Biological catalytic film as well as preparation method and application thereof

Abstract

The invention provides a biological catalytic film as well as a preparation method and application thereof, and belongs to the technical field of biological catalysis. The preparation method of the biological catalytic film provided by the invention comprises the following steps: pretreating a substrate to remove surface impurities and grease to obtain a pretreated substrate; ru, B and V serve as target materials, the pretreated base serves as a substrate, deposition is conducted on the pretreated base through a magnetron sputtering method under the conditions that the sputtering power of Ru and B ranges from 50 W to 80 W, and the sputtering voltage of V ranges from 200 V to 430 V, in the deposition process, V, Ru and B are regulated and controlled through the sputtering voltage of V and the sputtering power of Ru and B to generate a ternary phase, the electronic configuration of V is regulated and controlled, and the ternary phase is obtained. The activity of catalase is improved, and the biological catalytic film is prepared. The prepared biological catalytic film is good in stability and high in catalytic activity.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and more particularly to a biocatalytic film and its preparation method and application. Background Art

[0002] Reactive oxygen species (ROS) are oxygen-derived molecules produced by aerobic organisms during metabolism. They play a dual role: at moderate, balanced levels, ROS are involved in cell signaling and maintain physiological balance, while excessive ROS can induce oxidative stress, damaging proteins, lipids, and DNA, ultimately leading to cell dysfunction or death. An elevated ROS level is a hallmark of infectious and diabetic wounds and a core mechanism underlying poor wound healing. Hyperglycemia in diabetic patients induces osmotic imbalance, vascular dysfunction, neuropathy, and local infection, in which ROS plays a central role. Hyperglycemia significantly increases ROS levels mainly by activating the mitochondrial electron transport chain and oxidases. At the same time, hyperglycemia also impairs the antioxidant defense system, reducing the ability to eliminate ROS. Excessive ROS oxidize cellular components, causing DNA damage and cell death, and exacerbating the inflammatory response, thus having an adverse effect on chronic wound healing. Inflammatory cells such as neutrophils release more ROS, leading to endothelial dysfunction and barrier damage. This facilitates further infiltration of inflammatory cells, forming a vicious cycle, aggravating tissue damage, reducing local blood supply, inhibiting angiogenesis, and delaying healing. Therefore, formulating an intervention strategy targeting ROS is crucial for treating diabetic ulcers, pressure sores, and other chronic wounds.

[0003] Current ROS control strategies mainly rely on natural antioxidants, including enzyme preparations such as superoxide dismutase and catalase, and non-enzyme preparations such as vitamin C and vitamin E. However, natural enzymes are limited by factors such as unstable biological activity, sensitivity to environmental factors, and production challenges. To overcome these limitations, artificial enzymes mimicking the ROS-scavenging activity of natural enzymes have become the focus of research.

[0004] Currently, the catalyst materials used in the market for removing ROS mainly include noble metal catalysts and oxide catalysts, but these materials generally have problems such as low catalytic efficiency, poor stability, and high cost, and cannot meet the actual application requirements. Therefore, developing a new catalyst material with high catalytic performance and moderate cost has become a current research hotspot and difficulty. Summary of the Invention

[0005] In view of the above problems, the present invention provides a biocatalytic film, a preparation method thereof and an application thereof. The present invention prepares a biocatalytic film by magnetron sputtering. This film can significantly reduce the ROS level at the wound site, accelerate wound healing, and maintain superhydrophobic properties. By providing a stable loading mode and an antioxidant microenvironment, this film can effectively alleviate oxidative stress and provide a promising solution for the treatment of chronic diabetic wounds.

[0006] The first object of the present invention is to provide a preparation method of a biocatalytic film, comprising the following steps: Pretreat the substrate to remove surface impurities and grease to obtain a pretreated substrate.

[0007] Using Ru, B, and V as targets and the pretreated substrate as the substrate, deposit on the pretreated substrate by magnetron sputtering under the conditions that the sputtering power of Ru is 50 W - 80 W, the sputtering power of B is 50 W - 80 W, and the sputtering voltage of V is 200 V - 430 V. During the deposition process, regulate the formation of a ternary phase of V, Ru, and B by the sputtering voltage of V and the sputtering powers of Ru and B, and regulate the electronic configuration of V to enhance the activity of catalase-like, thereby preparing a biocatalytic film.

[0008] In a preferred embodiment of the present invention, the sputtering powers of Ru and B are 80 W.

[0009] In a preferred embodiment of the present invention, the sputtering voltage is 430 V In a preferred embodiment of the present invention, the sputtering power of V is 50 W - 80 W.

[0010] In a preferred embodiment of the present invention, the mass flow rate of the sputtering gas is 30 sccm - 50 sccm, and the sputtering pressure is 0.2 Pa - 0.4 Pa.

[0011] In a preferred embodiment of the present invention, the angle of the Ru target facing the substrate is 45° - 60°, the angle of the B target facing the substrate is 45° - 60°, and the angle of the V target facing the substrate is 45° - 60°.

[0012] In a preferred embodiment of the present invention, the sputtering temperature is room temperature.

[0013] In a preferred embodiment of the present invention, the distance from the substrate to the target is 15 cm - 20 cm.

[0014] The second object of the present invention is to provide a biocatalytic film prepared by the above preparation method.

[0015] The third object of the present invention is to provide an application of the above biocatalytic film in the preparation of materials for eliminating reactive oxygen species.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention selects three kinds of atoms, Ru, V, and B, with relatively low costs. The low electronegativity and stable oxidation state of Ru provide highly efficient redox active centers. The d-electron configuration of V enhances catalytic kinetics. The electron-deficient characteristic of B induces local charge redistribution by forming Ru-B bonds, accelerating electron transfer. During preparation, the magnetron sputtering method is used. During the deposition process, the catalytic efficiency of active sites is enhanced by B. The ternary phase of V, Ru, and B is generated by the sputtering power of Ru and B and the sputtering voltage of V. The electron configuration of V is regulated by the sputtering voltage of V to enhance the activity of catalase-like enzymes. The VRuB ternary phase is obtained by co-sputtering. VRuB has a high ROS scavenging efficiency and various enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD). This coating has nanoscale uniformity, abundant active sites, and a large specific surface area. While improving the ROS scavenging performance, it can also maintain the biomechanical properties of the superhydrophobic surface.

[0017] (2) The preparation method of the present invention adopts a one-step preparation method, which has a simple process, short time consumption, low cost, high repeatability, stable composition, and can be mass-produced. Avoiding sample contamination under ultra-high vacuum and not introducing too many impurities, it has a high cleanliness. The obtained biocatalytic thin film has a dense structure and a high binding force with the substrate.

[0018] (3) Experimental studies and density functional theory (DFT) calculations confirm that the VRuB biocatalyst synthesized by magnetron sputtering has excellent CAT-like reaction kinetics, with a Michaelis constant of 371.13×10 - 3 M; the maximum reaction rate is 48.53×10 -6 M s -1 ; the turnover number is 7.66 s -1 , exceeding that of natural antioxidants (such as antioxidant enzymes, vitamin C, etc.) and previously reported ROS scavenging biocatalysts (such as catechol-based polymers, carbon nanomaterials, small molecule-based reducing agents, etc.). In vivo and in vitro studies show that the modified coating can significantly reduce the ROS level at the wound site, accelerate wound healing, and maintain the superhydrophobic property. By providing a stable loading mode and an antioxidant microenvironment, this coating can effectively reduce oxidative stress and improve cell regeneration conditions, providing a promising solution for the treatment of diabetic chronic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 24 shows the CAT-like oxygen production curves of different catalysts.

[0020] Figure 2 H2O2 elimination curves for different catalysts.

[0021] Figure 3 K for different catalysts m diagram.

[0022] Figure 4 Antioxidant test diagram of the catalyst. Among them, a is the relationship diagram between the absorbance and wavelength of different catalysts, and b is the elimination rate diagram of the SOD-like enzyme activity of different catalysts.

[0023] Figure 5 Hydrophobicity test of the catalyst.

[0024] Figure 6 Wound healing test of different catalysts. Detailed implementation mode

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] Among transition metal-based artificial enzymes, ruthenium (Ru) stands out due to its low electronegativity and stable redox properties. The d-electron configuration of ruthenium enhances the bonding ability, while the vacant orbitals optimize the adsorption and desorption kinetics of redox reactions. Changing the d-electron configuration of the vanadium (V) center can adjust the adsorption energy between H2O2 and metal sites, and adding boron (B) can further change the electronic structure of the catalyst, improve the electron transfer efficiency, and optimize the performance of active sites. Based on this, the present invention prepares a biocatalytic film with these three atoms of Ru, V, and B. When preparing the biocatalytic film of the present invention, the synergistic effect of sputtering power and voltage is regulated as follows:

[0027] a. The power of the Ru / B target is set to 50 W - 80 W By adjusting the sputtering power (50 W - 80 W) of the Ru and B targets, the sputtering rate and energy of the target atoms are controlled. At a lower power (such as 50 W), Ru-B intermetallic compounds can be formed; at a higher power (such as 80 W), the sputtering yields of Ru and B can be increased, further promoting the formation of Ru-B intermetallic compounds. The Ru-B bond induces local charge redistribution, accelerates electron transfer, is beneficial to the adsorption and dissociation of raw materials and the generation of catalytic products. At the same time, the introduction of B can optimize the electron transfer path and enhance the catalytic efficiency of active sites.

[0028] b. Voltage regulation of the V target (200 V - 430 V): The sputtering voltage of V (200 V - 430 V) directly affects the plasma energy and the intensity of ion bombardment. A high voltage (such as 430 V) can enhance the kinetic energy of V atoms. When the sputtering power of Ru / B and the sputtering voltage of V are respectively in appropriate ranges, the sputtering power of Ru / B can endow the film with efficient electron transfer ability and good active site structure, while the sputtering voltage of V plays a key role in enhancing the chemical stability of the film and further finely regulating the electronic structure. Thus, the synergistic optimization of the two can achieve more efficient and stable catalytic performance for electrocatalytic processes such as hydrogen peroxide generation, making it easier to combine with Ru and B to form a stable ternary phase (such as VRuB). At the same time, it regulates the d-electron configuration of V, optimizes the adsorption / desorption kinetics of H2O2, and enhances the activity of catalase-like (CAT).

[0029] Furthermore, the present invention synergistically regulates the sputtering gas and pressure.

[0030] The argon flow rate is set to 30 sccm - 50 sccm, and the pressure is set to 0.2 Pa - 0.4 Pa. The argon flow rate and pressure jointly determine the plasma density and the mean free path of sputtered particles. At medium and low pressures of 0.2 - 0.4 Pa, the bombardment efficiency of argon ions on the target is relatively high. At the same time, the gas flow rate (such as 50 sccm) can maintain a stable plasma environment to ensure uniform deposition of the thin film. Under these conditions, the formation of nanoscale grains (<20 nm) can significantly increase the specific surface area and expose more active sites.

[0031] Furthermore, the distance between the substrate and the target is set to 15 cm - 20 cm. The distance between the substrate and the target directly affects the deposition rate and the thickness distribution of the thin film. A distance of 15 cm can balance the kinetic energy and diffusion ability of sputtered particles, enabling the thin film to uniformly cover the silica gel substrate (thickness error <5%), avoiding local stress concentration, and maintaining the continuity of the structure at the same time.

[0032] Furthermore, the angles of the Ru target and the B target facing the substrate are set to 45° - 60°, and the angle of the V target facing the substrate is set to 45° - 60°.

[0033] The low electronegativity and stable oxidation state of Ru provide redox active centers; the d-electron configuration of V regulates the adsorption energy of H2O2 and reduces the reaction energy barrier; the electron-deficient property of B optimizes the metal-nonmetal interface through charge redistribution and accelerates electron transfer. By adjusting the orientation of the target holder and the substrate, the element ratio (such as Ru 72 V 21 B7) is regulated. Through the synergistic effect of Ru, V, and B, precise matching of the active site density and the electronic structure can be achieved, thereby increasing the turnover number (TON) and catalytic stability.

[0034] Example 1 This embodiment provides a method for preparing a biocatalytic film, comprising the following steps: (1) Pretreat the substrate.

[0035] Select distilled water and anhydrous ethanol to clean the surface of the silica gel substrate in sequence to remove surface impurities and grease, and dry it in vacuum at 60 °C.

[0036] (2) Specific preparation process of the biocatalytic film Use Ru, B, and V as target materials; before deposition, adjust the distance between the substrate and the target materials to 15 cm, evacuate the magnetron sputtering system to make the vacuum degree in the cavity reach 5×10 -5 Pa; before starting deposition, fill the sputtering cavity with the sputtering gas high-purity argon to make the working pressure reach 0.2 Pa, and set the mass flow rate of the sputtering gas argon to 50 sccm; keep the substrate temperature at 25 °C; start sputtering, set the sputtering power of the Ru and B targets to 80 watt, and the angle of the Ru and B targets facing the substrate is 60° (based on the angle between the tabletop and the target holder); use V as the target material, in the experiment, the sputtering power is 80 watt and the sputtering voltage is 360 V, and the angle of the V target facing the substrate is 45° (based on the angle between the tabletop and the target holder); under the action of the electric field, electrons will collide with argon atoms during the flight to the substrate, causing the argon gas to be ionized (under the action of high voltage, Ar atoms are ionized into Ar + ions and electrons), and the incident ions (Ar + ) bombard the target materials under the action of the electric field, so that the neutral atoms or molecules on the surface of the target materials obtain sufficient kinetic energy to break away from the surface of the target materials and deposit on the surface of the substrate to form a film. Set the deposition rate to 12.5 nm / min and the deposition time to 30 min to realize the deposition of a crystal-crystal nano-dual-phase structure film on the carbon cloth substrate, and obtain a biocatalytic film, that is, Ru 72 V 21 B7 hydrogen evolution catalyst.

[0037] Example 2 This embodiment provides a method for preparing a biocatalytic film, comprising the following steps: (1) Pretreat the substrate.

[0038] Select distilled water and anhydrous ethanol to clean the surface of the silica gel substrate in sequence to remove surface impurities and grease, and dry it in vacuum at 60 °C.

[0039] (2) Specific preparation process of the biocatalytic film Use Ru, B, and V as target materials; before deposition, adjust the distance between the substrate and the target materials to 15 cm, evacuate the magnetron sputtering system to make the vacuum degree in the cavity reach 5×10 -5Pa; Before starting deposition, the sputtering chamber was filled with high-purity argon as the sputtering gas to bring the working pressure to 0.2 Pa, and the mass flow rate of the sputtering gas argon was set at 50 sccm; the substrate temperature was maintained at 25 °C; sputtering was started, and the sputtering power of the Ru, B target was set at 80 watt, and the angle of the Ru, B target facing the substrate was 45° (based on the angle between the tabletop and the target holder); using V as the target material, the sputtering power was 80 watt, the sputtering voltage was 360 V, and the angle of the V target facing the substrate was 60° (based on the angle between the tabletop and the target holder); under the action of the electric field, electrons will collide with argon atoms during their flight towards the substrate, ionizing the argon gas (under high voltage, Ar atoms are ionized into Ar + ions and electrons), and the incident ions (Ar + ) bombard the target material under the action of the electric field, enabling neutral atoms or molecules on the surface of the target material to obtain sufficient kinetic energy to break away from the surface of the target material and deposit on the surface of the substrate to form a thin film. The set deposition rate was 12.5 nm / min, and the deposition time was 30 min to achieve the deposition of a crystal-crystal nanobiphasic structure thin film on the carbon cloth substrate, obtaining a biocatalytic thin film, namely V 65 Ru 27 B8 hydrogen evolution catalyst.

[0040] Example 3 This example provides a method for preparing a biocatalytic thin film, including the following steps: (1) Pretreat the substrate.

[0041] The surface of the silica gel substrate was successively cleaned with distilled water and absolute ethanol to remove surface impurities and grease, and then dried in vacuum at 60 °C.

[0042] (2) The specific preparation process of the biocatalytic thin film Using Ru, B, V as the target materials; before deposition, the distance between the substrate and the target materials was adjusted to 20 cm, and the magnetron sputtering system was evacuated to bring the vacuum degree in the cavity to 5×10 -5 Pa; Before starting deposition, the sputtering chamber was filled with high-purity argon as the sputtering gas to bring the working pressure to 0.4 Pa, and the mass flow rate of the sputtering gas argon was set at 30 sccm; the substrate temperature was maintained at 25 °C; sputtering was started, and the sputtering power of the Ru, B target was set at 80 watt, and the angle of the Ru, B target facing the substrate was 45° (based on the angle between the tabletop and the target holder); using V as the target material, the sputtering power was 50 watt and the sputtering voltage was 430 V in the experiment, and the angle of the V target facing the substrate was 60° (based on the angle between the tabletop and the target holder); under the action of the electric field, electrons will collide with argon atoms during their flight towards the substrate, ionizing the argon gas (under high voltage, Ar atoms are ionized into Ar + ions and electrons), and the incident ions (Ar+ Under the action of an electric field, the target material is bombarded, so that neutral atoms or molecules on the surface of the target material gain sufficient kinetic energy to break away from the surface of the target material and deposit on the surface of the substrate to form a thin film. The set deposition rate is 12.5 nm / min, and the deposition time is 40 min, so as to realize the deposition of the crystal-crystal nanobiphase structure thin film on the carbon cloth substrate and obtain the biocatalytic thin film.

[0043] Example 4 This example provides a method for preparing a biocatalytic thin film, which includes the following steps: (1) Pretreat the substrate.

[0044] The surface of the silica gel substrate is successively cleaned with distilled water and absolute ethanol to remove surface impurities and grease, and then dried in vacuum at 60 °C.

[0045] (2) Specific preparation process of the biocatalytic thin film Using Ru, B, and V as target materials; before deposition, adjust the distance between the substrate and the target material to 18 cm, evacuate the magnetron sputtering system to make the vacuum degree in the cavity reach 5×10 -5 Pa; before starting deposition, fill the sputtering chamber with the sputtering gas high-purity argon to make the working pressure reach 0.3 Pa, and set the mass flow rate of the sputtering gas argon to 40 sccm; keep the substrate temperature at 25 °C; start sputtering, set the sputtering power of the Ru and B targets to 60 watt, and the angle of the Ru and B targets facing the substrate is 50° (based on the angle between the desktop and the target seat); using V as the target material, in the experiment, the sputtering power is 60 watt, the sputtering voltage is 200 V, and the angle of the V target facing the substrate is 50° (based on the angle between the desktop and the target seat); under the action of the electric field, electrons will collide with argon atoms during the flight to the substrate, causing the argon gas to be ionized (under the action of high voltage, Ar atoms are ionized into Ar + ions and electrons), and the incident ions (Ar + ions) bombard the target material under the action of the electric field, so that neutral atoms or molecules on the surface of the target material gain sufficient kinetic energy to break away from the surface of the target material and deposit on the surface of the substrate to form a thin film. The set deposition rate is 12.5 nm / min, and the deposition time is 35 min, so as to realize the deposition of the crystal-crystal nanobiphase structure thin film on the carbon cloth substrate and obtain the biocatalytic thin film.

[0046] Comparative Example 1 This comparative example provides a method for preparing a biocatalytic thin film, including the following steps: (1) Pretreat the substrate.

[0047] The surface of the silica gel substrate is successively cleaned with distilled water and absolute ethanol to remove surface impurities and grease, and then dried in vacuum at 60 °C.

[0048] (2)Specific preparation process of the biocatalytic film Using Ru, B, and V as the targets; before deposition, adjust the distance between the substrate and the targets to 15 cm, evacuate the magnetron sputtering system to make the vacuum degree in the cavity reach 5×10 -5 Pa; before starting deposition, fill the sputtering chamber with the sputtering gas high-purity argon to make the working pressure reach 0.2 Pa, and set the mass flow rate of the sputtering gas argon to 50 sccm; keep the substrate temperature at 25 °C; start sputtering, set the sputtering power of the Ru and B targets to 80 watt, and the angles of the Ru and B targets facing the substrate are 45° (based on the angle between the desktop and the target holder); using V as the target, the sputtering power is 80 watt, the sputtering voltage is 360 V, and the angle of the V target facing the substrate is 60° (based on the angle between the desktop and the target holder); under the action of the electric field, electrons will collide with argon atoms during the flight to the substrate, causing the argon gas to ionize (under high voltage, Ar atoms ionize into Ar + ions and electrons), and the incident ions (Ar + ) bombard the targets under the action of the electric field, so that the neutral atoms or molecules on the target surface obtain sufficient kinetic energy to break away from the target surface and deposit on the substrate surface to form a film. Set the deposition rate to 12.5 nm / min and the deposition time to 30 min to achieve the deposition of a crystal-crystal nano-dual-phase structure film on the carbon cloth substrate, and obtain the biocatalytic film, that is, V 70 Ru 30 hydrogen evolution catalyst.

[0049] Comparative Example 2 This comparative example provides a method for preparing a biocatalytic film, including the following steps: (1) Pretreat the substrate.

[0050] Select distilled water and absolute ethanol to wash the surface of the silica gel substrate in turn to remove surface impurities and grease, and dry it in vacuum at 60 °C.

[0051] (2) Specific preparation process of the biocatalytic film Using Ti and V as the targets; before deposition, adjust the distance between the substrate and the targets to 15 cm, evacuate the magnetron sputtering system to make the vacuum degree in the cavity reach 5×10 -5Pa; Before starting the deposition, the sputtering chamber was filled with high-purity argon as the sputtering gas to make the working pressure reach 0.2 Pa. The mass flow rate of the sputtering gas argon was set at 50 sccm; the substrate temperature was maintained at 25 °C; sputtering was started. The sputtering power of the Ti target was set at 80 watt, the sputtering voltage was 290 V, and the angle of the Ti target facing the substrate was 45° (based on the angle between the tabletop and the target holder); Using V as the target material, the sputtering power was 80 watt, the sputtering voltage was 360 V, and the angle of the V target facing the substrate was 60° (based on the angle between the tabletop and the target holder); Under the action of the electric field, electrons will collide with argon atoms during their flight towards the substrate, causing the argon gas to ionize (under high voltage, Ar atoms are ionized into Ar + ions and electrons), and the incident ions (Ar + ) bombard the target material under the action of the electric field, enabling the neutral atoms or molecules on the surface of the target material to obtain sufficient kinetic energy to break away from the surface of the target material and deposit on the surface of the substrate to form a thin film. The set deposition rate was 12.5 nm / min, and the deposition time was 30 min, realizing the deposition of a crystal-crystal nanodual-phase structure thin film on the carbon cloth substrate to obtain a biocatalytic thin film, that is, V 70 Ti 30 hydrogen evolution catalyst.

[0052] Next, the catalysts prepared in Examples 1 to 2 and Comparative Example 1 were subjected to performance tests.

[0053] The CAT-like activity was evaluated by assessing the H2O2 scavenging ability and O2 generation ability. When evaluating the H2O2 scavenging ability, 20 µL of H2O2 (1 M), 1.97 mL of phosphate buffered saline (PBS, pH 7.4), and 1 cm 2 of the hydrogen evolution catalyst were mixed. After 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes, 100 µL of this solution was mixed with 100 µL of Ti(SO4)2 solution (13.9 mM), and the absorbance at 405 nm was recorded using a microplate reader.

[0054] In 20 mL of PBS (pH 7.4) containing 1 cm 2 of the hydrogen evolution catalytic coating, 200 µL of H2O2 (10 M) was added to test the O2 generation ability. By changing the concentration of H2O2, the steady-state kinetic parameters of the CAT-like activity were determined. All experiments were carried out in 50 mL centrifuge tubes containing 20 mL of PBS (pH 7.4) and 20 µL of the biocatalyst (10 mg·mL -1). PBS containing a hydrogen evolution catalyst was mixed with different concentrations of H2O2 (100 mM - 900 mM), and a dissolved oxygen meter was used to track the change in O2 solubility for 3 minutes. The "absorbance vs. time" curve for each H2O2 concentration was used to calculate the initial reaction rate (V0). The reaction rate curve was plotted against the H2O2 concentration and fitted to the Michaelis - Menten equation (Equation S1). Additionally, a Lineweaver - Burk plot (Equation S2) was used to obtain the maximum reaction rate (V max ) and the Michaelis - Menten constant (K m ). The catalytic efficiency was expressed as the turnover number (TON), calculated using Equation S3, where [E0] represents the concentration of catalytic centers in the biocatalyst. The TON / K m ratio was used to measure the catalytic efficiency.

[0055] (S1) (S2) TON = V max / [E0] (S3) The Michaelis constant (K m ) is an important parameter in enzyme kinetics, which represents that in an enzyme - catalyzed reaction, when the substrate concentration reaches K m , the reaction rate reaches half of the maximum reaction rate (V max ). The K m value reflects the affinity between the enzyme and the substrate. A smaller K m value means a higher affinity of the enzyme for the substrate, while a larger Km value indicates a lower affinity.

[0056] The maximum reaction rate (V max ) refers to the maximum rate that an enzyme - catalyzed reaction can reach when the enzyme is completely saturated with the substrate. V max reflects the catalytic ability of the enzyme, that is, when the substrate concentration is high enough, the highest reaction rate that the enzyme can reach. V max is proportional to the concentration of the enzyme, and increasing the enzyme concentration can increase V max .

[0057] The turnover number (TON) refers to the number of substrate molecules that each enzyme molecule can catalytically convert per unit time. It is also known as the catalytic constant (kcat). TON represents the catalytic efficiency of the enzyme, that is, how many substrate molecules each enzyme molecule can convert per second. A larger value indicates a higher catalytic efficiency of the enzyme. TON can comprehensively evaluate the catalytic performance of the enzyme, especially the efficiency at low substrate concentrations.

[0058] The experimental results are as Figure 1As shown, the Ru prepared in Example 1 72 V 21 The Michaelis constant calculated for the B7 catalyst ( K m ) is 371.13×10 -3 M; the maximum reaction rate (V max ) is 48.53×10 -6 M·s -1 ; the turnover number (TON) is 7.66 s -1 .

[0059] The Michaelis constant calculated for the V 65 Ru 27 B8 catalyst prepared in Example 2 (K m ) is 271.13×10 -3 M; the maximum reaction rate (V max ) is 33.4×10 -6 M·s -1 ; the turnover number (TON) is 5.82 s -1 .

[0060] The Michaelis constant calculated for the V 70 Ru 30 catalyst prepared in Comparative Example 1 (K m ) is 563.6×10 -3 M; the maximum reaction rate (V max ) is 26.4 × 10 -6 M·s -1 ; the turnover number (TON) is 3.11 s -1 .

[0061] The Michaelis constant calculated for the V 70 Ti 30 catalyst prepared in Comparative Example 2 (K m ) is 675.98×10 -3 M; the maximum reaction rate (V max ) is 10.56×10 -6 M·s -1 ; the turnover number (TON) is 4.12 s -1 .

[0062] In the present invention, three atoms of ruthenium (Ru), vanadium (V) and boron (B) form a ternary intermetallic compound (VRuB) through a specific preparation process (magnetron sputtering method), and their synergistic effect significantly improves the active oxygen (ROS) scavenging ability and stability of the catalytic film.

[0063] The functions of the three atoms in the present invention are as follows: Redox activity of Ru: The low electronegativity and stable oxidation states of Ru (such as Ru³⁺ / Ru 4 ⁺) provide efficient redox active centers, promoting the decomposition of H2O2 (catalase-like activity) and the disproportionation of O2⁻ (superoxide dismutase-like activity).

[0064] d-electron regulation of V: The d-electron configuration of V (such as V 4 ⁺ / V 5 ⁺) optimizes the adsorption energy of H2O2 at the active site and reduces the reaction energy barrier. DFT calculations show that the doping of V reduces the adsorption energy of H2O2 from -1.2 eV to -1.8 eV, significantly enhancing the catalytic kinetics.

[0065] Charge redistribution of B: The electron-deficient property of B induces local charge redistribution by forming Ru-B bonds, accelerating electron transfer. The introduction of B increases the electron density gradient at the metal-nonmetal interface, and the turnover number (TON) increases from 3.11 s⁻¹ (VRu) to 7.66 s⁻¹ (VRuB).

[0066] To evaluate the catalytic elimination ability of ·O2- radicals, 1 mg of KO2 was dissolved in 1 mL of dimethyl sulfoxide (DMSO) solution containing 3 mg of 18-crown-6. Subsequently, a 1 cm 2 hydrogen evolution catalyst was introduced into the prepared KO2 / DMSO solution. After the reaction proceeded for 5 minutes, 10 μL of nitroblue tetrazolium (NBT)-DMSO solution (10 mg mL -1 NBT) was added to evaluate the remaining ·O2- radicals. The absorbance of the reaction mixture at λ max = 680 nm was immediately recorded and compared with the initial -O2- concentration to determine the ·O2- scavenging efficiency.

[0067] Male BALB / c mice aged 6 - 8 weeks were induced with a high-fat diet for 4 weeks, and then intraperitoneally injected with 200 mg / kg streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA). Blood glucose levels were measured using a blood glucose meter (Roche Diagnostics, Mannheim, Germany). Animals with blood glucose levels exceeding 20 mmol / L for two consecutive days were considered diabetic animals and underwent skin wound surgery. The central area of the back of the diabetic model mice was depilated using depilatory cream and a depilatory device. A circular wound with a diameter of 8 mm was made using a skin biopsy punch, and the wound edges were sharp and clean. The wound was pressed with a cotton swab to stop bleeding.

[0068] It should be noted that Figures 2 to 6In this, VRuB refers to the sample prepared in Example 1, VRu refers to the sample prepared in Comparative Example 1, and the control group (Control) refers to the pure silica gel substrate.

[0069] Figure 2 The results of the elimination of hydrogen peroxide (H2O2) over time (0 to 30 minutes) under three different experimental conditions are shown: VRuB shows the fastest elimination rate of H2O2, and the elimination percentage of H2O2 drops sharply at the beginning of the experiment. By 5 minutes, the elimination percentage decreases significantly and continues to decrease steadily within 30 minutes, stabilizing at around 20%. VRu has a similar trend, but is slower compared to VRuB. The elimination rate of VRu also drops rapidly in the first few minutes, but the decrease is smaller, and it stabilizes at around 30% at 30 minutes. The control group has the worst H2O2 elimination effect. The curve remains almost flat, and the elimination rate of H2O2 only decreases slightly within 30 minutes and finally drops to around 85%. The data indicate that VRuB is the most effective in eliminating H2O2, followed by VRu, while the control group has little effect on reducing the H2O2 level.

[0070] Figure 3 This figure shows the enzyme kinetic parameters under two experimental conditions: V max and K m , corresponding to VRuB and VRu respectively. In terms of V max (maximum reaction rate), the value of VRuB is higher, around 30 μM·s⁻¹, while the value of VRu is slightly lower, about 25 μM·s⁻¹. This indicates that VRuB is superior to VRu in the maximum reaction rate. In terms of K m (Michaelis constant), the value of VRuB is lower, close to 350 mM, indicating its higher affinity for the substrate. While the K m value of VRu is slightly higher, close to 400 mM, indicating that VRu has a weaker substrate affinity. In summary, VRuB shows higher efficiency in both reaction rate and substrate affinity.

[0071] Figure 4 In the graph of the relationship between absorbance and wavelength, the absorbances of VRuB and VRu are lower than that of the control group (Control), indicating that they have a better scavenging effect on superoxide anion (·O2⁻). Especially the absorbance of VRuB is the lowest, further indicating its higher scavenging rate of ·O2⁻, which can effectively reduce the concentration of free radicals and thus improve its antioxidant activity. VRuB and VRu show similar performance in the elimination rate of SOD-like enzyme activity, both approaching 80%, showing strong scavenging ability with almost no significant difference. Therefore, the low value of absorbance reflects a higher scavenging rate of ·O2⁻, indicating that VRuB shows a stronger antioxidant effect in this test.

[0072] Figure 5 shows the droplet contact angle image taken by a contact angle tester. The contact angle formed between the droplet and the solid surface can be used to characterize the wettability between the liquid and the solid. In the figure, it can be seen that the bottom of the droplet contacts the solid surface, and the degree of the contact angle is calculated by measuring the angle between the edge of the droplet and the solid surface. According to Figure 5 the measurement data in, the contact angle of the biocatalytic film in Example 1 is about 150°. A high contact angle value (e.g., 150°) indicates that the droplet exhibits strong water-repellent behavior on the solid surface, that is, the droplet does not spread rapidly but remains relatively spherical. This usually represents that the surface has strong hydrophobicity.

[0073] Figure 6 shows the wound healing process at different time points or treatment conditions. The image of the wound is on the left, and the corresponding analysis diagram of the healing area is on the right. SWD refers to the silicone + coating composite structure, and Gauze refers to the gauze. Wound image: The wound heals gradually over time or under different treatment conditions. Each row shows the wound pictures of different treatment groups at different time points. From top to bottom, it can be seen that the wound changes from initial redness, exudation to contraction and surface changes after healing. The appearance and size of the wound have changed significantly over time. Healing area analysis diagram: On the right side of each wound is the color distribution of the healing area obtained through image analysis. Different colors represent different healing states or different types of wound areas. Generally, blue represents relatively healthy tissue areas, and yellow and green may represent different stages of healing or tissue changes.

[0074] By comparison, it can be seen that with time or different treatment conditions, the degree of wound healing is different, the healing area gradually increases, the wound becomes more contracted, and the healing state gradually improves. The overall trend is that the wound heals gradually within a certain time, and the color of the area gradually transitions from a darker color to a lighter color, reflecting the healing process.

[0075] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0076] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A method for preparing a biocatalytic film, characterized in that, It includes the following steps: Pre-treat the substrate to remove surface impurities and grease to obtain a pre-treated substrate; Using Ru, B, and V as targets and the pre-treated substrate as the substrate, deposit on the pre-treated substrate by magnetron sputtering under the conditions that the sputtering power of Ru and B is 50W - 80W and the sputtering voltage of V is 200V - 430V. During the deposition process, regulate the formation of the ternary phase of V, Ru, and B and regulate the electronic configuration of V through the sputtering voltage of V and the sputtering power of Ru and B to enhance the activity of the catalase-like enzyme and prepare a biocatalytic film.

2. The preparation method of a biocatalytic film according to claim 1, characterized in that, The sputtering power of both Ru and B is 80W.

3. The preparation method of a biocatalytic film according to claim 1, wherein The sputtering voltage of V is 360V.

4. The preparation method of a biocatalytic film according to claim 1, wherein, The sputtering power of V is 50W - 80W.

5. The preparation method of a biocatalytic film according to claim 1, wherein, The mass flow rate of the sputtering gas is 30sccm - 50sccm, and the sputtering pressure is 0.2Pa - 0.4Pa.

6. The preparation method of a biocatalytic film according to claim 1, wherein The angle of the Ru target facing the substrate is 45° - 60°, the angle of the B target facing the substrate is 45° - 60°, and the angle of the V target facing the substrate is 45° - 60°.

7. The preparation method of a biocatalytic film according to claim 1, characterized in that The sputtering temperature is room temperature, and the deposition time is 30min - 40min.

8. The preparation method of a biocatalytic film according to claim 1, characterized in that The distance from the substrate to the target is 15cm - 20cm.

9. A biocatalytic film prepared by the preparation method according to any one of claims 1 - 8.

10. An application of the biocatalytic film according to claim 9 in the preparation of reactive oxygen species eliminating materials.

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