Gold nanowire micro-nano probe as well as preparation method and application thereof

Abstract

The invention discloses a gold nanowire micro-nano probe as well as a preparation method and application thereof, and belongs to the technical field of nano electrochemical sensors and biomolecule detection. The gold nanowire micro-nano probe comprises a glass capillary substrate, a gold nanowire layer and an insulation sealing layer, the glass capillary base body is provided with a tip area, the tail end of the tip area is provided with an electroactive area, the surface of the tip area is wrapped with a gold nanowire layer, and the surface of the gold nanowire layer is wrapped with an insulating sealing layer except the electroactive area. According to the method, a three-dimensional gold nanowire network is grown on the surface of an insulating glass needle tip in situ through amino silanization modification, gold nanoparticle self-assembly seed crystal and a seed-mediated growth method. The insulating substrate is adopted to reduce background current, and the signal-to-noise ratio is high; the accurate control of the electroactive region is realized through a photoresist patterning process; the preparation process is simple, the cost is low, and the method can be widely applied to the single cell analysis fields of intracellular active oxygen species detection, neurotransmitter detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of nanoelectrochemical sensors and biomolecular detection technology, specifically relating to a gold nanowire micro / nano probe, its preparation method, and its application. Background Technology

[0002] Micro and nano probes, due to their superior properties such as small size, fast mass transfer rate, small iR drop, and low time constant, have shown broad application prospects in single-cell analysis and microscale electrochemical detection. Gold probes, with their good biocompatibility, conductivity, and ease of chemical modification via gold-sulfur bonds, have become a hot topic in micro and nanoelectrochemical sensing research. Traditional analytical methods typically perform average measurements on populations of millions of cells, which often masks intercellular heterogeneity. However, many key processes of life (such as redox homeostasis, neurotransmitter dynamics, and ion oscillations) occur within single cells (typically 10-20 µm in size) or even organelles. To "invade" these tiny spaces for real-time in-situ analysis without causing fatal cell damage, the size of the electrodes must be reduced to the micrometer or even nanometer scale.

[0003] Currently, common strategies for constructing gold micro / nano probes at the micro / nano scale mainly fall into two categories: one is to introduce gold nanostructures on conductive substrates through electrochemical deposition or chemical modification. For example, Chinese patent CN105842312B discloses a "nanoflower-shaped ultramicro gold electrode and its preparation and application," which uses carbon fiber as a substrate and prepares nanoflower-shaped gold structures at the tips of the carbon fibers through electrophoretic coating and electrochemical deposition. However, the carbon fiber substrate itself is a conductive material, which generates background current and affects the detection signal-to-noise ratio. The other strategy is to use microfabrication techniques such as photolithography to prepare gold electrode arrays on planar substrates. For example, Chinese patent CN107561140A discloses a "carbon nitride-modified ultramicro electrode, its preparation method and its application," which prepares ultramicro electrodes on ITO conductive glass. However, planar electrodes are difficult to apply to in-situ detection at the microscale, such as single-cell analysis, and microfabrication techniques require advanced equipment, are cumbersome to operate, and are expensive.

[0004] In addition, the insulating encapsulation of micro-nano probes in the prior art is mostly done by manual coating or dip coating, which makes it difficult to accurately control the length of the exposed electroactive region, resulting in poor geometric consistency and detection reproducibility of the probe.

[0005] In summary, the existing technology has the following shortcomings: First, the conductive substrate itself generates background current, which affects the detection signal-to-noise ratio; second, planar electrodes are difficult to apply to in-situ detection at the microscale; third, the existing preparation methods are complex and require high-end equipment.

[0006] Therefore, how to prepare gold nanowire micro / nano probes with good mechanical stability, controllable size, and precise electroactive regions is a technical problem that urgently needs to be solved. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the first objective of this invention is to provide a gold nanowire micro / nano probe that uses borosilicate or quartz glass micro / nano tips as a substrate and grows gold nanowires in situ using a seed-mediated growth method, exhibiting excellent mechanical stability and electrochemical performance.

[0008] The second objective of this invention is to provide a method for preparing gold nanowire micro / nano probes. By combining seed-mediated growth technology, conductive silver paste encapsulation process, and photoresist micro / nano fabrication technology, the method achieves simple encapsulation of the probes and precise control of the electroactive regions.

[0009] The third objective of this invention is to provide an application of gold nanowire micro / nano probes. The gold nanowire micro / nano probes provided by this invention can be applied to single-cell or cell spheroid analysis and highly sensitive electrochemical sensing, and have the characteristics of wide application range.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The present invention discloses a gold nanowire micro / nano probe, comprising a glass capillary substrate, a gold nanowire layer, and an insulating sealing layer; the glass capillary substrate has a tip region, the diameter of the tip region end being nanometer-scale and / or micrometer-scale, the tip region end having an electroactive region, the surface of the tip region being coated with a gold nanowire layer, and the surface of the gold nanowire layer being coated with an insulating sealing layer except for the electroactive region.

[0012] The gold nanowire micro / nano probe provided by this invention uses a glass capillary substrate, which completely eliminates the background current of the substrate itself, significantly improves the signal-to-noise ratio and sensitivity of electrochemical detection, and has good mechanical strength and chemical inertness. It is extremely stable in the growth solution, ensuring the mechanical strength and integrity of the probe when penetrating living cells. A gold nanowire layer is wrapped in the tip area of ​​the glass capillary substrate as a conductive layer. The gold nanowire layer has the following advantages: (1) Excellent conductivity: gold has extremely low resistivity, ensuring efficient transmission of electrical signals; (2) High specific surface area: the three-dimensional nanowire network structure greatly increases the electrochemical active area, improving detection sensitivity; (3) Good biocompatibility: gold material is non-toxic and non-immunogenic, suitable for in situ detection in cells; (4) Easy functionalization modification: various functional molecules (such as enzymes, aptamers, antibodies, DNA probes, etc.) can be easily connected through gold-sulfur bonds (Au-S), expanding the application range of the probe. Then, an insulating sealing layer is used to wrap the probe, exposing only the electroactive region at the end, which improves the geometric consistency of the electrode and the reproducibility of the detection data.

[0013] In a preferred embodiment, the glass in the glass capillary matrix is ​​quartz glass or borosilicate glass.

[0014] In a preferred embodiment, the diameter of the tip region is 50 nm-500 μm.

[0015] Further preferably, the diameter of the tip region is 50 nm to 999 nm, or 1 μm to 200 μm. Experiments have shown that when the diameter of the tip region is 50 nm to 999 nm, the probe exhibits extremely high spatial resolution, allowing for non-destructive penetration into single cells and enabling highly sensitive detection of the microenvironment of organelles (such as mitochondria and lysosomes) or the release of single vesicles within single cells. When the diameter of the tip region is 1 μm to 200 μm, the probe possesses high mechanical rigidity and a large electroactive area, making it suitable for puncture analysis or surface scanning of larger-scale samples such as multicellular spheres, brain slices, and tissue sections.

[0016] In a preferred embodiment, the gold nanowire layer has a three-dimensional nanowire network structure, with each gold nanowire having a diameter of 20-100 nm and a length of 200 nm-5 μm. The gold nanowire layer provided by this invention has a three-dimensional nanowire network structure, forming numerous nanoscale pores. This three-dimensional porous network structure significantly increases the effective specific surface area of ​​the probe, improving the density of electrochemical active sites and detection sensitivity.

[0017] In a preferred embodiment, the length of the electroactive region is 100 nm-100 μm.

[0018] Preferably, the surface of the electroactive region is further modified with a functionalized layer, the material of which is selected from at least one of Pt nanoparticles, enzymes, or aptamers.

[0019] In a preferred embodiment, the insulating sealing layer is made of negative photoresist or positive photoresist.

[0020] Negative photoresists such as the SU-8 series, or positive photoresists such as the AZ series.

[0021] In a preferred embodiment, the gold nanowire micro / nano probe further includes a conductive connector, which comprises a metal wire and a conductive silver paste. The metal wire is electrically connected to the gold nanowire layer located at the non-end portion of the tip region via the conductive silver paste.

[0022] This invention also provides a method for preparing gold nanowire micro / nano probes. A continuous gold nanowire layer is grown in the tip region of a glass capillary substrate using a seed-mediated growth method. Photoresist is coated on the surface of the gold nanowire layer. The photoresist located at the tip end of the glass capillary substrate is patterned, exposed, and developed. The photoresist at the tip end is selectively removed to expose the electroactive region.

[0023] In a preferred embodiment, the glass capillary substrate is obtained by drawing a quartz glass capillary or a borosilicate glass capillary using a laser needle drawing instrument, based on the designed diameter of the tip region.

[0024] Further optimization involves using the following parameters when the diameter of the tip region is 50 nm to 999 nm: Heat: 350 to 850 °C, Pull: 150 to 220 °C, Velocity: 50 to 80 °C, Delay: 130 to 150 °C, and Filament: 4 to 5 °C. This relatively high laser heating temperature and large pull force are used to form a slender taper.

[0025] Further preferred, when drawing quartz glass capillaries using a laser needle drawing instrument, the heating value (Heat) is 750~850, and when drawing borosilicate glass capillaries using a laser needle drawing instrument, the heating value (Heat) is 350~450.

[0026] Further preferred, when the diameter of the tip region is 1 μm ~ 200 μm, the drawing parameters are: Heat: 300 ~ 750, Pull: 50 ~ 100, Velocity: 20 ~ 50, Delay: 110 ~ 128, Filament: 2 ~ 3.

[0027] Further preferred, when drawing quartz glass capillaries using a laser needle drawing instrument, the heating value (Heat) is 650~750, and when drawing borosilicate glass capillaries using a laser needle drawing instrument, the heating value (Heat) is 300~380.

[0028] In a preferred embodiment, the process of growing a continuous gold nanowire layer in the tip region of a glass capillary substrate using a seed-mediated growth method is as follows: the tip of the glass capillary substrate is immersed in an alkaline solution for activation treatment, then neutralized with an acid solution, washed and dried, and then the activated tip region of the glass capillary substrate is immersed in a silane solution to introduce amino functional groups. Then, the tip region modified with amino functional groups is immersed in a gold nanoparticle solution, allowing the gold nanoparticles to self-assemble on the surface of the tip region to form a seed layer. Finally, the tip region containing the seed layer is immersed in a growth solution to react, and gold nanowires are grown on the surface of the tip region to form a gold nanowire layer.

[0029] In a further preferred embodiment, the alkaline solution is a NaOH solution with a NaOH concentration of 1-5 M, and the acid solution is an HCl solution with a dissolved HCl concentration of 0.05-0.5 M.

[0030] In a further preferred embodiment, the activation treatment time is 1 to 5 minutes.

[0031] In a further preferred embodiment, the silane solution is an ethanol solution containing 3-aminopropyltrimethoxysilane, wherein the volume ratio of 3-aminopropyltrimethoxysilane to anhydrous ethanol is (0.3~1.5) mL: (40~60) mL.

[0032] In a further preferred embodiment, the growth solution is obtained by mixing anhydrous ethanol, ultrapure water, 4-MBA solution, HAuCl4 solution, and ascorbic acid solution, wherein the volume ratio is: anhydrous ethanol: ultrapure water: 4-MBA solution: HAuCl4 solution: ascorbic acid solution = (4~7): (1~3): (0.2~0.5): (8~12): (1.5~2.5).

[0033] In a further preferred embodiment, the 4-MBA solution is obtained by dissolving 4-mercaptobenzoic acid powder in anhydrous ethanol, wherein the solid-liquid mass-volume ratio of 4-mercaptobenzoic acid powder to anhydrous ethanol is (15~30) mg: 1 mL.

[0034] More preferably, the concentration of the HAuCl4 solution is 10~30 mM.

[0035] In a further preferred embodiment, the ascorbic acid solution is obtained by dissolving L-ascorbic acid powder in ultrapure water, wherein the solid-liquid mass-volume ratio of L-ascorbic acid powder to ultrapure water is (60~120) mg: 1 mL.

[0036] Experiments revealed that glass capillary substrates, being insulating materials, cannot directly grow gold nanostructures via electrochemical deposition, necessitating a complete chemical reduction method. Furthermore, the strong chemical inertness of the glass surface lacks active sites for direct binding with gold nanoparticles, making it difficult for seed crystals to adhere uniformly and firmly. Additionally, the poor wettability of the glass surface hinders the uniform coverage of the micro / nano tips by the growth solution, and the grown gold nanowire layer is prone to detachment. To overcome these shortcomings, this invention employs a high-concentration NaOH solution (1-5 M) for activation treatment, hydrolyzing the glass surface to generate a large number of Si-OH hydroxyl groups, increasing the density of surface active sites and providing anchoring groups for subsequent silanization modification. 3-Aminopropyltrimethoxysilane is used to form a self-assembled monolayer on the glass surface, introducing -NH2 amino functional groups. The amino groups can firmly bind to negatively charged gold nanoparticles (stable by sodium citrate) through electrostatic interactions, solving the problem of poor seed crystal adhesion. The -SH group at one end of the 4-mercaptobenzoic acid molecule forms an Au-S covalent bond with the gold surface, while the -COOH group at the other end faces the solution. 4-MBA molecules act as "structure-directing agents," guiding gold to preferentially grow along specific crystal planes, forming one-dimensional nanowire structures rather than amorphous particle aggregates. L-ascorbic acid is used as a reducing agent; its moderate reduction potential allows for the slow reduction of Au at room temperature. 3+ The presence of ions facilitates the controllable growth of gold nanowires and avoids the disordered aggregation of nanoparticles caused by rapid reduction. Through the above-mentioned technical improvements, this invention successfully overcomes the technical difficulties of growing gold nanostructures on insulating glass substrates and achieves the controllable fabrication of high-quality three-dimensional gold nanowire networks.

[0037] In a preferred embodiment, one end of a metal wire is placed on the surface of a gold nanowire layer in the non-terminal portion of the tip region, coated with conductive silver paste, and then cured to achieve electrical connection.

[0038] In actual operation, after the electrical connection is completed, photoresist is coated on the surface of the gold nanowire layer and the electrical connection surface.

[0039] In a preferred embodiment, after exposing the electroactive region, a diamond micro / nano probe is obtained through hard baking. The insulating sealing layer is then cured by hard baking.

[0040] This invention also provides an application of diamond micro / nano probes, which are used in electrochemical sensors, biomolecule detection, and heavy metal ion detection.

[0041] In a preferred embodiment, the biomolecule detection is selected from one of the following: single-cell detection, cell spheroid detection, and tissue microenvironment (such as ROS, RNS, and neurotransmitters).

[0042] The abbreviations used in this invention are as follows: APTMS is the abbreviation for 3-Aminopropyltrimethoxysilane; 4-MBA is the abbreviation for 4-mercaptobenzoic acid; and AuNPs is the abbreviation for gold nanoparticles.

[0043] Beneficial effects

[0044] Compared with the prior art, the present invention has the following significant advantages:

[0045] Innovative Substrate, High Signal-to-Noise Ratio: This invention is the first to propose the direct growth of gold nanowires on an insulating borosilicate glass tip substrate. Compared with existing techniques that use conductive substrates (such as carbon fibers or ITO conductive glass), this invention utilizes the insulating properties of glass to completely eliminate the background current of the substrate itself, significantly improving the signal-to-noise ratio and sensitivity of electrochemical detection. Experimental results show that the probe of this invention exhibits extremely low background current in potassium ferricyanide detection, displaying a typical "S"-shaped steady-state voltammetry curve.

[0046] High mechanical stability: This invention uses borosilicate glass capillary nanoneedles as the matrix. Compared with carbon fiber or metal wire, borosilicate glass has good mechanical strength and chemical inertness, and is extremely stable in the growth solution, ensuring the mechanical strength and integrity of the probe when it penetrates living cells.

[0047] Wide size range and strong adaptability: The diameter of the micro-nano probe tip of this invention can be adjusted within the range of 50 nm-50 μm to meet the needs of detection in different micro-regions. It can be used to prepare nanoscale probes for detection inside single cells, as well as micron-scale probes for analysis of larger-scale samples such as cell spheres and tissue sections, with a wide range of applications.

[0048] Precise and controllable electroactive regions: This invention innovatively introduces photoresist as an insulating layer and combines it with laser direct-write lithography technology. Compared with the traditional dip-coating insulating method, this method utilizes the photosensitive properties of photoresist to precisely control the length of the exposed micro-nano tips (e.g., controlled at 1 μm, 2 μm, 10 or 50 μm, etc.) by adjusting the exposure area, which greatly improves the geometric consistency of the electrodes and the reproducibility of detection data.

[0049] The packaging process is simplified and reliable: This invention abandons the complex "external capillary + paraffin + liquid metal" packaging method and adopts a "conductive silver paste + photoresist full-encapsulation" solution. The conductive silver paste provides a stable ohmic contact, and the photoresist not only acts as an insulation layer but also seals the connection points as a protective layer, greatly simplifying the preparation process and improving the yield.

[0050] Large specific surface area and high sensitivity: This invention constructs a three-dimensional gold nanowire network structure on the probe tip surface using a seed-mediated growth method, which greatly increases the effective specific surface area of ​​the probe. Combined with subsequent functionalization modifications (such as Pt nanoparticles), the electrocatalytic activity and detection sensitivity for molecules such as H2O2 can be significantly improved.

[0051] Excellent electrochemical performance: The prepared gold nanowire micro / nano probes retain the excellent conductivity and electrochemical activity of gold materials. Combined with the high mass transfer rate of micro / nano electrodes, they enable highly sensitive detection of single cells, cell spheres, or microenvironments within tissues. Attached Figure Description

[0052] Figure 1 The image shows the tip morphology of the gold nanowire micro / nano probe prepared in Example 1 of this invention.

[0053] Figure 2 This is a process flow diagram of the gold nanowire micro / nano probes prepared according to the present invention.

[0054] Figure 3 This is an optical microscope image of the gold nanowire micro-nano probe prepared in Example 1 of the present invention after gold nanowires have been grown and before encapsulation. It can be seen that the gold nanowire layer is uniformly covered on the surface of the borosilicate glass tip.

[0055] Figure 4 This is a microscopic morphology (SEM) image of the gold nanowire micro / nano probe in Example 1 of the present invention.

[0056] Figure 5 This is the X-ray energy dispersive spectroscopy (EDS) diagram of the gold nanowire micro / nano probe in Example 1 of the present invention.

[0057] Figure 6 This is a cyclic voltammogram of the gold nanowire micro / nano probe in 5 mM potassium ferricyanide solution in Example 1 of the present invention.

[0058] Figure 7 The image shows the cyclic voltammetric response curve of the gold nanowire micro / nano probe used to detect NaNO2 in Example 1 of this invention.

[0059] Figure 8 In Example 2 of this invention, Pt-modified gold nanowire micro / nano probes were used to detect the cyclic voltammetric response curve of H2O2. Detailed Implementation

[0060] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0061] Example 1: Fabrication of gold nanowire micro / nano probes on a borosilicate glass substrate based on positive photoresist insulation

[0062] Step 1: Preparation of borosilicate glass nanopipette substrate

[0063] Material preparation: Standard borosilicate glass capillary tubes with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm were selected as raw materials.

[0064] Laser drawing: Using a commercial laser drawing instrument (such as Sutter P-2000), the parameters are set as follows: Heat = 350, Filament = 3, Velocity = 30, Delay = 120, Pull = 80, borosilicate glass capillary is drawn into a conical nanopipette with a tip diameter of about 4 μm.

[0065] Cleaning process: The drawn substrate is immersed in acetone, anhydrous ethanol and ultrapure water in sequence and ultrasonically cleaned for 10 minutes each to thoroughly remove surface organic contaminants. Finally, it is dried with high-purity nitrogen gas for later use.

[0066] Step 2: Gold Nanowire Growth

[0067] Pretreatment: Immerse the substrate tip in 3 M NaOH solution for 2 min for activation treatment, remove it and immerse it in 0.1 M HCl solution to neutralize the residual alkali, and finally rinse it thoroughly with ultrapure water 3 times and blow it dry with nitrogen.

[0068] Aminosilanization modification: Prepare an APTMS-ethanol solution (500 μL APTMS + 50 ml anhydrous ethanol, mix well). Immerse the activated matrix tip in the APTMS solution and soak at room temperature for 1 h. After removal, gently wash three times with anhydrous ethanol and dry with nitrogen.

[0069] Assemble the seed crystal: Immerse the silanized substrate tip in AuNPs solution and soak at room temperature for 1 hour. After removal, gently rinse three times with ultrapure water and dry with nitrogen.

[0070] Preparation of growth medium: In a clean glass container, add the following in sequence: 5.4 ml anhydrous ethanol, 1.8 ml ultrapure water, 0.3 ml 4-MBA solution (200 mg 4-mercaptobenzoic acid powder dissolved in 10 ml anhydrous ethanol, sonicated for 1 min), 10 ml 25 mM HAuCl4 solution, and 2.0 ml ascorbic acid solution (900 mg L-ascorbic acid powder dissolved in 10 ml ultrapure water, sonicated for 1 min). Mix gently and let stand for later use.

[0071] Nanowire growth: Immerse the tip of the substrate assembled with seed crystals into the above growth solution and allow it to react at room temperature for 10 minutes. After removal, gently wash with anhydrous ethanol and dry with nitrogen gas. Figure 3This is an optical microscope image of the actual object before encapsulation after the growth of gold nanowires. It can be seen that the surface of the borosilicate glass needle tip is uniformly covered with a layer of gold nanowires, exhibiting a characteristic golden yellow color, indicating that the gold nanowires have been successfully grown and are evenly distributed. Figure 4 SEM images of the microstructure of gold nanowires grown from borosilicate glass nanotubes are shown, revealing a three-dimensional network structure of gold nanowires. Figure 5 The corresponding EDS spectrum shows obvious characteristic peaks of gold.

[0072] Step 3: Conductive connection and positive photoresist insulating encapsulation

[0073] Conductive connection: Place one end of the copper wire in the non-tip region (such as the tail or middle section) of the borosilicate glass capillary on which the gold nanowire layer has grown, and apply conductive silver paste to firmly bond the copper wire to the gold nanowire layer. After the silver paste has completely cured, a stable electrical connection is achieved.

[0074] Coating with photoresist: Fix the probe with the connected wires on the spin coater, add positive photoresist (such as AZ5214), and spin coat at 8000 rpm for 40 s to make the photoresist evenly coat the surface of the gold nanowire layer and the conductive connection, forming an insulating and sealing layer.

[0075] Pre-baking: Soft bake on a hot plate at 100 ℃ for 120 s to remove solvent.

[0076] Patterned exposure: Using a laser direct-write lithography machine with a preset pattern, laser exposure is performed only on the very tip of the micro-nano probe.

[0077] Development: The probe is immersed in an alkaline developer (such as TMAH solution) for approximately 50 seconds. Due to the use of positive photoresist, the photoresist in the laser-irradiated tip region undergoes a photochemical reaction and dissolves, exposing the underlying gold nanowire micro-tip, forming a precisely defined electroactive region, while the remaining portion remains insulated. A schematic diagram of the structure is shown below. Figure 1 As shown.

[0078] Hard baking: Hard bake at 120-130℃ for 30 minutes. The overall process flow is as follows: Figure 2 As shown.

[0079] Step 4: Electrochemical Characterization

[0080] A three-electrode system was used: the prepared gold nanowire micro / nano probe was used as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. Cyclic voltammetry was performed in a 5 mM K3[Fe(CN)6] solution containing 0.1 M KCl at a scan rate of 50 mV / s. The test results are as follows: Figure 6As shown, the probe exhibits a typical "S"-shaped steady-state current-voltage curve, indicating that it has excellent micro / nano electrode characteristics and extremely low background current.

[0081] NaNO2 detection verification: Figure 7 Cyclic voltammetric response curves of gold nanowire micro / nano probes in a solution containing 1 mM NaNO2 are shown. The obvious redox peaks demonstrate that the probes have good electrocatalytic activity towards NaNO2.

[0082] Prospects for intracellular detection: By inserting functionalized microprobes into target cell spheres and applying specific oxidation potentials for real-time monitoring (e.g., +600 mV for NaNO2 detection), real-time monitoring of intracellular reactive oxygen species can be achieved.

[0083] Example 2: Fabrication and Detection of Reactive Oxygen Species Using Pt-Modified Gold Nanowire Micro / Nano Probes Based on Negative Photoresist Insulation

[0084] Step 1: Preparation of quartz glass nanopipette substrate

[0085] Material preparation: Standard borosilicate glass capillary tubes with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm were selected as raw materials.

[0086] Laser drawing: Using a commercial laser drawing instrument (such as Sutter P-2000), the parameters are set as follows: Heat = 800, Filament = 5, Velocity = 80, Delay = 130, Pull = 200, to draw a quartz glass capillary into a tapered nanopipette with a tip diameter of approximately 200 nm.

[0087] Cleaning process: The drawn substrate is immersed in acetone, anhydrous ethanol and ultrapure water in sequence and ultrasonically cleaned for 10 minutes each to thoroughly remove surface organic contaminants. Finally, it is dried with high-purity nitrogen gas for later use.

[0088] Step 2: Gold Nanowire Growth

[0089] Same as step two in Example 1.

[0090] Step 3: Conductive connection and negative photoresist insulating encapsulation

[0091] Conductive connection: The copper wires are connected to the probe conductive layer using conductive silver paste and then cured.

[0092] Surface modification: UV ozone treatment for 30 min improves the adhesion between photoresist and gold surface.

[0093] Coating with photoresist: Apply negative photoresist (such as SU8-2035) by dotting or dipping to cover the probe surface and connection points.

[0094] Pre-baking: Baking in stages at 65 ℃ and 95 ℃ to slowly evaporate the solvent.

[0095] Patterned exposure: Using a laser direct-write lithography machine, laser exposure (50 mW, 10 s) is performed on the area far from the tip of the micro / nano probe (i.e., the non-tip area), while the tip area is left unexposed.

[0096] Development: Immerse in PGMEA developer for 60 seconds. Due to the use of negative photoresist, the exposed areas crosslink and solidify, while the photoresist in the unexposed tip areas is dissolved, thereby exposing the electroactive regions of the gold nanowire micro / nano probe.

[0097] Hard baking: Hard bake at 150℃ for 30 minutes.

[0098] Step 4: Functionalization and Detection Application of Reactive Oxygen Species (ROS)

[0099] Preparation of platinum electroplating solution: In a clean container, add 6.4 mL of PBS buffer (10 mM, pH 7.4), 125 µL of chloroplatinic acid solution (8 wt.% H2PtCl6), and 0.2 mg of lead acetate trihydrate (Pb(CH3COO)2·3H2O) sequentially. Mix ultrasonically for 1 min to obtain the platinum electroplating solution. Lead acetate acts as a grain refiner, resulting in finer and more uniform deposited platinum nanoparticles, thus increasing the specific surface area and electrocatalytic activity.

[0100] Surface activation: The encapsulated gold nanowire micro / nano probes were immersed in the aforementioned platinum electroplating solution. A three-electrode system was used (probe as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode). Cyclic voltammetry was applied for two cycles at a scan rate of 50 mV / s, with a potential range of 0 V to -0.35 V (vs. Ag / AgCl). This activation step cleans the electrode surface and enhances the adhesion of the platinum seed crystals.

[0101] Potentiostatic deposition: Immediately after activation, an amperometric method (it) was used to apply a constant potential of -0.15 V (vs. Ag / AgCl) for 200 s to reduce and deposit high-density Pt nanoparticles on the surface of gold nanowires. The presence of lead acetate inhibited the excessive growth of platinum particles, forming a uniformly sized nanoplatinum layer.

[0102] H2O2 detection verification: Figure 7 The cyclic voltammetric response curves of the Pt-modified gold nanowire micro / nano probe in a solution containing 1 mM H2O2 are shown. The obvious redox peaks demonstrate that the probe has good electrocatalytic activity for H2O2.

[0103] Prospects for intracellular detection: By inserting functionalized probes into target cells and applying specific oxidation potentials for real-time monitoring (e.g., +500 mV for H2O2 detection), it is possible to achieve real-time monitoring of intracellular reactive oxygen species.

[0104] Comparative Example 1

[0105] Other conditions are the same as in Example 1, except that the APTMS silanization modification step is omitted, that is, after alkaline activation and acid neutralization, the substrate tip is directly immersed in the gold nanoparticle solution.

[0106] Results: Due to the lack of amino functional groups on the glass surface, the negatively charged gold nanoparticles repel the negatively charged glass surface and cannot be effectively adsorbed, resulting in only a small number of gold nanoparticles being scattered. After reaction in the growth solution, the gold nanowires only grow at individual seed sites, forming isolated island structures and failing to form a continuous three-dimensional network. The probe conductivity is extremely poor, failing to meet the requirements for electrochemical detection.

[0107] Comparative Example 2

[0108] The other conditions were the same as in Example 1, except that 4-MBA solution was not added when preparing the growth medium.

[0109] Results: In the absence of 4-MBA structure-directing agents, ascorbic acid reduced Au. 3+ The resulting structure is a random aggregate of gold nanoparticles, rather than a one-dimensional nanowire structure. During subsequent encapsulation and use, the gold nanoparticles are prone to detachment. Electrochemical testing shows that the electroactive area of ​​this probe is only 30% of that in Example 1, resulting in a significant decrease in detection sensitivity.

Claims

1. A gold nanowire micro / nano probe, characterized in that: It includes a glass capillary substrate, a gold nanowire layer, and an insulating sealing layer; the glass capillary substrate has a tip region with a diameter at the end of the tip region being in the nanometer and / or micrometer range, the end of the tip region having an electroactive region, the surface of the tip region being coated with a gold nanowire layer, and the surface of the gold nanowire layer being coated with an insulating sealing layer except for the electroactive region.

2. The gold nanowire micro / nano probe according to claim 1, characterized in that: The glass in the glass capillary matrix is ​​quartz glass or borosilicate glass; The diameter of the tip region is 50 nm to 500 μm. The gold nanowire layer has a three-dimensional nanowire network structure, with the diameter of a single gold nanowire being 20~100 nm and the length being 200 nm~5 μm. The length of the electroactive region is 100 nm to 100 μm.

3. The gold nanowire micro / nano probe according to claim 1, characterized in that: The diameter of the tip region is 50 nm to 999 nm, or the diameter of the tip region is 1 μm to 200 μm. The surface of the electroactive region is further modified with a functionalized layer, the material of which is selected from at least one of Pt nanoparticles, enzymes, or aptamers. The insulating sealing layer is made of negative photoresist or positive photoresist; The gold nanowire micro / nano probe also includes a conductive connector, which comprises a metal wire and a conductive silver paste. The metal wire is electrically connected to the gold nanowire layer located at the non-end portion of the tip region via the conductive silver paste.

4. A method for preparing a gold nanowire micro / nano probe according to any one of claims 1-3, characterized in that: A continuous gold nanowire layer was grown at the tip of a glass capillary substrate using a seed-mediated growth method. Photoresist was coated on the surface of the gold nanowire layer. The photoresist at the tip of the glass capillary substrate was patterned, exposed, and developed. The photoresist at the tip was selectively removed to expose the electroactive region.

5. The method for preparing a gold nanowire micro / nano probe according to claim 4, characterized in that: The glass capillary substrate is obtained by drawing quartz glass capillary or borosilicate glass capillary using a laser needle drawing instrument, based on the designed diameter of the tip region.

6. The method for preparing a gold nanowire micro / nano probe according to claim 5, characterized in that: When the diameter of the tip region is 50 nm ~ 999 nm, the pulling parameters are: heating value: 350 ~ 850, pulling force: 150 ~ 220, speed: 50 ~ 80, delay: 130 ~ 150, filament scanning range: 4 ~ 5; When the diameter of the tip region is 1 μm ~ 200 μm, the drawing parameters are: heating value: 300 ~ 750, tension: 50 ~ 100, speed: 20 ~ 50, delay: 110 ~ 128, filament scanning range: 2 ~ 3.

7. The method for preparing a gold nanowire micro / nano probe according to claim 4, characterized in that: The process of growing a continuous gold nanowire layer in the tip region of a glass capillary substrate using a seed-mediated growth method is as follows: The tip of the glass capillary substrate is immersed in an alkaline solution for activation treatment, then neutralized with an acid solution, washed and dried. The activated tip region of the glass capillary substrate is then immersed in a silane solution to introduce amino functional groups. The amino functional group-modified tip region is then immersed in a gold nanoparticle solution, allowing the gold nanoparticles to self-assemble on the surface of the tip region to form a seed layer. Finally, the tip region containing the seed layer is immersed in a growth solution to react, and gold nanowires are grown on the surface of the tip region to form a gold nanowire layer.

8. The method for preparing a gold nanowire micro / nano probe according to claim 7, characterized in that: The alkaline solution is a NaOH solution with a concentration of 1-5 M, and the acid solution is an HCl solution with a concentration of 0.05-0.5 M. The activation treatment time is 1~5 min; The silane solution is an ethanol solution containing 3-aminopropyltrimethoxysilane, wherein the volume ratio of 3-aminopropyltrimethoxysilane to anhydrous ethanol is 0.3~1.5 mL: 40~60 mL. The growth medium is obtained by mixing anhydrous ethanol, ultrapure water, 4-MBA solution, HAuCl4 solution, and ascorbic acid solution, wherein, by volume ratio, the ratio of anhydrous ethanol: ultrapure water: 4-MBA solution: HAuCl4 solution: ascorbic acid solution is 4~7: 1~3: 0.2~0.5: 8~12: 1.5~2.

5. The 4-MBA solution is obtained by dissolving 4-mercaptobenzoic acid powder in anhydrous ethanol, wherein the solid-liquid mass-volume ratio of 4-mercaptobenzoic acid powder to anhydrous ethanol is 15~30 mg:1 mL. The concentration of the HAuCl4 solution is 10~30 mM; The ascorbic acid solution is obtained by dissolving L-ascorbic acid powder in ultrapure water, wherein the solid-liquid mass-volume ratio of L-ascorbic acid powder to ultrapure water is 60~120 mg:1 mL.

9. The method for preparing a gold nanowire micro / nano probe according to claim 4, characterized in that: Electrical connection is achieved by coating a conductive silver paste onto the surface of a gold nanowire layer at the non-end portion of the tip region with one end of a metal wire and then curing it. After exposing the electroactive region, diamond micro / nano probes are obtained by hard baking.

10. The application of the gold nanowire micro / nano probe according to any one of claims 1-3, characterized in that: One application of diamond micro / nano probes is in electrochemical sensors, biomolecular detection, and heavy metal ion detection.

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