Preparation method and application of titanium alloy surface antibacterial nano-enzyme coating

Abstract

The application relates to a preparation method and application of a titanium alloy surface antibacterial nanometer enzyme coating and relates to a titanium alloy surface modification method and application. The application aims to solve the problem of nanometer enzyme activity reduction caused by the shielding effect of a resin primer on active sites of nanometer enzymes in a traditional nanometer enzyme immobilization method. A porous ceramic coating is prepared on a titanium alloy surface through plasma electrolytic oxidation technology, active groups and nano structures are generated on the surface of the porous ceramic coating through alkali heat treatment, and cerium-based nanometer enzyme UiO-66 (Ce) with high HPO activity and high binding force is grown on the surface of the titanium alloy porous ceramic coating in situ; the PEO / UiO-66 (Ce) integral coating prepared in the in-situ growth mode has the advantages that the UiO-66 (Ce) nanometer enzyme is directly grown on the surface in situ, active sites can be fully exposed to a water environment, Br ‑ and H2O2 can easily enter the active sites to trigger a catalytic reaction to generate HOBr.

Description

Technical Field

[0001] This invention relates to a method and application for modifying the surface of titanium alloys. Background Technology

[0002] Titanium alloys, known as "marine metals" due to their high strength, light weight, high toughness, and resistance to seawater corrosion, are widely used in ships, nuclear submarines, and seawater desalination, making them one of the key new materials in marine engineering. However, compared to other metals, titanium alloys have a dense TiO2 film on their surface, giving them excellent biocompatibility. Almost all marine organisms can adhere to their surface, leading to serious biofouling problems. Marine biofouling not only increases ship drag and fuel consumption but also causes pipe blockage, affects the function of underwater equipment, and reduces equipment lifespan; biofouling has become a global problem. Based on the evolution of biofouling, research has shown that inhibiting the initial adhesion of bacteria and controlling biofilm formation can significantly reduce the subsequent adhesion of larger organisms, thus directly inhibiting biofouling in the initial stage. Therefore, constructing antibacterial functional coatings on titanium alloy surfaces to inhibit biofouling formation is of great significance for the development of marine equipment. Currently, the most economical, simple, and effective method is to apply antifouling coatings to material surfaces. These coatings release toxic substances to repel or kill organisms attached to the surface, achieving antifouling. Examples include antifouling release coatings such as cuprous oxide and tributyltin, and organotin self-polishing coatings. However, these coatings lack specificity, exhibit high toxicity to non-target marine organisms, and the continuous release of antifouling agents causes serious damage to the marine environment. Furthermore, research reports indicate that marine organisms develop resistance after long-term use of copper-based antifouling materials. Environmental concerns and stringent legislation have led to the gradual banning of these coatings globally. Therefore, developing novel antifouling coatings with broad-spectrum antifouling capabilities and strong environmental adaptability is of paramount importance.

[0003] In recent years, inspired by the antifouling strategy of vanadium halide peroxidase secreted by marine macroalgae to combat the attachment of fouling organisms on their surfaces, antifouling strategies based on nanozymes have attracted widespread attention from scholars at home and abroad due to their significant advantages of higher efficiency, greener and more environmentally friendly approaches. Among these, halogenated peroxidase (HPO) catalyzes the reaction of Br₂ in seawater in the presence of trace amounts of H₂O₂. - It is converted into the corresponding hypobromic acid (HOBr). HOBr selectively destroys proteins and halogenated bacterial signaling molecules in bacterial membranes, thereby inhibiting quorum sensing quenching and blocking intercellular communication, thus suppressing bacterial adhesion to material surfaces. Currently, various nanozymes such as CeO2 have been developed. 2-xNanomaterials with halogenated peroxidase activity, such as nanorods, NL-NiMoS2, and W-UiO, have been used in antibacterial and antifouling research. Among them, cerium-based MOFs have been shown to have excellent HPO properties and are expected to be used to prevent biofilm formation. Although HPO-active nanozymes have shown great promise in antibacterial and antifouling applications, current research typically uses resin as a dispersion medium to immobilize nanozymes on the substrate surface, a method with significant limitations. Since the HPO catalytic reaction of cerium-based nanozymes mainly occurs on their surface, and their activity depends on the diffusion of hydrophilic small molecules H2O2 and Br- from seawater to the surface active sites and the resulting charge transfer to form HOBr, the interfacial structure formed after mixing with resin severely hinders this crucial charge transfer process, weakening the catalytic activity of the nanozymes. Furthermore, the resin substrate covering hinders the outward diffusion of HOBr, further limiting enzyme activity. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of reduced nanozyme activity caused by the shielding effect of resin primer on the active sites of nanozymes in traditional nanozyme immobilization methods, and to provide a method for preparing and applying an antibacterial nanozyme coating on a titanium alloy surface.

[0005] Compared with the traditional method of dispersing nanozymes in resin, the present invention constructs an integral nanozyme coating in situ on the substrate surface, which can more effectively maintain the activity and reaction efficiency of nanozymes, thereby effectively exerting antibacterial and antifouling properties.

[0006] A method for preparing an antibacterial nanoenzyme coating on a titanium alloy surface is specifically carried out according to the following steps:

[0007] I. Pretreatment of Titanium Alloys:

[0008] The titanium alloy is polished with sandpaper and then cleaned to remove surface oil and dirt, resulting in a pre-treated titanium alloy.

[0009] II. Preparation of electrolyte:

[0010] Na2SiO3·9H2O, Na6O 18 P6, (NaPO3) n Dissolve NaOH in deionized water to obtain the electrolyte;

[0011] 3. Connect the positive terminal of the plasma electrolytic oxidation equipment power supply to the pretreated titanium alloy, and the negative terminal to the stainless steel electrolytic cell. Pour the electrolyte into the stainless steel electrolytic cell and stir continuously. Immerse the pretreated titanium alloy in the electrolyte at a current density of 6 A / cm². 2 ~10A / cm 2The titanium alloy was reacted for a period of time under the conditions of a duty cycle of 20% to 50% and a frequency of 500Hz to 1000Hz. After the reaction was completed, the titanium alloy was taken out, washed with deionized water, and then dried to obtain a titanium alloy containing a titanium oxide ceramic transition layer.

[0012] IV. The titanium alloy containing the titanium oxide ceramic transition layer is immersed in a NaOH solution at a temperature of 60℃~80℃ for a period of time for etching. After the etching is completed, the titanium alloy is taken out, cleaned with deionized water, and then dried to obtain a titanium alloy with a nanostructure on the surface. five,

[0014] ① Dissolve (NH4)2Ce(NO3)6 in deionized water to obtain (NH4)2Ce(NO3)6 solution;

[0015] ② Dissolve terephthalic acid in N,N-dimethylformamide to obtain a terephthalic acid solution;

[0016] ③ Immerse the titanium alloy with nanostructures on its surface in a (NH4)2Ce(NO3)6 solution for a period of time, then add terephthalic acid solution dropwise to the (NH4)2Ce(NO3)6 solution, so that the terephthalic acid reacts with the Ce on the surface of the titanium alloy. 4+ Complexation;

[0017] 6. Transfer the mixture from step 5.③ to a reaction vessel, with the titanium alloy placed at a 45-degree angle in the reaction vessel and subjected to hydrothermal reaction at 100℃~150℃ for a period of time. After cooling to room temperature, remove the titanium alloy, clean and dry it to obtain a titanium alloy with an antibacterial nanoenzyme coating on its surface.

[0018] The principle of this invention:

[0019] I. This invention proposes to prepare a porous ceramic coating on the surface of a titanium alloy using plasma electrolytic oxidation (PEO) technology. Then, alkaline heat treatment is applied to generate active groups and nanostructures on the surface of the porous ceramic coating. Finally, a cerium-based nanozyme, UiO-66(Ce), with high HPO activity and high binding strength is in situ grown on the surface of the porous ceramic coating of the titanium alloy. Because the UiO-66(Ce) nanozyme is directly grown in situ on the surface, the active sites can be fully exposed to the aqueous environment. - H2O2 can easily enter the active site to trigger the catalytic reaction to produce HOBr; in addition, the porous structure of the PEO coating can provide more growth sites, enabling the growth of more nanozymes per unit area. The synergistic effect of the excellent enzyme-like activity inherent in UiO-66(Ce) nanozymes and the structural advantages of in-situ growth gives the PEO / UiO-66(Ce) monolithic coating good halogenated peroxidase activity and antibacterial properties;

[0020] II. The porous ceramic coating prepared on the surface of titanium alloy using PEO technology mainly consists of TiO2 and amorphous SiO2. During the etching process, the TiO2 and SiO2 on the coating surface are affected by OH groups in the alkaline solution. - An attack will trigger the following reaction:

[0021] TiO2+OH - →HTiO2 -

[0022] SiO2 + NaOH + H2O → NaSiO3 + 2H2

[0023] During this process, a large number of active groups are generated on the coating surface, and numerous nanostructures are formed due to the etching of TiO2 and SiO2 on the coating surface. The active groups generated on the coating surface are used to adsorb Ce in the subsequent hydrothermal solvent. 4+ This allows UiO-66(Ce) nanozymes to grow in situ on their surface. The nanostructure provides a certain degree of confined growth for the UiO-66(Ce) nanozymes, preventing them from aggregating and stacking, and maximizing the exposure of active sites.

[0024] Advantages of this invention:

[0025] This invention provides a method for preparing nanozymes with halogenated peroxidase (HPO) activity in situ on the surface of titanium alloys and applying it to the fields of antibacterial and antifouling. The method uses TA1 as a substrate, prepares a porous ceramic coating on the TA1 surface using PEO technology, then etches the PEO coating in a sodium hydroxide solution to activate its surface, generating active groups and microstructures. Finally, a UiO-66(Ce) nanozyme with high binding strength and high HPO activity is constructed in situ on the etched porous surface using a solvothermal method. The in-situ growth of the nanozyme on the titanium alloy surface avoids the shielding effect of resin base materials on the active sites of the nanozyme in traditional coating methods, fully utilizing HPO activity to generate a large amount of hypobromous acid to inhibit bacteria and biofouling. Its advantages are as follows:

[0026] (1) Metal-organic frameworks (MOFs) are ideal enzyme mimics. UiO-66(Ce), as a cerium-based MOF, has primary and secondary coordination environments similar to natural enzymes and possesses high HPO activity, which can be used to mimic natural vanadium halide peroxidases and catalyze Br₂. - It reacts with H2O2 to form HOBr, which has good catalytic antibacterial ability and also has the advantages of being green, efficient, stable and low cost;

[0027] (2) A porous ceramic coating was prepared on TA1 using PEO technology. The surface was activated and a nanostructure was generated by alkaline heat treatment. As a transition layer, it further improved the corrosion resistance of TA1 and the bonding strength between the overall coating and the substrate. On the other hand, the active groups and microstructures on the surface provided active sites for the in-situ growth of UiO-66(Ce) nanozymes.

[0028] (3) The in-situ construction of UiO-66(Ce) nanozyme on the TA1 surface maximizes the HPO activity of UiO-66(Ce) while ensuring high binding strength. It fully exposes the catalytic active sites, enabling direct interaction and efficient charge transfer between the nanozyme and the substrate, and generating a large amount of hypobromic acid to inhibit bacteria and fouling organisms. Attached Figure Description

[0029] Figure 1 XRD patterns of the titanium alloy with a titanium oxide ceramic transition layer, the titanium alloy with a nanostructure on the surface, and the titanium alloy with an antibacterial nanoenzyme coating prepared in Example 1.

[0030] Figure 2 Infrared spectra of the titanium alloy containing a titanium oxide ceramic transition layer, the titanium alloy with nanostructures on its surface, and the titanium alloy with an antibacterial nanoenzyme coating on its surface prepared in Example 1.

[0031] Figure 3 SEM images of the titanium alloy with a titanium oxide ceramic transition layer, the titanium alloy with nanostructures on its surface, and the titanium alloy with an antibacterial nanoenzyme coating prepared in Example 1.

[0032] Figure 4 SEM images and EDS surface scans of the titanium alloy with an antibacterial nanoenzyme coating prepared in Example 1;

[0033] Figure 5 XPS image of the titanium alloy with an antibacterial nanoenzyme coating on its surface prepared in Example 1;

[0034] Figure 6 HPO activity test diagram of the titanium alloy with antibacterial nanoenzyme coating on the surface prepared in Example 1;

[0035] Figure 7 The HPO activity cycle stability diagram is shown for the titanium alloy with an antibacterial nanozyme coating prepared in Example 1. Figure 7 The left-middle graph shows the absorbance change at a wavelength of 432 nm, representing the degradation cycle performance of phenol red; the right-middle graph shows the absorbance change at a wavelength of 590 nm, representing the formation cycle performance of bromophenol blue.

[0036] Figure 8The graph shows the degradation performance of celestite blue by the titanium alloy with an antibacterial nanozyme coating prepared in Example 1.

[0037] Figure 9 This is a diagram showing the antibacterial properties.

[0038] Figure 10 HPO activity test diagrams of the titanium alloy with antibacterial nanoenzyme coating prepared in Example 1 and the conventional resin-dispersed coating prepared in Comparative Example 1.

[0039] Figure 11 The HPO activity test diagrams for the titanium alloy with an antibacterial nanoenzyme coating and the PEO titanium oxide coating prepared in Example 1 are shown. Detailed Implementation

[0040] Specific Implementation Method 1: This implementation method is a method for preparing an antibacterial nanoenzyme coating on a titanium alloy surface, specifically completed according to the following steps:

[0041] I. Pretreatment of Titanium Alloys:

[0042] The titanium alloy is polished with sandpaper and then cleaned to remove surface oil and dirt, resulting in a pre-treated titanium alloy.

[0043] II. Preparation of electrolyte:

[0044] Na2SiO3·9H2O, Na6O 18 P6, (NaPO3) n Dissolve NaOH in deionized water to obtain the electrolyte;

[0045] 3. Connect the positive terminal of the plasma electrolytic oxidation equipment power supply to the pretreated titanium alloy, and the negative terminal to the stainless steel electrolytic cell. Pour the electrolyte into the stainless steel electrolytic cell and stir continuously. Immerse the pretreated titanium alloy in the electrolyte at a current density of 6 A / cm². 2 ~10A / cm 2 The titanium alloy was reacted for a period of time under the conditions of a duty cycle of 20% to 50% and a frequency of 500Hz to 1000Hz. After the reaction was completed, the titanium alloy was taken out, washed with deionized water, and then dried to obtain a titanium alloy containing a titanium oxide ceramic transition layer.

[0046] IV. The titanium alloy containing the titanium oxide ceramic transition layer is immersed in a NaOH solution at a temperature of 60℃~80℃ for a period of time for etching. After the etching is completed, the titanium alloy is taken out, cleaned with deionized water, and then dried to obtain a titanium alloy with a nanostructure on the surface. five,

[0048] ① Dissolve (NH4)2Ce(NO3)6 in deionized water to obtain (NH4)2Ce(NO3)6 solution;

[0049] ② Dissolve terephthalic acid in N,N-dimethylformamide to obtain a terephthalic acid solution;

[0050] ③ Immerse the titanium alloy with nanostructures on its surface in a (NH4)2Ce(NO3)6 solution for a period of time, then add terephthalic acid solution dropwise to the (NH4)2Ce(NO3)6 solution, so that the terephthalic acid reacts with the Ce on the surface of the titanium alloy. 4+ Complexation;

[0051] 6. Transfer the mixture from step 5.③ to a reaction vessel, with the titanium alloy placed at a 45-degree angle in the reaction vessel and subjected to hydrothermal reaction at 100℃~150℃ for a period of time. After cooling to room temperature, remove the titanium alloy, clean and dry it to obtain a titanium alloy with an antibacterial nanoenzyme coating on its surface.

[0052] This invention combines PEO technology and chemical etching to in-situ grow a UiO-66(Ce)-loaded nanoenzyme coating (i.e., an antibacterial nanoenzyme coating) on ​​the surface of titanium alloy, achieving continuous and uniform growth of UiO-66(Ce) enzymes with high HPO activity on the titanium alloy surface. The porous ceramic coating prepared by PEO on the titanium alloy surface serves as a transition layer, further improving the corrosion resistance of the titanium alloy. Furthermore, the active groups and nanostructures generated on the surface of the porous ceramic coating after alkaline etching provide nucleation sites for the in-situ growth of UiO-66(Ce), enabling continuous and uniform growth of UiO-66(Ce) on the porous ceramic surface. The nanoenzyme is firmly fixed to the coating surface through the electrostatic adsorption of surface active groups and the mechanical "locking" mechanism of the nanostructures, fully utilizing the HPO activity of the UiO-66(Ce) nanoenzyme and avoiding the adverse effects of traditional coating methods on the active sites of the nanoenzyme. This method provides good inspiration and reference for the in-situ construction and design of other nanoenzymes on material surfaces and has promising application prospects.

[0053] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that: in step one, the titanium alloy is polished sequentially using 600#, 800#, 1200#, and 1500# sandpaper; the cleaning described in step one involves sequentially cleaning with anhydrous ethanol and acetone. Other steps are the same as in Specific Implementation Method One.

[0054] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the titanium alloy mentioned in step one is TA1, TA2, or TC4. The other steps are the same as in Specific Implementation Method One or Two.

[0055] Specific Implementation Method Four: The difference between this implementation method and Specific Implementation Methods One to Three is that the concentration of Na2SiO3·9H2O in the electrolyte mentioned in step two is 15g / L to 20g / L, and the concentration of Na6O is... 18 The concentration of P6 is 6 g / L to 10 g / L (NaPO3). n The concentration of the active ingredient is 2 g / L to 4 g / L, and the concentration of NaOH is 6 g / L to 8 g / L. Other steps are the same as in embodiments one through three.

[0056] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the reaction time in step three is 10 to 20 minutes; after the reaction in step three is completed, the titanium alloy is removed and washed with deionized water 3 to 5 times to obtain a titanium alloy containing a titanium oxide ceramic transition layer. Other steps are the same as in Specific Implementation Methods One to Four.

[0057] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: the concentration of the NaOH solution mentioned in step four is 1 mol / L to 3 mol / L; the etching time mentioned in step four is 6 h to 24 h. The other steps are the same as in Specific Implementation Methods One to Five.

[0058] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One through Six in that: after etching in step four, the titanium alloy is removed, washed 3 to 5 times with deionized water, and then dried at 60°C to 80°C for 6 to 12 hours to obtain a titanium alloy with a nanostructure on its surface. Other steps are the same as in Specific Implementation Methods One through Six.

[0059] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in the following ways: In step five ①, the mass ratio of (NH4)2Ce(NO3)6 to the volume ratio of deionized water is (3g-4g):(10mL-15mL); in step five ②, the mass ratio of terephthalic acid to the volume ratio of N,N-dimethylformamide is (1g-1.1g):(20mL-30mL); in step five ③, the volume ratio of (NH4)2Ce(NO3)6 solution to terephthalic acid solution is (10mL-15mL):(20mL-30mL); and in step five ③, the soaking time is 3h-6h. The other steps are the same as in Specific Implementation Methods One to Seven.

[0060] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in the following ways: the hydrothermal reaction time in step six is ​​2 to 6 hours; the titanium alloy is cleaned 2 to 4 times sequentially with deionized water, anhydrous ethanol, and acetone in step six; the drying temperature in step six is ​​60°C to 80°C, and the drying time is 6 to 12 hours. Other steps are the same as in Specific Implementation Methods One to Eight.

[0061] Specific Implementation Method 10: This implementation method is an application of an antibacterial nanoenzyme coating on a titanium alloy surface in the field of marine antifouling.

[0062] The beneficial effects of the present invention are verified using the following embodiments:

[0063] Example 1: A method for preparing an antibacterial nanoenzyme coating on a titanium alloy surface, specifically comprising the following steps:

[0064] I. Pretreatment of Titanium Alloys:

[0065] The titanium alloy was polished with 600#, 800#, 1200# and 1500# sandpaper in sequence, and then cleaned with anhydrous ethanol and acetone in sequence to remove the oil stains on the surface of the titanium alloy, thus obtaining the pretreated titanium alloy.

[0066] The titanium alloy mentioned in step one is TA1;

[0067] II. Preparation of electrolyte:

[0068] Na2SiO3·9H2O, Na6O 18 P6, (NaPO3) n Dissolve NaOH in deionized water to obtain the electrolyte;

[0069] The concentration of Na2SiO3·9H2O in the electrolyte mentioned in step two is 15 g / L, and the concentration of Na6O is... 18 The concentration of P6 is 6 g / L (NaPO3). n The concentration of the substance is 2 g / L, and the concentration of NaOH is 6 g / L;

[0070] 3. Connect the positive terminal of the plasma electrolytic oxidation equipment power supply to the pretreated titanium alloy, and the negative terminal to the stainless steel electrolytic cell. Pour the electrolyte into the stainless steel electrolytic cell and stir continuously. Immerse the pretreated titanium alloy in the electrolyte at a current density of 6 A / cm². 2 The reaction was carried out for 10 minutes under the conditions of a duty cycle of 20% and a frequency of 1000Hz. After the reaction was completed, the titanium alloy was taken out, washed three times with deionized water, and then dried at 60℃ for 12 hours to obtain a titanium alloy (PEO) containing a titanium oxide ceramic transition layer.

[0071] IV. The titanium alloy containing the titanium oxide ceramic transition layer was immersed in a NaOH solution at 60°C for 12 hours for etching. After etching, the titanium alloy was taken out, washed with deionized water 3 times, and then dried at 60°C for 8 hours to obtain a titanium alloy (PEO-Etch) with nanostructures on the surface.

[0072] The concentration of the NaOH solution mentioned in step four is 3 mol / L; five,

[0074] ① Dissolve 3.506g of (NH4)2Ce(NO3)6 in 12mL of deionized water to obtain (NH4)2Ce(NO3)6 solution;

[0075] ② Dissolve 1.062g of terephthalic acid in 24mL of N,N-dimethylformamide to obtain a terephthalic acid solution;

[0076] ③ Immerse the titanium alloy with nanostructures on its surface in a (NH4)2Ce(NO3)6 solution for 3 hours, then add terephthalic acid solution dropwise to the (NH4)2Ce(NO3)6 solution, allowing the terephthalic acid to react with the Ce on the titanium alloy surface. 4+ Complexation;

[0077] 6. Transfer the mixture from step 5.③ to a reaction vessel, with the titanium alloy placed at a 45-degree angle in the reaction vessel and subjected to hydrothermal reaction at 100°C for 3 hours. After cooling to room temperature, remove the titanium alloy and clean it three times each with deionized water, anhydrous ethanol, and acetone. Then dry it at 60°C for 12 hours to obtain a titanium alloy (PEO / UiO-66(Ce)) with an antibacterial nanoenzyme coating on its surface.

[0078] Figure 1 XRD patterns of the titanium alloy with a titanium oxide ceramic transition layer, the titanium alloy with a nanostructure on the surface, and the titanium alloy with an antibacterial nanoenzyme coating prepared in Example 1.

[0079] from Figure 1 As can be seen from the data, the titanium alloy (PEO / UiO-66(Ce)) with an antibacterial nanoenzyme coating prepared in Example 1 is composed of titanium and titanium oxide, wherein the titanium oxide includes rutile TiO2 and anatase TiO2. The XRD pattern of PEO / UiO-66(Ce) shows sharp reflections near 7.3°, 8.2°, 11.6°, 13.6°, and 14.3°, which correspond to the (111), (200), (220), (311), and (222) characteristic crystal planes of UiO-66(Ce), respectively. This is in good agreement with the simulation data, indicating that UiO-66(Ce) was successfully loaded onto the PEO coating surface.

[0080] Figure 2Infrared spectra of the titanium alloy containing a titanium oxide ceramic transition layer, the titanium alloy with nanostructures on its surface, and the titanium alloy with an antibacterial nanoenzyme coating on its surface prepared in Example 1.

[0081] from Figure 2 It can be seen that at 1623-1634 cm⁻¹, a value was observed on the PEO-Etch surface. -1 Within the range and 3300-3600cm -1 The characteristic absorption peaks of the OH stretching vibration over a wide bandwidth demonstrate that the chemically etched PEO-Etch surface contains a large number of hydroxyl groups. The presence of these active hydroxyl functional groups ensures the presence of Ce... 4+ The adsorption of cations on the coating surface provides the necessary conditions for the in-situ growth of UiO-66(Ce). Furthermore, at 1070 cm⁻¹... -1 An absorption peak for the PO bending vibration was observed, which is attributed to the presence of amorphous phosphate in the coating. PEO / UiO-66(Ce) was observed at 1563 cm⁻¹. -1 746cm -1 and 1380cm -1 The position represents the C=C vibration mode, CO bond extension mode, and Ce-O mode in UiO-66(Ce).

[0082] Figure 3 SEM images of the titanium alloy with a titanium oxide ceramic transition layer, the titanium alloy with nanostructures on its surface, and the titanium alloy with an antibacterial nanoenzyme coating prepared in Example 1.

[0083] from Figure 3 As can be seen, the PEO coating surface exhibits a large number of micron / nanopores, displaying a classic porous structure. After chemical etching, the PEO-Etch coating retains the porous characteristics of the PEO coating as a whole. At high magnification, small protrusions were observed on the PEO-Etch surface and the inner walls of the micropores after chemical etching, forming a complex secondary nanostructure. In fact, this unique secondary nanostructure provides anchoring points for the subsequent in-situ growth of UiO-66(Ce) to embed nanoparticles onto the coating surface. In the SEM image of PEO / UiO-66(Ce), a significant decrease in the porosity of the coating surface can be clearly seen. This is because the in-situ growth of UiO-66(Ce) sealed the smaller nanopores and defects in the PEO coating. Under high magnification, UiO-66(Ce) was observed to grow continuously and uniformly in situ on the surface of the PEO coating and the inner wall of the micropores. These nanoparticles exhibited regular polyhedral morphology, were relatively uniform in size, with a particle size of 150-200 nm, and were tightly bound to the coating surface through the mechanical "interlocking" mechanism of secondary nanostructures, forming a continuous and stable composite structure.

[0084] Figure 4 SEM images and EDS surface scans of the titanium alloy with an antibacterial nanoenzyme coating prepared in Example 1;

[0085] from Figure 4 As can be seen, the average thickness of the antibacterial nanoenzyme coating prepared in Example 1 is about 15 μm. The EDS spectrum clearly shows that the cross-section of the PEO / UiO-66(Ce) coating is mainly composed of elements such as Ti, O, Ce and Si. Among them, Ti comes from the TA1 substrate, Si comes from the electrolyte, and Ce comes from the UiO-66(Ce) grown in situ on the surface.

[0086] Figure 5 XPS image of the titanium alloy with an antibacterial nanoenzyme coating on its surface prepared in Example 1;

[0087] from Figure 5 The full spectrum shows peaks for C1s, Ti2p, O1s, and Ce3d around 285 eV, 460 eV, 532 eV, and in the range of 870-930 eV. Figure 5 The high-resolution spectrum of C1s in b shows three peaks at 284.79 eV, 286.36 eV, and 288.58 eV, which correspond to the C=C, CO, and C=O bonds in the PTA linker, respectively. (Ti2p spectrum) Figure 5 c) The peaks at 465.59 eV and 459.87 eV correspond to Ti-O bonds in TiO2. In the O1s spectrum ( Figure 5 d) The three peaks at 529.85 eV, 531.45 eV, and 532.45 eV correspond to OC=O, Ce-OH, and Ce-O-Ce bonds, respectively. The Ce3d spectrum reveals Ce... 3+ and Ce 4+ The coexistence of Ce on the UiO-66(Ce) surface, with peaks at 885.96 eV, 899.45 eV, and 904.61 eV, corresponds to Ce. 3+ The peaks at 882.81 eV, 888.88 eV, 901.84 eV, 907.55 eV, and 917.32 eV are related, while the peaks at these values ​​are attributed to Ce. 4+ .

[0088] HPO activity test:

[0089] This invention investigated the halogenated peroxidase activity of a monolithic PEO / UiO-66(Ce) coating using a phenol red bromination assay. The PEO / UiO-66(Ce) sample was suspended in a stirred beaker, and 25 mL of a mixed solution containing 50 μM phenol red (PR) and 25 mM ammonium bromide (NH4Br) was added to ensure complete immersion of the sample. Subsequently, 500 μM H2O2 was injected to trigger the bromination reaction. At room temperature, 2 mL of the suspension was taken at different time intervals, and the phenol red bromination reaction was tracked over time using UV-Vis absorption spectroscopy in the wavelength range of 300-700 nm. The monitoring wavelengths for phenol red (PR) and bromophenol blue (Br4PR) were 432 nm and 590 nm, respectively. The experiment was conducted for 480 minutes.

[0090] Figure 6 HPO activity test diagram of the titanium alloy with antibacterial nanoenzyme coating on the surface prepared in Example 1;

[0091] like Figure 6 As shown: In the presence of H2O2 and NH4Br, UiO-66(Ce) on the PEO / UiO-66(Ce) surface can catalyze the reaction of H2O2 and Br. - Hypobromic acid is generated, and the resulting HOBr reacts with phenol red (yellow in acidic solution) to form bromophenol blue, turning the solution blue-violet. Time-dependent catalytic behavior results of the PEO / UiO-66(Ce) monolithic coating show that the absorption peak intensity of phenol red at λ=430 nm gradually decreases over time, while the absorption peak intensity of bromophenol blue at λ=590 nm gradually increases. This clearly demonstrates the dynamic process of phenol red forming bromophenol blue, indicating that the PEO / UiO-66(Ce) monolithic coating exhibits good HPO activity.

[0092] HPO activity cycling stability test:

[0093] The cyclic stability of the PEO / UiO-66(Ce) monolithic coating was tested by 5 consecutive 8-hour cycles using phenol red bromination experiments. Absorbance changes were monitored at wavelengths of 432 nm and 590 nm during the tests. After each 8-hour cycle, the reaction solution was replaced to ensure that the concentrations of phenol red and ammonium bromide in the reaction solution were 50 μM and 25 mM, respectively, before each cycle. Subsequently, 500 μM H₂O₂ was injected as a trigger to initiate the single reaction.

[0094] Figure 7 The HPO activity cycle stability diagram is shown for the titanium alloy with an antibacterial nanozyme coating prepared in Example 1. Figure 7The left-middle graph shows the absorbance change at a wavelength of 432 nm, representing the degradation cycle performance of phenol red; the right-middle graph shows the absorbance change at a wavelength of 590 nm, representing the formation cycle performance of bromophenol blue.

[0095] Figure 7 The results of five consecutive 8-hour HPO activity cycling tests on the PEO / UiO-66(Ce) coating show that the enzyme-like catalytic performance decreased slightly after the first test, but remained basically stable in subsequent tests, indicating that the PEO / UiO-66(Ce) coating has good cycling stability.

[0096] Degradation of lapis lazuli blue:

[0097] This invention investigated the degradation effect of PEO / UiO-66(Ce) coating on azurite blue indicator, demonstrating that the coating catalyzes the degradation of Br. - The PEO / UiO-66(Ce) sample reacts with H₂O₂ to produce HOBr. The PEO / UiO-66(Ce) sample was suspended in a stirred beaker, and 25 mL of a mixed solution containing 50 μM azurite blue and 25 mM NH₄Br was added to ensure complete immersion of the sample. Then, 350 μM H₂O₂ was injected to trigger the catalytic reaction. The solution was collected every hour, and the time-dependent optical absorption spectrum was recorded in the wavelength range of 400-800 nm for 10 hours.

[0098] Figure 8 The graph shows the degradation performance of celestite blue by the titanium alloy with an antibacterial nanozyme coating prepared in Example 1.

[0099] Celestolite blue can only be bleached by HOBr and does not react with H2O2 or superoxide. In the presence of H2O2, the absorption intensity of celestolite blue at 640 nm gradually decreases, clearly indicating that the PEO / UiO-66(Ce) coating catalyzes Br. - It is oxidized to HOBr.

[0100] Antibacterial test:

[0101] The antibacterial properties of the composite coating were characterized using the plate coating method. The antibacterial steps are as follows:

[0102] Bacterial activation: The bacterial stock solution was removed from the -80℃ biomedical freezer and slowly thawed at room temperature. Then, in a laminar flow hood, 25 μL of the bacterial stock solution was pipetted into a sterile tube, and 150 mL of LB liquid bacterial culture medium was added. The sterile tube was then incubated on a shaker at 37℃ and 150 rpm for 12 h. After bacterial culture, the bacteria were washed three times with PBS solution to remove the culture medium. The bacterial solution was then diluted with PBS solution to a concentration of 1.0 × 10⁻⁶ in a laminar flow hood. 8 A bacterial suspension of CFU / mL.

[0103] Bubble tablets: The antibacterial system volume is 5 ml (ensuring the sample is completely submerged in the system), containing 125 μL of the above bacterial suspension (here, 125 μL is 1.0 × 10⁻⁶). 8 A bacterial suspension of CFU / mL was added to 5 mL of PBS solution to form a final concentration of 10. 6 The samples were immersed in a bacterial suspension (approximately 100 μM), NH4Br at 25 mM, and H2O2 at 500 μM, and cultured at 37 °C and 150 rpm for 24 h. A control group containing only bacteria, NH4Br, and H2O2 was set up for convenience.

[0104] Quantitative analysis of antibacterial activity: The above-mentioned reaction system after 24 hours of culture was serially diluted with PBS to 10⁻⁶. 4 The bacterial suspension was diluted 100 μL and then spread evenly on a plate culture medium. The plate was incubated at 37°C for 24 hours, and the inhibition rate was calculated by plate counting.

[0105] Figure 9 This is a diagram showing the antibacterial properties.

[0106] The tested bacterial species was *Escherichia coli*. Figure 9 The results show that PEO / UiO-66(Ce) itself exhibits an antibacterial rate of 43.3%, which is due to the inherent antibacterial activity of UiO-66(Ce). The system was further enhanced by the simultaneous addition of 500 μM H₂O₂ and 25 mM Br₂. - At that time, the antibacterial rate of PEO / UiO-66(Ce) climbed to 97.6%, indicating that the PEO / UiO-66(Ce) coating has excellent halogenated peroxidase catalytic antibacterial properties.

[0107] Comparative Example 1: The traditional method for preparing a coating after resin dispersion is as follows:

[0108] I. Preparation of UiO-66(Ce) nanoparticles by hydrothermal method:

[0109] 3.506 g of (NH4)2Ce(NO3)6 was dissolved in 12 mL of deionized water, and 1.062 g of terephthalic acid was dissolved in 24 mL of N,N-dimethylformamide. The two solutions were then mixed and stirred for 30 min before being transferred to a reaction vessel and heated at 100 °C for 3 h. After cooling to room temperature, the mixture was washed three times alternately with distilled water and ethanol, and then dried in a drying oven at 60 °C for 12 h to obtain UiO-66(Ce) nanoparticles.

[0110] 2. The prepared UiO-66(Ce) nanoparticles were thoroughly mixed with epoxy resin (E44) at a ratio of 5 wt.% and then coated onto a titanium alloy substrate (TA1) using a coating instrument to obtain a coating thickness of 200 μm, thus obtaining a conventional resin-dispersed coating.

[0111] HPO activity test:

[0112] This invention investigated the halogenated peroxidase activity of the monolithic PEO / UiO-66(Ce) coating prepared in Example 1 and the conventional resin-dispersed coating prepared in Comparative Example 1 using a phenol red bromination experiment. The PEO / UiO-66(Ce) sample and the conventional resin-dispersed coating prepared in Comparative Example 1 were suspended in stirred beakers, respectively. 25 mL of a mixed solution containing 50 μM phenol red (PR) and 25 mM ammonium bromide (NH4Br) was added to each beaker to ensure complete immersion of the sample. Subsequently, 500 μM H2O2 was injected to trigger the bromination reaction. At room temperature, 2 mL of the suspension was taken at different time intervals, and the phenol red bromination reaction was tracked over time using UV-Vis absorption spectroscopy in the wavelength range of 300-700 nm. The monitoring wavelengths for phenol red (PR) and bromophenol blue (Br4PR) were 432 nm and 590 nm, respectively. The experiment was conducted for 480 minutes. UiO-66(Ce) nanozyme powder was dispersed in a resin matrix and coated onto a substrate using conventional methods. The activity of the halogenated peroxidase in the nanozyme coating with resin as a dispersant was compared with that of the in-situ grown PEO / UiO-66(Ce) coating of this invention. The results are as follows: Figure 10 As shown;

[0113] Figure 10 HPO activity test diagrams of the titanium alloy with antibacterial nanoenzyme coating prepared in Example 1 and the conventional resin-dispersed coating prepared in Comparative Example 1.

[0114] Figure 10 UiO-66(Ce) / In-situ growth is a titanium alloy with an antibacterial nanoenzyme coating on its surface prepared in Example 1, and UiO-66(Ce) / Resin coated is a conventional resin-dispersed coating prepared in Comparative Example 1.

[0115] from Figure 10 It can be seen that almost no enzyme-catalyzed reaction is detected in nanozyme coatings using resin as the dispersion medium. This is because during the dispersion of UiO-66(Ce) nanozymes, the resin wets the surface of the nanozymes, covering their surface active sites, and the Br in the system... -H2O2 cannot enter the active sites to trigger the reaction. In contrast, the PEO / UiO-66(Ce) monolithic coating prepared by in-situ growth allows the UiO-66(Ce) nanozyme to grow directly on the surface without the need for anchoring to the substrate with resin materials, thus fully exposing the active sites to the system. - H2O2 can easily enter the active site to trigger the reaction and produce hypobromic acid. Furthermore, the porous structure of the PEO coating provides more growth sites, allowing for the growth of more nanozymes per unit area. The synergistic effect of the excellent intrinsic enzyme-like activity of UiO-66(Ce) nanozymes and the structural advantages of in-situ growth gives the PEO / UiO-66(Ce) monolithic coating good halogenated peroxidase activity.

[0116] The HPO activity of the PEO titanium oxide coating was tested according to the above method;

[0117] Figure 11 HPO activity test diagrams of titanium alloys with antibacterial nanoenzyme coatings and PEO titanium oxide coatings prepared in Example 1;

[0118] Figure 11 PEO / UiO-66(Ce) is a titanium alloy with an antibacterial nanoenzyme coating on its surface prepared in Example 1, where PEO is a titanium oxide coating.

[0119] from Figure 11 It can be seen that under the same test conditions, enzyme activity cannot be detected in a simple PEO titanium oxide coating.

Claims

1. A method for preparing an antibacterial nanoenzyme coating on a titanium alloy surface, characterized in that... The preparation method is specifically carried out according to the following steps: Step 1: Pretreatment of titanium alloys: The titanium alloy is polished with sandpaper and then cleaned to remove surface oil and dirt, resulting in a pre-treated titanium alloy. Step 2: Prepare the electrolyte: Na2SiO3·9H2O, Na6O 18 P6, (NaPO3) n Dissolve NaOH in deionized water to obtain the electrolyte; The concentration of Na2SiO3·9H2O in the electrolyte mentioned in step two is 15 g / L~20 g / L, and the concentration of Na6O is... 18 The concentration of P6 is 6 g / L~10 g / L, (NaPO3) n The concentration of the active ingredient is 2 g / L to 4 g / L, and the concentration of NaOH is 6 g / L to 8 g / L. Step 3: Connect the positive terminal of the plasma electrolytic oxidation equipment power supply to the pretreated titanium alloy, and the negative terminal to the stainless steel electrolytic cell. Pour the electrolyte into the stainless steel electrolytic cell and stir continuously. Immerse the pretreated titanium alloy in the electrolyte at a current density of 6 A / cm². 2 ~10A / cm 2 The reaction was carried out for 10 to 20 minutes under the conditions of a duty cycle of 20% to 50% and a frequency of 500 Hz to 1000 Hz. After the reaction was completed, the titanium alloy was taken out, washed with deionized water 3 to 5 times, and then dried to obtain a titanium alloy containing a titanium oxide ceramic transition layer. Step 4: Immerse the titanium alloy containing the titanium oxide ceramic transition layer in a NaOH solution at a temperature of 60℃~80℃ for 6h~24h for etching. After etching, remove the titanium alloy, clean it with deionized water 3~5 times, and then dry it at 60℃~80℃ for 6h~12h to obtain a titanium alloy with a nanostructure on the surface. The concentration of the NaOH solution mentioned in step four is 1 mol / L to 3 mol / L; Step 5 ① Dissolve (NH4)2Ce(NO3)6 in deionized water to obtain (NH4)2Ce(NO3)6 solution; ② Dissolve terephthalic acid in N,N-dimethylformamide to obtain a terephthalic acid solution; ③ Immerse the titanium alloy with nanostructures on its surface in a (NH4)2Ce(NO3)6 solution for 3-6 hours, then add terephthalic acid solution dropwise to the (NH4)2Ce(NO3)6 solution, allowing the terephthalic acid to react with the Ce on the titanium alloy surface. 4+ Complexation; In step 5①, the mass ratio of (NH4)2Ce(NO3)6 to the volume ratio of deionized water is (3g~4g):(10mL~15mL). The mass ratio of terephthalic acid to N,N-dimethylformamide mentioned in step 5② is (1g~1.1g):(20mL~30mL); The volume ratio of (NH4)2Ce(NO3)6 solution to terephthalic acid solution mentioned in step 5③ is (10mL~15mL):(20mL~30mL); Step 6: Transfer the mixture from Step 5 ③ to a reaction vessel, with the titanium alloy placed at a 45-degree angle in the reaction vessel, and perform a hydrothermal reaction at 100℃~150℃ for 2h~6h. After cooling to room temperature, remove the titanium alloy and then clean it 2~4 times with deionized water, anhydrous ethanol and acetone in sequence. After drying, a titanium alloy with an antibacterial nanoenzyme coating on the surface is obtained. The drying temperature described in step six is ​​60℃~80℃, and the drying time is 6h~12h.

2. The method for preparing an antibacterial nanoenzyme coating on a titanium alloy surface according to claim 1, characterized in that... In step one, the titanium alloy is polished with 600#, 800#, 1200# and 1500# sandpaper in sequence; the cleaning mentioned in step one is to clean with anhydrous ethanol and acetone in sequence.

3. The method for preparing an antibacterial nanoenzyme coating on a titanium alloy surface according to claim 1, characterized in that... The titanium alloy mentioned in step one is TA1, TA2, or TC4.

4. The application of an antibacterial nanoenzyme coating on a titanium alloy surface prepared by the method described in claim 1, characterized in that... An antibacterial nanoenzyme coating on a titanium alloy surface is applied in the field of marine antifouling.

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