the simultaneous detection of intracellular electrical signals, pH and biomolecules.

[0067] Example 3

[0068] This embodiment provides a method for preparing an intracellular implantable multifunctional carbon-coated nanoelectrode, which is performed according to the following steps:

[0069] S1, refer to Figure 1 As shown, a borosilicate glass tube is drawn with the aid of laser to obtain a nanopipette 1, in which a nanochannel 2 is formed. After its surface is dried and cleaned, metal nanoparticles 3 and a metal oxide sensing coating 4 are sputtered, and the silver wire is connected to the metal nanoparticles 3 with a conductive silver paste. Here, the material of the metal nanoparticles 3 is gold, and the raw material of the metal oxide sensing coating 4 is iridium oxide.

[0070] S2, reference Figure 1 and Figure 3 As shown, a low-temperature plasma jet processing platform is used to process precursors such as methane, hydrogen and argon to excite a plasma jet 8, and a carbon-based sensing coating 5 is deposited on the electrode surface. The carbon-based sensing coating here is a graphene coating with excellent conductive properties and a thickness of 100 nm.

[0071] S3, refer to Figure 1 and Figure 3 As shown, the graphene coating on the surface of the nanoelectrode is modified by a plasma jet excited by a mixed gas of oxygen and helium to obtain a roughened and modified graphene coating. The angle between the axis of the nanoelectrode tip and the axis of the plasma jet is 120°. At this time, the specific surface area of ​​the sensing coating is increased, the electrochemical performance is improved, and the introduction of oxygen-containing functional groups also improves the anti-biological contamination performance and biocompatibility of the nanoelectrode. At the same time, the nanochannel inside the electrode is exposed.

[0072] S4, ref. Figure 1 and Figure 3As shown, under the action of ±13kV triangular wave high voltage, the mixed gas of methane, hydrogen and argon stimulates plasma jet 8, and a diamond insulating coating 7 with a thickness of 200nm is deposited on the electrode surface to improve the long-term recording stability and anti-electromagnetic interference ability. Figure 2 The cross-sectional structure of the electrode is, from the inside out, a nanochannel 2, a nanopipette 1, metal nanoparticles 3 (gold), a metal oxide sensing coating 4 (iridium oxide), a roughened and modified carbon-based sensing coating 6 (graphene coating) and an insulating coating 7 (diamond insulating coating).

[0073] S5, refer to Figure 1 and Figure 3 As shown, the plasma jet is first excited on the stainless steel substrate, and then the tip of the nanoelectrode is controlled to gradually approach the excited jet. When the two are 3 mm apart, a thin branch of the plasma jet with a diameter of about 50 nm at the thinnest part will focus on the tip of the nanoelectrode and stay for 5 seconds. The diamond insulating coating is selectively removed, and the iridium oxide coating is exposed by 1000 nm, the boron-doped diamond coating is exposed by 500 nm, and the nanochannel is exposed by 100 nm, thereby obtaining an intracellular implantable multifunctional carbon-coated nanoelectrode. The electrode has the ability to detect targeted sites in specific intracellular regions. The nanochannel can detect intracellular electrical signals, the iridium oxide coating can detect intracellular pH, the roughened and modified graphene coating can detect reactive oxygen and reactive nitrogen, and the diamond insulating coating can ensure the long-term stability of the detection signal, thereby realizing the simultaneous detection of intracellular electrical signals, pH and biomolecules.

[0074] The above embodiment of the present invention is based on layer-by-layer deposition and low-temperature plasma jet processing technology. First, metal nanoparticles and metal oxide sensing coatings are sputtered on the surface of the drawn nanopipette. Then, the precursor is converted into a carbon-based sensing coating by using a low-temperature plasma jet. The surface of the carbon-based sensing coating is modified to roughen and introduce oxygen-containing functional groups. Finally, a nanodiamond insulating coating is deposited by using a plasma jet, and the electrode tip is selectively etched by using a jet branch to expose the sensing coating, the carbon-based sensing coating and the nanopipette at the same time, so that the electrical signals, pH and biomolecules such as oxygen implanted in cells can be detected simultaneously. In addition, the electrode has excellent electrochemical properties, anti-biological contamination performance, long-term stability and biocompatibility.

[0075] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or variations within the scope of the claims, which does not affect the essence of the present invention. The above preferred features can be used in any combination without conflicting with each other.

Claims

1. A method for preparing an intracellular implantable multifunctional carbon-coated nanoelectrode, characterized in that: include: Drawing a glass tube to form a nanopipette, wherein the nanopipette has a nanochannel inside, sequentially sputtering metal nanoparticles and a metal oxide sensing coating on the surface of the nanopipette, and wiring from the metal nanoparticles; After treating the precursor with a low-temperature plasma jet, a carbon-based sensing coating is deposited on the surface of the metal oxide sensing coating; Using a plasma jet to perform surface modification treatment on the carbon-based sensing coating to obtain a roughened and modified carbon-based sensing coating and expose the nanochannel; Depositing an insulating coating on the surface of the roughened and modified carbon-based sensing coating using a plasma jet; The electrode tip is selectively etched by using a branch of a plasma jet, so that the extent to which the metal oxide sensing coating, the carbon-based sensing coating and the nanopipette are exposed outside the insulating coating can be controlled.

2. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The method of drawing a glass tube to form a nanopipette comprises: drawing the glass tube by a heating drawing method to obtain the nanopipette.

3. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The method comprises using a low-temperature plasma jet to process a precursor, wherein the precursor is any one or more of methane, hydrogen, argon and boron oxide; A carbon-based sensing coating is deposited on the surface of the metal oxide sensing coating, wherein the carbon-based sensing coating is any one of graphene, carbon nanotubes, diamond-like carbon and boron-doped diamond.

4. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The surface modification treatment of the carbon-based sensing coating is performed by using a plasma jet, wherein the exciting gas of the plasma jet is a mixed gas of oxygen and helium.

5. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The surface modification treatment of the carbon-based sensing coating is performed using a plasma jet, wherein the angle between the axis of the electrode tip and the axis of the plasma jet is 0-360°.

6. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The method of using a plasma jet to deposit an insulating coating on the surface of the roughened and modified carbon-based sensing coating comprises: under the action of a sinusoidal high voltage of ±8-±15kV, a mixed gas of methane, hydrogen and argon excites a plasma jet to deposit a diamond insulating coating with a thickness of 0.05-10μm on the electrode surface.

7. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The method of selectively etching the electrode tip using a branch of a plasma jet includes: first exciting a plasma jet on a conductive substrate, then controlling the tip of the nanoelectrode to gradually approach the excited jet, and when a predetermined distance is reached, a small branch of the plasma jet is focused on the tip of the nanoelectrode to selectively remove the surface insulating coating, so that the exposure degree of other internal coatings can be controlled.

8. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 7, characterized in that: The plasma jet is first excited on a conductive substrate, wherein: the voltage waveform for exciting the plasma jet is any one of an AC pulse wave, a square wave, a triangle wave, a sound wave, a sine wave, a sawtooth wave and a trapezoidal wave; A thin branch of the plasma jet is focused on the tip of the nanoelectrode, wherein the diameter of the thinnest part of the branch of the plasma jet is 0.01-1000 μm.

9. The method for preparing the intracellular implantable multifunctional carbon-coated nanoelectrode according to claim 1, characterized in that: The total exposed length of the electrode tip was 0-100 μm.

10. An intracellular implantable multifunctional carbon-coated nanoelectrode, characterized in that: The nanoelectrode is prepared by the method for preparing an intracellularly implantable multifunctional carbon-coated nanoelectrode according to any one of claims 1 to 9.

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