An ultra-small size cerium-based metal-organic framework nanoscale enzyme and a preparation method and application thereof

Abstract

The application belongs to the field of nanobiotechnology, and discloses a super-small size cerium metal organic framework nanometer enzyme and a preparation method and application thereof. The nanometer enzyme is prepared by rapid self-assembly of cerium ions and azole ligands in an aqueous phase at room temperature. The catalyst is regulated by MOF nanoparticles, and the catalytic activity is enhanced. Compared with large size Ce-MOF, the super-small size (preferably 5-10 nm) Ce-MOF nanometer enzyme has more defect structures and dense active sites. Compared with natural enzymes, the Ce-MOF nanometer enzyme has the characteristics of simple synthesis, high yield, good stability, strong tolerance to extreme environments, high enzyme-like catalytic activity, and the like, and can complete the hydrolysis of organophosphorus pesticides, organic nerve toxic agents and phosphate biomolecules. The nanometer enzyme has high activity utilization rate in recycling, can adapt to various phosphate compounds, and provides an efficient catalyst for environmental treatment and biomedical treatment applications.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanobiotechnology, and particularly relates to a cerium-based metal-organic framework nanoenzyme with phosphate hydrolysis catalytic activity and size-adjusted activity, and a preparation method and application thereof. BACKGROUND

[0002] Enzymes are widely used, green and efficient biomacromolecular catalysts. The catalytic activity and specificity of enzymes are closely related to the structure of their active sites and the structure of the surrounding functional amino acid residues. Hydrolytic enzymes play an important role in environmental treatment, industrial production and scientific research. Phosphate ester compounds are a class of compounds with diverse functions and wide applications. Among them, organic phosphorus biomolecules containing phosphate bonds are involved in many important biological processes such as energy storage and conversion, cell division and growth, respiration and photosynthesis. In addition, organic phosphorus compounds are widely used in pesticides, petroleum additives and chemical reagents. In particular, organophosphorus trimers such as parathion, parathion and malathion are used as crop protection agents in agriculture, but they have potential toxicity, which can cause nerve shock, paralysis, respiratory failure and death. At present, enzyme-mediated dephosphorylation reaction is an important way to realize the inactivation and detection of harmful phosphorus chemicals. However, natural enzymes have poor resistance to external interference and cannot be reused, which limits their application range. Therefore, developing high-performance nano materials with enzyme-like activity to replace natural enzymes is an effective method to solve the dephosphorylation of organophosphorus compounds.

[0003] Metal-organic frameworks (MOFs) are hybrid porous coordination materials that exhibit astonishing performance due to their ultrahigh porosity, huge internal surface area, and high diversity and designability in structure and chemistry. These characteristics endow MOFs with a wide range of potential applications. Currently, the research of MOFs mainly focuses on the mixture of polydisperse crystals in multiple orders of magnitude. In recent years, MOF nanoparticles have attracted more attention. This is because at the nanoscale, materials can have size-dependent properties, such as high catalytic activity, accelerated adsorption / desorption kinetics, enhanced bioavailability, etc. With the decrease of particle size, the external surface area can be increased, while the internal surface area is retained, and the exposure of external active sites is enhanced. For example, it can be observed that with the decrease of particle size of ZIF-8 (J. Am. Chem. Soc. 2010, 132, 12365-12377) and MAF-4 (ACS Catal. 2011, 1, 120-127.), the catalytic activity of vegetable oil transesterification is significantly improved, because the acid-base sites are proved to be located on the external surface or defects. Therefore, it is expected that the size of MOF nanoparticles will be reduced to an extreme level, which can maximize the use of these active sites on both sides of the framework. The ultra-small size of MOFs makes the catalytic sites densely distributed, which improves the catalytic activity, which helps to solve the problem of low activity of nanoscale enzymes. Therefore, exploring a green and controllable way to synthesize ultra-small MOF nanoparticles with good dispersion and clear surface chemistry as catalysts is an effective way to improve the low catalytic activity of nanoscale enzymes.

[0004] The multifunctionality of metal nodes and organic ligands in MOFs can mimic the active center of natural enzymes, endowing MOFs with catalytic activity of mimetic enzymes. Inspired by the catalytic structure of natural metal enzymes, a metal-organic framework with phosphatase catalytic activity was assembled by biomimetic strategy. In nature, the active site of organophosphorus hydrolase (OPH) exists in the form of a Lewis acid with a hydrogen-oxygen bridged dinuclear Zn(II) center, which is connected to histidine, aspartate, and lysine residues. Through biomimetic strategy, a MOF-808 with similar Zr metal-Lewis acid catalytic site was synthesized, and MOF-808-NH2 was prepared by amine functionalization modification, which has better catalytic activity than non-functionalized nanoscale enzyme (J. Mater. Chem. A, 2023, 11, 13300-13308). In addition, a simple soaking procedure was developed to re-activate the active sites of spent nanoscale enzymes after catalysis of organophosphate using alkaline aqueous solution, which has excellent repeated use performance. Although this study improves the catalytic activity and repeated use performance of nanoscale enzymes, the synthesis method of the nanoscale enzyme requires a complex modification process to improve the catalytic activity, which is high in cost and complicated in operation process. SUMMARY

[0005] The application provides a method for preparing a cerium-based metal organic framework nanometer enzyme with a super-small size based on a biomimetic design.

[0006] Research finds that the activation active site of the Zn-OH Lewis acid of the catalytic center plays a key role in the hydrolysis of phosphate esters. The Ce-OH also has a Lewis acid effect. The application can advantageously utilize the structural advantages of MOFs to biomimetically construct an active center similar to a natural phosphate esterase, self-assemble MOFs with Ce as a metal center, and improve the hydrolysis catalytic efficiency by regulating the size of the MOF particles. Compared with nanometer enzymes prepared from traditional materials (such as metal oxides, carbon nanomaterials, noble metal nanoparticles, etc.), the nanometer enzyme obtained in the application is prepared according to a biomimetic strategy and has a clear chemical composition and structure, so that the catalytic mechanism can be more clearly studied, the size regulation is simple and feasible to enhance the catalytic activity, and the application range can be improved and a large amount of cost can be saved.

[0007] According to the application, different types of azole compound ligands can be used.

[0008] The azole compound ligand is an imidazole, a triazole, or a tetrazole.

[0009] The skilled person in the art understands that the active center in a phosphatase is composed of histidine and metal coordination, and azole compounds are analogues of histidine and all have imidazole groups. Azole compounds are stably coordinated with metals to assemble MOFs with catalytic activity.

[0010] The azole ligand in the application can realize defect coordination and expose more active sites under specific metal ion concentrations and synthesis methods. Therefore, selecting a suitable type of azole compound ligand and using a suitable synthesis method can efficiently construct a defective MOF.

[0011] According to the application, different cerium source compounds can be used as metal centers.

[0012] The cerium source compound is a metal salt, a metal oxide, and a metal complex of a combination of cerium ions and different anion groups. By changing the type of cerium source, the catalytic activity of the simulation product can be reasonably adjusted. Determining a suitable cerium source is beneficial to preparing an artificial enzyme with high catalytic performance. The catalytic activities of cerium metal centers with different valences are quite different. The catalytic activity of the nanometer enzyme is also highly affected by the anion of the metal salt, thereby affecting the crystal structure of the MOF. At the same time, the valence of the metal center with the same anion also produces a large activity difference.

[0013] According to the application, different synthesis solvents and synthesis methods can be used.

[0014] The different synthesis solvents greatly affect the crystallization degree of the MOF due to the difference in polarity. Different synthesis methods result in differences in nucleation and crystal growth.

[0015] Therefore, the application provides a preparation method of an ultra-small-size MOF nanozyme. The metal center catalytic site formed by coordination of a metal center and an azole ligand simulates a double-bridge zinc Lewis acid catalytic center of a phosphoric ester enzyme. The size is regulated by the mixing of a synthesis solvent and a synthesis method, and the defect exposes the active site. Due to the reduction in the particle size of the MOF nanozyme, the external surface area can be increased. While the internal surface area is retained, the exposure of the external active site is enhanced, and the dense catalytic sites are provided. Therefore, the synthesized ultra-small-size MOF-based nanohydrolytic enzyme has excellent catalytic activity, high stability under extreme operating environments, and good reusability, thereby providing new technologies for biomimetic catalysis under industrial scale conditions, environmental remediation, biological medicine treatment, and biosensors.

[0016] The key technical problem to be solved by the application for constructing the ultra-small-size nanozyme is to regulate and construct a cerium-based metal organic framework nanozyme with a defect structure. The application uses a green and environmentally friendly solvent at room temperature, and realizes the regulation of the size of the cerium-based metal organic framework in a very short time by optimizing the composition of the synthesis solvent and the molar ratio of cerium ions to azole ligands, thereby enhancing the exposure of the external active site and improving the catalytic activity. The green synthesis method at room temperature and the regulation of the ultra-small size can make up for the deficiencies in the prior art research.

[0017] Compared with the prior art, the application has the following advantages and beneficial effects:

[0018] (1) The synthesis method at room temperature can quickly form a metal organic framework, greatly saving energy consumption and synthesis time, and meeting the green chemistry concept.

[0019] (2) The application realizes the regulation of the size of the nanozyme and improves the catalytic activity by adjusting the complete process conditions and the synthesis process parameters.

[0020] (3) The ultra-small-size cerium-based metal organic framework nanozyme has good dispersity, clear surface chemistry, accelerated adsorption / desorption kinetics, enhanced exposure of the external active site, and can adapt to the hydrolysis of various organophosphorus compounds. DETAILED DESCRIPTION

[0021] Figure 1 The scanning electron microscope (SEM, a) and the transmission electron microscope (TEM, b) images of the surface morphology of Ce-MOF (aq) are shown.

[0022] Figure 2The X-ray diffraction (XRD) pattern of Ce-MOF(aq) is shown.

[0023] Figure 3 The Fourier transform infrared (FT-IR) spectrum of Ce-MOF(aq) is shown.

[0024] Figure 4 The X-ray diffraction (XRD) pattern of Ce-MOF(MeOH) is shown.

[0025] Figure 5 The surface morphology of Ce-MOF(MeOH) is shown in a scanning electron microscope (SEM) image.

[0026] Figure 6 The catalytic activity of MOF preparation with different molar ratios of 2-Hmim to Ce ions is compared.

[0027] Figure 7 X-ray diffraction (XRD) patterns of Ce-MOF(aq) at different synthesis times are shown.

[0028] Figure 8 The catalytic activity of Ce-MOF(MeOH) at different synthesis times is shown.

[0029] Figure 9 A comparison of the catalytic activities of Ce-MOF(aq) nanozymes prepared from cerium salts of different valence states is shown.

[0030] Figure 10 A comparison of the catalytic activities of Ce-MOF(aq) nanozymes prepared with different azole compound ligands is shown.

[0031] Figure 11 The catalytic activities of Ce-MOF nanozymes of different sizes are compared.

[0032] Figure 12 Scanning electron microscope (SEM) images of Ce-MOFs of different sizes are shown.

[0033] Figure 13 The catalytic activities of Ce-MOF nanozymes prepared with different organic ligands are compared. Detailed Implementation

[0034] The present invention provides a method of preparing ultra-small size metal organic framework nanoszymes with phosphonate hydrolytic activity. Green and mild catalytic conditions, along with fast and stable catalytic efficiency, make the MOF-based nanoszymes of the present invention very versatile as catalysts, expanding to the application of multiple classes of phosphonate compounds. The phosphonate compounds include p-nitrophenyl phosphate disodium, bis(p-nitrophenyl) phosphate, methyl parathion, ethyl parathion, methylthiophos, phorate, phosmet, chlorpyrifos, monocrotophos, adenosine triphosphate, adenosine diphosphate, cytidine-5'-triphosphate, cytidine-5'-diphosphate, 6-phosphogluconate, phosphoglycerate, pyridoxal-5'-phosphate, pyridoxamine-5'-phosphate, guanosine-5'-triphosphate, and the like.

[0035] The MOF-based nanohydrolytic enzymes of the present invention include at least one metal compound, at least one organic ligand.

[0036] As used herein, "metal compound" represents a chemical moiety containing at least one atom or ion in a cerium source compound, including trivalent or tetravalent cerium ions. Thus, the expression of metal compound includes, for example, salts, oxides, and complexes.

[0037] Suitable metal source compounds forming part of the MOF-based nanohydrolytic enzymes can be selected from metal salts of cerium ions in combination with different anion groups, metal oxides, and metal complexes. The cerium compounds can be selected from, but not limited to, cerium sulfate, cerium oxide, cerium nitrate, cerium ammonium nitrate, cerium carbonate, cerium chloride, cerium ammonium sulfate, cerium acetate, cerium oxalate, cerium hydroxide, cerium phosphate, cerium triflate, cerium sulfate, cerium perchlorate, and the like.

[0038] Suitable azole ligands for the present invention are selected from, but not limited to, imidazole, 2-methylimidazole, 2-nitroimidazole, 2-bromo-lH-imidazole, imidazole-2-carboxylic acid ethyl ester, 2-mercapto-imidazole, 2-propylimidazole, 2-ethylimidazole, 2-butylimidazole, lH-imidazole-4-carboxylic acid, 2-methyl-5-nitroimidazole, 1-octyl-3-methylimidazole bromide, imidazole-4,5-dicarboxylic acid, 4-phenylimidazole 5-amino-4-imidazole formamide, 4-nitroimidazole, 4-(hydroxymethyl)imidazole, 4-chloroimidazole, 4-methyl-5-hydroxymethylimidazole, 4-imidazole carboxaldehyde, 4-imidazole carboxylic acid ethyl ester, 2-imidazole carboxaldehyde, lH-tetrazole, lH-tetrazole-5-acetic acid, 5-(4-pyridyl)-lH-tetrazole, 5-(3-pyridyl)-lH-tetrazole, lH-tetrazole-5-acetic acid ethyl ester, l-methyl-lH-tetrazole, 5-(ethylthio)-lH-tetrazole, 5-(4-nitrophenyl)-lH-tetrazole, 5-(2-pyridyl)-lH-tetrazole, 5-chloromethyl-lH-tetrazole, 5-(4-carboxyphenyl)-lH-tetrazole, 8-chlorotetrazolo[l,5-A]pyridine, 5-propyl-lH-tetrazole.

[0039] In some embodiments, solvents that can be used for synthesizing MOFs include at least one of N,N-dimethylformamide, N,N-dimethylacetamide, water, methanol, ethanol, diethylformamide, toluene, chlorobenzene, or a mixed solution thereof. The synthesis method uses a solvothermal method, a hydrothermal method, or a diffusion method to prepare MOF nanoscale enzymes, and a single solvent or a mixed solvent can be used as the solvent as long as the MOF can be formed, and there is no limitation on the type of solvent.

[0040] Synthesis at room temperature is mainly green and simple, and water or methanol, ethanol is usually used as a solvent to quickly synthesize MOF. In the hydrothermal or solvothermal synthesis method, the solvent plays a crucial role. The effect of the solvent on the structure of the metal-organic framework compound mainly manifests in the solubility of the reactants in the solvent, the polarity of the solvent itself, the coordination ability of the solvent, and the template effect.

[0041] In some embodiments, at 25°C, water, methanol, and DMF are used as solvents to form MOFs of different sizes and different types. The reaction basically occurs immediately, the metal salt solution is mixed with the organic ligand, and a light yellow precipitate is immediately formed. Prolonging the synthesis time, the MOF tends to develop integrity.

[0042] In some embodiments, the metal-organic framework synthesized according to the method of the present application is synthesized by a solvent method to synthesize metal-organic frameworks of different scales. According to the type of metal-organic framework precursor used, a suitable solvent and synthesis temperature are selected. Therefore, according to the XRD spectrum analysis of the synthesized metal-organic framework, the approximate size of the metal-organic framework particles is determined according to the SEM and TEM maps.

[0043] In some embodiments, cerium-based metal-organic frameworks are used as nanoscale enzymes to realize the active center of the simulated natural phosphatase. Since the active center of the phosphatase is the imidazole of histidine and the carboxylate of aspartic acid and lysine based on the double-bridge zinc coordination to generate a stable coordination structure. Its activity mainly benefits from the Lewis acid site of Zn-OH catalyzing the hydrolysis of phosphoric acid ester compounds, and the coordination of azole compounds with cerium can also produce high catalytic activity Ce-OH Lewis site, which exactly simulates the active structure of phosphoric acid esterase. In addition, Ce-MOF has strong tolerance to harsh environments and high stability, and most importantly, it can still maintain excellent catalytic activity when recycled multiple times in an aqueous reaction system. The artificial enzyme prepared by the present application can be a cerium imidazole framework, a cerium triazole framework, and a cerium tetrazole framework, the ligand can be imidazole, triazole, and tetrazole, and the size can be adjusted in different ways to improve the catalytic activity.

[0044] In some embodiments, MOF nanohydrolyases are prepared using azole compounds and coordination modes of cerium. The concentration of the cerium salt and azole compound ligand used can include the following ranges: cerium salt between about 0.5 mmol to 1 mmol; azole compound between about 1.0 mmol to 6.0 mmol. The synthesis volume of the cerium salt is between about 0.1 to 10 mL; the volume of the azole compound is between about 0.1 to 10 mL.

[0045] In some embodiments, the size of the MOF particles is precisely controlled in the range of 5-10 nm by controlling the synthesis conditions, so that the external surface area is increased, the exposure of the external active sites is enhanced, the catalytic sites are dense, and thus the catalytic activity of the MOF nanohydrolyase is greatly improved.

[0046] The ratio of different MOF precursors has a direct impact on the crystallinity and yield of the MOF, and due to the difference in coordination ability of different types, the impact brought by different ratios of precursors is the content of active centers, leading to differences in the catalytic activity of the MOF nanohydrolyase.

[0047] Example 1

[0048] Synthesis of Ce-MOF(aq), including the following operation steps:

[0049] Take 1 mL of 2-methylimidazole (2.5 mmol) and uniformly drop it into a glass reaction bottle containing 10 mL of cerium ammonium nitrate (0.6 mmol), and stir at 500 rpm for 5 h. Then centrifuge the mixture to remove the supernatant, then wash with deionized water three times, centrifuge to collect the precipitate, and dry at 60°C under vacuum. Used for subsequent hydrolysis of phosphate ester compounds.

[0050] The crystal properties of Ce-MOF(aq) were verified by SEM, TEM, PXRD and FT-IR measurements, and the MOF particle size was confirmed, as shown in Figures 1-3 From Figure 1 it can be seen that the scanning electron microscope image of Ce-MOF(aq) shows uniform spherical particles, and the transmission electron microscope image shows that the nanoparticle diameter is about 5 nm, with obvious lattice fringes. As shown in Figure 2 , the XRD pattern of Ce-MOF(aq) shows sharp characteristic peaks at 28.5°, 47.2°, and 55.9°, indicating that Ce-MOF(aq) has a good crystal structure. From Figure 3 the FT-IR spectrum of Ce-MOF(aq) can be seen that the peak value of C=N vibration of 2-methylimidazole synthesized at 1677 cm -1 disappears, while peaks at 1042 cm -1 and 912 cm -1The appearance of new peaks between 530 and 490 cm -1 The band at 650 cm"1originates from Ce-N stretching vibrations, confirming the successful synthesis of Ce-MOF(aq).

[0051] Example 2

[0052] Synthesis of Ce-MOF(MeOH) including the following steps:

[0053] To synthesize Ce-MOF with larger size at room temperature, the solvent was changed from water to methanol. 1 mL of 2-methylimidazole (2.5 mmol) methanol solution was dropped evenly into a glass reaction bottle containing 10 mL of cerium ammonium nitrate (0.6 mmol) methanol solution, which was stirred at 500 rpm for 5 h. The light yellow powder was collected by centrifugation and washed with deionized water for three times. The collected precipitate was dried at 60 °C under vacuum for 12 h and was labeled as Ce-MOF(MeOH). The collected powder was used to evaluate the hydrolysis activity of p-nitrophenol disodium phosphate salt.

[0054] After changing the solvent, the powder XRD was used to confirm that the formed crystals have similar peaks to Ce-MOF(MeOH), as shown in Figure 2. Figure 4 In addition, SEM was used to confirm that the Ce-MOF(MeOH) synthesized at room temperature in methanol solution is a larger size cubic-shaped MOF particle with a single particle size of about 600-1000 nm, as shown in Figure 3. Figure 5

[0055] Example 3

[0056] To study the effect of the molar ratio of different metal salts to azole ligands on the catalytic activity. By adjusting the synthesis ratio of 2-methylimidazole aqueous solution and cerium ammonium nitrate aqueous solution under the synthesis conditions of Ce-MOF(aq), the catalytic activity of the products with the molar ratio of 2-methylimidazole to cerium nitrate of 5:1, 25:6, 10:3, 5:2, 5:3, 5:6, 5:12, and 5:14 was studied, as shown in Figure 4. From the figure, it can be seen that as the molar amount of cerium ions increases, the catalytic activity of the synthesized Ce-MOF(aq) also shows a clear upward trend. When the molar ratio of 2-methylimidazole to cerium ions is 5:12, the catalytic activity reaches the best state, however, continuing to increase the molar amount of cerium ions can obtain a small degree of catalytic activity improvement. Continuing to increase the concentration of cerium salt will lead to the failure to form MOF material, and too high concentration of metal salt will destroy the skeleton structure, so that the metal ions and ligands cannot react to generate MOF. Figure 6 Example 4

[0057]

[0058] ​​To optimize the catalytic activity of the nanohydrolyase, the effect of different synthesis time on the catalytic activity was studied. The group with the best catalytic activity in Example 3 was selected, and the synthesis process was consistent, only the synthesis time was changed, and the synthesis time was set to 15 min, 30 min, 60 min, 120 min, 240 min, and 720 min. The obtained powder was evaluated for its hydrolysis catalytic activity by hydrolyzing the model substrate p-nitrophenol disodium phosphate.

[0059] The crystal characteristics of the nanohydrolyase with different synthesis times were characterized by powder XRD, Figure 7 showed that the crystal forms of different synthesis times were basically consistent, indicating that changing the synthesis time could maintain the structure of the crystal, but there were certain differences in the catalytic activity. Figure 8 It can be seen that the catalytic activity of the nanohydrolyase was significantly improved when the synthesis time increased from 15 min to 30 min, and the catalytic activity improved less when the synthesis time continued to increase. This is because in the synthesis process, cerium ions are coordinated with water to form Ce-H2O, and as the synthesis time increases, the unsaturated sites are basically occupied, and prolonging the reaction time will not cause a large change in the catalytic activity.

[0060] Example 5

[0061] To explore the effect of different valence states of cerium salt on the catalytic activity of MOF nanohydrolyase, different sources of cerium salt, cerium oxide, and cerium hydroxide or cerium complex were dissolved in water. The cerium source compounds used in this example include cerium sulfate, cerium ammonium nitrate, cerium ammonium sulfate, cerium nitrate, cerium chloride, and cerium acetate. Each cerium source compound was subjected to an independent experiment, and 2-methylimidazole was used as the organic ligand. A total of 6 experiments were conducted. After centrifugation, the solid was collected and washed with deionized water 3 times, and the light yellow solid (tetravalent cerium) or light white solid (trivalent cerium) was collected and dried at 60°C under vacuum. The hydrolysis catalytic activity was evaluated by hydrolyzing the model substrate p-nitrophenol disodium phosphate. Figure 9 It can be seen that compared with trivalent cerium metal salts such as cerium nitrate, cerium chloride, and cerium acetate, the cerium metal organic frameworks prepared from tetravalent cerium-containing metal salts such as cerium ammonium nitrate, cerium sulfate, and cerium ammonium sulfate all exhibit very excellent catalytic activity. Different chemical valence of Ce makes water and hydrolysis substrate preferentially bind. 4+ ions can activate Lewis acids, and there are a large number of Ce 3+The Ce-MOF selectively adsorbs water molecules to form a Ce-H2O material. In a typical catalytic process, in an alkaline environment, the Ce-H2O is activated to form a Lewis acid active site with strong affinity of Ce-OH, and then the nucleophilic oxygen performs an effective nucleophilic attack on the phosphorus center of the substrate, resulting in the rupture of the P-O bond and the release of the hydrolysis product. Due to the lack of activation of tetravalent cerium, its catalytic activity is relatively low. By properly adjusting the ratio of Ce(III) / Ce(IV) in the cerium-based metal organic framework, the catalytic activity can be greatly improved.

[0062] Example 6

[0063] In order to study the effect of different azole ligands on the catalytic activity of MOF nanoscale enzymes, the Ce-MOF nanoscale enzyme with the best activity in Example 4 was selected, and the azole compound ligand was replaced to prepare Ce-X, X representing the azole compound ligand. The best cerium source in Example 5, the best synthesis time in Example 4, and the best synthesis ratio in Example 3 were selected. 1 mL of azole compound (2.5 mmol) aqueous solution (which can assist in dissolving) and 10 mL of cerium salt (0.6 mmol) aqueous solution were mixed under continuous stirring, and the precipitate was collected after 1 h of reaction. Washed with deionized water 3 times, centrifuged to collect the solid, and used for subsequent catalytic activity evaluation. The hydrolysis catalytic activity was evaluated by hydrolyzing the model substrate p-nitrophenyl phosphate disodium salt. From Figure 10 It can be seen that the cerium-based metal organic framework nanoscale enzyme formed by using Ce(IV) ions as metal centers and different azole ligands has certain phosphoric ester hydrolysis activity, and the imidazole derivative ligands selected by the present application all exhibit good catalytic activity.

[0064] Comparative Example 1

[0065] Comparison of catalytic activities of Ce-MOF nanoscale enzymes with different sizes.

[0066] Three Ce-MOF nanozymes of different sizes were prepared according to the standard procedure described herein. 1 mL of 2-methyl-imidazole aqueous solution (2.5 mmol) was added dropwise to an aqueous solution containing 10 mL of cerium ammonium nitrate (0.6 mmol). The reaction was carried out with continuous stirring at room temperature for 1 h. The precipitate was collected to obtain Ce-MOF(aq) with a particle size of 3–10 nm. 1 mL of 2-methyl-imidazole methanol solution (2.5 mmol) was added dropwise to a methanol solution containing 10 mL of cerium ammonium nitrate (0.6 mmol). The reaction was carried out with continuous stirring at room temperature for 1 h. The precipitate was collected to obtain Ce-MOF(MeOH) with an average particle size of 800–1000 nm. 1 mL of 2-methyl-imidazole (1 mmol) DMF solution was added dropwise to a DMF solution containing 10 mL of cerium ammonium nitrate (0.6 mmol), mixed thoroughly, and reacted at 100 °C for 12 h. The precipitate was collected to obtain Ce-MOF (DMF) with a particle size of 50–100 nm. 150 μg of Ce-MOF nanozyme was added to 2 mL of CHES solution (pH 9.0) containing 0.5 mM disodium p-nitrophenol phosphate, and reacted at 25 °C for different times (single experiments were conducted at 0, 10, 20, 30, and 40 min). The concentration of p-nitrophenol in the product was assessed and compared using UV-Vis spectroscopy. Figure 11 As shown, ultra-small Ce-MOF(aq) (5–10 nm) exhibits the best catalytic activity, while large-sized Ce-MOF(MeOH) (800–1000 nm) shows lower catalytic activity compared to smaller Ce-MOFs. Ce-MOF(DMF) with a size of around 50–100 nm also exhibits a certain degree of catalytic activity. Compared to existing technologies, its catalytic activity can be improved through functional modification, etching, and other methods. Ce-MOF(aq) has an initial hydrolysis rate 3–15 times higher than other sizes, which confirms that controlling the size of MOF nanozymes can enhance hydrolysis efficiency. Reducing the particle size increases the external surface area, enhancing the exposure of external active sites while retaining the internal surface area. Simultaneously, the ultra-small metal-organic framework accelerates adsorption / desorption kinetics, enhances substrate utilization, and the ultra-small nanoparticles may maximize the utilization of these active sites on both sides of the framework, resulting in a dense distribution of catalytic sites and improved catalytic activity. Figure 12The scanning electron microscope pictures show that the average size of Ce-MOF(aq) is 5-10 nm spherical particles, the average size of Ce-MOF(DMF) is 50-100 nm spherical particles, and the average size of Ce-MOF(MeOH) is 800-1000 nm cubic morphology. The polarity of the solvent is one of the key factors to regulate the size of Ce-MOFs. The order of the polarity of the three solvents for synthesizing Ce-MOFs is water > DMF > methanol. According to the SEM characterization results, under the same metal and ligand molar ratio, the greater the polarity of the synthesis solvent, the smaller the size of the prepared Ce-MOFs. The small-sized Ce-MOFs have dense catalytic sites and can more quickly adsorb substrates, thus having better catalytic activity.

[0067] Comparative Example 2

[0068] Comparison of catalytic activity by changing organic ligands

[0069] According to the standard procedure of Ce-MOF(aq) described herein, Ce-X MOF nanoszymes were prepared by changing the organic ligands, and the catalytic activity was compared. Sodium terephthalate (BDC), sodium benzenetricarboxylate (BTC), sodium glutamate (Gly), and cysteine (Cys) were selected as four kinds of organic ligands that can easily form metal-organic frameworks in aqueous solution at room temperature. The synthesis conditions of Ce-BTC MOF were as follows: 1 mL of sodium benzenetricarboxylate (0.5 mmol) and 10 mL of cerium ammonium nitrate solution (0.6 mmol) were uniformly mixed, and the reaction was continuously stirred for 1 h to obtain a solid precipitate, which was named Ce-BTC. The synthesis methods of Ce-BDC MOF and Ce-Gly MOF were consistent with Ce-BTC, except that the ligands were replaced by sodium terephthalate and monosodium glutamate. The synthesis of Ce-Cys MOF was basically consistent with the above synthesis method, and cysteine was dissolved in 1 M sodium hydroxide solution to assist dissolution. 150 μg of Ce-X nanoszyme was added to 2 mL of CHES solution containing 0.5 mM of p-nitrophenol disodium phosphate salt (pH 9.0) at 25°C, and the reaction was carried out for 5 min. The catalytic activity was evaluated by comparing the concentration of the product p-nitrophenol using a UV-Vis spectrometer. As shown in Figure 13 , the catalytic activity of the nanoszyme synthesized by the carboxylic acid ligand was lower than that of the nanoszyme synthesized according to the biomimetic strategy, and Ce-Cys synthesized by Cys with a reducing group exhibited very low catalytic activity, which also confirmed that the biomimetic design of MOF nanoszyme can well simulate the catalytic activity of natural enzymes.

[0070] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but the 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 application, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. The application of an ultra-small cerium-based metal-organic framework nanozyme with phosphatase-like catalytic activity in the hydrolysis of phosphate ester compounds, characterized in that, The ultra-small cerium-based metal-organic framework nanozyme is a porous nanomaterial containing azole ligands and tetravalent cerium ions that self-assemble, wherein cerium ions and azole ligands are combined in a coordination manner, and the nanozyme has a size of 5~10 nm. The preparation method of the ultra-small cerium-based metal-organic framework nanozyme includes: using azole ligands and cerium source compounds as raw materials, cerium ions are self-assembled with azole ligands under solution conditions to form an ultra-small cerium-based metal-organic framework nanozyme with a stable framework structure; the cerium source compound is selected from at least one of cerium sulfate, cerium oxide, cerium ammonium nitrate, and cerium ammonium sulfate; the solvent used is water.

2. The application according to claim 1, characterized in that, The azole ligands 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, 4-nitroimidazolium, 4-(hydroxymethyl)imidazolium, 4-chloroimidazolium, 4-methyl-5-hydroxymethylimidazolium, 4-imidazolium carboxaldehyde, and 4-imidazolium carboxylic acid. Ethyl ester, 2-imidazolium carboxaldehyde, 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.

3. The application according to claim 1, characterized in that, The molar ratio of azole ligands to cerium source compounds is 5:1 to 5:

14.

4. The application according to claim 1, characterized in that, The synthesis temperature of the ultra-small cerium-based metal-organic framework nanozymes is room temperature, and the synthesis time is 15-30 min.

5. The application according to claim 1, characterized in that, The synthesis process of the ultra-small cerium-based metal-organic framework nanoenzyme is as follows: first, a cerium source compound solution is added to a reaction flask, and then an azole ligand solution is added dropwise. The entire reaction process is carried out with magnetic stirring at a speed of 500 rpm to ensure uniform particle formation.

6. The application according to claim 1, characterized in that, The phosphate compound is selected from at least one of the following: disodium p-nitrophenolate phosphate, bis(p-nitrophenyl) phosphate, methyl parathion, ethyl parathion, methyl parathion, phorate, aniline thiophanate, malathion, chlorpyrifos, phorate, adenine nucleoside triphosphate, adenine nucleoside diphosphate, cytidine-5'-triphosphate, cytidine-5'-diphosphate, glucose-6-phosphate, glycerol phosphate, pyridoxal-5'-phosphate, pyridoxamine-5'-phosphate, and guanosine-5'-triphosphate.

Images