Catalyst material integrating au-ir cascade catalytic sites and preparation method and application thereof

Abstract

The application belongs to the technical field of catalyst materials, and particularly relates to a catalyst material integrated with Au-Ir cascade catalytic sites and a preparation method and application thereof. The catalyst material comprises micron-scale urchin-like carbonaceous particles loaded with cascade Au clusters and Ir monatomic material, has glucose oxidase-like activity, oxidase-like activity, peroxidase-like activity and NADH oxidase-like activity, and can combine metabolic inhibition and active oxygen attack to eliminate stubborn caries-related biofilms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of catalyst materials, and particularly relates to a catalyst material integrating Au-Ir cascade catalytic sites and a preparation method and application thereof. BACKGROUND

[0002] Dental caries is a widespread, biofilm-mediated and diet-regulated disease that affects 3.1 billion people worldwide and causes more than $290 billion in annual losses, and is a major public health problem that has not yet been solved. The pathological core is that cariogenic bacteria (such as Streptococcus mutans) metabolically assemble extracellular matrix rich in extracellular polysaccharides to form a dense biofilm barrier, and rely on glycolysis metabolism to continuously secrete lactic acid, causing local low pH and enamel demineralization. Current clinical treatment of cariogenic biofilm infection mainly relies on surgical debridement. However, surgical resection can temporarily remove visible lesions, but it inevitably damages healthy tooth tissue and even dental pulp; and it is a post-damage repair measure and cannot fundamentally prevent it. In addition, current high-dose fluoride and antibiotic non-surgical treatments cannot effectively prevent caries in high-risk groups (such as those with low saliva secretion and poor buffering capacity) because their biofilms rapidly form under high-sugar diets and poor oral hygiene, and drugs are difficult to penetrate the extracellular polysaccharide barrier, ultimately failing to block the demineralization process; at the same time, antibiotic use is also associated with an increased risk of drug resistance. Therefore, developing a new treatment strategy that can both efficiently remove stubborn biofilms and eliminate pathogenic bacteria while avoiding drug resistance and damaging healthy tissue has become an urgent need for current caries prevention and treatment.

[0003] In recent years, "starvation therapy" from the field of anti-tumor has attracted widespread attention through two major metabolic killing mechanisms of nutrient deprivation and energy system collapse. For example, glucose oxidase is used to deplete the glucose required by tumor cells, thereby achieving nutrient intake blockage; or NADH oxidase is used to destroy the NADH / NAD + cycle, interfere with the function of the electron transport chain to block ATP synthesis, and thus inhibit basic biological functions and cell growth. In view of the unique polysaccharide-high-sugar environment of dental caries, "starvation therapy" also shows potential in killing cariogenic bacteria: by cutting off the substrate supply of the glycolysis pathway of cariogenic bacteria and disrupting the ATP production chain, the growth and metabolism of bacteria are inhibited. However, directly transplanting the treatment paradigm of tumor treatment faces double challenges: on the one hand, cariogenic bacteria can maintain basic energy supply through metabolic dormancy; on the other hand, simply inhibiting energy metabolism is difficult to completely destroy the physical barrier (such as the extracellular polysaccharide matrix) of the stubborn biofilm of dental caries. Therefore, a breakthrough treatment plan needs to integrate "starvation therapy" and matrix degradation strategies to comprehensively achieve the complete eradication and long-term control of the stubborn biofilm of dental caries from the inside out.

[0004] Active oxygen therapy can directly disintegrate the physical barrier of biofilm by oxidative damage (such as degrading polysaccharides and destroying membrane integrity). Based on this, the dual strategy combining "endogenous metabolic interference-exogenous active oxygen introduction" is expected to not only block the energy supply of bacteria, but also trigger irreversible biofilm disintegration through oxidative damage, which is expected to achieve deep removal of biofilm. However, this aspect is rarely studied in the field of dental caries repair. In recent years, the breakthrough of artificial enzyme catalysis medicine - biomimetic artificial enzyme has both the high catalytic properties of natural enzymes and the designability advantages of artificial materials, which provides a platform for the implementation of various treatment strategies. For example, metal oxides, metal organic frameworks and noble metal nanoparticles and other biological catalytic artificial enzyme materials have shown significant efficacy in the fields of anti-tumor and wound repair by simulating the catalytic activity of natural peroxidase and oxidase. However, there are still two key scientific problems to be overcome for its reasonable application in dental caries biofilm treatment: 1) The multi-enzyme active site needs to be accurately designed to achieve the optimal coupling of "metabolic inhibition-active oxygen generation" function; 2) Due to the short diffusion distance (<0.1 μm) and transient life (~ ns level) of active oxygen, combined with the dynamic and complex saliva environment in the oral cavity, the material must have precise adhesion, penetration, biofilm penetration and active oxygen delivery capabilities. SUMMARY

[0005] To solve the above problems, the present application designs a micron urchin-like carbon particle (UCP) loaded cascade Au cluster and Ir single atom material (referred to as UCP-Au / Ir in this paper), which has glucose oxidase-like activity, oxidase-like activity, peroxidase-like activity and NADH oxidase-like activity, so as to combine metabolic inhibition with active oxygen attack to eliminate stubborn caries-related biofilm.

[0006] In a first aspect, the present application provides a catalyst material integrating Au-Ir cascade catalytic sites, the catalyst material comprising a carbon skeleton with urchin-like structure and gold clusters and iridium single atoms loaded on the surface thereof, and the catalyst material having glucose oxidase-like activity, oxidase-like activity, peroxidase-like activity and NADH oxidase-like activity.

[0007] Further, the carbon skeleton with urchin-like structure is a micron urchin-like carbon particle obtained by hydrothermal conversion of a zinc-based metal organic framework in the presence of urea to obtain a urchin-like precursor, and then carbonization and zinc sublimation.

[0008] Further, the organic ligand of the zinc-based metal organic framework is 2,5-dihydroxyterephthalic acid.

[0009] Further, the gold clusters are deposited by adsorbing gold precursors on the surface of the carbon skeleton and reducing with a reducing agent.

[0010] Further, the iridium single atom is in-situ coated on the surface of the carbon skeleton by forming an iridium-ligand complex with the nitrogen-containing ligand, and is obtained by high-temperature carbonization in an inert atmosphere.

[0011] In a second aspect, the present application provides a preparation method of the catalyst material integrating Au-Ir cascade catalytic sites as described herein, comprising the following steps:

[0012] (1) reacting a zinc salt with an organic ligand in a solvent to form a zinc-based metal organic framework;

[0013] (2) hydrothermally treating the zinc-based metal organic framework in a urea solution to obtain a precursor with urchin-like morphology;

[0014] (3) carbonizing the precursor, which simultaneously sublimates the zinc component in the precursor, to obtain micron-sized urchin-like carbonaceous particles;

[0015] (4) reducing a gold precursor on the micron-sized urchin-like carbonaceous particles using a reducing agent to obtain micron-sized urchin-like carbonaceous particles with gold clusters deposited on the surface;

[0016] (5) in-situ coating a metal-ligand complex formed by iridium ions and a nitrogen-containing ligand on the micron-sized urchin-like carbonaceous particles with gold clusters deposited on the surface, and then high-temperature carbonizing in an inert atmosphere to obtain the catalyst material integrating Au-Ir cascade catalytic sites.

[0017] Further, the organic ligand is 2,5-dihydroxyterephthalic acid.

[0018] Further, the zinc salt in step (1) is at least one of zinc nitrate, zinc acetate, zinc chloride or a hydrate thereof.

[0019] Further, the solvent is an alcohol solvent, including methanol or ethanol.

[0020] Further, the reaction of the zinc salt with the organic ligand is carried out under ultrasonic, and the time is 0.5-3 hours.

[0021] Further, after the reaction of the zinc salt with the organic ligand is completed, the reaction product is centrifuged and precipitated, and is washed with deionized water.

[0022] Further, the mass concentration of the urea solution in step (2) is 0.1-0.3%.

[0023] Further, the temperature of the hydrothermal treatment is 170-180 ℃, and the reaction time is 12-36 h.

[0024] Further, step (2) further comprises washing and drying the product of the hydrothermal treatment.

[0025] Further, the temperature of the carbonization treatment in step (3) is 900-1100 ℃, and the reaction time is 1-3 h.

[0026] Further, step (3) further comprises acid washing, water washing and drying treatment of the product after the carbonization treatment.

[0027] Further, step (4) comprises dispersing the micron-sized urchin-like carbonaceous particles in deionized water, adding an alcohol solution of a gold precursor, and reacting under the action of a reducing agent.

[0028] Further, the reducing agent is selected from one or more of sodium borohydride, hydrogen, ascorbic acid, and sodium citrate.

[0029] Further, the gold precursor is selected from one or more of chloroauric acid, gold chloride, or hydrates thereof.

[0030] Further, the reduction reaction time is 30-180 minutes.

[0031] Further, step (5) comprises dispersing the micron-sized urchin-like carbonaceous particles with gold clusters on the surface in an alcohol solvent, adding an alcohol solution containing an iridium precursor and a nitrogen-containing ligand, ultrasonic dispersion, and drying the dispersion and then heat treating it under an inert atmosphere.

[0032] Further, the iridium precursor is selected from one or more of iridium acetylacetonate, chloro iridic acid, iridium trichloride, or hydrates thereof.

[0033] Further, the nitrogen-containing ligand is selected from one or more of 1,10-phenanthroline, triphenylphosphine, or bipyridine compounds.

[0034] Further, the heat treatment temperature is 400-800 ℃, and the time is 1-5 hours.

[0035] In a third aspect, the present application provides use of the catalyst material integrating Au-Ir cascade catalytic sites as described herein in the preparation of antibacterial, antibiofilm, and anticaries materials.

[0036] Further, the antibacterial includes anti-Streptococcus mutans.

[0037] Further, the biofilm includes caries-associated biofilm.

[0038] Further, the catalyst material integrating Au-Ir cascade catalytic sites is used in combination with hydrogen peroxide.

[0039] Further, the catalyst material integrating Au-Ir cascade catalytic sites achieves antibacterial, antibiofilm, and anticaries by interfering with the energy metabolism of bacteria and generating reactive oxygen species.

[0040] Advantages of the present application

[0041] The present application has found that in the micron-sized urchin-like carbonaceous particles (hereinafter referred to as UCP-Au / Ir) of the present application loaded with cascaded Au clusters and Ir single atoms, the Au clusters mimic glucose oxidase activity to consume glucose, while the Ir single atoms exhibit NADH oxidase, peroxidase and oxidase mimic activities, so that NADH / NAD + cycle and catalytic active oxygen (including •O2 - and 1 O2) production can be simultaneously destroyed. Notably, the two catalytic sites produce a synergistic effect through the glucose oxidase-peroxidase cascade reaction, further improving the efficiency of active oxygen generation. Therefore, UCP-Au / Ir can destroy the bacterial energy system by inducing nutrient deprivation and inhibiting ATP synthesis, induce metabolic disorders, and combine with oxidative stress, resulting in a Streptococcus mutans and biofilm clearance rate close to 100%, without causing tissue damage or promoting drug resistance. The in vitro tooth model confirms that UCP-Au / Ir can effectively eradicate Streptococcus mutans, prevent biofilm formation on the enamel surface, and reduce enamel demineralization. By combining endogenous metabolic regulation with exogenous active oxygen generation, this material establishes a new framework for the development of advanced anti-biofilm materials and provides broad application prospects in the management of biofilm-related infections. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Scanning electron microscope images of UCP, UCP-Au, UCP-Ir and UCP-Au / Ir prepared in Example 1 are shown.

[0043] Figure 2 Aberration-corrected high-angle annular dark-field scanning transmission electron microscope images of UCP-Au / Ir prepared in Example 1 are shown.

[0044] Figure 3 Energy dispersive element distribution maps of UCP-Au / Ir prepared in Example 1 are shown.

[0045] Figure 4 Raman spectra of different materials are shown: (a) D peak represents disordered carbon peak, G peak represents graphitized carbon peak; (b) X-ray photoelectron spectroscopy full spectrum element composition comparison of different catalyst samples; (c) high-resolution X-ray photoelectron spectroscopy spectra of Ir 4f in different catalysts, (d) Au 4f and (e) N 1s.

[0046] Figure 5(a) oxido-enzyme activity of different catalysts; (b) peroxido-enzyme activity of different catalysts; (c) radical quenching experiments: t-butanol quenching of •OH, benzoquinone quenching of •O2 - , sodium azide quenching 1 O2; (d) comparison of NADH-oxidase like activity of different catalysts; (e) glucose-oxidase like activity of different catalysts; (f) H2O2 concentration change over 120 minutes in glucose substrate for UCP-Au, and continued monitoring of H2O2 level after addition of UCP-Ir at 120 minutes.

[0047] Figure 6 (a) OD absorbance values of S. mutans colonies under different concentrations of UCP-Au / Ir treatment. (b) OD absorbance values of planktonic S. mutans under different material treatment. (c) Scanning electron microscope images and (d) Transmission electron microscope images showing the bacterial capture ability of different materials towards planktonic S. mutans. 600 600

[0048] Figure 7 (a) Alexa Fluor 647 staining confocal microscope images showing the interaction of different materials with S. mutans biofilm. (b) Alexa Fluor 647 relative fluorescence intensity statistics. (c) Propidium iodide relative fluorescence intensity statistics.

[0049] Figure 8 (a) S. mutans cell membrane fluorescence CLSM images of different material treatment. (b) Alexa Fluor 647 fluorescence relative intensity statistical analysis. (c) Propidium iodide fluorescence relative intensity statistical analysis.

[0050] Figure 9 (a) Micro-CT images of treated tooth samples. (b) Scanning electron microscope images of tooth surface under different treatment in caries prevention study. (c) Surface microhardness change rate of enamel samples, (d) nanohardness change value, and (e) elastic modulus change value. (f) Caries lesion occurrence and severity quantification. Lesion staging was performed according to ICDAS criteria (0-6 scale), with higher scores indicating more severe tissue destruction. DETAILED DESCRIPTION

[0051] ​​To achieve the cascade catalytic amplification effect of physically penetrating the bacterial membrane structure, disrupting the bacterial metabolic process, and promoting the generation of reactive oxygen species, thereby achieving the goal of efficient sterilization and biofilm removal, the present application designs a urchin-like carbonaceous particle biological catalyst (named UCP-Au / Ir) which integrates cascade gold clusters and single-atom iridium catalytic sites. The preparation process starts from the hydrothermal conversion reaction of urea and zinc-based metal organic framework, first generating urchin-like precursors. Then through carbonization treatment and zinc sublimation process, the micro-sized urchin-like carbonaceous particles (UCP) with stable structure are obtained. Next, the gold cluster is deposited by sodium borohydride-mediated gold ion reduction method to obtain UCP-Au. Finally, by in-situ coating of metal-phenanthroline complex and carbonization treatment, single-atom Ir catalytic center is introduced, and finally UCP-Au / Ir catalyst is prepared.

[0052] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0053] Example 1: Preparation of micro-sized urchin-like carbonaceous particles integrating Au-Ir cascade catalytic sites (UCP-Au / Ir)

[0054] First, 0.3 grams of 2,5-dihydroxyterephthalic acid and 1.1 grams of zinc acetate dihydrate were dissolved in 70 milliliters of methanol, and ultrasonic treatment was performed for about 1 hour. Then the product after ultrasonic treatment was centrifuged and precipitated, the supernatant was discarded, and the precipitate was washed with deionized water three times. The precipitate was added to a urea solution (0.45 grams of urea dissolved in 220 milliliters of deionized water), stirred for 1 hour, and then transferred to a preheated 175°C polytetrafluoroethylene-lined high-pressure reaction kettle for reaction for 24 hours. The obtained precipitate was washed with water and ethanol and then vacuum dried. The prepared material was placed in a ceramic boat and placed in a tube furnace to heat to 1000°C at a rate of 5°C / min and hold for 1 hour. After the product cooled to room temperature, it was washed with 1 M hydrochloric acid solution at 80°C for 3 hours, then washed with water three times and vacuum dried, and finally the UCP material was obtained.

[0055] Then 32 mg of UCP was dispersed in 20 mL of deionized water, 800 µL of gold chloride trihydrate solution (10 mg / mL ethanol solution) was added, and 11 mg of sodium borohydride (NaBH4) was added. After stirring for 30 minutes, it was washed with water three times and vacuum dried to obtain the UCP-Au material.

[0056] To prepare UCP-Au / Ir, 10 mg of UCP-Au was dispersed in 1.25 mL of ethanol and 15 μΐ, of a mixture consisting of 75 mg of acetylacetonate iridium (III), 103 mg of 1,10-phenanthroline and 755 μΐ, of ethanol was added and sonicated for 30 minutes. The resulting dispersion was dried under vacuum overnight and heat treated at 600 °C for 2 hours under nitrogen atmosphere.

[0057] Additionally, 10 mg of UCP was dispersed in 1.25 mL of ethanol and 15 μΐ, of a mixture consisting of 75 mg of acetylacetonate iridium (III), 103 mg of 1,10-phenanthroline and 755 μΐ, of ethanol was added and sonicated for 30 minutes. The resulting dispersion was dried under vacuum overnight and heat treated at 600 °C for 2 hours under nitrogen atmosphere to obtain UCP-Ir material.

[0058] Example 2: Preparation of micron-sized urchin-like carbonaceous particles (UCP-Au / Ir) integrating Au-Ir cascade catalytic sites

[0059] First, 0.1 grams of 2,5-dihydroxyterephthalic acid and 1.1 grams of zinc acetate dihydrate were dissolved in 60 mL of methanol and sonicated for about 1 hour. The sonicated product was then centrifuged and the supernatant was discarded. The precipitate was washed with deionized water three times and then added to a urea solution (0.4 grams of urea dissolved in 220 mL of deionized water). After stirring for 1 hour, the mixture was transferred to a preheated 170 °C Teflon-lined autoclave and reacted for 24 hours. The resulting precipitate was washed with water and ethanol and then dried under vacuum. The prepared material was placed in a ceramic boat and inserted into a tube furnace, which was heated to 900 °C at a rate of 5 °C / min and held for 1 hour. After the product cooled to room temperature, it was washed with a 1 M hydrochloric acid solution at 80 °C for 3 hours, washed with water three times and dried under vacuum to obtain the UCP material.

[0060] Subsequently, 32 mg of UCP was dispersed in 20 mL of deionized water, 700 μΐ, of a gold chloride trihydrate solution (10 mg / mL in ethanol) was added, followed by the addition of 6 mg of sodium borohydride (NaBH4). After stirring for 30 minutes, the mixture was washed with water three times and dried under vacuum to obtain the UCP-Au material.

[0061] To prepare UCP-Au / Ir, 10 mg of UCP-Au was dispersed in 1.25 mL of ethanol and 15 μΐ, of a mixture consisting of 75 mg of acetylacetonate iridium (III), 103 mg of 1,10-phenanthroline and 755 μΐ, of ethanol was added and sonicated for 30 minutes. The resulting dispersion was dried under vacuum overnight and heat treated at 600 °C for 2 hours under nitrogen atmosphere.

[0062] Example 3: Preparation of micron-sized urchin-like carbon particles integrated with Au-Ir cascade catalytic sites (UCP-Au / Ir)

[0063] First, 0.5 g of 2,5-dihydroxyterephthalic acid and 1.1 g of zinc acetate dihydrate were dissolved in 80 mL of methanol and ultrasonically treated for about 1 hour. The ultrasonically treated product was then centrifuged and precipitated, the supernatant was discarded and the precipitate was washed with deionized water three times. The precipitate was added to a urea solution (0.5 g of urea dissolved in 220 mL of deionized water), stirred for 1 hour, and then transferred to a preheated 180°C Teflon-lined high-pressure reactor for 24 hours. The obtained precipitate was washed with water and ethanol and then vacuum dried. The prepared material was placed in a ceramic boat and placed in a tube furnace to heat to 1100°C at a rate of 5°C / min and held for 1 hour. After the product cooled to room temperature, it was washed with a 1 M hydrochloric acid solution at 80°C for 3 hours, washed with water three times, and vacuum dried to obtain the UCP material.

[0064] Then, 32 mg of UCP was dispersed in 20 mL of deionized water, 900 μL of gold chloride trihydrate solution (10 mg / mL ethanol solution) was added, and 16 mg of sodium borohydride (NaBH4) was added. After stirring for 30 minutes, it was washed with water three times and vacuum dried to obtain the UCP-Au material.

[0065] When preparing UCP-Au / Ir, 10 mg of UCP-Au was dispersed in 1.25 mL of ethanol, 20 μL of a mixture consisting of 75 mg of acetylacetonate iridium (III), 103 mg of 1,10-phenanthroline, and 755 μL of ethanol was added, and ultrasonically treated for 30 minutes. The obtained dispersion was dried overnight under vacuum and heat treated at 700°C for 2 hours under a nitrogen atmosphere.

[0066] Test Example 1: Structural characterization

[0067] The UCP-Au / Ir prepared in Example 1 and other intermediate materials were subjected to structural characterization.

[0068] The morphology of the UCP-Au / Ir was characterized by scanning electron microscopy. As shown in FIG. 1, the UCP-Au / Ir exhibited a micron-sized urchin-like structure, which is advantageous for bacterial capture and cell membrane penetration, thereby enhancing the efficiency of active oxygen delivery and improving the accessibility of the catalytic reaction. Figure 1 High-angle annular dark field scanning transmission electron microscopy images showed that the gold clusters were densely distributed in the UCP-Au / Ir, with an average cluster size of about 5.11 nm (FIG. 2). Figure 2 In addition, a large number of bright atomic clusters and pairs of bright atomic points were observed, confirming the coexistence of Au clusters and single-atom iridium catalytic sites (FIG. 3). Figure 2The elemental distribution map of energy-dispersive X-ray spectroscopy further corroborates the coexistence of aggregated gold atoms and monodisperse iridium atoms in UCP-Au / Ir. Figure 3 ).

[0069] The valence state and electronic structure of UCP-Au / Ir were then investigated using X-ray photoelectron spectroscopy and Raman spectroscopy. Raman spectroscopy showed that all samples exhibited the same degree of defect and graphitization. Figure 4 a). X-ray photoelectron spectroscopy elemental analysis confirmed the presence of gold in UCP-Au / Ir and UCP-Au, and the presence of iridium in UCP-Au / Ir and UCP-Ir, thus verifying the successful elemental incorporation. Figure 4 b). The high-resolution X-ray photoelectron spectroscopy spectra show characteristic peaks at 60.92 eV and 63.88 eV, indicating that iridium in UCP-Au / Ir and UCP-Ir is in a positive valence state, suggesting that iridium has a single-atom structure. Figure 4 c). The Au 4f spectrum further confirms the presence of metallic Au in UCP-Au / Ir and UCP-Au. 0 This verifies the formation of gold clusters. Figure 4 d). It is worth noting that a small amount of gold exhibits a positive price state (Au). n+ This indicates an electronic interaction between the outer atoms of the gold cluster and the UCP substrate. Furthermore, the N 1s spectra of UCP-Au / Ir, UCP-Au, and UCP-Ir show a positive binding energy shift of pyridine nitrogen relative to UCP, indicating the formation of metal-nitrogen coordination bonds in the structure. Figure 4 e).

[0070] Test Example 2: Characterization of Catalytic Activity

[0071] Based on a detailed characterization of the morphology and electronic structure of UCP-Au / Ir (especially its gold clusters and iridium single-atom sites), this invention systematically evaluated and compared its enzyme-mimicking activity and cascade reactive oxygen species (ROS) generation capacity. First, the classic 3,3′,5,5′-tetramethylbenzidine colorimetric method was used to detect the oxidase-like activity of all samples. UCP-Au / Ir exhibited catalytic performance comparable to UCP-Ir, indicating that the iridium atom sites are the main contributors to achieving highly efficient oxidase-like activity. Figure 5 a). Similarly, peroxidase-like activities also showed the same trend, further confirming that the iridium atom site plays a key role in catalyzing the generation of reactive oxygen species mediated by hydrogen peroxide. Figure 5 b). Free radical quenching experiments confirmed that, in the presence of hydrogen peroxide, superoxide anions (•O2) quenched... - ) and singlet oxygen ( 1 O2) is the main reactive oxygen species produced by UCP-Au / Ir. Figure 5c). Considering that nicotinamide adenine dinucleotide (NADH) imbalance can disrupt bacterial energy metabolism and induce severe oxidative stress, ultimately leading to bacterial death through DNA and cell membrane damage, the present application evaluated the NADH-like oxidase activity of different samples. As shown in Figure 5 d, after UCP-Au / Ir or UCP-Ir treatment, the NADH level showed a time-dependent decrease, indicating that the iridium atomic site has strong NADH oxidation ability.

[0072] In the evaluation of the glucose oxidase-like activity of UCP-Au / Ir, UCP-Au, UCP-Ir and UCP, the present application found that UCP-Au showed the highest glucose oxidation and hydrogen peroxide generation ability (A652= 0. 48, 0. 36, 0. 24 and 0. 18, respectively). Figure 5 e). Notably, the hydrogen peroxide concentration decreased after the addition of UCP-Ir (A652= 0. 36, 0. 24, 0. 18 and 0. 12, respectively), indicating that there is an effective cascade catalysis between the gold clusters and the iridium atomic site. Figure 5 f), which further confirmed the cascade catalysis between these active centers (A652= 0. 48, 0. 36, 0. 24 and 0. 18, respectively). Figure 5 f). In summary, the synergistic effect of gold clusters and iridium atomic sites endows UCP-Au / Ir with multi-enzyme biomimetic and cascade catalytic ability, which can simultaneously consume oxygen, glucose and NADH, while enhancing the generation of reactive oxygen species. These multi-dimensional catalytic activities are expected to disrupt bacterial energy metabolism and ATP synthesis, induce oxidative stress, and ultimately produce a strong antibacterial effect.

[0073] The experimental methods involved in this test example are described as follows.

[0074] Oxidase-like activity analysis: The biological catalyst solution (10 mg / mL, 10 μL) was added to the acetic acid-sodium acetate buffer (100 mM, pH 4.5) containing 3,3',5,5'-tetramethylbenzidine (TMB, 10 mg / mL, 24 μL). The mixed system was made up to 2 mL with acetic acid-sodium acetate buffer, and the final sample concentration was 50 μg / mL. 200 μL of the mixed solution was taken, and the absorbance was measured at 652 nm wavelength using a multifunctional enzyme marker.

[0075] Peroxidase-like activity analysis: The biological catalyst solution (10 mg / mL, 10 μL) was added to the acetic acid-sodium acetate buffer (100 mM, pH 4.5) containing hydrogen peroxide (0.1 M, 25 μL) and 3,3',5,5'-tetramethylbenzidine (10 mg / mL, 24 μL). The mixed system was made up to 2 mL with acetic acid-sodium acetate buffer, and the final sample concentration was 50 μg / mL. 200 μL of the mixed solution was taken, and the absorbance was measured at 652 nm wavelength using a multifunctional enzyme marker.

[0076] Radical quenching experiment analysis: To detect •OH, 5 μL of catalyst (10 mg / mL), 25 μL of H2O2(0.1 M), 200 μL of tert-butyl alcohol (TBA), and 25 μL of TMB (10 mg / mL) were added to 1745 μL of acetate-sodium acetate buffer (100 mM, pH 4.5), and the absorbance at 652 nm wavelength was recorded after 10 minutes of reaction.

[0077] To detect •O2 - , 5 μL of catalyst (10 mg / mL), 25 μL of H2O2(0.1 M), 100 μL of p-benzoquinone, and 25 μL of TMB (10 mg / mL) were added to 1845 μL of acetate-sodium acetate buffer (100 mM, pH 4.5), and the absorbance at 652 nm wavelength was recorded after 10 minutes of reaction.

[0078] To detect 1 O2, 5 μL of catalyst (10 mg / mL), 25 μL of H2O2(0.1 M), 100 μL of sodium azide, and 25 μL of TMB (10 mg / mL) were added to 1845 μL of acetate-sodium acetate buffer (100 mM, pH 4.5), and the absorbance at 652 nm wavelength was recorded after 10 minutes of reaction.

[0079] Nicotinamide adenine dinucleotide oxidase-like activity evaluation: 10 μL of catalyst (final concentration 50 μg / mL) was added to 300 μL of NADH (2 mM) in 1690 μL of HEPES buffer (10 mM, pH 6.5), and the absorbance at 334 nm wavelength was measured using a multifunctional enzyme marker at 5, 10, and 15 minutes of reaction, respectively.

[0080] Glucose oxidase-like activity evaluation: 50 μL of catalyst (10 mg / mL) was added to 70 μL of glucose in 880 μL of phosphate buffer (PBS, pH 6.5) and reacted in a 42°C water bath. 50 μL of supernatant was transferred to a 96-well plate at 0, 30, 60, 90, and 120 minutes, respectively, and 150 μL of potassium iodide (1 M) solution was added to detect the concentration of hydrogen peroxide. After observing the color reaction, the absorbance at 350 nm wavelength was measured using a multifunctional enzyme marker.

[0081] To evaluate the cascade effect of the gold site and the iridium atom site, 10 μL of UCP-Ir was added after 120 minutes of UCP-Au reaction. Subsequently, 50 μL of the supernatant was taken again at time points 1, 2, 3 and 4 minutes and added to a 96-well plate. 150 μL of potassium iodide (KI, 1 M) solution was added to detect the H2O2 concentration.

[0082] Test Example 3: Scavenging effect of bacteria and related biofilms

[0083] It is known that excessive dietary sugar intake is metabolized into acid, promoting the colonization of Streptococcus mutans (a major pathogen causing dental caries) in dental plaque; therefore, inhibiting Streptococcus mutans colonization and biofilm formation is a key objective in dental caries prevention and control. Inspired by the unique urchin-like morphology, sugar consumption and NADH oxidation capacity of UCP-Au / Ir, as well as the reactive oxygen species generated by its multi-enzyme biomimetic cascade, this invention investigated its potential to capture and remove Streptococcus mutans and related biofilms. Preliminary evaluation of bacterial activity after incubation with different concentrations of UCP-Au / Ir determined its minimum inhibitory concentration to be 100 µg / mL. Figure 6 a). Subsequent evaluations under different incubation conditions showed that, in the presence of hydrogen peroxide, UCP-Au / Ir treatment significantly reduced bacterial counts, outperforming treatments using UCP-Au / Ir, UCP-Au, or UCP-Ir alone. Figure 6 b). For example Figure 6 As shown in c and 6d, scanning electron microscopy and transmission electron microscopy images show that the spike-like morphology of UCP-Au / Ir can effectively capture Streptococcus mutans and penetrate the bacterial cell wall and lipid membrane, causing irreversible damage to the bacteria, such as visible deformation and morphological collapse.

[0084] Subsequent analysis of the biofilm matrix labeled with Alexa Fluor 647-dextran showed that the UCP-Au / Ir-hydrogen peroxide group significantly reduced extracellular polymer levels (visible with blue fluorescence), with a clearance rate approaching 100%, which was superior to the UCP-Au and UCP-Au / Ir treatment groups. Figure 7 a, b). Furthermore, quantitative analysis of propidium iodide fluorescence intensity showed that the UCP-Au / Ir-hydrogen peroxide group exhibited the strongest red fluorescence, indicating that this group had the highest bacterial mortality rate (a, b). Figure 7c). Based on these findings and the previous enzyme mimic activity test results, the present application elucidates a stepwise optimized antibacterial mechanism: UCP-Au generates a basic antibacterial effect through sugar consumption and subsequent hydrogen peroxide generation at its gold site; after the introduction of the iridium site to form UCP-Au / Ir, a cascade reaction is established to convert hydrogen peroxide into reactive oxygen species, thereby enhancing the antibacterial activity; in addition, the exogenous addition of hydrogen peroxide (UCP-Au / Ir-hydrogen peroxide group) significantly amplifies the generation of reactive oxygen species, achieving almost complete antibacterial efficacy. This multi-stage amplified reactive oxygen species generation effect, in combination with the unique urchin-like morphology of the material (whose sharp spike structure promotes local delivery of reactive oxygen species), ultimately achieves effective clearance of S. mutans and its biofilm.

[0085] S. mutans, as the main pathogenic bacteria of dental biofilm formation, is the primary cause of irreversible dental caries. UCP-Au / Ir has shown significant inhibitory effect on S. mutans and potential in clearing existing biofilm and preventing tooth decay. Therefore, the present application further evaluates the clearance efficacy of UCP-Au / Ir on biofilm formed on the surface of human teeth. As shown in Figure 8 a), after co-culturing with the material, the biofilm formed on the surface of the teeth was subjected to fluorescence microscope staining using a Live / Dead BacLight bacterial viability kit, and at the same time, 10 mM Alexa Fluor 647-dextran solution was used for staining for 30 minutes to label extracellular polymeric substances, and images were collected by confocal laser scanning microscopy. The biofilm matrix labeled by Alexa Fluor 647-dextran showed that UCP-Au / Ir-hydrogen peroxide treatment could significantly reduce the EPS level (indicated by blue fluorescence), with a clearance rate close to 100% - this effect was better than that of UCP-Au and UCP-Au / Ir treatment groups (indicated by green fluorescence) (Fig. 6a). Figure 8 b). At the same time, the number of dead bacteria in the UCP-Au / Ir-hydrogen peroxide group increased significantly (indicated by red fluorescence) (Fig. 6b). Figure 8 c).

[0086] The experimental methods involved in this test example are described as follows.

[0087] In vitro antibacterial experiment: S. mutans UA159 (ATCC 700610) was used as a representative pathogenic bacteria to study the bacterial capture and clearance ability of the UCP-Au / Ir catalytic system. The antibacterial performance was evaluated by 600 nm absorbance (OD 600 ) and plate colony counting method. The bacteria were co-cultured with different final concentrations (0, 25, 50, 75, 100 and 150 μg / mL) of UCP-Au / Ir, and the system contained 0.2 mM hydrogen peroxide and 10 6 CFU·mL -1Bacteria were incubated at 37 °C for 12 h. The OD values of the bacterial suspensions after treatment with different concentrations of the system were recorded 600 The minimum inhibitory concentration was calculated. The active oxygen generation biocatalyst (UCP-Au, UCP-Ir, and UCP-Au / Ir) system (100 μg / mL) was co-incubated with 1 mL of bacterial suspension (10 6 CFU·mL -1 After 12 h of co-incubation at 37 °C, the culture solution was diluted 10 5 times for plate colony counting. Meanwhile, the bacterial suspension after incubation was diluted 10 3 , 10 4 , 10 5 , and 10 6 times, respectively, for plate colony counting. The agar plates were further incubated at 37 °C for 12 h, and the final number of colonies was counted. The OD 600 values were monitored by a UV-visible spectrometer (UV-3600, Shimadzu), and the antibacterial performance was evaluated by comparing the OD 600 values of each system with the control group. The protein leakage amount of the treated bacteria was measured using a BCA protein quantification kit (Solarbio). The bactericidal performance and bacterial capture ability of the system were observed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscopy. The bacterial suspensions treated with different systems were fixed with 2.5 wt% glutaraldehyde and dehydrated with a gradient ethanol / water solution, and the bacterial capture ability and morphological changes were observed by SEM and TEM images. In addition, the bacteria were stained using a LIVE / DEAD BacLight bacterial viability kit (SYTO-9 staining of live cells and iodine propyl phenyl staining of dead cells), and observed by fluorescence microscopy. To culture the biofilm, S. mutans was cultured in BHI medium with the addition of 1% (w / v) sucrose to 1 x 10 8 CFU·mL -1 , and then added to a 96-well plate for static culture at 37 °C for 24 h. Subsequently, the biofilm was treated with different systems, and the biofilm biomass was detected by crystal violet staining. Meanwhile, the untreated biofilm after treatment was fixed and dehydrated according to the aforementioned method, and then observed by scanning electron microscopy. In addition, the capture and killing behavior of different samples were stained using a LIVE / DEAD BacLight bacterial viability kit (SYTO-9 staining of live cells and iodine propyl phenyl staining of dead cells), and observed by confocal laser scanning microscopy (CLSM, St5, Leica). The extracellular polymeric substance was detected using dextran Alexa Fluor 647 (Thermo, D22914).

[0088] Test Example 4: Anti-cariogenic effect

[0089] Significant differences in bacterial density and structural organization may lead to varying degrees of damage to mineralized tooth tissue beneath different biofilms. Macroscopic observation revealed extensive surface demineralization within the carious enamel; close examination showed chalky white patchy demineralization patterns, similar to those commonly seen in early caries lesions. Further characterization of tooth samples was performed using micro-CT. Figure 9 a): Significant tooth defects were observed in the UCP-Au and UCP-Au / Ir treatment groups, while the enamel surface of the UCP-Au / Ir-hydrogen peroxide group remained largely intact and smooth, with subsurface demineralization limited to a confined area. It is important to note that subsurface demineralization is an early stage of caries; if bacterial infection persists, it may develop into substantial caries. Furthermore, this invention uses scanning electron microscopy (SEM) to assess the degree of enamel damage in each treatment group. Figure 9 As shown in b, erosion-induced microcavities appeared on the enamel surfaces of the UCP-Au and UCP-Au / Ir groups; in contrast, the enamel surface treated with UCP-Au / Ir-hydrogen peroxide remained largely intact and smooth. The effects of different materials on enamel demineralization were further evaluated using surface microhardness (SMH), nanoindentation hardness, and elastic modulus analysis. Compared to the UCP-Au and UCP-Au / Ir groups, the UCP-Au / Ir-hydrogen peroxide group showed lower surface microhardness (SMH). Figure 9 c) Nano-indentation hardness ( Figure 9 d) and elastic modulus ( Figure 9 All aspects (e) showed minimal changes. These results indicate that even under significant cariogenic stress induced by daily sucrose exposure, UCP-Au / Ir-hydrogen peroxide treatment significantly reduced demineralization. This study used the International Caries Detection and Assessment System (ICDAS) standard as a reference due to its well-validated diagnostic reliability in the literature. This system is currently recognized as the gold standard for caries detection and grading, used not only in research and epidemiological surveys but also widely in clinical and teaching fields. Quantitative caries scoring analysis showed that, compared with the carrier control group, UCP-Au / Ir-hydrogen peroxide significantly reduced the incidence and severity of caries lesions. Figure 9 f), effectively inhibiting enamel damage and cavity formation. In summary, these results demonstrate that UCP-Au / Ir, through its multi-enzyme biomimetic and cascade catalytic capabilities, can effectively disrupt the development of cariogenic biofilms and prevent localized demineralization and caries-like lesions on the enamel surface.

[0090] The experimental methods involved in this test case are described below.

[0091] Caries Prevention Experiment: All extracted teeth used in this experiment were healthy, caries-free samples. The teeth were placed in a suspension containing 2 mL of Streptococcus mutans (10... 8After 24 h of incubation at 37 °C, the centrifuge tubes were washed twice with physiological saline to remove non-adherent cells and immersed in fresh medium. UCP-Au, UCP-Ir and UCP-Au / Ir + hydrogen peroxide systems were then added to the centrifuge tubes, repeating the procedure every 12 h. After 6 weeks of incubation, the dental biofilm was collected and the live and dead cells were labeled with SYTO 9 and propidium iodide, respectively. Extracellular polymeric substances were labeled and visualized using Alexa Fluor 647-dextran conjugate (647 / 668 nm; Molecular Probes). After imaging by confocal laser scanning microscopy and analysis by Image J software, the teeth were cleaned and the enamel morphology was observed by scanning electron microscopy and micro-CT (Quantum GX, PerkinElmer). Based on the diagnostic reliability verified in the literature, the International Caries Detection and Assessment System (ICDAS) criteria were used as a reference standard. The main examiner was fully trained to combine the ICDAS criteria with clinical examination and reference images representing different ICDAS caries severity grades (0-6). The teeth samples subjected to bacterial co-cultures and material interaction were systematically evaluated by the trained ICDAS method. The teeth samples were prepared by cross-sectioning, resin embedding and stepwise grinding / polishing. Microhardness and nanoindentation analysis were performed on the enamel regions: microhardness was tested using a Vickers hardness tester (load 2 N, dwell time 15 s), while the elastic modulus and nano-hardness were measured using an in situ nanoindentation system (Hysitron TI950 Triboindenter, Bruker, USA) under controlled parameters (maximum load 2000 μN, dwell time 30 s, loading / unloading rate 400 μN / s).

[0092] It should be noted that the preferred embodiments of the present application are shown in the description and drawings of the present application, but the present application can be implemented in many different forms, and is not limited to the embodiments described in the specification. These embodiments are not additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, the above technical features continue to be combined with each other, forming various embodiments not listed above, which are all considered to be within the scope of the present application. Furthermore, for those skilled in the art, the above description can be improved or changed, and all these improvements and changes should be within the scope of the appended claims of the present application.

Claims

1. A catalyst material integrating Au-Ir cascade catalytic sites, characterized in that, The catalyst material comprises a carbon framework with a sea urchin-like structure and gold clusters and iridium single atoms supported on its surface. The carbon skeleton with the sea urchin-like structure is obtained by hydrothermal conversion of a zinc-based metal-organic framework in the presence of urea to obtain a sea urchin-like precursor, followed by carbonization and zinc sublimation to obtain micron-sized sea urchin-like carbon particles. The organic ligand of the zinc-based metal-organic framework is 2,5-dihydroxyterephthalic acid.

2. A method for preparing a catalyst material with integrated Au-Ir cascade catalytic sites according to claim 1, characterized in that, Includes the following steps: (1) The zinc salt is reacted with the organic ligand 2,5-dihydroxyterephthalic acid in a solvent to form a zinc-based metal-organic framework; (2) The zinc-based metal-organic framework is subjected to hydrothermal treatment in urea solution to obtain a precursor with a sea urchin-like morphology; (3) The precursor is subjected to carbonization treatment, which simultaneously sublimates the zinc component in the precursor to obtain micron-sized sea urchin-shaped carbon particles. (4) The gold precursor was reduced on the micron-sized sea urchin-shaped carbon particles using a reducing agent to obtain micron-sized sea urchin-shaped carbon particles with gold clusters deposited on the surface. (5) The metal-ligand complex formed by iridium ions and nitrogen-containing ligands is in situ coated on the micron-sized sea urchin-shaped carbonaceous particles with gold clusters deposited on the surface, and then carbonized at high temperature under an inert atmosphere to obtain the catalyst material with integrated Au-Ir cascade catalytic sites.

3. The preparation method according to claim 2, characterized in that, The zinc salt mentioned in step (1) is at least one of zinc nitrate, zinc acetate, zinc chloride, or their hydrates; The solvent is an alcohol solvent, including methanol or ethanol.

4. The preparation method according to claim 2, characterized in that, The mass concentration of the urea solution in step (2) is 0.1-0.3%; The hydrothermal treatment temperature is 170-180 ℃, and the reaction time is 12-36 h.

5. The preparation method according to claim 2, characterized in that, The carbonization temperature in step (3) is 900-1100 ℃, and the reaction time is 1-3 h.

6. The preparation method according to claim 2, characterized in that, Step (3) also includes acid washing, water washing and drying of the carbonized product.

7. The preparation method according to claim 2, characterized in that, Step (4) includes dispersing the micron-sized sea urchin-like carbonaceous particles in deionized water, adding an alcoholic solution of the gold precursor, and reacting it under the action of a reducing agent; The reducing agent is selected from one or more of sodium borohydride, hydrogen, ascorbic acid, and sodium citrate. The gold precursor is selected from one or more of chloroauric acid, gold chloride, or their hydrates.

8. The preparation method according to claim 2, characterized in that, Step (5) includes dispersing the micron-sized sea urchin-like carbonaceous particles of the gold clusters deposited on the surface in an alcohol solvent, adding an alcohol solution containing an iridium precursor and a nitrogen-containing ligand, ultrasonically dispersing, drying the dispersion, and then heat-treating it under an inert atmosphere. The iridium precursor is selected from one or more of iridium acetylacetonate, iridium chloro-iridium acid, iridium trichloride, or their hydrates; The nitrogen-containing ligand is selected from one or more of 1,10-phenanthroline, triphenylphosphine, or bipyridine compounds; The heat treatment temperature is 400-800℃, and the time is 1-5 hours.

9. Use of the catalyst material with integrated Au-Ir cascade catalytic sites according to claim 1 in the preparation of materials for antibacterial, anti-biofilm and anti-caries purposes.

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