Preparation method and application of phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity

Abstract

The invention discloses a preparation method and application of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity, belonging to the field of nanozyme functional materials. The composite nanozyme is simply and efficiently prepared by a two-step method of preparing a bimetallic organic framework precursor, mixing and grinding it with an equal mass of tributyl phosphorotrithioate, and calcining it. The composite nanozyme is used as a peroxidase-like catalyst, a haloperoxidase-like catalyst, and a glutathione peroxidase-like catalyst for sterilization or colorimetric sensing of H2O2 by using a TMB color reaction. The invention solves the problems of few types of biological-like activities, low catalytic activity, and poor enzymatic catalytic effect at low temperature in the existing nanozymes, and can be applied to fields such as colorimetric sensing, medical dressings, bionic systems, and advanced functional material coatings.

Description

Technical Field

[0001] The present invention belongs to the field of nanozyme functional materials, and relates to a preparation method and application of an antibacterial nanozyme, specifically a preparation method and application of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activities. Background Art

[0002] Nanozymes are a class of nanomaterials with enzyme-like catalytic activities developed inspired by natural enzymes and traditional artificial enzymes. Compared with natural enzymes, nanozymes not only have advantages such as low cost, high stability, and easy large-scale production, but also the unique physical and chemical properties of nanomaterials endow nanozymes with multiple functions, providing more possibilities for the design, development, and future applications of nanozymes. Metal-organic frameworks (MOFs) are coordination polymers assembled from organic ligands and transition metal ions, which have advantages such as multi-porosity, large specific surface area, and many metal sites. With the in-depth research, many MOFs have also been found to have the characteristics of nanozymes, making them have more application values and being widely used in the field of nanozymes.

[0003] At present, the enzyme-like activities of most MOF nanozymes mainly focus on peroxidase-like activity. The Chinese patent application with publication number CN 115227719A discloses a calcium-phosphorus nanozyme with excellent peroxidase activity, which is prepared by uniformly mixing a calcium source, a phosphorus source, urea, and a surfactant, followed by suction filtration, rinsing, drying, and grinding. This nanozyme only has peroxidase activity, and the preparation process is complex; the Chinese patent application with publication number CN116003818A discloses a method for preparing a functionalized multi-metal organic framework nanozyme and its application in peroxidase activity, which is to synthesize a functionalized multi-metal organic framework nanozyme by using solvent thermal method in N,N-dimethylformamide solution with aminoterephthalic acid, nickel chloride hexahydrate, and ferric chloride hexahydrate. This nanozyme only has peroxidase activity; the Chinese patent with publication number CN115368579B discloses a preparation method and application of a manganese porphyrin-based metal organic framework nanozyme, which is a nano material with superoxide dismutase-like and catalase activities synthesized by using copper nitrate trihydrate, meso-tetrakis(4-carboxyphenyl) porphine, and manganese chloride through two steps.

[0004] In summary, at present, the types of bio-like activities of metal organic framework nanozymes are few, mainly concentrated in peroxidase activity, with low catalytic activity, limited by temperature, poor enzyme catalytic effect at low temperature, and the preparation methods are cumbersome. Summary of the Invention

[0005] To address the above deficiencies in the existing technology, the present invention aims to provide a preparation method and application of a phosphorus and sulfur-doped bimetallic MOF composite nanozyme with multi-enzyme activity. The method involves doping phosphorus and sulfur elements into the prepared MOF precursor to obtain a phosphorus and sulfur-doped bimetallic MOF composite nanozyme with multi-enzyme activity. This composite nanozyme exhibits three enzyme activities: peroxidase-like, haloperoxidase-like, and glutathione peroxidase-like. At the same time, it can achieve the goals of simple preparation process, high catalytic activity, unchanged catalytic effect of the enzyme at low temperature, high bactericidal rate, and H2O2 colorimetric sensing.

[0006] To achieve the above objectives, the technical solutions adopted in the present invention are as follows:

[0007] A preparation method of a phosphorus and sulfur-doped bimetallic MOF composite nanozyme with multi-enzyme activity, comprising the following steps carried out in sequence:

[0008] S1. Preparation of MOF precursor

[0009] Dissolve polyvinylpyrrolidone (PVP), bimetallic raw material A, and bimetallic raw material B completely in anhydrous methanol to obtain solution I;

[0010] Dissolve 2-methylimidazole completely in anhydrous methanol to obtain solution II;

[0011] After the first stirring of solution I, add it to solution II, carry out the second stirring, add an alkali solution dropwise, and after the third stirring, centrifuge and dry to obtain the MOF precursor;

[0012] Among them, the bimetallic raw material A is zinc nitrate;

[0013] The bimetallic raw material B includes ferric nitrate, cerium nitrate, cobalt nitrate, or copper nitrate;

[0014] The drying method is freeze-drying;

[0015] After forming solution I and solution II, adding solution I to solution II is because metal ions serve as the core of MOF and link with organic ligands to form MOF, which is beneficial for nucleation. Therefore, the substances cannot be directly added to anhydrous methanol for mixing; stirring is to accelerate the formation rate of MOF;

[0016] S2. Preparation of a phosphorus and sulfur-doped bimetallic MOF composite nanozyme with multi-enzyme activity

[0017] Mix and grind the MOF precursor and an equal mass of n-butylphosphorothioic triamide (NBPT), and calcine it in an inert gas atmosphere to obtain a phosphorus and sulfur-doped bimetallic MOF composite nanozyme with multi-enzyme activity, labeled as ZFPS.

[0018] Among them, NBPT is a doping reagent for doping phosphorus and sulfur elements at one time;

[0019] The mass of both the MOF precursor and NBPT is 0.1 - 1 g;

[0020] The inert gas is nitrogen or argon;

[0021] The calcination temperature is 650 - 1000 °C, and the calcination time is 0.5 - 4 h.

[0022] As a limitation of the present invention, the average molar ratio of the bimetallic raw material A, bimetallic raw material B, and polyvinylpyrrolidone is 1:0.1 - 9:0.01 - 0.05. PVPK30 has several macromolecular chains with an average molar mass of 40,000, so it is the average molar ratio.

[0023] As the second limitation of the present invention, in step S1, the content of 2-methylimidazole is 0.4 - 2.5 g.

[0024] As the third limitation of the present invention, in step S1, the alkali solution includes NaOH solution, KOH solution, or ammonia water.

[0025] As the fourth limitation of the present invention, in step S1, the molar concentration of the alkali solution is 1 - 10 mol / L, and the dropping amount is 1 - 20 ml.

[0026] As the fifth limitation of the present invention, in step S1, the molar volume ratio of solution I, solution II, and the alkali solution is 1:4:2 - 6.

[0027] As the sixth limitation of the present invention, in step S1, the time of the first stirring and the second stirring is both 10 - 180 min, and the time of the third stirring is 15 - 24 h.

[0028] The present invention also provides an application of a phosphorus and sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity. The phosphorus and sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity is used as a peroxidase-like catalyst, a haloperoxidase-like catalyst, and a glutathione peroxidase-like catalyst for sterilization or colorimetric sensing of H2O2.

[0029] The principle of the present invention is:

[0030] The phosphorus and sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity prepared by the present invention has three enzyme activities of peroxidase-like, haloperoxidase-like, and glutathione peroxidase-like, and can achieve the effect of efficient sterilization. Among them, the doped phosphorus and sulfur elements provide electron donation for zinc ions and iron ions, so that more zinc ions and iron ions are retained in the MOF carbon framework to generate Fe4P6N 12S, FeP, and ZnS. This enables the prepared ZFPS to provide more catalytic sites for various enzyme-like substances, such as peroxidase-like, haloperoxidase-like, and glutathione peroxidase-like, thus enhancing the catalytic activity. Among them, Fe4P6N 12 There is a P-Fe-S bond between S and FeP, which provides a fast channel for electron transfer, thereby enhancing the enzyme-like activity; at the same time, it reduces the temperature required for mimetic enzyme catalysis.

[0031] The catalytic principles of peroxidase-like, haloperoxidase-like, and glutathione peroxidase-like are as follows:

[0032] Peroxidase-like: H2O2 + ZFPS → O 2-

[0033] Haloperoxidase-like: H2O2 + ZFPS + Cl - → HClO

[0034] Glutathione peroxidase-like: H2O2 + ZFPS → GSSG + 2H +

[0035] Peroxidase-like can efficiently and rapidly catalyze low-concentration H2O2, thereby generating a large amount of reactive oxygen species (ROS). The generated ROS can efficiently and rapidly damage cell membranes, proteins, etc., thus killing bacteria; at the same time, peroxidase-like can catalyze H2O2 and halide ions to produce hypohalous acid, which can more efficiently damage cell membranes and bacterial walls; glutathione peroxidase-like can consume glutathione, block the internal nutrient supply of bacteria, and cooperate in sterilization. At the same time, Zn + is trapped within the MOF carbon framework. Zn + has a bactericidal effect. Therefore, ZFPS also has an ion bactericidal effect.

[0036] Due to the peroxidase-like activity of ZFPS, it can catalyze hydrogen peroxide to produce ROS, thereby oxidizing colorless TMB into blue oxidized TMB, thus achieving the purpose of colorimetric sensing.

[0037] Due to the adoption of the above technical solution, compared with the prior art, the beneficial effects obtained by the present invention are:

[0038] (1) The ZFPS prepared by the present invention has three enzyme activities: peroxidase-like, haloperoxidase-like, and glutathione peroxidase-like;

[0039] (2) The ZFPS prepared by the present invention is doped with phosphorus and sulfur elements, which significantly enhances the three enzyme-like activities, and solves the problem of insufficient catalytic activity sites in the prior art resulting in low catalytic activity;

[0040] (3) The catalytic activity of the ZFPS enzyme prepared by the present invention is less restricted by temperature and can achieve the same enzyme catalytic effect even under low temperature conditions;

[0041] (4) The ZFPS prepared by the present invention can achieve the purpose of efficient and rapid sterilization with less ZFPS. When the concentration of the ZFPS aqueous solution is as low as 10 μg / ml, the sterilization rates against Escherichia coli and Staphylococcus aureus reach 99.5%;

[0042] (5) The ZFPS prepared by the present invention has an increased affinity for H2O2. Through the TMB color reaction, it can visually detect low concentrations of H2O2, achieving the purpose of rapid detection;

[0043] (6) The preparation method of the present invention is simple, and ZFPS is simply and efficiently prepared by a one-pot method of high-temperature calcination. Description of the Drawings

[0044] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0045] Figure 1 are high-magnification scanning electron microscope images of the MOF precursor, ZFPS-1, and ZFNPS prepared in Example 1. Among them, Figure 1 a is the high-magnification scanning electron microscope image of the MOF precursor, Figure 1 b is the high-magnification scanning electron microscope image of ZFPS-1, Figure 1 c is the high-magnification scanning electron microscope image of ZFNPS;

[0046] Figure 2 is the X-ray diffraction pattern of the MOF precursor, ZFPS-1, and ZFNPS prepared in Example 1;

[0047] Figure 3 is the experimental result graph of the peroxidase-like catalytic activity verification of ZFNPS, ZFPS-1, and the blank group prepared in Example 1;

[0048] Figure 4 is the experimental result graph of the haloperoxidase-like catalytic activity verification of ZFNPS, ZFPS-1, and the blank group prepared in Example 1;

[0049] Figure 5 is the experimental result graph of the glutathione peroxidase-like catalytic activity verification of ZFNPS, ZFPS-1, and the blank group prepared in Example 1;

[0050] Figure 6 is the experimental result graph of the H2O2 colorimetric test verification of ZFPS-1 prepared in Example 1. Among them, Figure 6a is the concentration-ultraviolet absorbance graph of ZFPS-1 for H2O2, Figure 6 b is the concentration-TMB color graph of H2O2;

[0051] Figure 7 It is the antibacterial verification experimental result graph of Escherichia coli of ZFNPS, ZFPS-1 and the blank group prepared in Example 1;

[0052] Figure 8 It is the antibacterial verification experimental result graph of Staphylococcus aureus of ZFNPS, ZFPS-1 and the blank group prepared in Example 1. Detailed implementation mode

[0053] The present invention will be further described in detail below through specific examples. It should be understood that the preferred embodiments described herein are only used to illustrate and understand the present invention, and are not used to limit the present invention.

[0054] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels without special instructions. The experimental methods without specific conditions in the examples usually follow conventional conditions or the conditions recommended by the manufacturer.

[0055] Example 1 A preparation method of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity

[0056] This example is a preparation method of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity, including:

[0057] S1. Preparation of MOF precursor

[0058] Dissolve 0.1895 g of zinc nitrate (i.e., bimetallic raw material A), 0.242 g of iron nitrate (i.e., bimetallic raw material B) and 0.4 g of polyvinylpyrrolidone completely in 50 ml of anhydrous methanol to obtain solution I;

[0059] Dissolve 1.642 g of 2-methylimidazole completely in 100 ml of anhydrous methanol to obtain solution II;

[0060] After the first stirring of solution I for 1 h, slowly add it under the stirring state of solution II, carry out the second stirring for 1 h, dropwise add 5 ml of 5 mol / L NaOH solution under the stirring state, carry out the third stirring for 24 h, centrifuge and wash with anhydrous ethanol and water 3 times in sequence, and then freeze-dry to obtain the MOF precursor;

[0061] Among them, the average molar ratio of bimetallic raw material A, bimetallic raw material B and polyvinylpyrrolidone is 1:1:0.01;

[0062] The molar volume ratio of solution I, solution II and the alkali solution is 1:4:4;

[0063] S2. Preparation of phosphorus and sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity

[0064] Grind 0.5 g of MOF precursor and 0.5 g of tributyl thiophosphate in a mortar for 10 min to mix evenly, transfer to a crucible, and calcine for 2 h in a nitrogen gas atmosphere at 850 °C to obtain a phosphorus and sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity, labeled as ZFPS-1.

[0065] Set a control group: The control group is a bimetallic MOF composite nanozyme without phosphorus and sulfur doping, and its component content and preparation method are basically the same as those in Example 1, except that: in step S2, tributyl thiophosphate is not added to obtain a bimetallic MOF composite nanozyme without phosphorus and sulfur doping, labeled as ZFNPS.

[0066] Perform high-magnification scanning electron microscope observation on the prepared MOF precursor, ZFPS-1 and ZFNPS, and the observation results are as Figure 1 shown, where Figure 1 a is the high-magnification scanning electron microscope image of the MOF precursor, Figure 1 b is the high-magnification scanning electron microscope image of ZFPS-1, Figure 1 c is the high-magnification scanning electron microscope image of ZFNPS. It is found by comparison that, compared with the MOF precursor, doping with phosphorus and sulfur elements does not change the rhombic dodecahedron structure of the MOF precursor, so that the bivalent metal ions are captured by the phosphorus and sulfur elements and limited within the MOF framework, preventing the escape of Zn + and some iron ions during the calcination process, thereby increasing the active center, exposing the catalytic active center of the prepared nanozyme, increasing the catalytic sites, and thus enhancing the enzyme catalytic activity.

[0067] Perform X-ray diffraction structure characterization on the theoretical MOF model, the prepared bimetallic MOF precursor, ZFPS-1 and ZFNPS, and the results are as Figure 2 shown. It is found by comparison that the peak positions of the bimetallic MOF precursor are consistent with the crystal structure of the theoretical MOF model, indicating that the introduction of Fe element does not destroy the MOF crystal structure and a bimetallic MOF structure is formed; ZFPS-1 is composed of ZnS, FeP and Fe4P6N 12 S, indicating that after calcination, phosphorus and sulfur elements are successfully doped to form ZFPS-1; while ZFNPS is Fe3C / C, indicating that ZFNPS does not contain phosphorus and sulfur elements, resulting in the escape of zinc ions during calcination.

[0068] Verification experiment

[0069] I) Enzyme-like activity test

[0070] This verification test conducted an enzyme-like activity test on ZFNPS and ZFPS-1 prepared in Example 1.

[0071] 1) Peroxidase-like activity test:

[0072] ① Group setting

[0073] ZFPS-1 H Group: Add 50 μg / ml ZFPS-1 aqueous solution, 5 mmol / L H2O2, and 0.2 mmol / L TMB into an acetic acid-sodium acetate buffer solution with a pH of 5.0 respectively;

[0074] ZFNPS H Group: Add 50 μg / ml ZFNPS aqueous solution, 5 mmol / L H2O2, and 0.2 mmol / L TMB into an acetic acid-sodium acetate buffer solution with a pH of 5.0 respectively;

[0075] C H Group: Add 5 mmol / L H2O2 and 0.2 mmol / L TMB into an acetic acid-sodium acetate buffer solution with a pH of 5.0 respectively.

[0076] ② Test method

[0077] After each group was incubated at 37 °C for 10 min, the characterization was carried out by the oxidation of colorless TMB to blue oxidized state by reactive oxygen species, and its ultraviolet-visible absorption spectrum was measured using an ultraviolet-visible spectrophotometer. The peroxidase-like catalytic activity results of ZFPS-1 and ZFNPS are as Figure 3 shown.

[0078] ③ Test results

[0079] It can be seen from Figure 3 that ZFPS-1 prepared by the present invention has obvious peroxidase-like activity compared with the blank C H group, and the peroxidase-like activity of ZFPS-1 doped with phosphorus and sulfur is significantly better than that of ZFNPS not doped with phosphorus and sulfur.

[0080] 2) Haloperoxidase-like activity test:

[0081] ① Group setting

[0082] ZFPS-1 P Group: Add 50 μg / ml ZFPS-1 aqueous solution, 5 mmol / L H2O2, 4 mmol / L azure blue solution, and 5 mmol / L NaCl into an acetic acid-sodium acetate buffer solution with a pH of 6.0 respectively;

[0083] ZFNPS PGroup: Add 50 μg / ml ZFNPS aqueous solution, 5 mmol / L H2O2, 4 mmol / L Brilliant Celestine Blue solution, and 5 mmol / L NaCl into an acetic acid-sodium acetate buffer solution with a pH of 6.0 respectively;

[0084] C P Group: Add 5 mmol / L H2O2, 4 mmol / L Brilliant Celestine Blue solution, and 5 mmol / L NaCl into an acetic acid-sodium acetate buffer solution with a pH of 6.0 respectively.

[0085] ② Test method

[0086] After incubating each group at 37 °C for 20 min, the peroxidase-like catalytic activity of ZFPS-1 and ZFNPS was visually characterized by the change of Brilliant Celestine Blue to pink through peroxidase-like catalysis, and its UV-visible absorption spectrum was measured using a UV-visible spectrophotometer, as shown in Figure 4 shown.

[0087] ③ Test results

[0088] It can be seen from Figure 4 that ZFPS-1 prepared by the present invention has obvious peroxidase-like activity compared with the blank C P group, and the peroxidase-like activity of ZFPS-1 doped with phosphorus and sulfur is significantly better than that of ZFNPS without doping phosphorus and sulfur.

[0089] 3) Test for glutathione peroxidase-like activity:

[0090] ① Group setting

[0091] ZFPS-1 G Group: Add 50 μg / ml ZFPS-1 aqueous solution and 2 mmol / L GSH into an acetic acid-sodium acetate buffer solution with a pH of 7.0 respectively;

[0092] ZFNPS G Group: Add 50 μg / ml ZFNPS aqueous solution and 2 mmol / L GSH into an acetic acid-sodium acetate buffer solution with a pH of 7.0 respectively;

[0093] C G Group: Add 2 mmol / L GSH into an acetic acid-sodium acetate buffer solution with a pH of 7.0.

[0094] ② Test method

[0095] After incubation at 37 °C for 10 min, 100 μl of the mixture was added to an acetic acid-sodium acetate buffer solution with a pH of 7.0 containing 0.1 mmol / L DTNB, and its ultraviolet-visible absorption spectrum was measured using an ultraviolet-visible spectrophotometer. The glutathione peroxidase-like catalytic activities of ZFPS-1 and ZFNPS were as Figure 5 shown.

[0096] ③ Test results

[0097] As can be seen from Figure 5 it, compared with the blank C G group, ZFPS-1 prepared by the present invention has obvious glutathione peroxidase-like activity, and the glutathione peroxidase-like activity of ZFPS-1 doped with phosphorus and sulfur is significantly better than that of ZFNPS without doping phosphorus and sulfur.

[0098] In summary, ZFPS-1 prepared by high-temperature calcination and doping with transition elements phosphorus and sulfur has peroxidase-like, haloperoxidase-like, and glutathione peroxidase-like activities, and all three enzyme activities have been greatly improved.

[0099] II) H2O2 colorimetric test

[0100] This verification test performed an H2O2 colorimetric test on ZFPS-1 prepared in Example 1.

[0101] ① Test method

[0102] Based on the principle that ROS generated by the catalytic activity of peroxidase-like can oxidize colorless TMB into blue, a 50 μg / ml aqueous solution of ZFPS-1, a 5 mmol / L colorless TMB solution, and H2O2 solutions with different concentrations were mixed, and the absorbance at 652 nm was detected using an ultraviolet-visible spectrophotometer. The ultraviolet-visible absorbance response of ZFPS-1 to H2O2 with different concentrations was as Figure 6 shown.

[0103] ② Test results

[0104] As can be seen from Figure 6 a, within a certain range of the concentration of H2O2, the absorbance is linearly related to the concentration, and the linear regression equation is: y = 0.03258x + 0.635, R 2 = 0.989. At the same time, through visual observation, as Figure 6 shown in b, it was found that as the concentration of H2O2 increased, the color it exhibited also deepened.

[0105] Therefore, the prepared ZFPS-1 of the present invention can be used for H2O2 colorimetric sensing.

[0106] III) Antibacterial performance test

[0107] 1) Antibacterial performance test of Escherichia coli

[0108] This verification test conducted an antibacterial performance test of Escherichia coli on ZFNPS and ZFPS-1 prepared in Example 1.

[0109] ① Sample pretreatment

[0110] Take Escherichia coli out of the -80 °C ultra-low temperature refrigerator and place it in a laminar flow hood. After the bacterial strain melts, use a sterilized inoculation loop to pick up the frozen bacterial mass in the test tube and put it into 20 mL of Luria-Bertani (LB) medium. Incubate overnight at 37 °C and 150 rpm. Take the overnight bacterial liquid and measure its OD with an enzyme-linked immunosorbent assay (ELISA) reader. 630 , and dilute the bacterial liquid concentration to 10 6 CFU / mL for standby.

[0111] ② Group setting

[0112] ZFPS group: Add 10 μg / ml ZFPS-1 and 10 6 CFU / ml bacterial liquid into a 12-well plate, mix well, and place it in an incubator at 37 °C for 4 h to obtain the co-cultured bacterial liquid of the ZFPS group;

[0113] ZFPS + H2O2 group: Add 10 μg / ml ZFPS-1, 10 6 CFU / ml bacterial liquid and 100 μmol / L H2O2 into a 12-well plate, mix well, and place it in an incubator at 37 °C for 4 h to obtain the co-cultured bacterial liquid of the ZFPS + H2O2 group;

[0114] ZFNPS group: Add 10 μg / ml ZFNPS and 10 6 CFU / ml bacterial liquid into a 12-well plate, mix well, and place it in an incubator at 37 °C for 4 h to obtain the co-cultured bacterial liquid of the ZFNPS group;

[0115] ZFNPS + H2O2 group: Add 10 μg / ml ZFNPS, 10 6 CFU / ml bacterial liquid and 100 μmol / L H2O2 into a 12-well plate, mix well, and place it in an incubator at 37 °C for 4 h to obtain the co-cultured bacterial liquid of the ZFNPS + H2O2 group;

[0116] Group C: Add 10 6 CFU / ml bacterial liquid into a 12-well plate, mix well, and place it in an incubator at 37 °C for 4 h to obtain the co-cultured bacterial liquid of Group C;

[0117] C + H2O2 group: Add 10 6 CFU / ml bacterial liquid and 100 μmol / L H2O2 into a 12-well plate, mix well, and place it in an incubator at 37 °C for 4 h to obtain the co-cultured bacterial liquid of the C + H2O2 group.

[0118] ③Testing method

[0119] Take 100 μl of the co-cultured bacterial solution of each group for plate coating respectively. Make three parallel samples for each sample. Put the coated agar plates into an incubator at 37 °C and incubate overnight. After incubation, place the agar plates under a colony counter for photographing. The experimental result diagram of the antibacterial verification of Escherichia coli is as Figure 7 shown.

[0120] ④Testing results

[0121] It can be seen from Figure 7 that the antibacterial rate of the ZFPS-1 group is higher than that of the ZFNPS group, indicating that the antibacterial performance of ZFPS-1 against Escherichia coli is better than that of ZFNPS, and the bactericidal rate reaches 60%; the antibacterial rate of the ZFPS + H2O2 group is higher than that of the ZFPS-1 group, indicating that after adding H2O2, the bactericidal rate of ZFPS-1 is significantly increased, reaching 99.5%.

[0122] 2) Antibacterial performance test of Staphylococcus aureus

[0123] This verification test conducts the antibacterial performance test of Staphylococcus aureus on ZFNPS and ZFPS-1 prepared in Example 1.

[0124] The contents of ① sample pretreatment, ② group setting, and ③ testing method are basically the same as those of the antibacterial performance test of Escherichia coli, except that Staphylococcus aureus is selected as the sample. The experimental result diagram of the antibacterial verification of Staphylococcus aureus is as Figure 8 shown.

[0125] ④Testing results

[0126] It can be seen from Figure 8 that the antibacterial rate of the ZFPS-1' group is higher than that of the ZFNPS' group, indicating that the antibacterial performance of ZFPS-1 against Staphylococcus aureus is better than that of ZFNPS, and the bactericidal rate reaches 60%; the antibacterial rate of the ZFPS' + H2O2 group is higher than that of the ZFPS'-1 group, indicating that after adding H2O2, the bactericidal rate of ZFPS-1 reaches 99.5%.

[0127] Therefore, ZFPS-1 prepared by the present invention has a good bactericidal effect and can effectively inhibit the growth of Escherichia coli and Staphylococcus aureus.

[0128] IV) Low-temperature performance test

[0129] 1) Low-temperature performance test of peroxidase-like enzyme

[0130] 50 μg / ml ZFPS-1 aqueous solution and 50 μg / ml ZFNPS aqueous solution were respectively mixed with 5 mmol / L H2O2 and 0.2 mmol / L TMB, and then added to an acetic acid-sodium acetate buffer solution with a pH of 5.0. After incubation at different temperatures for 10 min, the colorless TMB was oxidized by reactive oxygen species to the blue oxidized state for characterization. The absorbance at 652 nm was measured using a microplate reader. The higher the absorbance value, the better the enzyme-like performance of the mimic enzyme. Then, by comparing the absorbance values, the low-temperature performance of ZFPS and ZFNPS was obtained.

[0131] Table 1 Experimental test on the low-temperature performance of peroxidase mimics

[0132]

[0133] From the above experimental results, it can be seen that compared with ZFNPS, the peroxidase mimic of ZFPS-1 has good low-temperature performance.

[0134] 2) Test on the low-temperature performance of haloperoxidase mimics

[0135] 50 μg / ml ZFPS-1 aqueous solution and 50 μg / ml ZFNPS aqueous solution were respectively mixed with 5 mmol / L H2O2, 4 mmol / L azure blue solution, and 5 mmol / L NaCl, and then added to an acetic acid-sodium acetate buffer solution with a pH of 6.0. After incubation at different temperatures for 20 min, the catalytic conversion of azure blue to pink by the enzyme mimic was used for visual characterization. The absorbance at 645 nm was measured using a microplate reader. The lower the absorbance, the better the catalytic effect of the mimic enzyme. By comparing the absorbance values, the low-temperature performance of ZFPS and ZFNPS was obtained.

[0136] Table 2 Experimental test on the low-temperature performance of haloperoxidase mimics

[0137]

[0138] From the above experimental results, it can be seen that compared with ZFNPS, the haloperoxidase mimic of ZFPS-1 has good low-temperature performance.

[0139] 3) Test on the low-temperature performance of glutathione peroxidase mimics

[0140] 50 μg / ml aqueous solution of ZFPS-1 and 50 μg / ml aqueous solution of ZFNPS were respectively added to an acetic acid-sodium acetate buffer solution with a pH of 7.0 containing 2 mmol / L GSH; after incubation at different temperatures for 10 min, 100 μl of each mixture was then added to an acetic acid-sodium acetate buffer solution with a pH of 7.0 containing 0.1 mmol / L DTNB, and the absorbance at 410 nm was measured using a microplate reader. The lower the absorbance value, the better the enzyme-like performance of the mimetic enzyme. The low-temperature performance of ZFPS and ZFNPS was obtained by comparing the absorbance values.

[0141] Table 3 Experimental test on the low-temperature performance of glutathione peroxidase-like

[0142]

[0143] From the above experimental results, it can be seen that compared with ZFNPS, the glutathione peroxidase-like of ZFPS-1 has good low-temperature performance.

[0144] In summary, the enzyme catalytic activity of ZFPS-1 prepared in the present invention is less restricted by temperature and can achieve the same enzyme catalytic effect under low-temperature conditions.

[0145] Preparation methods of phosphorus-sulfur doped bimetallic MOF composite nanozymes with multi-enzyme activities in Examples 2 to 5

[0146] Examples 2 to 5 are respectively preparation methods of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activities. The content is basically the same as that of Example 1, and the differences are only in the specific components and the method parameters of each step in the method. See Table 4 for details:

[0147] Table 4 Component and method parameter table of Examples 2 to 5

[0148]

[0149]

[0150] Through verification experiments, the test results of the enzyme-like activity test, H2O2 colorimetric test, antibacterial performance test, and low-temperature performance test of Examples 2 to 5 are consistent with the results of Example 1.

[0151] It should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions recorded in the above embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity, characterized in that, It includes the following steps carried out in sequence: S1. Prepare the MOF precursor After dissolving polyvinylpyrrolidone, bimetallic raw material A and bimetallic raw material B in anhydrous methanol and mixing them evenly, solution I is obtained; After dissolving 2-methylimidazole in anhydrous methanol and mixing it evenly, solution II is obtained; After adding solution I to solution II, mix it with an alkali solution, centrifuge and dry to prepare the MOF precursor; Among them, the bimetallic raw material A is zinc nitrate; The bimetallic raw material B includes ferric nitrate, cerium nitrate, cobalt nitrate or copper nitrate; S2. Prepare a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity Mix and grind the MOF precursor and an equal mass of tributyl phosphorotrithioate, and calcine it in an inert gas atmosphere to prepare a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity; The calcination temperature is 650 - 1000 °C, and the calcination time is 0.5 - 4 h; In step S1, the average molar ratio of the bimetallic raw material A, the bimetallic raw material B and polyvinylpyrrolidone is 1:0.1 - 9:0.01 - 0.05; in step S1, the molar volume ratio of solution I, solution II and the alkali solution is 1:4:2 - 6.

2. The preparation method of the phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity according to claim 1, characterized in that, In step S1, the mass of the 2-methylimidazole is 0.4 - 2.5 g.

3. The preparation method of the phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity according to claim 1, characterized in that, In step S1, the alkali solution includes a NaOH solution, a KOH solution or ammonia water.

4. The preparation method of the phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity according to claim 1 or 3, characterized in that, In step S1, the molar concentration of the alkali solution is 1 - 10 mol / L, and the volume is 1 - 20 ml.

5. Application of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity, characterized in that, The phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity according to any one of claims 1 - 4 is used as a peroxidase-like catalyst, a haloperoxidase-like catalyst and a glutathione peroxidase-like catalyst for sterilization.

6. Application of a phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity, characterized in that, The phosphorus-sulfur doped bimetallic MOF composite nanozyme with multi-enzyme activity according to any one of claims 1 - 4 is used as a peroxidase-like catalyst, a haloperoxidase-like catalyst and a glutathione peroxidase-like catalyst for the colorimetric sensing of H2O2.

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