Self-driven catalytic halloysite-supported metal nanoszyme, preparation method and application thereof

Abstract

The application discloses a self-driven catalytic halloysite metal nanometer enzyme and a preparation method and application thereof, and comprises the following steps: acidizing and activating halloysite, and then modifying the halloysite by using a silane coupling agent; reacting the halloysite and dopamine hydrochloride in a buffer solution to realize PDA modification; then loading Fe3O4 and MnO2 nanoparticles; finally loading Ag nanoparticles to obtain HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag. The application constructs a self-driven-catalytic sterilization-magnetic recovery integrated system, MnO2 and Ag synergistically catalyze the decomposition of H2O2 to generate O2 bubbles, which drive the material to migrate autonomously; Fe3O4 realizes magnetic control recovery; Ag + releases Fe 2+ and initiates Fenton reaction to generate active oxygen such as OH, thereby synergistically killing microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of water pollution control and environmental catalytic materials technology, and particularly relates to a self-driven catalytic halloysite-supported metal nanozyme, its preparation method and application. Background Technology

[0002] With increasing water pollution, waterborne infectious diseases remain a global public health challenge. More than 300 water-related microorganisms can cause 115 infectious diseases, especially since bacterial biofilms can enhance pathogens' resistance to disinfection. Traditional disinfection methods have significant drawbacks: chlorination disinfection is ineffective against drug-resistant pathogens such as Pseudomonas aeruginosa and produces carcinogenic byproducts such as chloroform; ultraviolet light and ozone oxidation technologies rely on energy-intensive equipment, are complex to maintain, and lack sustained sterilization capabilities.

[0003] The fusion of nanozymes and micro / nanomotor technologies offers a new direction for water disinfection. Halloysite (HNTs), as a natural nanotube material, possesses advantages such as geometric asymmetry, low cost, and good biocompatibility, making it an ideal carrier for micro / nanomotors. In existing technologies, Chinese invention patent publication number CN112516998A discloses an antibacterial material with halloysite-loaded silver nanoparticles, but its contact with microorganisms is insufficient; the iron-based nanozyme in Chinese invention patent publication number CN113813967A requires external energy such as light, limiting its application scenarios. Furthermore, the chemical inertness of the halloysite surface leads to low loading of metal nanoparticles and easy aggregation; some supported nanozymes suffer from poor catalytic stability, difficulty in recovery, and potential secondary pollution.

[0004] Therefore, developing a novel halloysite-based composite nanoenzyme to address the shortcomings of existing disinfection materials, such as low efficiency, difficulty in recycling, and poor stability, has become an urgent need in the field of water pollution control. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a self-driven catalytic halloysite-supported metal nanozyme, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A method for preparing a self-driven catalytic halloysite-supported metal nanozyme includes the following steps:

[0008] (1) Halloysite was acidified and activated by soaking in acid to obtain acidified halloysite, and then reacted with silane coupling agent KH-550 in solvent to achieve silane coupling agent modification and obtain HNTs@KH550;

[0009] (2) HNTs@KH550 and dopamine hydrochloride are reacted in a buffer solution to polymerize and form PDA, thereby achieving PDA modification and obtaining HNT@KH550@PDA;

[0010] (3) Ferrous salt and ferric salt were mixed with HNT@KH550@PDA under alkaline conditions and stirred in an aqueous solution for aging. Then the pH was adjusted to neutral, permanganate and manganese salt were added, and the reaction was stirred to achieve the loading of Fe3O4 and MnO2 nanoparticles, resulting in HNT@KH-550@PDA / Fe3O4 / MnO2.

[0011] (4) HNT@KH550@PDA / Fe3O4 / MnO2, silver salt and dispersant were dispersed in water, ultrasonically treated, and then a reducing agent was added to react and achieve Ag nanoparticle loading to obtain HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag.

[0012] As a further improvement, the mass ratio of KH550 to acidified halloysite in step (1) is 10:1-20:1.

[0013] As a further improvement, the reaction temperature in step (1) is 65-70℃.

[0014] As a further improvement, the mass ratio of HNTs@KH550 to dopamine hydrochloride in step (2) is 1:1-2:1.

[0015] As a further improvement, the pH of the buffer solution in step (2) is 8 to 9.

[0016] As a further improvement, the reaction temperature in step (2) is 65-75℃.

[0017] As a further improvement, the aging temperature in step (3) is 50~80℃, and the reaction temperature is 70~90℃.

[0018] The present invention also provides a self-driven catalytic halloysite-supported metal nanozyme prepared by the method described above.

[0019] The present invention also provides an application of the self-driven catalytic halloysite-loaded metal nanozyme for the disinfection of microorganisms in water.

[0020] As a further improvement, the self-driven catalytic halloysite-supported metal nanozyme was combined with H2O2 for microbial disinfection in water.

[0021] The improvement of the present invention compared with the prior art is as follows:

[0022] (1) To address the problem that metal nanozymes are prone to aggregation, reducing the contact between the material and bacteria and resulting in low antibacterial efficiency, halloysite-loaded metal nanozymes are introduced. In this invention, Ag and MnO2 promote the generation of reactive oxygen species through electron transfer, forming Ag... + / Mn 3+ The redox cycle synergistically enhances antibacterial properties and synergistically enhances catalytic activity to catalyze the generation of bubbles from H2O2, giving the material self-driving properties and improving catalytic sterilization kinetics.

[0023] (2) To address the problem of low loading capacity and easy agglomeration of metal nanoparticles due to the chemical inertness of halloysite surface, this invention uses silane coupling agent modification and dopamine self-polymerization modification to construct PDA functional layer. Through the coordination of catechol or amino with metal ions, uniform and firm loading of Fe3O4, MnO2 and Ag nanoparticles is achieved, thereby improving the stability of the material.

[0024] (3) In view of the problem that existing nanozymes are difficult to recycle and are prone to secondary pollution, this invention introduces Fe3O4 nanoparticles to give the material strong magnetic responsiveness, which can be quickly recycled and reused through external magnetic force, reducing environmental risks.

[0025] (4) In view of the limited removal effect of existing disinfection materials on drug-resistant bacteria and biofilms, the present invention utilizes Ag + Release (H2O2 oxidation and dissolution release Ag) + The synergistic effect of reactive oxygen species (ROS) generation and self-driven permeation enables highly efficient killing of multidrug-resistant bacteria (such as MRSA) and biofilms.

[0026] (5) In view of the problem of complex existing preparation processes, the present invention adopts a one-pot method to load Fe3O4 and MnO2, combined with in-situ reduction and immobilization of Ag. The process is simple and mild and easy to scale up.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) High efficiency of self-driven and catalytic sterilization: The synergistic catalytic effect of Ag and MnO2 on H2O2 generates continuous bubble thrust, endowing the material with autonomous migration ability. Under the combined action of 1% hydrogen peroxide, the average movement speed reaches 15 μm / s, significantly increasing the probability of contact with microorganisms; combined with Ag + Release and Fe 2+ The reactive oxygen species generated by the Fenton reaction have a significant impact on 10 6 The kill rate of CFU / mL E. coli can reach 100% within 1 hour, and it is effective against 10... 6 The kill rate of MRSA at CFU / mL can reach 100% within 3 hours. This represents a significant improvement in efficiency compared to traditional static antibacterial materials.

[0029] (2) Broad spectrum of bactericidal activity and resistance to drug resistance: It has a high killing effect on both Gram-negative and Gram-positive bacteria such as Escherichia coli and Staphylococcus aureus (MRSA). In particular, it can solve the drug resistance problem mediated by biofilm. Through the dual action of physical penetration and chemical degradation, it can completely remove biofilm and residual bacteria in the biofilm, thereby reducing the risk of waterborne infectious diseases from the root.

[0030] (3) Inhibits the aggregation of metal nanoparticles and has excellent stability: The tubular structure and high specific surface area of ​​natural halloysite nanotubes provide uniform loading sites for metal nanoparticles (Fe3O4, MnO2, Ag), effectively inhibiting particle aggregation and ensuring the full play of the catalytic and antibacterial activities of each component.

[0031] (4) Good biocompatibility and low cytotoxicity: In the range of catalyst concentration (10-40 μg / L) in practical applications, the material exhibits low toxicity or even no toxicity to normal cells, avoiding the biosafety risks caused by excessive concentration or particle agglomeration of traditional metal-based antibacterial materials, and is suitable for scenarios with high biocompatibility requirements such as drinking water disinfection.

[0032] (5) Magnetic response and recyclability, green and environmentally friendly: The introduction of Fe3O4 gives the material strong magnetic response. After disinfection, it can be quickly separated by an external magnet, with a recycling rate of over 95% and no risk of secondary pollution.

[0033] (6) Synergistic stabilization of multiple components: The PDA functional layer modified by silane coupling agent and dopamine binds firmly to metal ions through coordination, and increases the loading of metal nanozymes, thereby improving antibacterial efficiency. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The characterization spectra of HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag in Example 1 are: (a) Infrared (b) XRD (c) XPS;

[0036] Figure 2 Here are the SEM images of Example 1: (a) HNTs (b) HNT@Kh550@PDA (c) HNT@Kh550@PDA / Fe3O4 / MnO2 (d) HNT@Kh550@PDA / Fe3O4 / MnO2 / Ag;

[0037] Figure 3 These are TEM and EDS images (Si, Al, N, O, Fe, Mn, Ag) of HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag from Example 1.

[0038] Figure 4 Here are the simulated activity diagrams for CAT and POD: (a) kinetic parameters; (b) UV absorbance; (c) oxygen production.

[0039] Figure 5 These are antibacterial experiments under different treatment conditions;

[0040] Figure 6 These are antibacterial experiments using different materials;

[0041] Figure 7 The anti-biofilm (E. coli) test was performed using: (a) SEM; (b) inverted fluorescence microscopy; (c) confocal microscopy; and (d) crystal violet staining.

[0042] Figure 8 The tests for resistance to biofilm (methicillin-resistant Staphylococcus aureus) were: (a) SEM; (b) fluorescence inverted microscope; (c) confocal microscope; and (d) crystal violet staining.

[0043] Figure 9 It refers to the toxicity of materials at different concentrations to cells;

[0044] Figure 10 It is a material-driven performance diagram;

[0045] Figure 11 This is a diagram of the material recycling experiment process;

[0046] Figure 12 It has antibacterial and anti-biofilm properties of HNT / Fe3O4 / MnO2 / Ag;

[0047] Figure 13 This is a reaction diagram for the synthesis of HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag with self-driven catalytic function. Detailed Implementation

[0048] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0049] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0050] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0051] This invention provides a method for preparing a self-driven catalytic composite nanozyme based on dopamine-modified halloysite (HNTs) loaded with Fe3O4, MnO2, and Ag nanoparticles, and its application in the field of microbial disinfection in water. The method first uses natural halloysite nanotubes as a carrier, and constructs a functionalized surface through silane coupling agent modification and forms a polydopamine (PDA) coating through dopamine (DA) self-polymerization for surface functionalization. Fe3O4 and MnO2 nanoparticles are in-situ loaded onto the modified halloysite surface using a one-pot method to construct a magnetic response and catalytic synergistic system. Ag nanoparticles are then immobilized on the modified halloysite surface using an in-situ reduction method to enhance antibacterial activity. The synergistic catalytic effect of MnO2 and Ag on H2O2 generates bubble propulsion to achieve self-drive, and the magnetic response properties of Fe3O4 facilitate recovery. Finally, a composite nanozyme material with self-drive, highly efficient catalytic sterilization, magnetic recyclability, and environmental stability is obtained, which can rapidly degrade bacteria, biofilms, and other microorganisms in water, achieving green and efficient disinfection.

[0052] In some specific embodiments, the preparation method of the self-driven catalytic halloysite-supported metal nanozyme of the present invention includes the following steps:

[0053] (1) Halloysite is acidified and activated by soaking in acid to obtain acidified halloysite, and then reacted with silane coupling agent KH-550 in a solvent (e.g., toluene) to modify it with silane coupling agent to obtain HNTs@KH550.

[0054] In some embodiments, the acid may be hydrochloric acid or the like, with a molar concentration of 0.5–1.5 mol / L. The soaking time is 3–5 days. Acidification is performed to remove impurities from the halloysite surface, increase the number of active hydroxyl groups, and provide more reaction sites for the subsequent grafting of the silane coupling agent KH-550.

[0055] In some embodiments, the mass ratio of KH550 to acidified halloysite can be 10:1-20:1.

[0056] In some embodiments, the silane coupling agent modification reaction temperature can be 65-70°C, and the reaction time can be 5-8 hours.

[0057] (2) HNTs@KH550 and dopamine hydrochloride are reacted in a buffer solution to polymerize and form polydopamine (PDA). PDA modification is performed to obtain HNT@KH550@PDA.

[0058] In some embodiments, the mass ratio of HNTs@KH550 to dopamine hydrochloride can be 1:1 to 2:1. The pH of the buffer solution is 8 to 9.

[0059] In some embodiments, the PDA modification reaction temperature can be 65-75℃, and the reaction time can be 20-28 h.

[0060] In some embodiments, the PDA-modified product is separated by centrifugation and repeatedly washed with deionized water, and then dried. Preferably, the centrifugation speed is not less than 8000 rpm, the washing is performed at least 3 times, and the drying temperature is controlled at 45-55℃.

[0061] (3) Ferrous salt (e.g., ferrous chloride) and ferric salt (e.g., ferric chloride) are stirred and aged with HNT@KH550@PDA in an aqueous solution under alkaline conditions. Then, the pH is adjusted to neutral, and permanganate (e.g., potassium permanganate) and manganese salt (e.g., manganese sulfate) are added and stirred to react, so as to obtain HNT@KH-550@PDA / Fe3O4 / MnO2 loaded with Fe3O4 and MnO2 nanoparticles.

[0062] In some embodiments, the molar ratio of FeCl2·4H2O to FeCl3·6H2O can be 1:1.5-1:2.0. The mass ratio of FeCl2·4H2O to HNT@KH550@PDA is 1:10-1:15.

[0063] In some embodiments, the alkaline conditions are adjusted by adding an ammonia solution 10-11, the ammonia solution concentration is 25-28 wt%, and the dropping rate is controlled to be 1-2 drops / second.

[0064] In some embodiments, the aging temperature is 50~80℃ and the time is 2-4 h.

[0065] In some embodiments, the molar ratio of KMnO4 to MnSO4·H2O can be optimized to 1:1.5-2:3. The mass ratio of KMnO4 to HNT@KH550@PDA is 1:12-1:18.

[0066] In some embodiments, after adding potassium permanganate and manganese sulfate, the reaction temperature is 70~90℃ and the reaction time is 1.5-2.5 h to carry out MnO2 deposition.

[0067] (4) HNT@KH550@PDA / Fe3O4 / MnO2, silver salt (e.g., silver nitrate) and dispersant (e.g., PVP) are dispersed in water, ultrasonically treated, and then a reducing agent (e.g., sodium borohydride) is added to react and load Ag nanoparticles to obtain HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag.

[0068] In some embodiments, the mass ratio of HNT@KH-550@PDA / Fe3O4 / MnO2 to silver nitrate is 0.5g / 15-25 mg, the mass ratio of HNT@KH-550@PDA / Fe3O4 / MnO2 to PVP is 0.5g / 200-300 mg, and the mass ratio of MnO2, PVP, and both is 1:10-1:12.5.

[0069] In some embodiments, the ultrasonic homogenization time is 10-20 min.

[0070] In some embodiments, the mass ratio of sodium borohydride to silver nitrate is 4-6:20, and the stirring time after adding sodium borohydride is 20-40 min.

[0071] In some embodiments, after the reaction, the mixture is centrifuged and washed with deionized water at a speed of not less than 8000 rpm and washed at least 3 times.

[0072] This invention uses natural halloysite as the core carrier, modifies the surface of halloysite with a silane coupling agent, and coats it with a polydopamine (PDA) functional layer. Fe3O4 (magnetically responsive component), MnO2 (catalytic and self-driven component), and Ag (antibacterial component) are sequentially loaded to form a multilayer composite structure of "haloysite-KH550-PDA-Fe3O4 / MnO2-Ag". This yields a halloysite-based composite nanozyme based on dopamine modification, synergistic effects of multiple metal components, and possessing both self-driven and magnetic recovery functions. This invention constructs an integrated system of "self-driven-catalytic sterilization-magnetic recovery". MnO2 and Ag synergistically catalyze the decomposition of H2O2 to generate O2 bubbles, driving the autonomous migration of materials; Fe3O4 achieves magnetically controlled recovery; Ag... + Release and Fe 2+ The Fenton reaction it triggers produces reactive oxygen species such as OH, which synergistically kill microorganisms.

[0073] Example 1 Synthesis of HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag with self-driven catalytic function:

[0074] 1.1. Silane coupling agent modified HNTs surface: First, dried halloysite was soaked in 1.0 mol / L hydrochloric acid at room temperature for 4 days with stirring to complete acidification and activation. After filtration and washing until neutral, acidification and activation were completed. Then, 1g of acidified halloysite was dispersed in 50mL of toluene, 10 times the excess of KH-550 was added, and the mixture was refluxed at 65℃ for 6h. The precipitate was washed three times with anhydrous ethanol and deionized water, and then vacuum dried at 50℃ for 8h to obtain KH550 modified halloysite HNTs@KH550.

[0075] 1.2. 0.5 g of HNTs@KH550 from 1.1 and 0.5 g of dopamine hydrochloride were dispersed in 250 mL of Tris (0.394 g) buffer solution at pH 8.5 and stirred at 70 °C for 24 h. The resulting product was separated by centrifugation and repeatedly washed with deionized water, then dried overnight in an oven at 50 °C to obtain HNT@KH550@PDA.

[0076] 1.3. FeCl2·4H2O (0.04 g) and FeCl3·6H2O were mixed in a molar ratio of 1:1.75 and dissolved in 120 mL of distilled water. The mixture was stirred vigorously under a nitrogen atmosphere. 0.5 g of HNT@KH550@PDA was added, and 5 mL of ammonia solution was added dropwise in a 60℃ water bath to adjust the pH of the solution to 10-11. The mixture was stirred and aged for 3 h. The pH was adjusted to 7.0, the temperature was raised to 80℃, and KMnO4 (0.035 g) and MnSO4·H2O were added in a molar ratio of 2:3. The mixture was stirred and reacted for 2 h. The product was separated by magnetic separation, washed 4 times with distilled water, and dried under vacuum at 80℃ for 3 h to obtain HNT@KH-550@PDA / Fe3O4 / MnO2.

[0077] 1.4. First, 0.5 g of the previously prepared HNT@KH-550@PDA / Fe3O4 / MnO2 material was dispersed in 10 ml of water with 20 mg of silver nitrate and 250 mg of PVP, and homogenized by sonication for 15 min. Then, 5 mg of sodium borohydride was added dropwise, and the mixture was stirred for 30 min. After completion, the mixture was centrifuged and washed with deionized water. Finally, it was vacuum dried for 12 hours to obtain HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag.

[0078] Infrared, XRD, XPS, SEM, TEM, and EDS images of the product are shown below. Figure 1 , Figure 2 , Figure 3 This indicates that HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag was obtained.

[0079] Example 2: Simulated Activity of CAT and POD

[0080] Oxygen generation detection: The efficiency of HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag (hereinafter referred to as the material) in catalyzing the decomposition of hydrogen peroxide (H2O2) to produce oxygen was determined using a dissolved oxygen analyzer (JPSJ-605F). 50 μg·mL⁻¹ -1The materials were mixed with hydrogen peroxide of different concentrations (0, 25, 50, 100, 150, 200, 250, 300 mM) and dissolved in 1 mL of phosphate-buffered saline (PBS) at pH 6.5. Oxygen production was then measured using a dissolved oxygen meter (JPSJ-605F). Figure 4 c. The addition of materials and H2O2 causes DO to increase with time, and the higher the concentration of materials, the more O2 is produced.

[0081] Detection of hydroxyl radical (·OH) generation: A stock solution was prepared by dissolving 3,3',5,5'-tetramethylbenzidine (TMB) in dimethyl sulfoxide (DMSO), and then diluted to a final concentration of 3 mM with phosphate-buffered saline (PBS) at pH 6.0. 600 μL of a 3 mM TMB solution, 600 μL of a 3 M hydrogen peroxide (H2O2) solution, and 100-300 μg of the material were added and allowed to react for 10 minutes. The reacted solution was centrifuged, and the absorbance curve of the supernatant was measured using a UV-3600 UV-Vis spectrophotometer. Figure 4 b. The higher the material concentration, the higher the peak value, which proves that more ·OH is generated in the same amount of time, that is, the greater the POD activity.

[0082] POD kinetics simulation: 50 μL of 10 mM DMSO system TMB solution, different concentrations of H2O2 (0, 2, 4, 6, 12, 18, 20, 100 mM), and 100 μg·mL⁻¹ were used. -1 The materials were mixed and brought to a final volume of 1 mL of pH 6.5 PBS. The absorbance of oxidized TMB at 620 nm was monitored in real time using a microplate reader, and the kinetic parameters were calculated (see [link to microplate reader]). Figure 4 a). Calculate the maximum initial velocity (V). max ) and Michaelis-Menten constant (K M The values ​​were 0.90 mM / s and 3.35 mM, respectively. K M The relatively low value indicates that the material has high POD activity, while the high Vmax confirms its good catalytic rate.

[0083] Example 3 Antibacterial test of catalysts at different concentrations

[0084] This experiment uses 10 6Using *E. coli* and MRSA at concentrations of CFU / mL as the research subjects, four material concentration gradients were set up: 0 μg / mL (control group), 10 μg / mL, 20 μg / mL, and 40 μg / mL. 100 μM H2O2 was added to each concentration group. *E. coli* was incubated at 37℃ with shaking for 1 h, and MRSA was incubated at 37℃ with shaking for 3 h. After incubation, plates were plated, and colony counts were recorded after 18 h of incubation at 37℃. By comparing the colony growth of different concentration groups, the antibacterial effect of the combined action of the materials and 100 μM H2O2 on the two strains was analyzed (see...). Figure 5 ), only under the action of 10 μg / mL of material, 10 6 Escherichia coli at CFU / mL (1h) and MRSA (3h) can be completely inactivated.

[0085] Example 4 Antibacterial test of different materials

[0086] This experiment uses 10 6 Using CFU / mL *E. coli* and MRSA as the research subjects, six experimental groups were set up: Group A (control group), Group B (H2O2 only), Group C (material only), Group D (Nano-Ag + H2O2), Group E (AgNO3 + H2O2), and Group F (material + H2O2). *E. coli* was incubated at 37℃ with shaking for 1 h, and MRSA was incubated at 37℃ with shaking for 3 h. After incubation, plates were spread and incubated at 37℃ for 24 h. Colony counts were recorded for each group. By comparing the colony growth of different groups, the antibacterial effects of each group combined with (or alone) H2O2 on the two strains were analyzed (see [link to study]. Figure 6 Both AgNO3 and the material have the ability to kill 10 in 1 hour. 6 CFU / mL E. coli, and completely inactivated MRSA within 3 hours, while AgNPs showed significantly weaker antibacterial activity.

[0087] Example 5: Anti-biofilm test

[0088] Experimental activation of Escherichia coli and preparation of 10 8 After sterilizing experimental equipment and adjusting relevant instruments, CFU / mL bacterial suspension was inoculated into well plates and cultured at 37℃ for 48 hours to form a stable biofilm. Six experimental groups were set up (Group A: control group, Group B: H2O2 only, Group C: material only, Group D: Nano-Ag + H2O2, Group E: AgNO3 + H2O2, Group F: material + H2O2). After incubation at 37℃ for 24 hours, the biofilm was fixed and then observed using a scanning electron microscope (SEM). Aseptic technique was followed throughout the process. Figure 7(a) After material treatment, the number of bacteria was significantly reduced compared to the control group. In addition, the outer surface of the bacteria ruptured, and the tightly packed bacterial aggregates were observed to disperse. The bacteria that destroyed the biofilm were killed by the production of ROS and the bacteria that removed the biofilm were removed. This confirmed that H2O2 catalyzed by HNT@KH550@PDA / Fe3O4 / MnO2 / Ag can destroy the biofilm formed by Escherichia coli.

[0089] Experimental activation of Escherichia coli and preparation of 10 8 CFU / mL bacterial suspension was prepared, and 0 μg / mL (control group), 10 μg / mL, 20 μg / mL, and 40 μg / mL materials and 100 μM H2O2 solution were prepared and aseptically treated. After sterilizing equipment, preparing SYTO9 / PI staining solution, and adjusting the instrument, the bacterial suspension was inoculated into culture plates and incubated at 37℃ for 48 h to form a stable biofilm. Four concentration groups (3 replicates per group) were set up. After adding the corresponding materials and H2O2, the samples were incubated at 37℃ for 24 h. After washing with PBS to remove impurities, SYTO9 / PI staining solution was added (SYTO9 stains live bacteria green fluorescence, and PI stains dead bacteria red fluorescence; the Merge figure shows the superposition of the two) and incubated in the dark. After rinsing off excess staining solution, the fluorescence differences were observed and analyzed using a fluorescence inverted microscope. Figure 7 (b) The green fluorescence gradually weakened while the red fluorescence gradually strengthened, confirming that the higher the material concentration, the stronger its ability to destroy the biofilm and kill E. coli within 24 hours.

[0090] Experimental activation of Escherichia coli and preparation of 10 8 After sterilizing experimental equipment and adjusting relevant instruments, CFU / mL bacterial suspension was inoculated into culture plates and incubated at 37℃ for 48 hours to form a stable biofilm. Six experimental groups were set up (Group A: control group, Group B: H2O2 only, Group C: material only, Group D: Nano-Ag + H2O2, Group E: AgNO3 + H2O2, Group F: material + H2O2). Unbound bacteria and reagents were removed by rinsing with sterile PBS. SYTO9 / PI staining solution was added for staining in the dark. After rinsing off excess staining solution, the fluorescence of the biofilm was observed using a confocal microscope, and the differences between groups were analyzed. Aseptic operation was followed throughout the process. Figure 7 (c) Untreated biofilms exhibit high-intensity green fluorescence, while the intensity of green fluorescence in the treated group gradually decreases, while red fluorescence gradually increases. It is clearly observed that the material exhibits the strongest red fluorescence and the weakest green fluorescence after treatment, indicating that the HNT@KH550@PDA / Fe3O4 / MnO2 / Ag nanomotors can wet and penetrate biofilms, thereby further killing bacteria and improving antibacterial efficiency.

[0091] Experimental activation of Escherichia coli and preparation of 10 8After sterilizing experimental equipment and adjusting relevant instruments, CFU / mL bacterial suspension was inoculated into culture plates and incubated at 37℃ for 48 hours to form a stable biofilm. Six experimental groups were set up (Group A: control group, Group B: H2O2 only, Group C: material only, Group D: Nano-Ag + H2O2, Group E: AgNO3 + H2O2, Group F: material + H2O2). After incubation at 37℃ for 24 hours, unbound bacteria and reagents were removed by rinsing with sterile PBS. Crystal violet staining solution was added, and after staining, excess staining solution was rinsed off. After drying, the crystal violet was dissolved in glacial acetic acid, and the absorbance of the dissolved solution was measured using an ELISA reader to analyze the biofilm formation and destruction in each group. Aseptic operation was followed throughout the process. Figure 7 (d) It can be seen that the crystal violet color of the material + H2O2 group is the lightest, indicating that this group of E. coli has the highest degree of biofilm damage, verifying that the nanomotor has the ability to efficiently remove biofilms. The degree of biofilm damage can be quantitatively analyzed by the absorbance of each component, such as... Figure 7 (d) Bar chart, quantitative analysis of biofilm removal shows that the material + H2O2 group can remove 85% of the biomass, which is significantly better than other groups.

[0092] The antibacterial test for methicillin-resistant Staphylococcus aureus (MRSA) was the same as above (see above). Figure 8 ).

[0093] Example 6 Cytotoxicity Test

[0094] Huvec cells were cultured in 96-well plates at 37°C, 5% CO2, and 90% humidity for 24 hours to allow cell adhesion and exponential growth phase. 10 μL of the drug was added to each well to achieve concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, and 100 μg / mL, with three or more replicates. A control group without the drug and a blank group without cells were also included. The 96-well plates were incubated for another 24 hours to allow for sufficient interaction between the drug and cells. The plates were then removed, and 10 μL of CCK-8 solution was added to each well. The plates were gently shaken to mix, avoiding air bubbles. The plates were then incubated again for 0.5–4 hours until the desired colorimetric depth was reached. Finally, the absorbance of each well was measured at 450 nm using a microplate reader to analyze the cytotoxicity of the drug (see [link to microplate reading]). Figure 9 ).

[0095] Example 7 Material-driven performance

[0096] 50 μg / mL of the material was mixed with hydrogen peroxide at concentrations of 1%, 2%, 5%, 10%, and 15%, respectively. The trajectories were observed and recorded under a microscope. Analysis of the trajectories revealed… Figure 10The figures show (a) the trajectory diagram, (b) the DSM diagram, and (c) the average velocity v. As H2O2 is gradually added, the material trajectory gradually increases. It can be seen that the MSD curve exhibits a parabolic trend under different H2O2 concentrations, indicating that the O2 generated by the catalytic H2O2 provides the material with a driving force, enabling autonomous and continuous motion. The average velocity of the material is affected by the H2O2 concentration; the higher the H2O2 concentration, the greater the average velocity, reaching a maximum of 100 μm / s at a concentration of 10%.

[0097] Example 8 Material Recycling Efficiency

[0098] The material was placed in water and thoroughly mixed, and then separated and recovered using a magnetic adsorption method (see...). Figure 11 ).

[0099] Example 9: Comparison of the performance of HNT / Fe3O4 / MnO2 / Ag

[0100] Omitting steps 1.1 and 1.2 of Example 1, we obtain HNT / Fe3O4 / MnO2 / Ag.

[0101] Under the same conditions, the antibacterial and anti-biofilm properties of HNT / Fe3O4 / MnO2 / Ag were tested (see...). Figure 12 The images shown are (a) anti-E. coli experimental image, (b) anti-MRSA experimental image, and (c) anti-biofilm confocal image. It can be seen that the unmodified HNT / Fe3O4 / MnO2 / Ag can achieve 10 6 CFU / mL E. coli inactivation (1h), but poor antibacterial performance against MRSA, and almost no damage to bacteria that form biofilms.

[0102] Table 1 lists the cost calculation information for 1g of HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag, which has a low cost.

[0103] Table 1

[0104]

[0105] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for preparing a self-driven catalytic halloysite-supported metal nanozyme, characterized in that, Includes the following steps: (1) Halloysite is acidified and activated by soaking in acid to obtain acidified halloysite, and then reacted with silane coupling agent KH-550 in solvent to achieve silane coupling agent modification and obtain HNTs@KH550; (2) HNTs@KH550 and dopamine hydrochloride are reacted in a buffer solution to polymerize and form PDA, thereby achieving PDA modification and obtaining HNT@KH550@PDA; (3) Ferrous salt and ferric salt were mixed with HNT@KH550@PDA under alkaline conditions and stirred in an aqueous solution for aging. Then the pH was adjusted to neutral, permanganate and manganese salt were added, and the reaction was stirred to achieve the loading of Fe3O4 and MnO2 nanoparticles, resulting in HNT@KH-550@PDA / Fe3O4 / MnO2. (4) HNT@KH550@PDA / Fe3O4 / MnO2, silver salt and dispersant were dispersed in water, ultrasonically treated, and then a reducing agent was added to react and achieve Ag nanoparticle loading to obtain HNT@KH-550@PDA / Fe3O4 / MnO2 / Ag.

2. The method for preparing self-driven catalytic halloysite-supported metal nanozymes according to claim 1, characterized in that, In step (1), the mass ratio of KH550 to acidified halloysite is 10:1-20:

1.

3. The method for preparing self-driven catalytic halloysite-supported metal nanozymes according to claim 1, characterized in that, The reaction temperature in step (1) is 65-70℃.

4. The method for preparing self-driven catalytic halloysite-supported metal nanozymes according to claim 1, characterized in that, In step (2), the mass ratio of HNTs@KH550 to dopamine hydrochloride is 1:1-2:

1.

5. The method for preparing self-driven catalytic halloysite-supported metal nanozymes according to claim 1, characterized in that, The pH of the buffer solution in step (2) is 8-9.

6. The method for preparing self-driven catalytic halloysite-supported metal nanozymes according to claim 1, characterized in that, The reaction temperature in step (2) is 65-75℃.

7. The method for preparing self-driven catalytic halloysite-supported metal nanozymes according to claim 1, characterized in that, The aging temperature in step (3) is 50~80℃, and the reaction temperature is 70~90℃.

8. A self-driven catalytic halloysite-supported metal nanozyme prepared by the method according to any one of claims 1 to 7.

9. An application of the self-driven catalytic halloysite-supported metal nanozyme according to claim 8, characterized in that, It is used for disinfection of microorganisms in water.

10. The application according to claim 9, characterized in that, The self-driven catalytic halloysite-supported metal nanozyme was combined with H2O2 for microbial disinfection in water.

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