Metal organic framework peroxidase nano-enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano biology, and discloses a metal organic framework peroxidase nano enzyme as well as a preparation method and application thereof. The preparation method comprises the following steps: by taking an azole compound as an organic ligand, an iron-based element as a metal center and a mixture of deionized water and an organic solvent in a certain proportion as a solvent, mixing a metal ion solution and an organic ligand solution at normal temperature, reacting through a solvothermal method, and carrying out coordination assembly to form the metal organic framework peroxidase nano-enzyme with a stable structure. The metal organic framework peroxidase nano-enzyme with the optimal pH being neutral or near-neutral is constructed by adjusting the ratio of water to an organic solvent in the preparation process. The preparation method of the nano-enzyme is simple and controllable, low in cost and easy for large-scale synthesis, and provides a thought for design synthesis and application of the novel MOF nano-enzyme.

Description

Technical Field

[0001] The present invention relates to the field of nanobiotechnology, and in particular to a metal-organic framework peroxidase nanozyme having high catalytic activity under neutral or near-neutral pH conditions, and a preparation method and application thereof. Background Art

[0002] As highly efficient catalysts in nature, enzymes have advantages such as high catalytic activity, high selectivity and mild reaction conditions. They are widely used in biomedicine, food production and environmental governance. However, when enzymes are used in high temperature 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 and provide a new approach to solving the above problems. Among the existing nanozyme materials, metal-organic frameworks (MOFs) have become an important carrier for nanozyme research due to their highly stable structure, adjustable components, controllable structure and rich porous structure. MOFs represented by iron-based elements Fe, Co and Ni can act as electron transfer media due to the unique redox properties of the metal ions in them, accepting electrons from the substrate and transferring electrons to another substrate, thereby realizing a catalytic process similar to that of natural peroxidase.

[0003] However, the MOFs peroxidase nanozymes reported in the current literature have optimal catalytic activity (3.0-4.0) under acidic pH conditions, and their activity is significantly reduced under neutral or near-neutral conditions, which limits their application in different 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 does not match the neutral pH conditions during food processing and storage. In blood glucose detection systems, glucose oxidase often synergistically catalyzes with peroxidase under neutral conditions to produce detection signals, while peroxidase nanozymes are usually less active at this time and cannot achieve rate matching in the cascade enzyme catalysis process, limiting their application in biomedical testing. Although increasing the catalyst dosage of nanozymes can compensate for their insufficient activity to a certain extent, it usually brings about the adverse effects of biological toxicity or increased cost. Therefore, the development of peroxidase nanozymes with high catalytic activity at near-physiological pH values ​​is of great significance for broadening their applications.

[0004] Researchers have synthesized a sulfur-doped single-atom catalyst, Fe@CN-S, that modulates the spin state of the active site through sulfur doping, thereby enhancing the peroxidase activity of the nanozyme under neutral conditions (Small, 2024, 20, 2311848). Although this strategy effectively improves the catalytic activity and atom utilization of the nanozyme, the synthesis method requires high-temperature calcination, which is more complex, has poor reproducibility and stability, is costly, and is difficult to scale up. Summary of the Invention

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

[0006] The present invention provides a metal-organic framework peroxidase nanozyme. The peroxidase nanozyme is a metal-organic framework material composed of an iron-based element and an azole compound ligand, exhibiting peroxidase-like catalytic activity with an optimal pH of 5-6; in the peroxidase nanozyme, the azole compound ligand binds to the metal ion in a coordinated manner.

[0007] The present invention provides a method for preparing a metal-organic framework peroxidase nanozyme, which uses an azole compound as an organic ligand, an iron-based element as a 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 reacted by a solvothermal method to perform coordination assembly to form a structurally stable metal-organic framework peroxidase nanozyme. The specific preparation steps are as follows:

[0008] The metal compound is dissolved in deionized water to obtain solution 1, and the azole compound is dissolved in an organic solvent to obtain solution 2. Solution 1 and solution 2 are mixed, and then the mixture is transferred to the inner lining of a Teflon high-pressure reactor, ultrasonicated until completely dissolved, and placed in an oven for heating and reaction. After the reaction is completed, it is naturally cooled to room temperature and centrifuged to obtain a powdered product. After washing, it is 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-based element, or a combination of multiple compounds. The iron-based element includes iron, cobalt, and nickel; and is 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 nickelous hydroxide.

[0010] Further, the azole compound is selected from imidazole, 2-methylimidazole, 2-nitroimidazole, 2-bromo-1H-imidazole, ethyl imidazole-2-carboxylate, 2-mercaptoimidazole, 2-propylimidazole, 2-ethylimidazole, 2-butylimidazole, 1H-imidazole-4-carboxylic acid, 2-methyl-5-nitroimidazole, 1-octyl-3-methylimidazole bromide, imidazole-4,5-dicarboxylic acid, 4-phenylimidazole-5-amino-4-imidazolecarboxamide, methyl imidazole-4-carboxylate, 4-nitroimidazole, 4-(hydroxymethyl)imidazole, 4-chloroimidazole, 4-methyl-5-hydroxymethylimidazole, 4-imidazolecarboxaldehyde, ethyl 4-imidazolecarboxylate, 2-imidazolecarboxaldehyde, benzimidazole, 5-amino-2-methylbenzimidazole, 2-(methylthio)benzimidazole, 2- Chloro-4-fluoro-(9CI)-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-carboxylate, 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, 1-bromo-1H-1,2,4-triazole-3-carboxylic acid ethyl ester, ethyl-1H-1,2,4-triazol-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-benzotriazol-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-formylphenyl)-1H-tetrazole, 8-chlorotetrazo[1,5-a]pyridine, 5-propyl-1H-tetrazole, 6-bromotetrazo[1,5-a]pyridine5-a] pyridine, 3-(1H-tetrazolyl)benzaldehyde, 3-tetrazol-1-yl-phenol, 5-(3-methoxyphenyl)-1H-tetrazol, 2-methyl-5-(1H-tetrazol-5-yl)aniline, 5-(4-hydroxyphenyl)-1H-tetrazol, 5-(2-methylphenyl)-1H-tetrazol, 1-benzyl-1H-tetrazol-5-thiol, 3-phenyl-2-(1H-tetrazol-1-yl)propionic acid, 3-methoxy-5-(5-methyl-tetrazol-1-yl)-aniline, methyl 4-(2H-1,2,3,4-tetrazol-5-yl)benzoate, 5-(3-nitrophenyl)-2H-tetrazol, 1H-tetrazol-1-acetamide or more.

[0011] Furthermore, 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 mixed solution thereof.

[0012] Furthermore, the molar ratio of the metal compound to deionized water in solution 1 is 9:1 to 1:2.25, preferably 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 1:3.2.

[0014] Furthermore, solution 1 and solution 2 are uniformly mixed in a volume ratio of solvent 2 to solution 1 of 1:3 to 3:1, preferably solvent 2 to solution 1 = 3:1.

[0015] Furthermore, the temperature of the heating reaction is 120-220° C., and the reaction time is 12-36 hours.

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

[0017] The present invention provides a method for preparing a metal-organic framework peroxidase nanozyme with high catalytic activity under neutral or near-neutral pH conditions. The metal center catalytic site formed by the coordination of the metal center and the azole ligand simulates the catalytic center site of the peroxidase. The catalytic center is regulated by the proportion of the synthetic solution. Therefore, the synthesized metal-organic framework peroxidase nanozyme has excellent catalytic activity, high stability and good reusability under extreme operating environments, providing new technology for biomedicine fields such as synthetic antibacterial preparations, colorimetric sensing and environmental governance.

[0018] The key to the metal organic framework peroxidase nanozyme with high catalytic activity at neutral or near-neutral pH constructed by the present invention is to regulate and construct an iron-based metal organic framework nanozyme with irregularly structured particle stacking. The present invention achieves regulation of the active center of the iron-based organic metal framework by optimizing the ratio of the synthetic solution and the molar ratio of iron ions to azole compounds, thereby improving the catalytic activity of the enzyme at neutral or near-neutral pH. The synthesis conditions are simple, controllable and reproducible. The metal organic framework peroxidase nanozyme synthesized by the present invention has high catalytic activity at neutral or near-neutral pH, and is more suitable for use in medical fields such as antibacterial treatment and colorimetric sensing and in environmental governance fields than existing peroxidases.

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

[0020] (1) The present 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 conditions 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 the present invention to exert peroxidase catalytic activity is 5-6.

[0022] (3) The preparation method of the metal organic framework peroxidase nanozyme provided by the present invention has low requirements for synthesis equipment, a simple synthesis method, controllable reaction conditions, and can achieve large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0026] Figure 4 A comparison of the catalytic activities and yields of FeMAF nanozymes synthesized at different solution volume ratios in Example 4 is shown.

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

[0028] Figure 6A comparison of the catalytic activities of FeMAF nanozymes synthesized with different azole compound ligands in Example 6 is shown.

[0029] Figure 7 The curve showing the change in relative absorbance of different FeMAF nanozymes over time in Example 7 is shown.

[0030] Figure 8 A graph showing the relationship between the catalytic activity of different FeMAFs synthesized in Example 8 and pH is shown.

[0031] Figure 9 Shown are the catalytic activities of FeMAF-25 and native horseradish peroxidase under different incubation temperature (a), organic solvent (b), incubation pH (c), and ionic strength (d) conditions.

[0032] Figure 10 A comparison of the catalytic activity of FeMAF nanozymes synthesized in a single solvent in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are not limited to the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention are equivalent replacement methods and are included in the scope of protection of the present invention.

[0034] The present invention is 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. Solution 2 was prepared by dissolving 1 mmol of benzotriazole in 24 mL of N,N-dimethylformamide. Solution 1 and Solution 2 were ultrasonically mixed in a glass bottle at a volume ratio of 1:3 to obtain a mixed solution. The resulting solution was then transferred to the inner liner of a reactor and heated at 140°C for 24 hours. After the reaction, the mixture was naturally cooled to room temperature, transferred to a centrifuge tube, washed three times with N,N-dimethylformamide and methanol, respectively, and centrifuged at 10,000 rpm for 3 minutes. The supernatant was removed and the solid was dried in a 60°C oven for 24 hours to obtain FeMAF-25 (the value 25 refers to the volume proportion of Solution 1 in the mixed solution as "25%").

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

[0038] Example 2

[0039] FeMAF-x (x refers to the volume ratio of solution 1 in the mixed solution) was synthesized according to the method of Example 1. In this example, five nanozymes, 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 a high-intensity diffraction peak and a crystalline structure. Figure 2 The Fourier transform infrared spectra of the five nanozymes are shown in b. -1 The characteristic peak of Fe-N at 3153 cm -1 ,1476cm -1 , and 1127cm -1 The vibrations of -NH, -N=N- and -CN- in benzotriazole confirmed the successful coordination of benzotriazole with iron ions.

[0041] Example 3

[0042] In order to study the effect of different types of iron-based element compounds on the catalytic activity of nanozymes, this example replaces ferrous chloride with ferrous sulfate, ferrous nitrate, ferrous acetate, and ferrous gluconate. 1mmol of the above iron salts were dissolved in 8mL of deionized water to obtain solution 1, and 1mmol of benzotriazole was dissolved in 24mL of N,N-dimethylformamide to obtain solution 2. Solution 1 and solution 2 were ultrasonically mixed in a glass bottle in a certain proportion to obtain a mixed solution, which was then transferred to the lining of the reactor and heated at 140°C for 24 hours. After the reaction was completed, the mixture was naturally cooled, centrifuged and washed, dried and collected to obtain FeMAF synthesized based on different types of iron salt compounds. Then, the catalytic performance of the nanozyme was verified in the TMB / H2O2 system.

[0043] Figure 3 The catalytic activity of FeMAF nanozymes synthesized based on different types of iron-based element compounds is shown. Different ferrous anions affect the catalytic activity of FeMAF through anion effects, solution pH regulation, redox stability and other pathways. Figure 3It can be seen that the FeMAF obtained with ferrous chloride as the precursor iron salt compound has the highest catalytic activity, followed by ferrous sulfate, ferrous nitrate, ferrous acetate and ferrous gluconate, so ferrous chloride is the best choice. This may be because the coordination ability of chloride ions is relatively weak, and it is easy to detach from the metal center during the synthesis process, and will not occupy too many metal coordination sites, thereby ensuring the effective coordination of organic ligands with Fe2+, forming a highly active catalytic structure, while the coordination of sulfate, nitrate, etc. will reduce the effective catalytic sites by delaying the nucleation rate. The steric hindrance of acetate and gluconate is large, which hinders the effective coordination of organic ligands with Fe2+, so its catalytic activity is relatively lower. In addition to iron salt compounds, nanozymes synthesized with other iron-based element compounds such as cobalt nitrate and nickel chloride as precursors also have certain peroxidase nanozyme activity. 2+ / Co 3+ or Ni 2+ / Ni 3+ The electron transfer pathway at the catalytic site activates H2O2 to produce reactive oxygen species, thereby exhibiting peroxidase nanozyme activity.

[0044] Example 4

[0045] To investigate the effects of different mixed solution ratios on the catalytic activity of the nanozyme, a series of volume ratios of the synthetic 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 synthetic solution ratios (volume ratio of solution 2: solution 1) were controlled to be 1:3, 1:2, 1:1, 2:1, and 3:1. The mixed solutions were transferred to the inner lining of a reactor and heated at 140°C for 24 hours. After the reaction, the mixture was naturally cooled, centrifuged, washed, and dried. The catalytic performance of the nanozyme was then verified in a TMB / H2O2 system.

[0046] Figure 4 The relative activity of FeMAF under different solution volume ratios is shown. As the proportion of deionized water in the mixed solution decreases, the catalytic activity and yield of the nanozyme also increase. When the volume ratio of solution 2: solution 1 is 3:1, the catalytic activity and yield of the nanozyme are the best. Compared with the volume ratio of solution 2: solution 1 = 1:3, its catalytic activity is increased by about 3 times. This performance improvement may be attributed to the regulatory effect of DMF's higher 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 center.

[0047] Example 5

[0048] To investigate the effects of varying metal ion to organic ligand molar ratios on the catalytic activity of the nanozyme, a range of metal ion to azole organic compound ligand ratios was investigated. The optimal catalytic activity was determined from Example 4, using the same synthesis method. Only the molar ratio of iron ion to azole compound ligand was varied to 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1. The resulting mixed solutions were then subjected to a TMB / H₂O₂ system to validate the nanozyme's catalytic performance.

[0049] from Figure 5 It can be seen that with the increase of the molar ratio of metal ions, the catalytic activity of nanozymes shows a trend of first increasing and then decreasing. 2+ The catalytic activity reached its peak when the molar ratio of metal ions to benzotriazole was 1:1, with a relative absorbance of 0.623. Further increasing the molar ratio of metal ions resulted in an excess of metal ions, preventing some metal sites from fully coordinating with organic ligands and making it difficult to form a catalytically active MOF material.

[0050] Example 6

[0051] To investigate the effects of different azole ligands on the catalytic activity of the nanozyme, the types of azole ligands listed above were varied. In this example, 2-methylimidazole, 2-ethylimidazole, benzimidazole, 5-benzotriazole carboxylate, and 1H-tetrazole were selected. The optimal metallic iron source, the optimal synthesis solution volume ratio, and the optimal synthesis raw material ratios described in Example 3 and Example 5 were selected. 1 mmol of ferrous chloride was dissolved in 8 mL of deionized water to obtain Solution 1. 1 mmol of each azole ligand was dissolved in 24 mL of N,N-dimethylformamide to obtain Solution 2. Solutions 1 and 2 were then mixed in a glass bottle at the optimal volume ratio (Solution 2:Solution 1 = 3:1), ultrasonically mixed, and transferred to the inner liner of a reactor. 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 with DMF and methanol each 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 the use of Fe 2+ The iron-based metal organic framework nanozymes formed by Fe ions as metal centers and different azole compound ligands all have peroxidase mimicking enzyme activity. 2+Ions coordinate with the N atoms in the azole compound ligands to form MOFs, but the catalytic reaction rate may vary due to differences in the ligand structure. The catalytic reaction rates are ranked from high to low as benzimidazole, 1H-tetrazole, 2-ethylimidazole, 2-methylimidazole, and 5-carboxylic acid benzotriazole. This is related to the electronic effect, steric hindrance, and coordination mode of the ligand. 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), thereby exhibiting a better reaction rate. The steric hindrance of 5-carboxylic acid benzotriazole is large, which hinders the effective coordination of the organic ligand with Fe2+, reduces the formation of catalytic sites, and thus its reaction rate is low.

[0053] Example 7

[0054] In order to evaluate the peroxidase mimetic activity of the nanozyme, the mimetic enzyme activity of FeMAF was determined by colorimetric analysis using TMB / H2O2 as a model substrate at room temperature. The specific experimental process is as follows: 12.02 mg of TMB was accurately weighed and dissolved in 2.0 mL of DMSO to obtain a 25 mM TMB working solution; 2.00 mg of FeMAF was ultrasonically dispersed in 4.0 mL of PBS buffer (50 mM, pH 6.0) to obtain a 500 μg / mL nanozyme dispersion; 20 μL of H2O2 stock solution (concentration of 10 mol / L) was taken with a pipette and dispersed in 1980 μL of deionized water to obtain a 100 mM H2O2 solution. An appropriate amount of PBS buffer was added to a 5 mL centrifuge tube in advance to make the final reaction volume 3.0 mL. 30 μL of TMB working solution was added first, followed by 90 μL of nanozyme dispersion, and finally 30 μL of H2O2 solution. The mixture was quickly mixed up and down, and then transferred to a quartz cuvette. The change in absorbance at 652 nm was monitored on a UV2500 UV-Vis spectrophotometer within 2 minutes.

[0055] Figure 7 The curves of the relative absorbance values ​​of different FeMAF nanozymes over time are shown. By comparing the change amplitude of the absorbance values ​​within the same time period, it can be seen that the relative absorbance value of FeMAF-25 is the highest per unit time, indicating that it has the best catalytic activity and the best substrate conversion ability. The catalytic activity of the remaining nanozymes decreases with the gradual increase of the water ratio in the organic solvent, in the order of FeMAF-33, FeMAF-50, FeMAF-66, and FeMAF-75. This is related to the solvent ratio of the synthesized nanozymes. The coordination environment of FeMAF-25 synthesized under low water ratio and high organic solvent conditions is more stable, and the low water ratio helps to reduce the Fe content in the catalytic site of the nanozyme. 2+ The hydrolysis or oxidation of FeMAF-75 may be due to the competition of water molecules for Fe coordination. 2+, which causes the structure of some MOFs to be unstable or even collapse, and even promotes the oxidation of Fe2+ to Fe3+, reducing the redox activity of Fe2+ / Fe3+, thereby weakening its peroxidase catalytic ability.

[0056] Example 8

[0057] In order to study the optimal catalytic pH of FeMAF nanozyme, according to the activity measurement method of Example 7, 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, and the group with the highest catalytic activity was set to 100%. The relative activity of FeMAF nanozyme at different catalytic pH was calculated.

[0058] Figure 8 The relative activities of different FeMAF nanozymes at different reaction pHs are shown. Among them, FeMAF-25 has the best catalytic activity at pH 6.0, while FeMAF-33, FeMAF-50, and FeMAF-66 have the best catalytic activity at pH 5.0, and FeMAF-75 has the best catalytic activity at pH 4.0. This may be related to the structure of the active site. The catalytic active site of FeMAF-25 synthesized under low water ratio and high organic solvent conditions is more stable, so it can still maintain high reducibility under neutral to acidic conditions (pH 6.0), ensuring an effective redox cycle between Fe2+ / Fe3+, and thus exhibiting the best activity under neutral to acidic conditions; while in FeMAF-75 synthesized under high water ratio and low organic solvent conditions, water promotes the oxidation of Fe2+ to Fe3+. Under acidic conditions (pH 4.0), high H+ concentration inhibits the accumulation of Fe3+ and maintains the redox cycle of Fe2+ / Fe3+, thus exhibiting the best catalytic activity under acidic conditions.

[0059] Example 9

[0060] In order to study the stability of nanozymes under different extreme conditions, the catalytic activity of nanozymes and natural horseradish peroxidase was investigated under different incubation pH, incubation temperature, organic solvent types and ionic strength.

[0061] 1. In terms of thermal stability, FeMAF-25 was placed in an oven for heat treatment for 2 hours, and the temperature was set to 20-80°C. Then, the pseudoenzyme 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 at different temperatures was calculated.

[0062] 2. In terms of solvent stability, FeMAF-25 was incubated in methanol, ethanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide and acetone for 8 hours (~5 mg / mL, 2 mL), and the precipitate was collected by centrifugation at 10000 rpm for 2 min. It was washed three times with deionized water and dried. The enzyme activity was determined according to the method of 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. In terms of pH tolerance, FeMAF-25 was incubated in pH 3.0-9.0 buffer (50 mM) for 2 hours, and the precipitate was collected by centrifugation at 10,000 rpm for 2 min. After washing and drying, the pseudoenzyme activity was determined according to the method of Example 6. The activity of the untreated sample was recorded as the relative activity of 100%, and the relative activity of the nanozyme after different pH treatments was calculated.

[0064] 4. In terms of ionic strength tolerance, FeMAF-25 was placed in 0-1000 mM sodium chloride solution, and the pseudoenzyme activity was determined according to the method of Example 6. The activity of the untreated sample was recorded as the relative activity of 100%.

[0065] Figure 9 The relative activities of nanozymes and natural enzymes under different treatment conditions are shown. Figure 9 It can be seen from a that in the range of 20-40°C, 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°C, 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 skeleton and the stability of the coordination bond make FeMAF-25 have satisfactory stability in various organic solvents, retaining more than 90% of the initial activity, while the natural enzyme only maintains less than 60% of its original activity in organic solvents. Figure 9 It can be seen from the c that FeMAF-25 nanozyme can still retain 80% of its initial activity in a strong acid environment of pH 3.0 and a strong alkaline environment of pH 9.0, while the natural enzyme only retains about 20% of its initial activity under acidic conditions. High ionic strength may affect the charge distribution and spatial structure of the nanozyme, which may easily lead to the loss of enzyme activity. Figure 9As shown in the d, the catalytic activities of both the FeMAF-25 nanozyme and the natural enzyme showed a downward trend in 300-1000mM NaCl solutions. At 1000mM NaCl, the nanozyme still retained approximately 80% of its initial activity, while the natural enzyme only retained 30%, indicating that FeMAF-25 has good stability against high ionic strength. In summary, the nanozyme exhibited higher catalytic activity than the natural enzyme at different temperatures, pH, organic solvents, and ionic strengths, indicating that the catalytic stability of the FeMAF-25 nanozyme is superior to that of the natural enzyme.

[0066] Comparative Example 1

[0067] Comparison of catalytic activity of MOFs synthesized in single solvents

[0068] According to 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 in Example 6, except that the mixed solvent was replaced with pure water or pure N,N-dimethylformamide. After synthesis, the mixture was collected by centrifugation and its catalytic activity was evaluated according to the activity measurement method in Example 7. Figure 10 As shown, the catalytic activity of the MOFs synthesized in a single solvent system of pure water or pure N,N-dimethylformamide is extremely low, less than 1% of that in a mixed solvent system (with a volume ratio of solution 1:solution 2 of 1:3 as a control), indicating 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 above examples only represent several embodiments of the present invention, and their descriptions are relatively specific and detailed, but their technical scope is not limited to the above 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 are all within the scope of protection of the present invention. Therefore, the scope of protection of the patent of this invention shall be based on the appended claims.

Claims

1. A metal-organic framework peroxidase nanozyme, characterized in that The peroxidase nanozyme is a metal-organic framework material composed of iron elements and azole compound ligands. The azole compound ligands in the peroxidase nanozyme are combined with metal ions in a coordinated form. It has peroxidase-like catalytic activity and the optimal pH is 5-6.

2. The method for preparing a metal organic framework peroxidase nanozyme according to claim 1, characterized in that: Using azole compounds as organic ligands, iron elements as metal centers, deionized water and organic solvents as solvents, the metal compound solution is dissolved in deionized water, and the organic ligand solution is dissolved in the organic solvent. The two solutions are then mixed at room temperature, reacted by solvothermal method, and coordinated assembly is performed to form a structurally stable metal-organic framework peroxidase nanozyme.

3. The preparation method according to claim 2, characterized in that The specific preparation steps are: dissolving the metal compound in deionized water to obtain solution 1, dissolving the azole compound in an organic solvent to obtain solution 2, mixing solution 1 and solution 2, and then transferring the mixture to the inner lining of a Teflon high-pressure reactor, ultrasonicating until completely dissolved, placing it in an oven for heating and reaction, and after the reaction is completed, naturally cooling to room temperature, centrifuging to obtain a powdered product, washing, and drying to obtain the metal-organic framework peroxidase nanozyme.

4. The preparation method according to claim 3, characterized in that The azole compound is selected from imidazole, 2-methylimidazole, 2-nitroimidazole, 2-bromo-1H-imidazole, ethyl imidazole-2-carboxylate, 2-mercaptoimidazole, 2-propylimidazole, 2-ethylimidazole, 2-butylimidazole, 1H-imidazole-4-carboxylic acid, 2-methyl-5-nitroimidazole, 1-octyl-3-methylimidazole bromide, imidazole-4,5-dicarboxylic acid, 4-phenylimidazole-5-amino-4-imidazolecarboxamide, methyl imidazole-4-carboxylate, 4-nitroimidazole, 4-(hydroxymethyl)imidazole, 4-chloroimidazole, 4-methyl-5-hydroxymethylimidazole, 4-imidazolecarboxaldehyde, ethyl 4-imidazolecarboxylate, 2-imidazolecarboxaldehyde, benzimidazole, 5-amino-2-methylbenzimidazole, 2-(methylthio)benzimidazole, 2-chloro- 4-Fluoro-(9CI)-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-carboxylate, 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, 1H-1,2,4-triazol-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-benzotriazol-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-carboxylic acid phenyl)-1H-tetrazole, 8-chlorotetrazo[1,5-a]pyridine, 5-propyl-1H-tetrazole, 6-bromotetrazo[1,5-a]pyridine5-a]pyridine, 3-(1H-tetrazolyl)benzaldehyde, 3-tetrazol-1-yl-phenol, 5-(3-methoxyphenyl)-1H-tetrazolyl, 2-methyl-5-(1H-tetrazol-5-yl)aniline, 5-(4-hydroxyphenyl)-1H-tetrazolyl, 5-(2-methylphenyl)-1H-tetrazolyl, 1-benzyl-1H-tetrazolyl-5-thiol, 3-phenyl-2-(1H-tetrazol-1-yl)propionic acid, 3-methoxy-5-(5-methyl-tetrazol-1-yl)-aniline, methyl 4-(2H-1,2,3,4-tetrazol-5-yl)benzoate, 5-(3-nitrophenyl)-2H-tetrazolyl, 1H-tetrazolyl-1-acetamide.

5. The preparation method according to claim 3, characterized in that The metal compound includes a compound containing at least one atom or ion of at least one iron-based element or a combination of multiple compounds; the iron-based element includes iron, cobalt, and nickel; and is 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 nickelous hydroxide.

6. The preparation method according to claim 3, characterized in that 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 mixed solution thereof.

7. The preparation method according to claim 3, characterized in that 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.

8. The preparation method according to claim 3, characterized in that Solution 1 and solution 2 are mixed evenly in a volume ratio of solvent 2 to solution 1 of 1:3 to 3:

1.

9. The preparation method according to claim 3, characterized in that The temperature of the heating reaction is 120-220° C., and the reaction time is 12-36 hours.

10. Application of the metal organic framework peroxidase nanozyme according to claim 1 in the synthesis of antibacterial preparations, colorimetric sensing and environmental management.

Images