Metal-organic framework peroxidase nanoszyme, preparation method and application thereof

Abstract

The application belongs to the field of nanobiotechnology, and discloses a metal organic framework peroxidase nanoenzyme, a preparation method and application thereof. The metal organic framework peroxidase nanoenzyme is prepared by using azole compounds as organic ligands, iron series elements as metal centers, and a mixture of deionized water and organic solvents in a certain proportion as a solvent. After mixing a metal ion solution and an organic ligand solution at room temperature, the metal organic framework peroxidase nanoenzyme is formed by reaction and coordination assembly through a solvothermal method. By adjusting the proportion of water and organic solvents in the preparation process, the metal organic framework peroxidase nanoenzyme with an optimal pH of neutral or near-neutral conditions is constructed. The nanoenzyme preparation method is simple and controllable, low in cost, and easy to synthesize in large quantities, thereby providing a train of thought for the design, synthesis and application of new MOF nanoenzymes.

Description

Technical Field

[0001] This invention relates to the field of nanobiotechnology, and more particularly to a metal-organic framework peroxidase nanozyme with high catalytic activity under neutral or near-neutral pH conditions, its preparation method, and its application. Background Technology

[0002] Enzymes, as highly efficient catalysts in nature, possess advantages such as high catalytic activity, high selectivity, and mild reaction conditions, and have wide applications in fields such as biomedicine, food production, and environmental remediation. However, when enzymes are applied in high-temperature environments or organic solvent conditions in vitro, their three-dimensional conformation is easily deformed, leading to inactivation. Nanomaterials with enzyme-like catalytic activity, represented by nanozymes, have the advantage of structural stability, providing a new approach to solving the above problems. Among existing nanozyme materials, metal-organic frameworks (MOFs) have become important carriers for nanozyme research due to their highly stable structure, tunable composition, controllable structure, and abundant porous structure. MOFs represented by iron-group elements Fe, Co, and Ni can act as electron transport media due to the unique redox properties of their metal ions, accepting electrons from the substrate and transferring them to another substrate, thereby achieving a catalytic process similar to that of natural peroxidases.

[0003] However, current literature reports that MOF-based peroxidase nanozymes exhibit optimal catalytic activity under acidic pH conditions (3.0–4.0), with activity significantly decreasing under neutral or near-neutral conditions. This limits their application in various practical scenarios. For example, peroxidase nanozymes only show good antibacterial activity against foodborne pathogens such as Staphylococcus aureus in acidic solutions of hydrogen peroxide, which is incompatible with the neutral pH conditions encountered during food processing and storage. In blood glucose detection systems, glucose oxidase often co-catalyzes with peroxidase under neutral conditions to generate a detection signal. However, peroxidase nanozymes typically exhibit low activity under these conditions, failing to achieve rate matching in the cascade enzyme catalysis process, thus limiting their application in biomedical detection. Although increasing the catalyst dosage of nanozymes can compensate for their insufficient activity to some extent, it usually leads to adverse effects such as biotoxicity or increased costs. Therefore, developing peroxidase nanozymes with high catalytic activity close to physiological pH is of great significance for broadening their applications.

[0004] Researchers have synthesized a sulfur-doped single-atom catalyst, Fe@CN-S, which enhances the peroxidase activity of nanozymes under neutral conditions by adjusting the spin state of the active sites through sulfur doping (Small, 2024, 20, 2311848). Although this strategy effectively improves the catalytic activity and atom utilization of nanozymes, the synthesis method requires high-temperature calcination, making the process more complex, with poor reproducibility, low stability, high cost, and difficulty in large-scale production. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a metal-organic framework peroxidase nanozyme with high catalytic activity under neutral or near-neutral pH conditions, as well as its preparation method and application.

[0006] This invention provides a metal-organic framework peroxidase nanozyme. The peroxidase nanozyme is a metal-organic framework material composed of iron-based elements and azole compound ligands, exhibiting peroxidase-like catalytic activity with an optimal pH of 5-6; in the peroxidase nanozyme, the azole compound ligands are coordinated with metal ions.

[0007] This invention provides a method for preparing metal-organic framework peroxidase nanozymes. Using azole compounds as organic ligands and iron-based elements as the metal center, and deionized water and an organic solvent as solvents, the metal compound is dissolved in deionized water, and the organic ligand is dissolved in the organic solvent. The two solutions are then mixed at room temperature, and a solvothermal reaction is carried out to achieve coordination assembly, forming a structurally stable metal-organic framework peroxidase nanozyme. The specific preparation steps are as follows:

[0008] The metal compound was dissolved in deionized water to obtain solution 1, and the azole compound was dissolved in an organic solvent to obtain solution 2. Solution 1 and solution 2 were mixed, and then the mixture was transferred to the inner lining of a Teflon high-pressure reactor, sonicated until completely dissolved, and placed in an oven for heating and reaction. After the reaction was completed, it was naturally cooled to room temperature, centrifuged to obtain a powdered product, washed, and dried to obtain the metal-organic framework peroxidase nanozyme.

[0009] Furthermore, the metal compound includes a compound containing at least one atom or ion of at least one iron-group element, or a combination of multiple compounds. The iron-group element includes iron, cobalt, and nickel; selected from ferrous chloride, ferrous sulfate, ferrous nitrate, ferrous acetate, ferrous gluconate, cobalt bromide, cobalt iodide, cobalt hydroxide, cobalt carbonate, cobalt nitrate, cobalt sulfate, nickel chloride, nickel sulfate, nickel nitrate, nickel bromide, and nickel hydroxide.

[0010] Further, the azole compound is selected from imidazole, 2-methylimidazazole, 2-nitroimidazazole, 2-bromo-1H-imidazazole, ethyl imidazazole-2-carboxylate, 2-mercaptoimidazazole, 2-propylimidazazole, 2-ethylimidazazole, 2-butylimidazazole, 1H-imidazazole-4-carboxylic acid, 2-methyl-5-nitroimidazazole, 1-octyl-3-methylimidazazole bromide, imidazazole-4,5-dicarboxylic acid, 4-phenylimidazazole-5-amino-4-imidazazole carboxamide, methyl imidazazole-4-carboxylate, 4-nitroimidazazole, 4-(hydroxymethyl)imidazazole, 4-chloroimidazazole, 4-methyl-5-hydroxymethylimidazazole, 4-imidazazole formaldehyde, ethyl imidazazole carboxylate, 2-imidazazole formaldehyde, benzimidazole, 5-amino-2-methylbenzimidazole, 2-(methylthio)benzimidazole, 2- Chloro-4-fluoro-(9Cl)-1H-benzimidazole, 2-mercaptobenzimidazole, 5-aminobenzimidazole, 5-methylbenzimidazole, 5,6-dimethylbenzimidazole, 2-methylbenzimidazole, 1H-benzimidazole-2-carboxylic acid, 4-(1H-imidazol-4-yl)piperidine, 2-ethylbenzimidazole, 5-chloro-7-methyl-1H-benzimidazole, 4-methylbenzimidazole, 5-carboxybenzimidazole, 1H-benzimidazole-2-sulfonic acid, 6-nitrobenzimidazole, methyl benzimidazole-7-carboxylic acid, 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid, 4-aminobenzimidazole, 5-methoxybenzimidazole, 6-bromo-1H-benzimidazole-4-carboxylic acid, benzotriazole, 1,2,4-triazole 3-Amino-5-methyl-4H-1,2,4-triazole, 3-Amino-5-methylthio-1H-1,2,4-triazole, 3,5-dimethyl-1,2,4-triazole, ethyl 1-bromo-1H-1,2,4-triazole-3-carboxylate, ethyl-1H-1,2,4-triazole-5-ylacetate, 1-methyl-1,2,4-triazole, 3-chloro-1,2,4-triazole, 1,2,3-triazole, 4-nitro-2H-1,2,3-triazole, benzotriazole, 1-hydroxybenzotriazole, 5-carboxylic acid benzotriazole, 7-Boc-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazine, 6-chloro-1-hydroxybenzotriazole, 5-chlorobenzotriazole, 5-methylbenzotriazole 7-Bromo-1H-benzotriazole, ethyl benzotriazole-5-carboxylate, benzotriazole-4-sulfonic acid, 5-Bromo-1H-benzotriazole, 1H-1,2,3-benzotriazole-5-ylboronic acid, 1H-tetrazole, 1H-tetrazole-5-acetic acid, 5-(4-pyridyl)-1H-tetrazole, 5-(3-pyridyl)-1H-tetrazole, 1H-tetrazole-5-ethyl acetate, 1-methyl-1H-tetrazole, 5-(ethylthio)-1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, 5-(2-pyridyl)-1H-tetrazole, 5-chloromethyl-1H-tetrazole, 5-(4-carboxyphenyl)-1H-tetrazole, 8-chlorotetrazole[1,5-A]pyridine, 5-propyl-1H-tetrazole, 6-bromotetrazole[1,The following are one or more of the following: [5-a]pyridine, 3-(1H-tetrazole)benzaldehyde, 3-tetrazole-1-ylphenol, 5-(3-methoxyphenyl)-1H-tetrazole, 2-methyl-5-(1H-tetrazole-5-yl)aniline, 5-(4-hydroxyphenyl)-1H-tetrazole, 5-(2-methylphenyl)-1H-tetrazole, 1-benzyl-1H-tetrazole-5-thiol, 3-phenyl-2-(1H-tetrazole-1-yl)propionic acid, 3-methoxy-5-(5-methyl-tetrazole-1-yl)aniline, methyl 4-(2H-1,2,3,4-tetrazole-5-yl)benzoate, 5-(3-nitrophenyl)-2H-tetrazole, and 1H-tetrazole-1-acetamide.

[0011] Further, the organic solvent is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, water, methanol, ethanol, diethylformamide, toluene, dimethyl sulfoxide, chlorobenzene, or a mixture thereof.

[0012] Furthermore, the molar ratio of the metal compound to deionized water in solution 1 is 9:1 to 1:2.25. Preferably, it is 1:2.25.

[0013] Furthermore, the molar ratio of the azole compound to the organic solvent in solution 2 is 12.8:1 to 1:3.2. Preferably, it is 1:3.2.

[0014] Furthermore, solution 1 and solution 2 are mixed evenly at a solvent 2:solution 1 volume ratio of 1:3 to 3:1. Preferably, solvent 2:solution 1 = 3:1.

[0015] Furthermore, the heating reaction temperature is 120–220°C, and the reaction time is 12–36 h.

[0016] Furthermore, the cleaning is performed using N,N-dimethylformamide and methanol. The drying temperature is 60°C.

[0017] This invention provides a method for preparing a metal-organic framework peroxidase nanozyme with high catalytic activity under neutral or near-neutral pH conditions. The catalytic site of the metal center, formed by coordination of the metal center with an azole ligand, mimics the catalytic center site of peroxidase. The catalytic center is controlled by adjusting the ratio of the synthesis solution. Therefore, the synthesized metal-organic framework peroxidase nanozyme has excellent catalytic activity, high stability under extreme operating conditions, and good reusability, providing a new technology for biomedical fields such as the synthesis of antibacterial agents, colorimetric sensing, and environmental remediation.

[0018] The key to constructing this invention of a metal-organic framework peroxidase nanozyme with high catalytic activity under neutral or near-neutral pH conditions lies in the regulation and construction of an iron-based metal-organic framework nanozyme with irregularly structured, stacked particles. This invention achieves regulation of the active site of the iron-based organometallic framework by optimizing the proportions of the synthesis solution and the molar ratio of iron ions to azole compounds, thereby improving the enzyme's catalytic activity at neutral or near-neutral pH. The synthesis conditions are simple, controllable, and reproducible. The metal-organic framework peroxidase nanozyme synthesized in this invention exhibits high catalytic activity under neutral or near-neutral pH conditions, making it more suitable than existing peroxidases for medical applications such as antibacterial therapy and colorimetric sensing, as well as environmental remediation.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] (1) This invention utilizes iron-based elements and azole compound ligands as precursors and constructs a metal-organic framework peroxidase nanozyme with an optimal pH of neutral or near-neutral by adjusting the volume ratio of the two solutions during the preparation process.

[0021] (2) The optimal pH for the metal-organic framework peroxidase nanozyme synthesized in this invention to exert its peroxidase catalytic activity is 5-6.

[0022] (3) The method for preparing metal-organic framework peroxidase nanozymes provided by the present invention has low requirements for synthesis equipment, simple synthesis method, controllable reaction conditions, and can realize large-scale production. Attached Figure Description

[0023] Figure 1 A scanning electron microscope image of the surface morphology of FeMAF-25 synthesized in Example 1 is shown.

[0024] Figure 2 The X-ray diffraction pattern (a) and Fourier transform infrared spectrum (b) of FeMAF-25 synthesized in Example 1 are shown.

[0025] Figure 3 A comparison diagram of the catalytic activities of FeMAF nanozymes synthesized based on different iron-based element compounds in Example 3 is shown.

[0026] Figure 4 The diagram shows a comparison of the catalytic activity and yield of FeMAF nanozymes synthesized with different solution volume ratios in Example 4.

[0027] Figure 5 The catalytic activity of FeMAF nanozymes synthesized in different molar ratios of metal ions and azole compound ligands in Example 5 is shown.

[0028] Figure 6A comparison graph showing the catalytic activity of FeMAF nanozymes synthesized with different azole compound ligands in Example 6 is presented.

[0029] Figure 7 The curves showing the relative absorbance values ​​of different FeMAF nanozymes over time in Example 7 are shown.

[0030] Figure 8 The graph shows the relationship between the catalytic activity and pH of the different FeMAFs synthesized in Example 8.

[0031] Figure 9 The catalytic activities of FeMAF-25 and natural horseradish peroxidase under different incubation temperatures (a), organic solvents (b), incubation pH (c), and ionic strengths (d) are shown.

[0032] Figure 10 The diagram shows a comparison of the catalytic activity of FeMAF nanozymes synthesized in a single solvent in Comparative Example 1. Detailed Implementation

[0033] The embodiments of the present invention are not limited to the above-described embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention are equivalent substitutions and are included within the protection scope of the present invention.

[0034] The present invention will be further described in detail with reference to specific embodiments:

[0035] Example 1

[0036] Solution 1 was prepared by dissolving 1 mmol of ferrous chloride in 8 mL of deionized water, and solution 2 was prepared by dissolving 1 mmol of benzotriazole in 24 mL of N,N-dimethylformamide. Solutions 1 and 2 were mixed in a glass bottle at a volume ratio of 1:3, and then sonicated to obtain a mixed solution. The mixture was then transferred to the lining of a reaction vessel and heated at 140 °C for 24 hours. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, transferred to centrifuge tubes, washed three times each with N,N-dimethylformamide and methanol, centrifuged at 10,000 rpm for 3 minutes, and the supernatant was removed. The solid was then transferred to a 60 °C oven and dried for 24 hours to obtain FeMAF-25 (the value 25 refers to the volume percentage of solution 1 in the mixed solution, which is "25%").

[0037] Figure 1 The image shows a scanning electron microscope image of FeMAF-25. When the volume of solution 1 in the mixed solution is relatively low, the solvent water molecules in solution 1 can reduce the association of the organic ligand BTA and promote faster deprotonation and nucleation reactions. Therefore, FeMAF-25 exhibits an irregular morphology with a rough surface, consisting of small particles of about 200 nm stacked into larger solid particles.

[0038] Example 2

[0039] FeMAF-x (x refers to the volume percentage of solution 1 in the mixed solution) was synthesized according to the method of Example 1. In this example, five nanozymes, namely FeMAF-25, FeMAF-33, FeMAF-50, FeMAF-66 and FeMAF-75, were synthesized.

[0040] Figure 2 Figure a shows the X-ray powder diffraction results of the above five nanozymes, indicating that FeMAF has high-intensity diffraction peaks and a crystalline structure. Figure 2 Figure b shows the Fourier transform infrared spectra of the five nanozymes mentioned above, 550 -1 The characteristic peak of Fe-N at 3153 cm⁻¹ -1 1476cm -1 , and 1127cm -1 The vibrations of -NH, -N=N- and -CN- in benzotriazole confirm the successful coordination of benzotriazole with iron ions.

[0041] Example 3

[0042] To investigate the effects of different iron-based compounds on the catalytic activity of nanozymes, ferrous chloride was replaced with ferrous sulfate, ferrous nitrate, ferrous acetate, and ferrous gluconate in this embodiment. 1 mmol of each of the above iron salts was dissolved in 8 mL of deionized water to obtain solution 1, and 1 mmol of benzotriazole was dissolved in 24 mL of N,N-dimethylformamide to obtain solution 2. Solutions 1 and 2 were ultrasonically mixed in a glass bottle at a certain ratio and then transferred to the lining of a reaction vessel. The mixture was heated at 140°C for 24 hours. After the reaction, the mixture was allowed to cool naturally, centrifuged, washed, dried, and collected to obtain FeMAF synthesized based on different iron salt compounds. The catalytic performance of the nanozyme was then verified in a TMB / H₂O₂ system.

[0043] Figure 3 The catalytic activities of FeMAF nanozymes synthesized based on different types of iron-based compounds are shown. Different ferrous salt anions affect the catalytic activity of FeMAF through anionic effects, solution pH regulation, and redox stability. Figure 3It is known that FeMAF synthesized from ferrous chloride as a precursor exhibits the highest catalytic activity, followed by ferrous sulfate, ferrous nitrate, ferrous acetate, and ferrous gluconate. Therefore, ferrous chloride is the optimal choice. This may be because chloride ions have weaker coordination ability and easily detach from the metal center during synthesis, avoiding excessive occupation of metal coordination sites. This ensures effective coordination between the organic ligand and Fe2+, forming a highly active catalytic structure. Sulfate and nitrate ions, on the other hand, reduce effective catalytic sites by slowing down the nucleation rate. Acetate and gluconate ions have greater steric hindrance, hindering effective coordination between the organic ligand and Fe2+, thus resulting in relatively lower catalytic activity. Besides ferrous salt compounds, nanozymes synthesized from other iron-based element compounds such as cobalt nitrate and nickel chloride as precursors also possess certain peroxidase nanozyme activity. 2+ / Co 3+ or Ni 2+ / Ni 3+ The electron transfer pathway at the catalytic site activates H2O2 to generate reactive oxygen species, thereby exhibiting peroxidase nanozyme activity.

[0044] Example 4

[0045] To investigate the effect of different mixed solution ratios on the catalytic activity of nanozymes, a series of volume ratios of the synthesis solvents were set. 1 mmol of ferrous chloride was dissolved in deionized water to obtain solution 1, and 1 mmol of benzotriazole was dissolved in N,N-dimethylformamide to obtain solution 2. The volume ratio of the synthesis solutions (solution 2:solution 1) was controlled at 1:3, 1:2, 1:1, 2:1, and 3:1. The mixed solutions were transferred to the lining of a reaction vessel and heated at 140 °C for 24 hours. After the reaction, the solutions were allowed to cool naturally, centrifuged, washed, dried, and collected. The catalytic performance of the nanozymes was then verified in a TMB / H₂O₂ system.

[0046] Figure 4 The relative activity of FeMAF under different solution volume ratios was shown. As the proportion of deionized water in the mixed solution decreased, the catalytic activity and yield of the nanozyme also increased. When the volume ratio of solution 2: solution 1 was 3:1, the catalytic activity and yield of the nanozyme were the best. Compared with the volume ratio of solution 2: solution 1 was 1:3, its catalytic activity increased by about 3 times. This performance improvement may be attributed to the regulatory effect of DMF's high dielectric constant and coordination ability on the nucleation and growth process of nanoparticles, as well as its optimization effect on the microenvironment of the enzyme active site.

[0047] Example 5

[0048] To investigate the effect of different molar ratios of metal ions to organic ligands on the catalytic activity of nanozymes, a series of ratios of metal ions to azole organic compound ligands were set. The group with the best catalytic activity from Example 4 was selected, and the synthesis method was the same, only the molar ratio of iron ions to azole compound ligands was changed. The ratio was set to 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1. The catalytic performance of the nanozyme was verified in a TMB / H2O2 system using the mixed solution.

[0049] from Figure 5 It can be seen that as the molar ratio of metal ions increases, the catalytic activity of nanozymes shows a trend of first increasing and then decreasing. When Fe 2+ The catalytic activity is optimal when the molar ratio of metal ions to benzotriazole is 1:1, with a relative absorbance of 0.623. Further increasing the molar ratio of metal ions leads to a metal ion excess, preventing some metal sites from fully coordinating with the organic ligands, thus hindering the formation of catalytically active MOF materials.

[0050] Example 6

[0051] To investigate the effects of different azole ligands on the catalytic activity of nanozymes, the types of azole ligands were varied (listed above). This example used 2-methylimidazole, 2-ethylimidazole, benzimidazole, 5-carboxylic acid benzotriazole, and 1H-tetrazole. The optimal iron source from Example 3, the optimal volume ratio of the synthesis solution from Example 4, and the optimal ratio of the synthesis raw materials from Example 5 were selected. 1 mmol of ferrous chloride was dissolved in 8 mL of deionized water to obtain solution 1, and 1 mmol of different azole ligands was dissolved in 24 mL of N,N-dimethylformamide to obtain solution 2. Solutions 1 and 2 were then mixed in an optimal volume ratio (solution 2:solution 1 = 3:1) in a glass bottle, ultrasonically mixed, and transferred to the lining of a reaction vessel. The mixture was heated at 140°C for 24 h. After cooling to room temperature, the solid product was collected by centrifugation. The powder was washed three times each with DMF and methanol, and dried under vacuum at 60°C. The catalytic performance of the nanozyme was then verified in a TMB / H₂O₂ system.

[0052] from Figure 6 It can be seen that, using Fe 2+ Iron-based metal-organic framework nanozymes, formed by ions as metal centers and various azole compound ligands, all exhibit peroxidase-mimicking enzyme activity. 2+Ions coordinate with the nitrogen atoms in azole ligands to form MOFs, but the catalytic reaction rates may vary due to differences in ligand structure. The catalytic reaction rates, from highest to lowest, are benzimidazole, 1H-tetrazole, 2-ethylimidazole, 2-methylimidazole, and 5-carboxylic acid benzotriazole. This is related to the electronic effects, steric hindrance, and coordination mode of the ligands. For example, the benzene ring structure of benzimidazole can provide additional substrate adsorption sites (π-π stacking), promoting the oxidation of organic substrates (such as TMB), thus exhibiting a better reaction rate. On the other hand, 5-carboxylic acid benzotriazole has greater steric hindrance, hindering the effective coordination of organic ligands with Fe2+, reducing the formation of catalytic sites, and therefore its reaction rate is lower.

[0053] Example 7

[0054] To evaluate the peroxidase mimicry activity of nanozymes, the enzyme mimicry activity of FeMAF was determined by colorimetric analysis using TMB / H2O2 as a model substrate at room temperature. The specific experimental procedure is as follows: 12.02 mg TMB was accurately weighed and dissolved in 2.0 mL DMSO to obtain a 25 mM TMB working solution; 2.00 mg FeMAF was weighed and ultrasonically dispersed in 4.0 mL PBS buffer (50 mM, pH 6.0) to obtain a 500 μg / mL nanozyme dispersion; 20 μL of H2O2 stock solution (concentration 10 mol / L) was pipetted and dispersed in 1980 μL of deionized water to obtain a 100 mM H2O2 solution. Add an appropriate amount of PBS buffer to a 5 mL centrifuge tube to make the final reaction volume 3.0 mL. First, add 30 μL of TMB working solution, then add 90 μL of nanozyme dispersion, and finally add 30 μL of H2O2 solution. Mix quickly up and down, then transfer to a quartz cuvette. Monitor the change in absorbance at 652 nm within 2 minutes using a UV2500 UV-Vis spectrophotometer.

[0055] Figure 7 The relative absorbance values ​​of different FeMAF nanozymes over time are shown. By comparing the changes in absorbance values ​​within the same time period, it can be seen that FeMAF-25 has the highest relative absorbance value per unit time, indicating that it has the best catalytic activity and optimal substrate conversion ability. The catalytic activity of the other nanozymes decreases with the gradual increase of the water ratio in the organic solvent, in the following order: FeMAF-33, FeMAF-50, FeMAF-66, and FeMAF-75. This is related to the solvent ratio in the synthesis of the nanozymes. FeMAF-25 synthesized under conditions of low water ratio and high organic solvent has a more stable coordination environment. The low water ratio helps to reduce the Fe content of catalytic sites in the nanozyme. 2+ The hydrolysis or oxidation of FeMAF-75 synthesized under conditions of high water ratio and low organic solvent may be due to water molecules competing for coordination with Fe. 2+This leads to instability or even collapse of some MOF structures, and may even promote the oxidation of Fe2+ to Fe3+, reducing Fe2+ / Fe3+ redox activity and thus weakening its peroxidase catalytic ability.

[0056] Example 8

[0057] To investigate the optimal catalytic pH of FeMAF nanozymes, following the activity assay method in Example 7, and keeping other reaction conditions unchanged, the buffer pH was set to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, with the group showing the highest catalytic activity set as 100%, and the relative activity of FeMAF nanozymes at different catalytic pH was calculated.

[0058] Figure 8 The relative activities of different FeMAF nanozymes at different reaction pH values ​​were shown. FeMAF-25 exhibited the best catalytic activity at pH 6.0, while FeMAF-33, FeMAF-50, and FeMAF-66 showed the best catalytic activity at pH 5.0, and FeMAF-75 showed the best catalytic activity at pH 4.0. This may be related to the structure of the active sites. FeMAF-25 synthesized under low water ratio and high organic solvent conditions had more stable catalytic active sites, thus maintaining high reducing power under neutral to slightly acidic conditions (pH 6.0) to ensure an effective redox cycle between Fe2+ and Fe3+, and therefore exhibited the best activity under neutral to slightly acidic conditions. On the other hand, in FeMAF-75 synthesized under high water ratio and low organic solvent conditions, water promoted the oxidation of Fe2+ to Fe3+. Under acidic conditions (pH 4.0), the high H+ concentration inhibited the accumulation of Fe3+, maintaining the redox cycle of Fe2+ / Fe3+, and therefore exhibited the best catalytic activity under acidic conditions.

[0059] Example 9

[0060] To investigate the stability of nanozymes under different extreme conditions, the catalytic activity of nanozymes and natural horseradish peroxidase was examined under different incubation pH, incubation temperature, organic solvent type and ionic strength.

[0061] 1. Regarding thermal stability, FeMAF-25 was placed in an oven for 2 hours and the temperature was set to 20-80℃. Then, the enzyme activity was measured according to the method in Example 6. The activity of the untreated sample was recorded as 100% relative activity. The relative activity of the nanozyme after treatment at different temperatures was calculated.

[0062] 2. Regarding solvent stability, FeMAF-25 was incubated for 8 hours (~5 mg / mL, 2 mL) in methanol, ethanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and acetone, respectively. The precipitate was collected by centrifugation at 10,000 rpm for 2 min, washed three times with deionized water, dried, and then the enzyme activity was determined according to the method in Example 6. The activity of the untreated sample was recorded as the relative activity of 100%, and the relative activity of the nanozyme after treatment with different solvents was calculated.

[0063] 3. Regarding pH tolerance, FeMAF-25 was incubated in buffer solutions (50mM) ranging from pH 3.0 to 9.0 for 2 hours, and the precipitate was collected by centrifugation at 10,000 rpm for 2 minutes. After washing and drying, the enzyme activity was measured according to the method in Example 6. The activity of the untreated sample was recorded as 100% relative activity, and the relative activity of the nanozyme after different pH treatments was calculated.

[0064] 4. Regarding ionic strength tolerance, FeMAF-25 was placed in sodium chloride solutions ranging from 0 to 1000 mM, and the enzyme-like activity was determined according to the method in Example 6. The activity of the untreated sample was recorded as 100% relative activity.

[0065] Figure 9 The relative activities of nanozymes and natural enzymes under different treatment conditions were shown. Figure 9 As can be seen from a, within the temperature range of 20–40℃, the catalytic activity of nanozymes and natural enzymes is basically unaffected by temperature. However, as the temperature gradually increases, the relative activity of natural enzymes shows a sharp decline, maintaining only about 20% of the initial activity at 80℃, while the catalytic activity of nanozymes only decreases by less than 20%, indicating that nanozymes have better hydrothermal stability. Figure 9 Figure b shows the catalytic stability of nanozymes and natural enzymes in common organic solvents. The rigid framework and coordination bond stability enable FeMAF-25 to exhibit satisfactory stability in various organic solvents, retaining more than 90% of its initial activity, while natural enzymes retain less than 60% of their original activity in organic solvents. Figure 9 As shown in equation c, the FeMAF-25 nanozyme retains 80% of its initial activity in both strongly acidic environments (pH 3.0) and strongly alkaline environments (pH 9.0), while natural enzymes retain only about 20% of their initial activity under acidic conditions. High ionic strength may affect the charge distribution and spatial structure of nanozymes, easily leading to enzyme activity loss. From... Figure 9As shown in d, the catalytic activity of both FeMAF-25 nanozymes and natural enzymes decreased in NaCl solutions ranging from 300 to 1000 mM. At a NaCl concentration of 1000 mM, the nanozymes retained approximately 80% of their initial activity, while the natural enzymes retained only 30%, indicating that FeMAF-25 exhibits better stability under high ionic strength. In conclusion, under different temperatures, pH values, organic solvents, and ionic strengths, the nanozymes demonstrated higher catalytic activity than the natural enzymes, indicating that the catalytic stability of FeMAF-25 nanozymes is superior to that of the natural enzymes.

[0066] Comparative Example 1

[0067] Comparison of catalytic activities for MOF synthesis in single solvents

[0068] Following the FeMAF standard procedure described herein, metal-organic framework nanozymes were formed in two single solvents: pure deionized water and pure N,N-dimethylformamide, and their catalytic activities were compared. The synthesis conditions were consistent with those of Example 6, except that the mixed solvent was replaced with pure water or pure N,N-dimethylformamide. After synthesis, the samples were collected by centrifugation, and their catalytic activity was evaluated according to the activity assay method of Example 7. Figure 10 As shown, MOFs synthesized in pure water or pure N,N-dimethylformamide single solvent systems exhibit extremely low catalytic activity, less than 1% of that in mixed solvent systems (with a volume ratio of 1:3 for solution 1:2 as a control). This indicates that the preferred mixed solution ratio of the present invention plays an important role in the formation of peroxidase nanozymes with high catalytic activity.

[0069] The examples described above are merely illustrative of several embodiments of the present invention, and while the descriptions are relatively specific and detailed, the technical scope is not limited to these embodiments. For those skilled in the art, various improvements and implementations can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for preparing a metal-organic framework peroxidase nanozyme, characterized in that, The peroxidase nanozyme exhibits peroxidase-like catalytic activity, with an optimal pH of 5-6. The specific preparation steps are as follows: dissolve a metal compound in deionized water to obtain solution 1, dissolve an azole compound in an organic solvent to obtain solution 2, mix solution 1 and solution 2, then transfer the mixture to a Teflon high-pressure reactor liner, sonicate until completely dissolved, place in an oven for heating and reaction, after the reaction is completed, naturally cool to room temperature, centrifuge to obtain a powdered product, wash, and dry to obtain the metal-organic framework peroxidase nanozyme; The metal compound includes a compound containing at least one atom or ion of at least one iron-group element, or a combination of multiple compounds; the iron-group element includes iron, cobalt, and nickel; selected from ferrous chloride, ferrous nitrate, cobalt bromide, cobalt iodide, cobalt hydroxide, cobalt carbonate, cobalt nitrate, cobalt sulfate, nickel chloride, nickel sulfate, nickel nitrate, nickel bromide, and nickel hydroxide. The molar ratio of the metal compound to deionized water in solution 1 is 9:1 to 1:2.25; the molar ratio of the azole compound to the organic solvent in solution 2 is 12.8:1 to 1:3.

2. Solution 1 and solution 2 are mixed thoroughly at a volume ratio of solvent 2:solution 1 of 1:3 to 3:

1. The reaction temperature is 120–220℃, and the reaction time is 12–36 h.

2. The preparation method according to claim 1, characterized in that, The azole compounds are selected from imidazole, 2-methylimidazolium, 2-nitroimidazolium, 2-bromo-1H-imidazolium, ethyl imidazolium-2-carboxylate, 2-mercaptoimidazolium, 2-propylimidazolium, 2-ethylimidazolium, 2-butylimidazolium, 1H-imidazolium-4-carboxylic acid, 2-methyl-5-nitroimidazolium, 1-octyl-3-methylimidazolium bromide, imidazolium-4,5-dicarboxylic acid, 4-phenylimidazolium-5-amino-4-imidazolium carboxamide, methyl imidazolium-4-carboxylate, 4-nitroimidazolium, 4-(hydroxymethyl)imidazolium, 4-chloroimidazolium, 4-methyl-5-hydroxymethylimidazolium, 4-imidazolium carboxaldehyde, ethyl imidazolium carboxylate, 2-imidazolium carboxaldehyde, benzimidazole, 5-amino-2-methylbenzimidazole, 2-(methylthio)benzimidazole, 2-chloro- 4-Fluoro-(9Cl)-1H-benzimidazole, 2-mercaptobenzimidazole, 5-aminobenzimidazole, 5-methylbenzimidazole, 5,6-dimethylbenzimidazole, 2-methylbenzimidazole, 1H-benzimidazole-2-carboxylic acid, 4-(1H-imidazol-4-yl)piperidine, 2-ethylbenzimidazole, 5-chloro-7-methyl-1H-benzimidazole, 4-methylbenzimidazole, 5-carboxybenzimidazole, 1H-benzimidazole-2-sulfonic acid, 6-nitrobenzimidazole, methyl benzimidazole-7-carboxylic acid, 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid, 4-aminobenzimidazole, 5-methoxybenzimidazole, 6-bromo-1H-benzimidazole-4-carboxylic acid, 1,2,4-triazole, 3-amino- 5-Methyl-4H-1,2,4-triazole, 3-amino-5-methylthio-1H-1,2,4-triazole, 3,5-dimethyl-1,2,4-triazole, 1-bromo-1H-1,2,4-triazole-3-carboxylic acid ethyl ester, ethyl-1H-1,2,4-triazole-5-yl acetate, 1-methyl-1,2,4-triazole, 3-chloro-1,2,4-triazole, 1,2,3-triazole, 4-nitro-2H-1,2,3-triazole, benzotriazole, 1-hydroxybenzotriazole, 5-carboxylic acid benzotriazole, 7-Boc-5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrazine, 6-chloro-1-hydroxybenzotriazole, 5-chlorobenzotriazole, 5-methylbenzotriazole, 7- Bromo-1H-benzotriazole, ethyl benzotriazole-5-carboxylate, benzotriazole-4-sulfonic acid, 5-bromo-1H-benzotriazole, 1H-1,2,3-benzotriazole-5-ylboronic acid, 1H-tetrazole, 1H-tetrazole-5-acetic acid, 5-(4-pyridyl)-1H-tetrazole, 5-(3-pyridyl)-1H-tetrazole, 1H-tetrazole-5-ethyl acetate, 1-methyl-1H-tetrazole, 5-(ethylthio)-1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, 5-(2-pyridyl)-1H-tetrazole, 5-chloromethyl-1H-tetrazole, 5-(4-carboxyphenyl)-1H-tetrazole, 8-chlorotetrazole[1,5-A]pyridine, 5-propyl-1H-tetrazole, 6-bromotetrazole[1,[5-a]pyridine, 3-(1H-tetrazole)benzaldehyde, 3-tetrazole-1-ylphenol, 5-(3-methoxyphenyl)-1H-tetrazole, 2-methyl-5-(1H-tetrazole-5-yl)aniline, 5-(4-hydroxyphenyl)-1H-tetrazole, 5-(2-methylphenyl)-1H-tetrazole, 1-benzyl-1H-tetrazole-5-thiol, 3-phenyl-2-(1H-tetrazole-1-yl)propionic acid, 3-methoxy-5-(5-methyl-tetrazole-1-yl)aniline, methyl 4-(2H-1,2,3,4-tetrazole-5-yl)benzoate, 5-(3-nitrophenyl)-2H-tetrazole, 1H-tetrazole-1-acetamide.

3. The preparation method according to claim 1, characterized in that, The organic solvent is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, methanol, ethanol, diethylformamide, toluene, dimethyl sulfoxide, and chlorobenzene, or a mixture thereof.

4. The metal-organic framework peroxidase nanozyme obtained by the preparation method according to any one of claims 1-3, characterized in that, The peroxidase nanozyme is a metal-organic framework material composed of iron-based elements and azole compound ligands, wherein the azole compound ligands in the peroxidase nanozyme are coordinated with metal ions.

5. The application of the metal-organic framework peroxidase nanozyme according to claim 4 in the synthesis of antibacterial agents, colorimetric sensing, and environmental remediation.

Images