Diamond micro-nano probe and preparation method and application thereof

Abstract

The invention discloses a diamond micro-nano probe as well as a preparation method and application thereof, and belongs to the technical field of electrochemical sensors and biomolecule detection. The diamond micro-nano probe comprises a quartz capillary base body, a diamond film layer and an insulation sealing layer, the quartz capillary base body is provided with a tip area, the tail end of the tip area is provided with an electroactive area, and the surface of the tip area is wrapped with the diamond film layer. In the preparation process, by combining a chemical vapor deposition technology, a conductive silver paste packaging technology and a photoresist micro-nano processing technology, simple and convenient packaging of the electrode and accurate control of an electroactive region are realized. According to the invention, the quartz matrix is adopted to avoid the problems that a metal matrix is easy to brittle and break and carbon fibers are easy to etch, accurate control of an electroactive area is realized through a photoresist patterning technology, the packaging process is simple, convenient and reliable, and the method can be used for high-sensitivity detection of active oxygen / nitrogen species, neurotransmitters, heavy metal ions and the like in cells.

Description

Technical Field

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

[0002] Boron-doped diamond (BDD) electrodes have become ideal materials for bioelectrochemical analysis due to their extremely wide electrochemical window, extremely low background current, excellent corrosion resistance, and biocompatibility. Traditional analytical methods typically involve averaging measurements on populations of millions of cells, which often masks intercellular heterogeneity. However, many critical processes of life (such as redox homeostasis, neurotransmitter dynamics, and ion oscillations) occur within individual cells (typically 10-20 µm in size) or even organelles. To perform real-time in-situ analysis in these tiny spaces without causing fatal cell damage, detector sizes must be reduced to the micrometer or even nanometer scale.

[0003] However, current diamond electrode technologies are mostly at the tens or even hundreds of micrometer scale, and the fabrication of micro- and nano-scale diamond probes faces enormous technical challenges, mainly due to the following drawbacks:

[0004] (1) Size limitations and brittleness issues: Existing technologies typically deposit BDD on micron-sized metal substrates (such as tungsten wires), but during the deposition process, brittle tungsten carbide transition layers are easily generated, causing the electrode to fracture brittlely during testing and resulting in secondary damage to biological tissues.

[0005] (2) Substrate etching problem: Traditional carbon fiber substrates are easily etched by active hydrogen free radicals during chemical vapor deposition (CVD) BDD thin film deposition, making it difficult to directly deposit high-quality thin films.

[0006] (3) Difficult to process: Diamond is extremely hard and chemically inert, and traditional mechanical stretching, polishing or wet etching are difficult to achieve micro-nano level fine processing.

[0007] (4) Difficulty in controlling geometric morphology: Existing “coating method” is difficult to accurately control the geometric morphology and size of the electroactive region of micro-nano tip, resulting in poor detection reproducibility.

[0008] Therefore, how to fabricate diamond micro / nano probes with good mechanical stability, controllable size, and precise electroactive regions is a pressing technical challenge. Thus, there is an urgent need to develop a diamond micro / nano probe with good mechanical stability, controllable size, precise definition of the electroactive region, and simple packaging process, along with its fabrication method. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the first objective of this invention is to provide a diamond micro / nano probe. The diamond micro / nano probe provided by this invention uses a quartz micro / nano tip as a substrate, which solves the problems of easy brittleness and fracture of metal substrates and easy etching of carbon fibers, and has excellent mechanical stability.

[0010] The second objective of this invention is to provide a method for preparing diamond micro / nano probes, which combines chemical vapor deposition (CVD) technology, conductive silver paste encapsulation process and photoresist micro / nano fabrication technology to achieve simple encapsulation of electrodes and precise control of electroactive regions.

[0011] The third objective of this invention is to provide an application of diamond micro / nano probes. The micro / nano probes provided by this invention can be applied to single-cell or cell spheroid analysis as well as highly sensitive electrochemical sensing, and have a wide range of applications.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] This invention provides a diamond micro / nano probe comprising a quartz capillary substrate, a diamond thin film layer, and an insulating sealing layer. The quartz 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 tip region has an electroactive region at its end. The surface of the tip region is coated with a diamond thin film layer, and the surface of the diamond thin film layer, except for the electroactive region, is coated with an insulating sealing layer.

[0014] The diamond micro / nano probe provided by this invention uses a quartz capillary matrix to ensure the mechanical strength and integrity of the probe when it penetrates living cells. A diamond thin film layer is wrapped around the tip area of ​​the quartz capillary matrix as a conductive layer, and then wrapped with an insulating sealing layer, exposing only the electrically active area at the end.

[0015] In a preferred embodiment, the diameter of the tip region is 50 nm-500 μm.

[0016] 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.

[0017] In a preferred embodiment, the diamond in the diamond film layer is boron-doped diamond with a grain size of 5 nm to 100 nm and a boron atom concentration ≥ 10. 20 cm -3 Preferably 10 20 ~10 21 cm -3 By employing the aforementioned nanocrystalline diamond thin film layer and controlling the boron doping concentration within the above-mentioned range, optimal conductivity and best detection sensitivity are achieved.

[0018] In a preferred embodiment, the length of the electroactive region is 100 nm-100 μm.

[0019] 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.

[0020] In a preferred embodiment, the insulating sealing layer is made of negative photoresist or positive photoresist.

[0021] Negative photoresists such as the SU-8 series, or positive photoresists such as the AZ series.

[0022] In a preferred embodiment, the diamond 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 a diamond film layer located at the non-end portion of the tip region via the conductive silver paste.

[0023] The present invention also provides a method for preparing a diamond micro / nano probe, wherein a diamond thin film layer is deposited in the tip region of a quartz capillary substrate, photoresist is coated on the surface of the diamond thin film layer, the photoresist located at the tip end of the quartz capillary substrate is patterned, exposed and developed, and the photoresist at the tip end is selectively removed to expose the electroactive region.

[0024] In a preferred embodiment, the quartz capillary substrate is obtained by drawing a quartz capillary using a laser needle drawing instrument, based on the designed diameter of the tip region.

[0025] In actual operation, the Sutter P-2000 laser needle drawing machine is used.

[0026] Further optimization, when the diameter of the tip region is 50 nm to 999 nm, the pulling parameters are: Heat: 750 to 850 °C, Pull: 150 to 220 °C, Velocity: 50 to 80 °C, Delay: 130 to 150 °C, and Filament: 4 to 5 °C. A slender taper is formed by using a higher laser heating temperature and a larger pulling force.

[0027] Further preferred, when the diameter of the tip region is 1 μm ~ 200 μm, the drawing parameters are: Heat value: 650 ~ 750, Pull force: 50 ~ 100, Velocity: 20 ~ 50, Delay: 110 ~ 128, Filament scanning range: 2 ~ 3.

[0028] The preferred method involves depositing a diamond film layer in the tip region of a quartz capillary substrate as follows: first, the tip region of the quartz capillary substrate is ground or spin-coated with a nanodiamond suspension to plant seed crystals; then, the quartz capillary substrate with the seed crystals is placed in a deposition furnace for chemical vapor deposition.

[0029] In a further preferred embodiment, the nanodiamond particles in the nanodiamond suspension have a particle size of 4 nm to 18 nm, preferably 5-10 nm.

[0030] In a further preferred embodiment, during the chemical vapor deposition, the gas introduced is in the following flow ratio: methane: hydrogen: borane: argon = 1~10: 40~100: 0.05~2: 20~60, preferably 3~5: 50~100: 0.1~2: 40~60; the growth pressure is 1.5~4 kPa, preferably 2~3 kPa; the growth temperature is 600~750℃, preferably 600~700℃; and the growth time is 5~60 min.

[0031] The quartz capillary substrate used in this invention faces significant challenges in depositing high-quality BDD thin films. Firstly, SiO2 is easily etched in hydrogen-rich plasma, leading to tip structure damage. Secondly, nucleation at heterogeneous interfaces is difficult and adhesion is poor. Thirdly, the tip effect of nano-tip leads to localized overheating. The method of this invention addresses these challenges by using 5-10 nm seed crystals to increase nucleation density, enabling rapid nucleation and growth under the pressure conditions of this invention. Simultaneously, the growth temperature is appropriately reduced, and a short growth time strategy is employed. This ensures film continuity while avoiding grain coarsening and tip passivation caused by excessively long growth times. Furthermore, a higher proportion of argon gas is used to refine the grains while reducing hydrogen concentration, effectively suppressing the etching of quartz tips by hydrogen plasma. Under the synergistic effect of these conditions, nanocrystalline diamond probes that retain the geometric morphology of quartz micro / nano needles are successfully prepared.

[0032] In a further preferred embodiment, the chemical vapor deposition method is selected from hot filament chemical vapor deposition (HFCVD).

[0033] In a preferred embodiment, one end of the metal wire is placed on the surface of a diamond film layer in the non-terminal part of the tip area, coated with conductive silver paste and cured to achieve electrical connection.

[0034] In actual operation, after the electrical connection is completed, photoresist is coated on the surface of the diamond thin film layer and the electrical connection surface.

[0035] 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.

[0036] This invention also provides an application of diamond micro / nano probes, which are used in electrochemical sensors, biomolecular detection, and heavy metal ion detection.

[0037] 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).

[0038] Beneficial effects

[0039] Compared with the prior art, the present invention has the following significant advantages:

[0040] High mechanical stability: This invention uses quartz capillary nanoneedles as the matrix, which avoids the formation of brittle carbides during CVD compared to tungsten wire, and solves the problem of electrode breakage. Compared to carbon fiber, quartz is extremely stable in hydrogen plasma, ensuring the mechanical strength and integrity of the probe when it penetrates living cells.

[0041] Wide size range and strong adaptability: The diameter of the micro-nano probe tip of the present invention can be adjusted in the range of 50 nm-50 μm. It can be used to prepare nanoscale probes for single cell internal detection, as well as micron-scale probes for the analysis of larger-scale samples such as cell spheres and tissue sections, with a wide range of applications.

[0042] 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.

[0043] 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 insulator but also acts as a protective layer to seal the connection points, greatly simplifying the preparation process and improving the yield.

[0044] Excellent electrochemical performance: The prepared diamond micro-nano probes retain the wide potential window and low background current characteristics of BDD materials. Combined with the high mass transfer rate of micro-nano electrodes, they achieve highly sensitive detection of single cells, cell spheres or tissue microenvironments (such as ROS, RNS, neurotransmitters). Attached Figure Description

[0045] Figure 1 The image shows the tip morphology of the diamond micro / nano probe prepared in Example 1 of this invention.

[0046] Figure 2 This is a process flow diagram for preparing diamond micro / nano probes according to an embodiment of the present invention.

[0047] Figure 3 Optical image of a diamond micro / nano probe prepared in Example 1 of this invention inserted into a single cell.

[0048] Figure 4 This is a microscopic morphology (SEM) image of the diamond micro / nano probe tip in Embodiment 1 of the present invention.

[0049] Figure 5 An optical image of the diamond micro / nano probe prepared in Example 1 of this invention inserted into a cell sphere.

[0050] Figure 6 The cyclic voltammetric response curve of the Pt-modified diamond micro / nano probe used to detect H2O2 in Example 3 of this invention is shown.

[0051] Figure 7 The cyclic voltammetric response curve of the Pt-modified diamond micro / nano probe used to detect NaNO2 in Example 3 of this invention is shown.

[0052] Figure 8 The image shows the microstructure (SEM) of the sample prepared in Comparative Example 2 without the introduction of argon gas during the chemical vapor deposition process. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0054] Example 1: Fabrication of low-boron-doped HFCVD diamond micro / nano probes based on a quartz substrate with positive photoresist insulation.

[0055] Step 1: Preparation of quartz nanopipette substrate

[0056] Material preparation: Standard quartz glass capillary tubes with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm are selected as raw materials.

[0057] Laser drawing: Using a Sutter P-2000 laser needle drawing instrument, the parameters were set as follows: Heat = 800, Filament = 4, Velocity = 60, Delay = 140, Pull = 180, to draw quartz capillaries into tapered nanopipettes with a tip diameter of approximately 300 nm.

[0058] 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.

[0059] Step 2: HFCVD deposition of low boron-doped diamond thin films

[0060] Pretreatment for crystal implantation: To improve the adhesion of diamond films to quartz surfaces, a nanodiamond suspension with a particle size of about 10 nm is used to grind or spin-coat the tip area of ​​the nanopipette, followed by air drying.

[0061] Chemical vapor deposition: The pretreated substrate was placed in a hot filament chemical vapor deposition (HFCVD) system. The process parameters were set as follows: gas flow rate ratio CH4:H2:B2H6:Ar = 5:50:0.1:40 (sccm), growth gas pressure 2 kPa, growth temperature 700 ℃, and growth time 20 min. After deposition, a continuous low-boron-doped diamond (BDD) film was formed at the tip of the quartz substrate. Figure 1 The image shows the morphology of the boron-doped diamond microprobe tip grown from quartz nanotubes.

[0062] Step 3: Conductive connection and positive photoresist insulating encapsulation

[0063] Conductive connection: Place one end of the copper wire in the non-end region (such as the tail or middle section) of the tip area of ​​the quartz capillary on which the BDD film is deposited, apply conductive silver paste to firmly bond the copper wire to the BDD film layer, and achieve a stable electrical connection after the silver paste has completely cured.

[0064] Coating photoresist: Fix the electrode 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 BDD film and the conductive connection to form an insulating sealing layer.

[0065] Pre-baking: Soft bake on a hot plate at 100 ℃ for 120 s to remove solvent.

[0066] 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.

[0067] Development: Immerse the electrode 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, thereby exposing the underlying BDD micro / nano tip, forming a precisely defined electroactive region, while the rest remains insulated.

[0068] Hard baking: Hard bake at 120-130℃ for 30 minutes. The overall process flow is as follows: Figure 2 As shown.

[0069] Step 4: Application of H2O2 detection in single cells

[0070] Positioning and puncture: Under an optical microscope, a precision micromanipulator is used to control the probe tip to penetrate the cell membrane and enter the cell without damage. Figure 3 Optical images show the insertion of diamond micro / nano probes into single cells under the control of precision micromanipulators.

[0071] H2O2 monitoring: Amperometric method (applied -0.4 V). When cells are stimulated with PMA, they release hydrogen peroxide. The hydrogen peroxide molecules are reduced on the surface of the nanoelectrode, generating a transient current spike, thereby enabling the monitoring of the single-cell oxidative stress response process.

[0072] Example 2: Fabrication and Neurotransmitter Detection of Highly Boron-Doped HFCVD Diamond Micro / Nano Probes Based on Negative Photoresist Insulation

[0073] Step 1: Preparation of quartz nanopipette substrate

[0074] Material preparation: Standard quartz glass capillary tubes with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm are selected as raw materials.

[0075] Laser drawing: Using a Sutter P-2000 laser needle drawing instrument, the parameters were set as follows: Heat = 800, Filament = 4, Velocity = 60, Delay = 140, Pull = 180, to draw quartz capillaries into tapered nanopipettes with a tip diameter of approximately 500 nm.

[0076] 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.

[0077] Step 2: HFCVD deposition of highly boron-doped diamond thin films

[0078] Crystal implantation: A layer of nanodiamond suspension with a particle size of 5 nm is spin-coated onto the substrate surface for crystal implantation.

[0079] Deposition: An HFCVD system was used, with diborane (B2H6) introduced as the dopant source. Process parameters: gas flow rate ratio CH4:H2:B2H6:Ar = 5:100:2:60 (sccm), growth gas pressure 3 kPa, growth temperature 600 ℃, growth time 40 min. Figure 4 The SEM images of diamond micro / nano probe nanocrystals are shown.

[0080] Step 3: Conductive connection and negative photoresist insulating encapsulation

[0081] Conductive connection: Copper wires are fixedly connected to the non-tip parts of the BDD electrode using conductive silver paste, and cured at room temperature or by heating to complete the electrode assembly.

[0082] Surface modification: UV ozone treatment for 30 min improves the adhesion between photoresist and diamond.

[0083] Coating photoresist: Apply negative photoresist (such as SU8-2035) by dotting or dipping to cover the electrode surface and connection points.

[0084] Pre-baking: Baking in stages at 65 ℃ and 95 ℃ to slowly evaporate the solvent.

[0085] 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 not exposed.

[0086] 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 BDD nanotip with a length of approximately 2 μm.

[0087] Hard baking: Hard bake at 150℃ for 30 minutes.

[0088] Step 4: Application of Intracellular Neurotransmitter Detection

[0089] Positioning and puncture: Under an optical microscope, a precision micromanipulator is used to control the probe tip to penetrate the cell membrane and enter the cell without damage. Figure 5 Optical images show diamond micro / nano probes inserted into cell spheres under the control of precision micromanipulators.

[0090] Dopamine monitoring: An amperometric method was used (applied +0.6 to +0.8 V). When cells exocytose to release neurotransmitters, dopamine molecules are oxidized on the surface of the nanoelectrode, generating a transient current spike, enabling quantitative analysis of the neurotransmitter release kinetics in the cellular sphere.

[0091] Example 3: Fabrication and Detection of Reactive Oxygen / Nitrogen Species of Pt-Modified Boron-Doped HFCVD Diamond Micro / Nano Probes Based on Negative Photoresist Insulation

[0092] Step 1: Preparation of quartz micropipette substrate

[0093] Material preparation: Standard quartz glass capillary tubes with an outer diameter of 1.0 mm and an inner diameter of 0.5 mm are selected as raw materials.

[0094] Laser drawing: Using a Sutter P-2000 laser needle drawing instrument, the parameters were set as follows: Heat = 700, Filament = 3, Velocity = 30, Delay = 128, Pull = 80, to draw quartz capillaries into micron-sized pipettes with a tip diameter of approximately 3 μm.

[0095] 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.

[0096] Step 2: HFCVD deposition of boron-doped diamond thin films

[0097] Crystal implantation: Spin-coating a 10 nm nanodiamond suspension.

[0098] Deposition: An HFCVD system was used, with diborane (B2H6) as the dopant source. Process parameters: gas flow rate ratio CH4:H2:B2H6:Ar = 5:50:0.5:40 (sccm), growth gas pressure 2 kPa, growth temperature 700 ℃, growth time 40 min.

[0099] Step 3: Conductive connection and negative photoresist insulating encapsulation

[0100] Conductive connection: The copper wires are connected to the electrode conductive layer using conductive silver paste and then cured.

[0101] Surface treatment: UV ozone treatment for 1 hour.

[0102] Coating with photoresist: Apply negative photoresist (such as SU8-2002) to the electrode.

[0103] Pre-baking: Bake at 90 ℃ for 100 s.

[0104] Patterned exposure: Using a CO2 laser, the main body area away from the tip is exposed (20 mW, 35 s), while the tip micro-area is left unexposed.

[0105] Development: Immerse in PGMEA developer for 20 seconds. The exposed area cures, and the photoresist in the unexposed tip area dissolves, exposing a BDD electroactive region approximately 100 μm in length.

[0106] Hard baking: Hard baking at 140℃ for 40 minutes.

[0107] Step 4: Functionalization and Application of Reactive Oxygen Species / Nitrogen Species (ROS / RNS) Detection

[0108] Electrode functionalization: The prepared micro / nano probes were immersed in chloroplatinic acid solution and high-density Pt nanoparticles were deposited by applying a constant potential of -2.0 V.

[0109] H2O2 detection verification: Figure 6 The cyclic voltammetric response curves of the Pt-modified diamond micro / nano probe in a solution containing hydrogen peroxide (H2O2) are shown. The obvious redox peaks demonstrate that the probe has good electrocatalytic activity for H2O2.

[0110] NaNO2 detection verification: Figure 7 The cyclic voltammetric response curves of the Pt-modified diamond micro / nano probe in a solution containing sodium nitrite (NaNO2) are shown. The obvious redox peaks demonstrate that the probe has good electrocatalytic activity for NaNO2.

[0111] Intracellular detection: Functionalized probes are inserted into target cells (such as breast cancer cells).

[0112] Amperometric detection: Real-time monitoring is performed by applying a specific oxidation potential (e.g., +500 mV for H2O2 detection, +600 mV for NO detection). Transient current signals generated during cellular oxidative stress are recorded, enabling real-time monitoring of intracellular ROS / RNS.

[0113] Comparative Example 1

[0114] Other conditions were the same as in Example 1, except that the quartz capillary matrix was replaced with carbon fiber filaments (approximately 7 μm in diameter) as the matrix. Results: During HFCVD deposition, the carbon fibers were severely etched by active hydrogen radicals in a hydrogen-rich plasma environment, resulting in obvious pits and grooves on the fiber surface, a significant reduction in diameter, and an inability to form a continuous and dense diamond film. In some samples, the carbon fibers were completely broken during deposition, leading to preparation failure. Even in a few samples where deposition was successfully completed, their mechanical strength was significantly reduced, making them highly susceptible to breakage during subsequent cell insertion, thus failing to meet the requirements for single-cell detection.

[0115] Comparative Example 2

[0116] Other conditions were the same as in Example 1, except that argon gas was not introduced during the chemical vapor deposition process (i.e., the gas flow ratio was adjusted to CH4:H2:B2H6 = 5:50:0.1 sccm, Ar = 0 sccm). Results: The deposited diamond film showed significantly coarser grain size, exhibiting a micron-sized morphology rather than the nanocrystals required in this invention, as observed by SEM. Figure 8 As shown, the coarsened grains and passivated tips prevent the probe from achieving non-destructive puncture of single cells, and the electrochemical background current increases by approximately three times, significantly reducing detection sensitivity. Simultaneously, due to the lack of argon dilution of hydrogen, the high-concentration hydrogen plasma significantly etches the quartz nanotip.

Claims

1. A diamond micro / nano probe, characterized in that: It comprises a quartz capillary matrix, a diamond thin film layer, and an insulating sealing layer. The quartz capillary matrix 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 has an electroactive region. The surface of the tip region is coated with a diamond thin film layer. The surface of the diamond thin film layer, except for the electroactive region, is coated with an insulating sealing layer.

2. The diamond micro / nano probe according to claim 1, characterized in that: The diameter of the tip region is 50 nm-500 μm; The diamond in the diamond film layer is boron-doped diamond with a grain size of 5 nm to 100 nm and a boron atom concentration ≥10. 20 cm -3 , The length of the electroactive region is 100 nm-100 μm.

3. A diamond micro / nano probe according to claim 1 or 2, 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 diamond 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 a diamond film layer located in the non-end portion of the tip region via the conductive silver paste.

4. A method for preparing a diamond micro / nano probe according to any one of claims 1-3, characterized in that: A diamond film layer is deposited in the tip region of a quartz capillary substrate. Photoresist is coated on the surface of the diamond film layer. The photoresist located at the tip end of the quartz capillary substrate is patterned, exposed, and developed. The photoresist at the tip end is selectively removed to expose the electroactive region.

5. The method for preparing a diamond micro / nano probe according to claim 4, characterized in that: The process of obtaining the quartz capillary matrix is ​​as follows: according to the diameter of the designed tip region end, a quartz capillary is drawn by a laser needle drawing instrument. When the diameter of the tip region is 50 nm ~ 999 nm, the pulling parameters are: heating value: 750 ~ 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: 650 ~ 750, pulling force: 50 ~ 100, speed: 20 ~ 50, delay: 110 ~ 128, filament scanning range: 2 ~ 3.

6. The method for preparing a diamond micro / nano probe according to claim 4, characterized in that: The process of depositing a diamond film layer in the tip region of a quartz capillary substrate is as follows: First, the tip region of the quartz capillary substrate is ground or spin-coated with a nanodiamond suspension to plant seed crystals. Then, the quartz capillary substrate with seed crystals is placed in a deposition furnace for chemical vapor deposition.

7. The method for preparing a diamond micro / nano probe according to claim 6, characterized in that: In the nanodiamond suspension, the nanodiamond particles have a diameter of 4 nm to 18 nm. During the chemical vapor deposition process, the gas introduced is in the following flow ratio: methane: hydrogen: borane: argon = 1~10: 40~100: 0.05~2: 20~60, the growth pressure is 1.5~4 kPa, the growth temperature is 600~750 ℃, and the growth time is 5~60 min.

8. The method for preparing a diamond micro / nano probe according to claim 6, characterized in that: Electrical connection is achieved by coating one end of a metal wire with conductive silver paste on the surface of a diamond film layer in the non-end portion of the tip region and then curing it.

9. The method for preparing a diamond micro / nano probe according to claim 6, characterized in that: After exposing the electroactive region, diamond micro / nano probes are obtained by hard baking.

10. The application of a diamond 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.

Images