Preparation method and application of electrochemical deposition metal nanostructure film based on air gold plating

Abstract

The invention relates to a preparation method and application of an electrochemical deposition metal nanostructure film based on air gold plating. The method is based on an electric capillarity phenomenon and electrochemical deposition, metal particles in a plating solution are pulled into air to carry out electrooxidation of metal through the action of an electric field by adopting a mode that an electrode base material is immersed in the plating solution at a low level, electrons are obtained on the surface of a semiconductor or metal so as to form a metal simple substance, and then the metal nanostructure film is generated. Due to the vacancy effect of hydrogen ions and the deposition mode of heterogeneous nucleation of gold particles, a porous gold nanostructure is formed. Meanwhile, the current direction is perpendicular to the metal deposition direction, so that the formed nanostructure is very thin. The gold nanostructure film developed by the invention can reduce the impedance of the electrode, not only has an antibacterial effect, but also reduces the inflammatory reaction after being implanted into a body, and is suitable for surface modification of an implantable biological electrode.

Description

Technical Field

[0001] The present invention relates to a method for creating a metal nanostructure thin film on the surface of a semiconductor or metal substrate, and its preparation method and application. Specifically, it relates to obtaining a metal nanostructure with a porous or particle structure by electrocapillarity and electrowetting effects using a semiconductor (such as silicon, germanium, gallium arsenide, indium phosphide, indium antimonide, silicon carbide, cadmium sulfide, and gallium arsenide silicon) or metal as the substrate, belonging to the technical field of material surface modification. Background Art

[0002] In the field of neuroscience, bioelectrodes are the basis for connecting excitable tissues and external devices. In recent years, bioelectrodes have been widely used as medical devices to monitor and regulate bioelectrical signals for the treatment of neurological diseases and tissue organ replacement (Auris Nasus Larynx 2023, 50: 321 - 326.). However, the clinical application of bioelectrodes still faces many challenges, mainly reflected in the low signal-to-noise ratio caused by high impedance and the fiber encapsulation effect induced by the foreign body reaction (Ieee Transactions on Magnetics 1996, 32: 5121 - 5123.).

[0003] The high impedance problem limits the signal conduction ability of bioelectrodes, resulting in a decrease in the fidelity of the recorded signals. The foreign body reaction after implantation leads to the formation of fibrous tissue encapsulation around the electrodes, thereby increasing the interfacial impedance and affecting the long-term stability (Biomaterials Science 2024, 12: 270 - 287). Optimizing the design of bioelectrodes requires starting from two aspects: reducing the electrode impedance and reducing the foreign body reaction after implantation to improve its effectiveness in clinical applications.

[0004] The sacrificial template method is a simple and efficient method for manufacturing nanostructure thin films, and the sizes of the pores and ligaments can be controlled by subsequent thermal annealing and laser melting (Nanoscale, 2016, 8(2): 785 - 795.), but hard templates often lead to incomplete removal of template impurities. The application of electrochemical deposition is more extensive and convenient, and can be controlled to a certain extent by changing the interaction between the crystal growth rate and the mass transfer rate (Accounts of Chemical Research, 2018, 51(8): 1764 - 1773.). However, in order to obtain the desired nanostructure thin film, additives are often added to the plating solution, which not only increases the cost but also affects the use of the electrode as a biomaterial. Therefore, it is necessary to develop a method for manufacturing nanostructure thin films that avoids introducing impurities and is economical. Summary of the Invention

[0005] Aiming at the problems existing in the current technology, the purpose of the present invention is to provide a preparation method and application of an electrochemically deposited metal nanostructure thin film based on air gold plating. This method is based on electrocapillarity and electrochemical deposition. By adopting the method of low immersion of the electrode substrate in the electroplating solution, under the action of an electric field, metal particles in the plating solution are attracted into the air for electrooxidation of the metal, obtaining electrons on the semiconductor or metal surface to form elemental metal, thereby preparing a metal nanostructure thin film on the semiconductor or metal surface. Due to the vacancy effect of hydrogen ions and the deposition mode of heterogeneous nucleation of gold particles, a porous gold nanostructure is formed. At the same time, since the current direction is perpendicular to the metal deposition direction, the formed nanostructure is very thin. The gold nanostructure thin film developed by the present invention can reduce the impedance of the electrode, not only has an antibacterial effect, but also reduces the inflammatory reaction after implantation into the body, and is applicable to the surface modification of implantable bioelectrodes.

[0006] The technical solution of the present invention is as follows:

[0007] A preparation method of an electrochemically deposited metal nanostructure thin film based on air gold plating, the method comprising the following steps:

[0008] Electroplating is carried out under two electrodes, the substrate is the working electrode, and graphite is the counter electrode; the electroplating solution is a single electrolyte solution, electroplating is carried out for 10 - 1000 s under constant current or constant voltage to obtain a metal nanostructure thin film;

[0009] Among them, the height of the substrate immersed in the electroplating solution is 0.01 - 1 cm;

[0010] The electrolyte is chloroauric acid, zinc chloride or aluminum sulfate solution, and the concentration of the electroplating solution is 0.01 mM - 10 mM;

[0011] When electroplating under constant current, the voltage is 0.1 - 30 V; when electroplating under constant current, the current is 0.001 - 1 mA;

[0012] The substrate is a semiconductor or a metal;

[0013] The semiconductor material is silicon, germanium, gallium arsenide, indium phosphide, indium antimonide, silicon carbide, cadmium sulfide or gallium arsenide silicon;

[0014] The metal is nickel, chromium, copper or zinc;

[0015] When the substrate is a semiconductor, the surface of the semiconductor to be plated needs to be sputtered with gold;

[0016] The sputtering parameters are: the working pressure is 0.5 - 5 Pa, the sputtering power for gold sputtering is 50 - 200 w, the target-substrate distance is 50 - 80 nm, the substrate temperature is 25 - 150 °C, and the deposition time is 1 - 10 min. The material for sputtering with gold is gold or platinum.

[0017] The substrate described above can also be ultrasonically cleaned before gold spraying or electroplating; the cleaning solution is a 60-80% ethanol solution, the ultrasonic temperature is 20-60°C, and the ultrasonic time is 10-50 min.

[0018] 1. The nanostructures in the obtained metal nanostructure film are single-element doped metal salts, with a porous or particulate structure. The particle size range of the nanoparticles is 1-500 nm, and the particle spacing can be controlled within 2-1000 nm. The metal is gold, aluminum or zinc.

[0019] After the electroplating of the electroplated part is completed, it is placed in an oven at 25°C to 50°C for drying treatment.

[0020] It also includes a reduction step: in a hydrogen environment, the obtained metal nanostructure film is annealed at 550-650°C for 0.5-3 h.

[0021] The application of the electrochemically deposited metal nanostructure film based on air gold plating prepared by the method is used in implantable bioelectrodes, antibacterial materials or anti-inflammatory materials.

[0022] The substantial features of the present invention are:

[0023] When performing traditional electroplating, in order to ensure the integrity and uniformity of the coating, various additives are usually added to the electroplating solution, such as defoamers, softeners and stabilizers. By utilizing their surface activity characteristics, the generation of bubbles (or ensuring), the stress of the coating and the stability of the electroplating solution are eliminated.

[0024] In the present invention, a single-component electrolyte (chloroauric acid solution / zinc chloride solution / aluminum sulfate solution) is used, and the contact area between the electroplated part and the electrolyte is reduced (only 1 mm). By utilizing the mechanism of electrocapillary action and electrowetting effect, the coating thickness is precisely controlled; through the method of the present invention, the deposited metal layer on the electrode is very thin, uniform and dense. It can not only achieve the purpose of reducing material consumption, but also greatly save precious metal electrolytes.

[0025] The beneficial effects of the present invention are:

[0026] The advantages of creating a nanostructured film on the surface of a bioelectrode are as follows: (1) the increase in surface area-to-volume ratio leads to a decrease in electrode impedance; (2) it can better simulate the natural extracellular environment and obtain closer contact with individual cells; (3) the increase in surface roughness is beneficial to reducing inflammatory factors secreted by macrophages. Therefore, an excellent method for creating a nanostructured film on the surface of an implantable bioelectrode should have the following characteristics: (1) the formed metal nanostructured film is uniformly distributed; (2) it does not introduce other impurities into the sample; (3) the formed film is as thin as possible to ensure that the properties of the substrate remain unchanged; (4) the formed metal nanostructured film has good antibacterial properties. For this purpose, the present invention proposes a new design concept, using air gold plating method, which draws the plating solution into the air for electrochemical deposition. Since the liquid on the surface of the sample in the air is very thin, a very uniform and relatively thin nanostructured film can be obtained without adding any additives ( Figure 1 ). Since the direction of the current is opposite to the direction of lattice deposition, the air gold plating method will form a relatively thin structure on the surface of the sample without irregular three-dimensional structures ( Figures 2 to 5 ). In Examples 2 to 4, the air gold plating method is not only applicable to the deposition of gold nanostructures, but also can be used for the preparation of nanostructures of other metals (such as zinc, copper, aluminum), demonstrating the wide applicability and versatility of this technology ( Figures 6 to 7 ). In Example 5, by adjusting the depth of immersion of the working electrode in the electrolyte solution (0.1 cm and 1 cm), it was found that the formed metal nanostructures were denser and more uniform when the immersion depth was shallower (0.1 cm), optimizing the process parameters of the air gold plating method ( Figure 8 ). In Example 6, the silicon wafer electrode modified by the air gold plating method showed lower electrochemical impedance, indicating that this technology significantly improved the electrochemical performance of the electrode and was applicable to highly sensitive electrochemical sensors or bioelectrodes ( Figure 9 ). In Example 7, the sample modified by the air gold plating method showed anti-protein adhesion performance, which could reduce the adsorption of bovine serum albumin and reduce the acute inflammatory reaction that might be caused when implanting biomaterials ( Figure 10 ). In Example 8, the sample modified by the air gold plating method showed significant antibacterial effects against Staphylococcus aureus and Escherichia coli, and the bacterial morphology was significantly damaged, indicating that this technology has potential application value in anti-infection ( Figure 11 ). In Example 9, the sample modified by the air gold plating method could induce macrophages to polarize towards an anti-inflammatory phenotype (M2 type), reduce the release of pro-inflammatory factors (such as TNF-α), and at the same time increase the expression of anti-inflammatory factors (such as IL-10), significantly reducing the inflammatory reaction ( Figure 12)。In Example 10, in a mouse air sac model, the sample modified by the air gold plating method significantly reduced the formation of fibrous capsules and the infiltration of inflammatory cells, indicating that this technique can effectively inhibit the chronic inflammatory response and tissue fibrosis after implanting biomaterials. Figure 13 )。Therefore, the present invention can obtain obvious benefits in the fields of implantable bioelectrodes, antibacterial materials, anti-inflammatory materials, etc. Description of the Drawings

[0027] Figure 1 It is a schematic diagram of air gold plating using an electrochemical workstation.

[0028] Figure 2 It is a scanning electron microscope morphology diagram of depositing a gold nanostructure thin film on the silicon wafer surface using the air gold plating method obtained in Example 1.

[0029] Figure 3 It is a high-resolution X-ray photoelectron spectroscopy diagram of depositing a gold nanostructure thin film on the silicon wafer surface using the air gold plating method obtained in Example 1.

[0030] Figure 4 It is an atomic force microscope image of depositing a gold nanostructure thin film on the silicon surface using the air gold plating method obtained in Example 1.

[0031] Figure 5 It is a comparison diagram of the gold element content of depositing a nanostructure thin film on the silicon wafer surface using the air gold plating method and a sputtered gold sample without treatment obtained in Example 1.

[0032] Figure 6 It is a scanning electron microscope morphology diagram of depositing zinc, copper, and aluminum nanostructure thin films on the silicon wafer surface using the air gold plating method obtained in Examples 2 to 4.

[0033] Figure 7 It is a high-resolution X-ray photoelectron spectroscopy diagram of depositing zinc, copper, and aluminum nanostructure thin films on the silicon wafer surface using the air gold plating method obtained in Examples 2 to 4.

[0034] Figure 8 It is a scanning electron microscope diagram of immersing the silicon wafer surface in chloroauric acid solution with a height of 0.1 cm and a height of 1 cm using the air gold plating method obtained in Example 5.

[0035] Figure 9 It is an electrochemical impedance curve diagram of depositing a gold nanostructure thin film on the silicon surface using the air gold plating method and a sputtered gold sample without treatment in Example 6.

[0036] Figure 10 It is a comparison diagram of the protein adsorption content of depositing a gold nanostructure thin film on the silicon surface using the air gold plating method and a sputtered gold sample without treatment in Example 7.

[0037] Figure 11Comparison diagram of antibacterial properties between the gold nanostructure thin film deposited on the silicon surface by air gold plating method in Example 8 and the gold-sprayed sample without treatment.

[0038] Figure 12 Comparison diagram of the polarization performance of macrophages between the gold nanostructure thin film deposited on the silicon surface by air gold plating method in Example 9 and the gold-sprayed sample without treatment.

[0039] Figure 13 Comparison diagram of the thickness of the fibrous layer formed after seven days of implanting the gold nanostructure thin film deposited on the silicon surface by air gold plating method in Example 10 and the gold-sprayed sample without treatment. Detailed implementation manners

[0040] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners. It should be understood that the following examples are only used to further illustrate the present invention, and cannot be construed as limiting the protection scope of the present invention. Those skilled in the art's non-essential improvements and adjustments made based on the above content of the present invention all fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific values in the following examples.

[0041] Example 1

[0042] A silicon wafer with a length and width of 10 mm and a thickness of 500 μm is first ultrasonically cleaned in an alcohol solution with a mass concentration of 75% at 37°C for 15 min, and then ultrasonically cleaned in deionized water at 37°C for 15 min until it is cleaned. The cleaned silicon wafer is placed in a room temperature environment and dried naturally, and the gold spraying element treatment is carried out by using a vacuum evaporation device. When spraying gold, the working pressure is 2.5 Pa, the gold spraying sputtering power is 100 w, the target-substrate distance is 50 nm, the substrate temperature is 25°C, and the gold spraying time is 5 min.

[0043] Using 1 mM chloroauric acid solution as the electrolyte, the gold-sprayed silicon wafer as the working electrode, and the graphite electrode as the counter electrode to form a two-electrode circuit cycle. The height of the gold-sprayed silicon wafer sample as the working electrode immersed vertically in the chloroauric acid solution is 0.1 cm (that is, the bottom of the silicon wafer is immersed 0.1 cm deep in the plating solution). Under the electrocapillary phenomenon and electrodeposition effect, that is, at the electrode-electrolyte interface, due to the change in potential leading to the change in interfacial tension. At the same time, through the electrochemical reaction, the metal ions in the solution are reduced and deposited on the electrode surface to form a solid metal. At the same time, the graphite electrode as the counter electrode is completely immersed in the chloroauric acid solution. The device schematic diagram is as Figure 1As shown, the current is applied at 0.01 mA, and this sample is electroplated with a deposition time of 5 min. Thereby, the electrolyte is drawn from the bottom to the air, enabling electron transfer and crystal deposition to be completed in the air. After gold plating, avoid any liquid contact with the sample surface and place it in an oven at 37 °C for drying. Hydrogen reduction is a key step in the post-treatment of air electroplating, which can better improve the density, adhesion, and conductivity of the coating, and can completely remove the risk of corrosion caused by the presence of hydrogen, such as bubbling or coating deterioration, thereby enhancing the corrosion resistance. The specific procedure is to anneal the obtained metal nanostructure thin film at 600 °C for 1 h in a hydrogen environment. Finally, in this example, the metal layer deposited on the sputtered gold silicon wafer serving as the working electrode is very dense and uniform, the coating is very thin, and the thickness of the surface nanostructure is only 24.35 nm.

[0044] Figure 2 Figure 4 is the scanning electron microscope images of the surface morphology of the sample modified in this example and the sample after hydrogen reduction. As can be seen from the figure, the formed gold nanostructures exhibit a porous or particulate shape. By reducing the salt substances on the sample surface through the hydrogen reduction method, the surface morphology of the treated sample will change and become more dense and uniform.

[0045] Figure 3 Figure 8 is the X-ray photoelectron spectrum of the thin film modified in this example. As can be seen from the figure, the chemical composition of the sample contains gold element. In addition, the plating solution we selected is chloroauric acid solution, and a large amount of chlorine element can be found in the X-ray photoelectron spectrum of the sample.

[0046] Figure 4 Figure 12 is the atomic force microscope image of the sample modified in this example. It can be seen that the formed nanostructures are very uniform and dense. Since the current direction is opposite to the metal deposition direction, the electric field for vertical metal nucleation only relies on the electrons on the surface, so the deposited metal layer is very thin, and the thickness of the surface nanostructure is only 24.35 nm.

[0047] Figure 5 Figure 16 is the comparison chart of the gold element content between the nanostructure thin film deposited on the silicon wafer surface by the air gold plating method modified in this example and the sputtered gold sample without treatment. As can be found from the figure, the gold element content increases after the thin film is created, indicating that air electroplating, as a surface modification technology, is beneficial to the deposition of gold element.

[0048] As described in Example 1, the metal nanostructured film obtained by the present invention is a metal element doped electrolyte salt film. Taking the deposition of a gold film using a chloroauric acid solution as an example, after power is applied, the chloroauric acid solution is pulled to the surface of the plated part in the air. When the electroplating solution contacts the plated part under the action of an applied electric field, the charge on the interface will affect the tension on the surface, causing the spreading state of the electrolyte on the surface of the plated part to change, thereby pulling the gold ions in the electroplating solution from the solution to the air, thereby performing electron transfer and lattice deposition in the air. At the same time, since the gold ions and hydrogen ions are co-deposited on the substrate, the hydrogen ions gain electrons to form hydrogen, which hinders the growth of the gold element; the gold ions gain electrons to form gold particles, and the particles that have been formed are more conducive to the deposition of other gold ions relative to the flat substrate, so they present a nano-column formed by 3D growth. Therefore, the formation of the nanostructure should be attributed to the vacancy effect of hydrogen and the heterogeneous nucleation effect produced by the first deposited gold element. It is the salts and uneven nanostructures on the surface that give the film the characteristics of reducing impedance, antibacterial and anti-inflammatory.

[0049] According to the vacancy effect of hydrogen, in theory, the lower the pH of the electrolyte, the larger the vacancies of the generated nanostructure. Therefore, the pH of the electrolyte should be kept as small as possible to avoid generating more vacancies.

[0050] The metal islands that have been deposited during electrochemical deposition are usually favorable to the electron transfer rate relative to the position of the bare silicon surface, thereby facilitating the growth of the islands. Therefore, it seems unlikely to grow flat nanostructures on silicon electrodes using electrochemical deposition. In the process of air gold plating to modify nanostructures, the direction of the current is perpendicular to the direction of metal deposition, which is the biggest difference from traditional electroplating. It is precisely because there is no electric field driving the nucleation growth, and the formation of the structure depends solely on the reduction of the charged substrate, so compared with the structure formed by traditional electroplating, the nanostructure modified by air gold plating is thin and flat. Air gold plating is an innovation based on traditional electrochemical deposition. It is not only that various additives do not need to be added to the plating solution, but also that it overcomes the uncontrollable three-dimensional growth of metals in electrochemical deposition. Since the direction of the current is opposite to the direction of metal deposition, the electric field for metal nucleation in the vertical direction depends only on the electrons on the surface, so the deposited metal layer is very thin.

[0051] Embodiments 2 to 4

[0052] The other steps are the same as those in Example 1, except that the 1 mM chloroauric acid solution as the electrolyte is replaced by a 1 mM zinc chloride solution, a cupric chloride solution or an aluminum sulfate solution. Figure 6 Scanning electron microscope images of zinc, copper and aluminum nanostructured films formed after the gold-sprayed silicon wafer samples were immersed in 1mM zinc chloride solution, copper chloride solution or aluminum sulfate solution. Figure 7High-resolution X-ray photoelectron spectroscopy of zinc, copper, and aluminum nanostructured thin films deposited on the surface of a silicon wafer using the air-gilding method. Therefore, it can be shown that air gilding, as a surface modification technique, can electroplate different metals such as zinc, copper, and aluminum.

[0053] Example 5

[0054] Other steps are the same as in Example 1, except that the height at which the sputtered gold silicon wafer sample as the working electrode is immersed in the 1 mM chloroauric acid solution is changed from 0.1 cm to 1 cm. Figure 8 Scanning electron microscopy comparison images of the sputtered gold silicon wafer sample immersed in the 1 mM chloroauric acid solution at heights of 0.1 cm and 1 cm. Compared with the sputtered gold silicon wafer immersed in the 1 mM chloroauric acid solution at a height of 1 cm, the metal nanostructures formed on the sputtered gold silicon wafer sample when immersed at 0.1 cm are denser and more uniform.

[0055] Example 6

[0056] The impedance of the sample processed in Example 1 was analyzed using an electrochemical workstation. Before the test, the sample was exposed to a phosphate buffer solution for one minute to establish a relatively stable open-circuit state. The test sample was used as the working electrode; a saturated calomel electrode was used as the reference electrode; and a platinum electrode was used as the counter electrode. All electrochemical tests were performed in a 0.01 M phosphate buffer solution at a temperature of 25 °C. The scanning rate was 0.01 V / s. The impedance test was performed in the frequency range of 0.1 - 100 kHz.

[0057] Figure 9 Electrochemical impedance curves of the gold nanostructured thin film deposited on the silicon wafer surface by the air-gilding method and the untreated sputtered gold sample. It can be found from the figure that the impedance of the electrode decreases after creating the thin film, manifested as a smaller radius of the impedance arc. Therefore, it shows that air gilding, as a surface modification technique, is beneficial to improving the electrochemical performance of the electrode.

[0058] Example 7

[0059] The sample processed in Example 1 was used for the protein adsorption content test experiment. Bovine serum albumin was selected as the model protein. 100 μL of 1.0 mg / mL bovine serum albumin stock solution was placed on the sample surface and incubated for 6 h. The sample surface was rinsed 3 times with phosphate buffer solution, and then immersed in 1 mg / ml sodium dodecyl sulfate solution for 1 h to dissociate the unadsorbed protein, and shaken with a gyratory shaker for 30 s to dissociate the unadsorbed protein, obtaining the standard protein solution to be measured. A modified Lowry protein quantification kit was used to evaluate the protein adsorption of the sample. The determination of the bovine serum albumin adsorption amount on the sample surface was to monitor the increase in the blue color intensity at 750 nm of the reaction between the complex of peptide bonds and copper ions in the protein and the Folin reagent by an enzyme-linked immunosorbent assay reader. The standard protein solution to be measured was mixed with the working solution provided in the modified Lowry protein quantification kit, reacted at 25 °C for 15 min, added the Folin reagent, and continued to react for 30 min. Then, the absorbance value was measured at a wavelength of 750 nm using an enzyme-linked immunosorbent assay reader to detect the color intensity of the blue complex.

[0060] Figure 10 It is a comparative chart of the relative adsorption content of bovine serum albumin on the sample surface. Compared with the untreated group, the sample modified by the air gold plating method showed a certain ability to resist the adsorption of bovine serum albumin. This phenomenon is related to the curvature of the surface nanoparticles. Compared with the flat surface, the rough surface affects the distance between the adsorbed molecules. The implantable bioelectrode with the ability to resist protein adhesion is beneficial to reducing the acute inflammatory reaction in the body after implantation.

[0061] Example 8

[0062] The sample processed in Example 1 was used for all the following in vitro bacterial experiments, using Staphylococcus aureus and Escherichia coli. The strains were cultured in Luria Bertani broth and stored in a refrigerator at 4 °C. Before use, they were gently shaken at 160 r / min on a shaker at 37 °C for 2 h to revive the bacteria. The bacterial suspension was diluted with broth to a final concentration of 10 7 CFU / mL. The surface contamination of the three groups of samples was removed by ultraviolet light irradiation. Subsequently, 100 μL of the bacterial suspension was placed on the sample surface and co-cultured at 37 °C for 24 h. After the co-culture was completed, 1 mL of glutaraldehyde solution was added to the sample to fix the bacteria on the sample surface and stored overnight in a refrigerator at 4 °C. The fixed samples were dehydrated with a gradient of alcohol. 500 μL of alcohol with a volume fraction of 30%, 50%, 70%, 80%, 85%, 90%, 95% and 100% was added to each sample for dehydration, and the time for each time was 10 min for the sample. Then it was dried at room temperature and observed by scanning electron microscopy.

[0063] Figure 11It is a comparison of the activities of Staphylococcus aureus and Escherichia coli on the surface of the sample. Through scanning electron microscope images, it can be clearly seen that the sample modified by air gold plating has a certain bactericidal effect. In the Escherichia coli group, the unmodified sample was covered with Escherichia coli, and the morphology of the bacteria showed complete rod shapes. The sample modified by air gold plating showed a bactericidal effect, with the shape of Escherichia coli on the sample being significantly distorted and obvious leakage of cytoplasm. In the Staphylococcus aureus group, the untreated sample was covered with staphylococci, and the morphology of the bacteria showed round spherical shapes and formed grape-like clusters, which are typical characteristics of active staphylococci. The sample modified by the air gold plating method still showed a certain bactericidal ability, with the staphylococcal membrane on the surface being damaged and the morphology being non-spherical.

[0064] Example 9

[0065] The samples treated in Example 1 were used for the following experiments, cell culture experiment: Mouse monocyte macrophage leukemia cells (RAW 264.7; cells from the Stem Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were inoculated into cell culture plates (Thermo Fisher Scientific, USA), in DMEM medium (high glucose; USA) with 15% heat-inactivated fetal bovine serum (Shanghai; USA) and 1% penicillin / streptomycin (antibiotic antifungal; Gibco, USA), in a humid atmosphere of 5% carbon dioxide at 37°C. These cells were passaged at a ratio of 1:3 every four days. Before the experiment, all were sterilized by ultraviolet light irradiation.

[0066] Cell proliferation and viability: Using Alamar Blue TM (Thermo Fisher Scientific, USA) to detect cell proliferation and viability. Cells with a density of 1×10 5 per well were inoculated on 24-well plates (3 replicates) for 4 hours, lasting for 4 hours, 1 day, and 4 days. At each incubation period, the samples were rinsed 2 times with 0.6 mL phosphate buffer saline, and then 0.5 mL of fresh medium containing 10% TM Alamar Blue was added, and cultured for another 2 hours. Then, 0.1 mL of the medium was transferred to a black 96-well plate, and the excitation wavelength of 560 nm and the emission wavelength of 590 nm were detected.

[0067] Enzyme-linked immunosorbent assay (ELISA): Macrophages with a density of 1×10 5 cells per well were inoculated on the samples for 4 days, and then the cell culture medium was collected to measure the concentrations of interleukin-4 (IL-4; Canada), interleukin-6 (IL-6; Canada), interleukin-10 (IL-10; USA), and tumor necrosis factor-α (TNF-α; Canada). The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay reader (Thermo Fisher Scientific, USA). The concentrations of the above factors were calculated using a standard curve.

[0068] Real-time polymerase chain reaction (RT-PCR) analysis: Cells were cultured in DMEM medium at an initial density of 5×10 5 cells per well. After 4 days of culture, total RNA was extracted using TRIzolTM reagent (Invitrogen, Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the First Strand cDNA Synthesis Kit (Roche, Switzerland). RT-PCR was performed on the system (Roche, Switzerland) using the FastStart Universal SYBR Green I Master Mix (Roche, Switzerland). The relative expression levels of target genes were analyzed using the 2-ΔΔCt method and normalized to the internal reference gene GAPDH after normalization. All primers were purchased from BioTNT Co., Ltd. The RT-PCR experiments were performed at least 2 times, and 3 analyses were performed for each sample. The relative expression levels were quantified using the comparative threshold method.

[0069] Figure 12 ELISAs and RT-PCR were used to detect the polarization state of macrophages on the sample surface. ELISAs were used to detect IL-10 and TNF-α released by macrophages. The content of IL-10, which inhibits the inflammatory response, released by cells on the air-gilded modified sample was the highest, and the content of IL-10, a pro-repair factor, released by the untreated sample was the lowest. In contrast, TNF-α promotes inflammation, and the amount released by cells on the air-gilded modified sample was lower compared to the unmodified sample. RT-PCR was used to detect the expression levels of immune-related genes. The expression level of the inflammatory-related gene INOS in the air-gilded modified sample was significantly lower than that in the untreated group. The expression trends of the anti-inflammatory-related genes IL-4 and CD206 were opposite to that of INOS, which was consistent with the ELISA results. These results can be interpreted as that the air-gilded modified sample can induce the polarization of macrophages into the M2 phenotype and reduce the inflammatory response.

[0070] Example 10

[0071] The following experiments were performed using the samples treated in Example 1. Mouse air pouch model construction experiment: 4 mL of sterile air was subcutaneously injected into the lower dorsal region of mice to form an air pouch, and 3 mL of sterile air was injected 4 days later to strengthen the air pouch. After 48 hours, 1% sodium pentobarbital solution (1 mL per 100 g body weight) was intraperitoneally injected, and the fur on the air pouch was completely shaved off. Under sterile conditions, a surgical incision was made and a sample was implanted into each mouse.

[0072] Air pouch tissue experiment: Tissues were recovered from the mouse air pouches at 1 day and 4 days after implantation. The air pouch tissues were fixed with 4% paraformaldehyde for histological analysis, and the samples were rinsed 2 times with phosphate buffer solution and then fixed with 4% paraformaldehyde for immunocytochemical analysis.

[0073] Immunocytochemical analysis experiment of implanted samples: 1 wt% bovine serum albumin was added to block Fc receptors. The samples were incubated overnight with primary antibodies against CD206 (Abcam, UK) and iNOS (Novus, USA) at 4 °C and then incubated with secondary antibodies for 2 h.

[0074] Histological analysis of airbag tissue: The airbag tissue was embedded in paraffin and sectioned to 4 μm. After dewaxing and dehydration, the sections were stained with hematoxylin-eosin and Masson's trichrome. The stained sections were observed under an optical microscope. The thickness of the fibrous capsule and the number of infiltrating cells at 4 random positions were evaluated using Image J software.

[0075] Figure 13 The HE staining image of the tissue on the 4th day shows the overall structure and layers of the airbag tissue, and the Masson's trichrome staining image clearly shows fibrous tissue and infiltrating cells. The corresponding fibrous layer thickness and quantity. Four days after implantation, the implants in the untreated group were surrounded by a fibrous layer mainly composed of neutrophils, macrophages and monocytes. While there were no obvious changes around the samples modified with air gold plating. Quantitative analysis showed that no fibrous layer was formed around the samples modified with air gold plating, while the fibrous layer thickness of the untreated samples was 53.83 ± 13.80 μm. This was consistent with the trend of the number of infiltrating inflammatory cells. These findings may be due to the fact that the samples modified with air gold plating can significantly promote the polarization of M2 macrophages, thus inhibiting the inflammatory response of biomaterials, including reducing inflammatory cell infiltration and inhibiting the formation of fibrous capsules.

[0076] From the above examples, we can see that the metal nanostructure thin film created on the surface of the silicon electrode by the air gold plating method is thin and uniform and does not introduce impurities into the system. The thin film created by air gold plating is beneficial to improving the working performance of the electrode. The impedance experiment results show that introducing nanostructures can reduce the impedance by increasing the specific surface area of the electrode surface. The protein adsorption experiment indicates that the nanostructures created by the air gold plating method can resist protein adsorption. The antibacterial experiment results show that the nanostructures created by the air gold plating method can have a certain killing ability against bacteria. At the same time, the nanostructures created by the air gold plating method can affect the polarization of macrophages, reducing the inflammatory response and fibrous encapsulation after implantation. In short, the air gold plating method can be used as a method for creating metal nanostructures and applied to the surface modification of implantable bioelectrodes.

[0077] The above is only the preferred implementation method of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

[0078] Matters not covered in this invention are well-known technologies.

Claims

1. A method for preparing an electrochemically deposited metal nanostructured film based on air gold plating, characterized in that: The method comprises the following steps: Electroplating is carried out under two electrodes, the substrate is a working electrode, and graphite is a counter electrode; the electroplating solution is a single electrolyte solution, and the electroplating is carried out under constant current or constant voltage for 10 to 1000 seconds to obtain a metal nanostructured film; The height of the substrate immersed in the electroplating solution is 0.01 to 1 cm; The electrolyte is chloroauric acid, zinc chloride or aluminum sulfate solution, and the concentration of the plating solution is 0.01mM~10mM; The substrate is a semiconductor or a metal.

2. The method for preparing a metal nanostructured film by electrochemical deposition based on air gold plating according to claim 1, characterized in that: When constant current electroplating is used, the voltage is 0.1 to 30 V; when constant current electroplating is used, the current is 0.001 to 1 mA.

3. The method for preparing a metal nanostructured film by electrochemical deposition based on air gold plating according to claim 1, characterized in that: The semiconductor material is silicon, germanium, gallium arsenide, indium phosphide, indium antimonide, silicon carbide, cadmium sulfide or gallium arsenic silicon; The metal is nickel, chromium, copper or zinc.

4. The method for preparing a metal nanostructured film by electrochemical deposition based on air gold plating according to claim 1, characterized in that: When the substrate is a semiconductor, gold is sprayed on the surface of the semiconductor to be plated; The gold spraying parameters are: working pressure of 0.5-5 Pa, gold spraying sputtering power of 50-200 W, target-substrate distance of 50-80 nm, substrate temperature of 25-150° C., and deposition time of 1-10 min.

5. The method for preparing a metal nanostructured film by electrochemical deposition based on air gold plating according to claim 1, characterized in that: The substrate can also be ultrasonically cleaned before gold spraying or electroplating; the cleaning solution is 60-80% ethanol solution, the ultrasonic temperature is 20-60° C., and the ultrasonic time is 10-50 minutes.

6. The method for preparing a metal nanostructured film by electrochemical deposition based on air gold plating according to claim 1, characterized in that: The nanostructure in the obtained metal nanostructure film is a single substance doped with metal salts, the structure is porous or granular, the particle size range of the nanoparticles is 1-500nm, the particle distance can be controlled at 2-1000nm, and the metal is gold, aluminum or zinc.

7. The method for preparing a metal nanostructured film by electrochemical deposition based on air gold plating according to claim 1, characterized in that: The method also includes a reduction step: annealing the obtained metal nanostructure film at 550-650° C. for 0.5-3 hours in a hydrogen environment.

8. Application of the electrochemically deposited metal nanostructured film based on air gold plating prepared by the method of claim 1, characterized in that: Used in implantable bioelectrodes, antibacterial materials or anti-inflammatory materials.

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