Biocatalyst Ni-MOFs-HRP-coated MAF-7 with core-shell structure as well as preparation method and application of biocatalyst

Abstract

The invention discloses a core-shell structure biocatalyst Ni-MOFs-HRP-coated MAF-7 and a preparation method and application thereof.The preparation method comprises the steps that in the enzyme immobilization stage, horseradish peroxidase is immobilized on a nickel-based metal organic framework through the inherent affinity of nickel ions and histidine residues, a chemical cross-linking agent is not needed, and efficient immobilization of HRP is achieved; in the core-shell structure construction stage, MAF-7 is encapsulated on the surface of the HRP functionalized Ni-MOFs in a layered manner under the coordination action of Zn < 2 + > and 3-methyl-1, 2, 4-triazole at normal temperature to form the core-shell structure, and the MAF-7 coating is used as a multifunctional protective layer with hydrophilicity, biocompatibility and acid-base stability. The immobilized HRP in the biocatalyst keeps nearly natural catalytic kinetic characteristics, and has strong tolerance to extreme conditions such as extreme pH, high temperature, organic solvents and the like. The problems that biosensor equipment is short in service life and high in operation cost can be effectively solved, support is provided for sustainable biosensor development, and the application prospect in the field of hydrogen peroxide detection is wide.

Description

Technical Field

[0001] This invention relates to the field of biocatalyst technology, and in particular to a core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, its preparation method, and its applications. Background Technology

[0002] In vitro diagnostics (IVD), as a core technological support for modern disease diagnosis and health management, has become an indispensable key link in the medical system. By detecting biomarkers in human body fluids, tissues and other samples, it provides accurate quantitative evidence for early disease screening, diagnosis, treatment plan formulation and prognosis assessment, directly affecting the scientific nature and timeliness of medical decisions, and playing an irreplaceable role in the fields of chronic disease management, infectious disease prevention and control, and tumor diagnosis and treatment.

[0003] Enzymes, as the most widely used core biological raw materials in the IVD field, are used in almost all IVD testing scenarios due to their high catalytic specificity and reaction efficiency: In biochemical detection, enzymes can be used for quantitative analysis of substrate concentration, providing a basis for the assessment of biochemical indicators such as liver and kidney function; in immunological detection (such as ELISA), detection systems constructed with enzyme-labeled antibodies or antigens are the core for achieving high-sensitivity detection of trace targets; in the field of point-of-care testing (POCT), the rapid response characteristics of enzyme-catalyzed reactions meet the needs of rapid on-site detection; in blood glucose monitoring, glucose oxidase, horseradish peroxidase (HRP), and other enzymes can convert blood glucose into detectable signals in real time, providing immediate data for blood glucose management in diabetic patients; in coagulation analysis, reaction systems involving enzymes such as thrombin can accurately assess blood coagulation function and guide clinical anticoagulation therapy.

[0004] However, the current application of enzymes in the IVD field mostly relies on free enzyme systems, which presents significant technical bottlenecks: on the one hand, free enzymes are difficult to separate and recover after catalytic reactions and can only be used once, which not only wastes a lot of enzyme resources but also significantly increases the production cost of detection reagents; on the other hand, the molecular structure of biological enzymes is extremely sensitive to the environment. In conventional detection systems, they are easily affected by pH fluctuations, temperature changes, organic solvents, and ionic strength, which can cause conformational changes, leading to a rapid decline in catalytic activity or even inactivation, severely limiting their applicability and detection stability.

[0005] To address these challenges, researchers have developed various enzyme immobilization strategies, including physical adsorption, chemical cross-linking, and embedding. Among these, metal-organic frameworks (MOFs) have become ideal carrier materials for enzyme immobilization due to their high specific surface area, controllable pore structure, and tunable surface chemistry. The high specific surface area provides ample enzyme binding sites, increasing enzyme loading; the unique pore structure creates spatial confinement for enzyme molecules, reducing conformational changes; and surface modifiability can optimize the catalytic microenvironment by regulating the interaction between the carrier and the enzyme. For example, studies have shown that immobilizing microperoxidase-8 in the mesopores of MIL-101(Cr) has achieved complete preservation of enzyme catalytic activity; constructing MOFs using bio-enzyme metal ion activators can enhance enzyme activity while protecting it from extreme conditions.

[0006] However, existing MOF-based enzyme immobilization technologies still have significant shortcomings: First, while some MOF materials (such as the widely used ZIF-8) possess mild synthesis conditions and good biocompatibility, their stability in acidic environments is poor, failing to meet the application requirements of acidic detection systems. Second, in traditional immobilization methods, chemical cross-linking requires the use of cross-linking agents (such as glutaraldehyde), which easily react with the amino and thiol groups of enzyme molecules, disrupting the enzyme's spatial conformation and leading to irreversible loss of enzyme activity. Physical adsorption methods, due to the weak binding force between the carrier and the enzyme, are prone to enzyme detachment, affecting the immobilization effect and detection repeatability. Third, although bimetallic MOFs can improve enzyme loading and activity, their preparation process is complex and costly, making large-scale application difficult.

[0007] Therefore, developing an enzyme immobilization technology that requires no chemical cross-linking agents, has excellent carrier stability (especially resistance to acids and alkalis, high temperatures and organic solvents), and can efficiently retain enzyme catalytic activity has become a key requirement to overcome the current bottlenecks in enzyme applications in the IVD field. Summary of the Invention

[0008] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, its preparation method, and its applications. This invention addresses the pain points of existing technologies by designing and constructing a core-shell structured Ni-MOFs-HRP@MAF-7 biocatalyst, using Ni… 2+ The inherent affinity of HRP histidine residues enables cross-link-free enzyme immobilization. Combined with the high stability and protective effect of the MAF-7 coating, this technology can solve the problems of easy enzyme conformational damage, poor tolerance of immobilized enzymes, and insufficient reusability in existing technologies, providing a new technical path for the development of high-performance enzyme reagents in the IVD field.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a method for preparing a core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, the method being based on Ni… 2+ The inherent affinity of histidine residues and Ni 2+ The coordination mechanism is utilized, and the preparation is completed in four steps without the introduction of any chemical cross-linking agents. The specific steps include:

[0010] S1. Ni precursor is prepared by hydrothermal reaction using nickel source, urea and deionized water;

[0011] S2. Nickel-based metal-organic frameworks (Ni-MOFs) are prepared by hydrothermal reaction of Ni precursor and 2,5-dihydroxyterephthalic acid.

[0012] S3. Horseradish peroxidase (HRP) was immobilized in Ni-MOFs to prepare HRP-functionalized Ni-MOFs: Ni-MOFs-HRP;

[0013] S4. Layered encapsulation of Zn on the Ni-MOFs-HRP surface 2+ The MAF-7 material, synthesized from 3-methyl-1,2,4-triazole, forms a core-shell structure, yielding the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

[0014] In the preparation method of this invention, during the enzyme immobilization stage, horseradish peroxidase (HRP) is immobilized onto nickel-based metal-organic frameworks (Ni-MOFs) using the inherent affinity between nickel ions and histidine residues, achieving efficient HRP immobilization without the need for chemical cross-linking agents; during the core-shell structure construction stage, Zn is used at room temperature... 2+ Through coordination with 3-methyl-1,2,4-triazole, MAF-7 is layered and encapsulated on the surface of HRP-functionalized Ni-MOFs to form a core-shell structure. The MAF-7 coating serves as a multifunctional protective layer with hydrophilicity, biocompatibility, and acid-base stability. The immobilized HRP in this biocatalyst maintains near-natural catalytic kinetics and exhibits strong tolerance to extreme conditions such as extreme pH, high temperature, and organic solvents.

[0015] Preferably, the preparation method of the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 includes the following steps:

[0016] S1. Preparation of Ni precursor: Mix NiSO4·H2O, urea and deionized water, heat to carry out hydrothermal reaction, separate the solid product after the reaction is completed and dry it to obtain Ni precursor;

[0017] S2. Preparation of Ni-MOFs: 2,5-Dihydroxyterephthalic acid and Ni precursor were dispersed in a mixture of DMF, ethanol and deionized water, and heated to carry out a hydrothermal reaction. After the reaction was completed, the solid product was separated and dried to obtain nickel-based metal-organic frameworks: Ni-MOFs.

[0018] S3. Preparation of Ni-MOFs-HRP: Ni-MOFs were made into an aqueous solution, and then HRP solution was added. After adsorption by shaking at room temperature, the solid product was separated and dried to obtain HRP-functionalized Ni-MOFs: Ni-MOFs-HRP.

[0019] S4. Preparation of core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7: Zn(NO3)2 solution, Ni-MOFs-HRP, 3-methyl-1H-1,2,4-triazole solution and NH3·H2O were mixed and stirred at room temperature. After the reaction was completed, the solid product was separated and dried to obtain the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

[0020] Preferably, in step S1, the molar ratio of NiSO4·H2O to urea is 0.5-2:1-4, the hydrothermal reaction temperature is 140-180℃, and the reaction time is 9-36h.

[0021] Preferably, step S1 specifically includes:

[0022] Add 0.5-2 mmol NiSO4·H2O and 1-4 mmol urea to 15-60 mL of deionized water, stir to dissolve, and then transfer to a polytetrafluoroethylene-lined stainless steel autoclave. React at 140-180 °C for 9-36 h. After the reaction is complete, cool naturally to room temperature, collect the product, wash with deionized water and ethanol in sequence, centrifuge to collect the precipitate, and vacuum dry at 50-70 °C overnight to obtain the Ni precursor.

[0023] Preferably, in step S2, the mass ratio of 2,5-dihydroxyterephthalic acid to Ni precursor is 10-40:18-72, the volume ratio of DMF:ethanol:deionized water in the mixture is 0.5-2:0.5-2:0.5-2, the hydrothermal reaction temperature is 100-140℃, and the reaction time is 12-48h.

[0024] Preferably, step S2 specifically includes:

[0025] Take 10-40 mg of 2,5-dihydroxyterephthalic acid and 18-72 mg of Ni precursor and add them to a mixed solution consisting of 1.25-5 mL DMF, 1.25-5 mL ethanol and 1.25-5 mL deionized water. Sonicate for 5-30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined stainless steel autoclave and react at 100-140 °C for 12-48 h. After the reaction is complete, cool to room temperature, collect the product, wash with DMF and methanol sequentially, centrifuge to collect the precipitate, and vacuum dry at 50-70 °C for 3-12 h to obtain nickel-based metal-organic frameworks: Ni-MOFs.

[0026] Preferably, step S3 specifically includes:

[0027] Ni-MOFs were dispersed in deionized water to prepare an aqueous solution of Ni-MOFs with a concentration of 0.5-2 mg / mL. HRP solution was added to make the concentration of HRP in the resulting mixture 10-220 U / mL. The mixture was shaken at room temperature for 15-60 min, centrifuged, the precipitate was collected and dried to obtain HRP-functionalized Ni-MOFs: Ni-MOFs-HRP.

[0028] Preferably, step S4 specifically includes:

[0029] Take 20-80 mL of Zn(NO3)2 solution with a concentration of 0.4-1.6 mol / L, 20-80 mL of 3-methyl-1H-1,2,4-triazole solution with a concentration of 0.12-0.48 mol / L, and 0.8-3.2 mL of NH3·H2O solution with a concentration of 5-20 wt%, mix them thoroughly, and then add 2.5-10 mg of Ni-MOFs-HRP. Stir the mixture at room temperature for 12-48 h. After the reaction is complete, collect the precipitate by centrifugation, wash it with deionized water, centrifuge it again, and dry the precipitate under vacuum at 50-70 °C for 3-12 h to obtain the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

[0030] In a second aspect, the present invention provides a core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, characterized in that it is prepared by the method described above.

[0031] In a third aspect, the present invention provides an application of the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 as described above in the detection of hydrogen peroxide. The application method is as follows: the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 and the colorimetric solution are added to the sample solution containing hydrogen peroxide, and after mixing, the absorbance of the product is measured. The concentration of hydrogen peroxide in the sample is obtained based on the absorbance analysis.

[0032] The beneficial effects of this invention are:

[0033] This invention provides a core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, which utilizes Ni 2+ The inherent affinity of HRP histidine residues enables cross-link-free enzyme immobilization. Combined with the high stability and protective effect of the MAF-7 coating, this invention solves the problems of easily disrupted enzyme conformation, poor tolerance of immobilized enzymes, and insufficient reusability in existing technologies, providing a new technical path for the development of high-performance enzyme reagents in the IVD field. This invention also effectively addresses the problems of short lifespan and high operating costs of biosensor devices, providing support for the development of sustainable biosensors and showing broad application prospects in the field of hydrogen peroxide detection. Specifically, this invention has the following advantages:

[0034] (1) Excellent enzyme immobilization effect: utilizing Ni 2+ Ni-MOFs immobilize HRP with their inherent affinity for histidine residues, eliminating the need for chemical cross-linking agents, thus avoiding enzyme conformational changes. Furthermore, Ni-MOFs exhibit an adsorption capacity of 85.8 U / mg for HRP, demonstrating excellent immobilization performance.

[0035] (2) Excellent stability

[0036] pH stability: Within a wide pH range of 1-14, the enzyme activity of Ni-MOFs-HRP@MAF-7 was retained at over 80% after incubation for 0.5h and 3h, which is far superior to that of free HRP and Ni-MOFs-HRP.

[0037] Temperature stability: After incubation at 60℃ for 3 hours, Ni-MOFs-HRP@MAF-7 still retained 86% of the enzyme activity, and its thermal stability was significantly higher than that of free HRP and Ni-MOFs-HRP.

[0038] Strong resistance to denaturants: It has good resistance to denaturants such as 500mM NaCl, methanol, DMF, 20wt% SDS and 1U / mL trypsin. Except for DMF, it retains more than 80% of its activity after treatment with other denaturants for 0.5h and 3h, and still retains 78% of its activity after treatment with DMF for 3h.

[0039] Good reusability: After 10 cycles of reuse, Ni-MOFs-HRP@MAF-7 can still retain 87% of its activity, which can significantly reduce the cost of use;

[0040] High storage stability: The freeze-dried powder and aqueous solution retain more than 80% of their activity after 8 weeks of storage at 4°C and 25°C, making them suitable for long-term storage and use.

[0041] (3) Excellent detection performance: When Ni-MOFs-HRP@MAF-7 is used for H2O2 detection, its linear response range is 4.032-2016 μmol / L (R2 With a value >0.99%, it is comparable to commercially available diagnostic kits, and has a wide linear range and strong applicability, providing support for the development of sustainable biosensors and showing broad application prospects in environmental monitoring and biomedicine. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the preparation process (A) and characterization (B) of Ni-MOFs-HRP@MAF-7 in this invention;

[0043] Figure 2 A schematic diagram of the simulated structure of Ni-MOFs-HRP@MAF-7;

[0044] Figure 3 The enzyme kinetic characteristics (C) of HRP (A), Ni-MOFs-HRP (B) and Ni-MOFs-HRP@MAF-7 obtained in the enzyme kinetic test are shown.

[0045] Figure 4 The pH stability test results of HRP (A), Ni-MOFs-HRP (B), Ni-MOFs-HRP@MAF-7, and (C) obtained in the pH stability test are shown.

[0046] Figure 5 The temperature stability test results of HRP, Ni-MOFs-HRP and Ni-MOFs-HRP@MAF-7 at 40℃ (A) and 60℃ (B) are shown in the temperature stability test.

[0047] Figure 6 The results show the reusability test results for Ni-MOFs-HRP and Ni-MOFs-HRP@MAF-7.

[0048] Figure 7 The graph shows the linear relationship between absorbance at 652 nm and hydrogen peroxide concentration when using Ni-MOFs-HRP(A) and Ni-MOFs-HRP@MAF-7(B) as catalysts. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0050] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0052] Example 1: Preparation of Ni precursor

[0053] Weigh 264 mg (1.0 mmol) NiSO4·H2O and 120 mg (2.0 mmol) urea, add 30 mL of deionized water, stir to dissolve, and transfer to a 50 mL PTFE-lined stainless steel autoclave. Seal and place in a 160 °C oven for 18 h. After the reaction is complete, allow to cool naturally to room temperature, remove the green precipitate, and wash three times each with deionized water and ethanol. After each wash, centrifuge at 8000 rpm for 5 min to collect the precipitate. Finally, place the precipitate in a vacuum drying oven and dry under vacuum at 60 °C overnight to obtain the Ni precursor.

[0054] Example 2: Preparation of Ni-MOFs

[0055] Weigh 20 mg of 2,5-dihydroxyterephthalic acid and 36 mg of the Ni precursor prepared in Example 1, add a mixed solution of 2.5 mL DMF, 2.5 mL ethanol, and 2.5 mL deionized water, and sonicate for 15 min to completely disperse the solid and form a homogeneous suspension. Transfer the suspension to a 50 mL polytetrafluoroethylene-lined stainless steel autoclave, seal it, and place it in a 120 °C oven for 1 day. After the reaction, cool to room temperature, collect the product, and wash it three times with DMF and methanol respectively to remove unreacted raw materials and impurities. Centrifuge after each wash (8000 rpm, 5 min), and finally dry under vacuum at 60 °C for 6 h to obtain nickel-based metal-organic frameworks: Ni-MOFs.

[0056] Example 3: Preparation of Ni-MOFs-HRP

[0057] The Ni-MOFs prepared in Example 2 were dispersed in deionized water to prepare an aqueous solution with a concentration of 1 mg / mL. 200 μL of this aqueous solution was placed in six centrifuge tubes, and then 50, 100, 150, 200, 250, and 300 μL of HRP (horseradish peroxidase, purchased from Michael Chemical Technology (Shanghai) Co., Ltd.) solution with a concentration of 100 U / mL was added sequentially to each centrifuge tube (to ensure the HRP concentration in the system covered the range of 10-220 U / mL). The mixture was gently shaken to ensure homogeneity. The centrifuge tubes were placed in a shaker at room temperature and shaken at 150 rpm for 30 min to carry out the adsorption reaction. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min, the precipitate was collected, and vacuum dried at -80℃ for 6 h to obtain a series of HRP-functionalized Ni-MOFs with different HRP concentrations: Ni-MOFs-HRP.

[0058] The concentration of HRP in the supernatant was determined using a UV-Vis spectrophotometer, according to formula Q. m =(c0-c e The adsorption capacity is calculated using V / m, where c0 is the initial HRP concentration, and c e The concentration of HRP after adsorption equilibrium is given, V is the total volume of the solution, and m is the mass of Ni-MOFs. The results show that when the HRP concentration reaches a certain value, adsorption reaches equilibrium, and the adsorption capacity of Ni-MOFs for HRP is 85.8 U / mg.

[0059] Example 4: Preparation of Ni-MOFs-HRP@MAF-7

[0060] Measure 40 mL of a 0.8 mol / L Zn(NO3)2 solution, 40 mL of a 0.24 mol / L 3-methyl-1H-1,2,4-triazole solution (solvent: deionized water), and 1.6 mL of a 10 wt% NH3·H2O solution, and add them to a 250 mL beaker. Stir well. Then add 5 mg of the Ni-MOFs-HRP prepared in Example 3 to the beaker, and continue stirring at 300 rpm for 24 h at room temperature (using Zn...). 2+ The coordination with 3-methyl-1H-1,2,4-triazole enabled MAF-7 to be encapsulated in layers on the surface of Ni-MOFs-HRP. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min, the precipitate was collected, and washed three times with deionized water, centrifuged (8000 rpm, 5 min) after each wash. Finally, the precipitate was dried in a vacuum drying oven at 60 °C for 6 h to obtain the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

[0061] Performance testing

[0062] The Ni-MOFs-HRP, Ni-MOFs-HRP@MAF-7, and free HRP prepared using the above embodiments were subjected to the following performance tests:

[0063] Enzyme kinetics assay: 10 μL of each of the following solutions (all deionized water) was mixed with 200 μL of TMB chromogenic solution. The absorbance was monitored continuously at 652 nm for 5 minutes at 5-second intervals using a UV-Vis spectrophotometer. K was calculated according to the Michaelis-Menten equation. m and V m Value. Test results are as follows: Figure 3 As shown, the results indicate that the K of Ni-MOFs-HRP m The value was 0.2674 mmol / L, V m The value was 0.0522 mmol / (L·s); the K of Ni-MOFs-HRP@MAF-7 m The value was 0.6629 mmol / L, V m The value was 0.0301 mmol / (L·s).

[0064] pH stability test: Buffer solutions with pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0 were prepared respectively. Appropriate amounts of free HRP solution, Ni-MOFs-HRP solution, and Ni-MOFs-HRP@MAF-7 solution were mixed with buffer solutions of different pH values ​​to ensure consistent enzyme concentration. The solutions were incubated at room temperature for 0.5 h and 3 h respectively. After incubation, 10 μL of each mixture was mixed with 200 μL of TMB chromogenic solution, and the absorbance at 652 nm was measured. Residual activity was calculated with the initial activity considered as 100%. The test results are as follows: Figure 4 As shown, the results indicate that Ni-MOFs-HRP@MAF-7 retained more than 80% of its activity after incubation at pH 1-14 for 0.5 h and 3 h.

[0065] Temperature stability test: Appropriate amounts of free HRP solution, Ni-MOFs-HRP solution, and Ni-MOFs-HRP@MAF-7 solution were placed in constant temperature water baths at 40℃ and 60℃, respectively, and incubated for 0.5 h and 3 h, respectively. After incubation, the solutions were cooled to room temperature, and 10 μL of each solution was mixed with 200 μL of TMB colorimetric solution. The absorbance at 652 nm was measured, and the residual activity was calculated. The test results are as follows: Figure 5 As shown, the results indicate that after incubation at 60℃ for 3 hours, Ni-MOFs-HRP@MAF-7 still retains 86% of its activity.

[0066] Reusability test: A certain amount of Ni-MOFs-HRP@MAF-7 was dispersed in 2 mL of deionized water and 500 μL of TMB colorimetric solution, and reacted at room temperature for 5 min. The absorbance at 652 nm was measured. After the reaction, the material was washed three times with deionized water to remove the adsorbed TMB. Then, 2 mL of deionized water and 500 μL of TMB colorimetric solution were added again to start the next cycle, which was repeated 10 times. The test results are as follows. Figure 6 As shown, the results indicate that after 10 cycles, Ni-MOFs-HRP@MAF-7 still retains 87% of its activity.

[0067] H2O2 Detection Application Test: A series of H2O2 standard solutions with concentrations of 4.032, 10, 50, 100, 500, 1000, 1500, and 2016 μmol / L were prepared. An appropriate amount of Ni-MOFs-HRP@MAF-7 was taken and mixed with different concentrations of H2O2 standard solutions and TMB colorimetric solution, respectively. The mixture was reacted at room temperature for 5 min, and the absorbance at 652 nm was measured. A standard curve was plotted with H2O2 concentration on the x-axis and absorbance on the y-axis. The test results are as follows: Figure 7 As shown, the results indicate that the linear range for detecting H2O2 using Ni-MOFs-HRP@MAF-7 is 4.032-2016 μmol / L, with a correlation coefficient R0. 2 >0.99.

[0068] The linear range comparison results of different catalysts for hydrogen peroxide detection methods are shown in Table 1 below:

[0069] Table 1. Comparison of linear ranges for different catalysts in hydrogen peroxide detection methods

[0070]

[0071]

[0072] Among them, Document 1:

[0073] Literature 2: R Bandi, M Alle, CW Park, SY Han, GJ Kwon, NH Kim, JC Kim, S HLee. Cellulose nanofibrils / carbon dots composite nanopapers for the smartphone-based colorimetric detection of hydrogen peroxide and glucose [J]. Sensors and Actuators B: Chemical, 2021, 330, 129330.

[0074] Literature 3: L Zhang,

[0075] Document 4: K Wang,

[0076] As can be seen from Table 1, the Ni-MOFs-HRP@MAF-7 provided in Embodiment 4 of the present invention has a wider detection range.

[0077] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing a core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, characterized in that, Includes the following steps: S1. Ni precursor is prepared by hydrothermal reaction using nickel source, urea and deionized water; S2. Nickel-based metal-organic frameworks (Ni-MOFs) are prepared by hydrothermal reaction of Ni precursor and 2,5-dihydroxyterephthalic acid. S3. Horseradish peroxidase (HRP) was immobilized in Ni-MOFs to prepare HRP-functionalized Ni-MOFs: Ni-MOFs-HRP; S4. Layered encapsulation of Zn on the Ni-MOFs-HRP surface 2+ The MAF-7 material, synthesized from 3-methyl-1,2,4-triazole, forms a core-shell structure, yielding the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

2. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 1, characterized in that, Includes the following steps: S1. Preparation of Ni precursor: Mix NiSO4·H2O, urea and deionized water, heat to carry out hydrothermal reaction, separate the solid product after the reaction is completed and dry it to obtain Ni precursor; S2. Preparation of Ni-MOFs: 2,5-Dihydroxyterephthalic acid and Ni precursor were dispersed in a mixture of DMF, ethanol and deionized water, and heated to carry out a hydrothermal reaction. After the reaction was completed, the solid product was separated and dried to obtain nickel-based metal-organic frameworks: Ni-MOFs. S3. Preparation of Ni-MOFs-HRP: Ni-MOFs were made into an aqueous solution, and then HRP solution was added. After adsorption by shaking at room temperature, the solid product was separated and dried to obtain HRP-functionalized Ni-MOFs: Ni-MOFs-HRP. S4. Preparation of core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7: Zn(NO3)2 solution, Ni-MOFs-HRP, 3-methyl-1H-1,2,4-triazole solution and NH3·H2O were mixed and stirred at room temperature. After the reaction was completed, the solid product was separated and dried to obtain the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

3. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 2, characterized in that, In step S1, the molar ratio of NiSO4·H2O to urea is 0.5-2:1-4, the hydrothermal reaction temperature is 140-180℃, and the reaction time is 9-36h.

4. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 3, characterized in that, Step S1 is as follows: Add 0.5-2 mmol NiSO4·H2O and 1-4 mmol urea to 15-60 mL of deionized water, stir to dissolve, and then transfer to a polytetrafluoroethylene-lined stainless steel autoclave. React at 140-180 °C for 9-36 h. After the reaction is complete, cool naturally to room temperature, collect the product, wash with deionized water and ethanol in sequence, centrifuge to collect the precipitate, and vacuum dry at 50-70 °C overnight to obtain the Ni precursor.

5. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 2, characterized in that, In step S2: the mass ratio of 2,5-dihydroxyterephthalic acid to Ni precursor is 10-40:18-72, the volume ratio of DMF:ethanol:deionized water in the mixture is 0.5-2:0.5-2:0.5-2, the hydrothermal reaction temperature is 100-140℃, and the reaction time is 12-48h.

6. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 5, characterized in that, Step S2 is as follows: Take 10-40 mg of 2,5-dihydroxyterephthalic acid and 18-72 mg of Ni precursor and add them to a mixed solution consisting of 1.25-5 mL DMF, 1.25-5 mL ethanol and 1.25-5 mL deionized water. Sonicate for 5-30 min. Transfer the resulting mixture to a polytetrafluoroethylene-lined stainless steel autoclave and react at 100-140 °C for 12-48 h. After the reaction is complete, cool to room temperature, collect the product, wash with DMF and methanol sequentially, centrifuge to collect the precipitate, and vacuum dry at 50-70 °C for 3-12 h to obtain nickel-based metal-organic frameworks: Ni-MOFs.

7. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 2, characterized in that, Step S3 is as follows: Ni-MOFs were dispersed in deionized water to prepare an aqueous solution of Ni-MOFs with a concentration of 0.5-2 mg / mL. HRP solution was added to make the concentration of HRP in the resulting mixture 10-220 U / mL. The mixture was shaken at room temperature for 15-60 min, centrifuged, the precipitate was collected and dried to obtain HRP-functionalized Ni-MOFs: Ni-MOFs-HRP.

8. The method for preparing the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 according to claim 2, characterized in that, Step S4 is as follows: Take 20-80 mL of Zn(NO3)2 solution with a concentration of 0.4-1.6 mol / L, 20-80 mL of 3-methyl-1H-1,2,4-triazole solution with a concentration of 0.12-0.48 mol / L, and 0.8-3.2 mL of NH3·H2O solution with a concentration of 5-20 wt%, mix them thoroughly, and then add 2.5-10 mg of Ni-MOFs-HRP. Stir the mixture at room temperature for 12-48 h. After the reaction is complete, collect the precipitate by centrifugation, wash it with deionized water, centrifuge it again, and dry the precipitate under vacuum at 50-70 °C for 3-12 h to obtain the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7.

9. A core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. An application of the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 as described in claim 9 in the detection of hydrogen peroxide, wherein the application method is as follows: the core-shell structured biocatalyst Ni-MOFs-HRP@MAF-7 and the colorimetric solution are added to the sample solution containing hydrogen peroxide, the absorbance of the product is measured after mixing, and the concentration of hydrogen peroxide in the sample is obtained based on the absorbance analysis.

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