Preparation method of polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase

Abstract

The application discloses a preparation method of polydopamine-coated mesoporous metal organic framework immobilized glucose oxidase, which uses nitrogen-containing heterocyclic organic ligand, divalent metal ions and glucose oxidase as raw materials, adds a double-end dopamine modified hydrophilic polymer, in-situ embeds the glucose oxidase, obtains mesoporous metal organic framework immobilized glucose oxidase, and then mixes and reacts with dopamine, so that the polydopamine-coated mesoporous metal organic framework immobilized glucose oxidase is obtained. The application belongs to the field of enzyme engineering, and the prepared immobilized enzyme has the advantages of high catalytic efficiency and high stability.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, and in particular to a method for preparing glucose oxidase immobilized on a polydopamine-coated mesoporous metal-organic framework. Background Technology

[0002] Glucose oxidase (GOx) catalyzes the conversion of glucose into gluconic acid and hydrogen peroxide. It is characterized by high catalytic efficiency, high selectivity, and mild reaction conditions, and is widely used in food, pharmaceutical, and fermentation industries. However, free glucose oxidase is easily inactivated, has poor stability, and low recovery rate during application, which limits its application to some extent.

[0003] Enzyme immobilization is a technology that, while maintaining the enzyme's biological activity, binds or confines the enzyme to a specific area using a solid material, allowing for enzyme recovery and reuse. Currently, there are four main methods for preparing immobilized glucose oxidase: adsorption, cross-linking, embedding, and covalent bonding. Immobilized glucose oxidases prepared using traditional methods exhibit relatively low enzyme activity recovery rates, and their activity declines significantly after a certain period of use. Therefore, it is necessary to seek suitable materials and simple methods to prepare immobilized glucose oxidases with good biological activity and stability, enabling the industrial production and application of immobilized glucose oxidases.

[0004] In recent years, commonly used carriers can be classified according to their composition into polymeric carriers, inorganic carriers, composite carriers, and novel carriers. Polymer materials have drawbacks such as short lifespan and poor mass transfer performance; inorganic carriers have advantages such as good stability, low cost, and long lifespan, but the loading rate of immobilized enzymes prepared using inorganic materials is generally low. In contrast, metal-organic frameworks (MOFs) are increasingly attracting attention as a new type of immobilization carrier. MOFs are coordination polymers with a three-dimensional porous structure. Compared with the aforementioned immobilization materials, MOFs have advantages such as high specific surface area and tunable pore structure and pore chemical environment. Furthermore, MOFs are prepared under mild conditions, avoiding damage to enzyme structure and function; they can interact with amino acid residues on enzyme molecules through -COOH or -NH2 on organic ligands, thereby preventing enzyme leaching during the reaction, and have broad application prospects in the field of immobilized enzymes. Summary of the Invention

[0005] The purpose of this invention is to select suitable organic ligands and metal ions to immobilize glucose oxidase, thereby obtaining an immobilized glucose oxidase with high enzyme activity and stability that can be recycled, thus solving the problems of low enzyme activity recovery rate and easy enzyme leakage of current immobilized enzymes.

[0006] In this invention, biomimetic mineralization technology is used to introduce glucose oxidase in situ into the pore structure of ZIF-7, a metal-organic framework material formed by divalent zinc ions and nitrogen-containing heterocyclic organic ligands (abbreviated as Bim), thereby achieving rapid one-step immobilization of the enzyme. The resulting immobilized enzyme overcomes the shortcomings of free glucose oxidase in terms of reusability and stability.

[0007] The technical solution of the present invention includes the following steps:

[0008] A method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, employing nitrogen-containing heterocyclic organic ligands and divalent metal ions (primarily Zn). 2+ Using glucose oxidase as a raw material, a hydrophilic polymer modified with dopamine at both ends (abbreviated as DA-PEG-DA) is added to in situ to immobilize glucose oxidase, resulting in a mesoporous metal-organic framework material immobilized glucose oxidase. Then, it is mixed and reacted with dopamine to obtain the polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0009] As a further improvement to the technical solution, the preparation method of the polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase described above specifically includes the following steps:

[0010] (1) Raw material preparation:

[0011] Preparation of aqueous solution of nitrogen-containing heterocyclic organic ligand: Dissolve the nitrogen-containing heterocyclic organic ligand in deionized water to prepare an aqueous solution of nitrogen-containing heterocyclic organic ligand;

[0012] Preparation of zinc nitrate hexahydrate with divalent metal ions: Dissolve divalent metal ions in deionized water to prepare an aqueous solution of divalent metal ions;

[0013] Preparation of a mixed solution of a dopamine-modified hydrophilic polymer and glucose oxidase: Place the dopamine-modified hydrophilic polymer and glucose oxidase lyophilized powder in a centrifuge tube, and add Tris-HCl buffer to prepare a mixed solution of the dopamine-modified hydrophilic polymer and glucose oxidase.

[0014] Preparation of dopamine solution: Dissolve dopamine in Tris-HCl buffer and mix well to obtain dopamine solution;

[0015] (2) Preparation of glucose oxidase immobilized by mesoporous metal-organic framework material: A hydrophilic polymer modified with dopamine ends was mixed with glucose oxidase solution, and an aqueous solution of nitrogen-containing heterocyclic organic ligand and divalent metal ion was added. A biomimetic mineralization reaction was carried out under the conditions of pH 3.0-8.0 and temperature 20-60℃ to coordinate polymerization of nitrogen-containing heterocyclic organic ligand and divalent metal ion and to embed glucose oxidase in situ. Then, the mixture was centrifuged, washed, and the precipitate was collected to obtain glucose oxidase immobilized by mesoporous metal-organic framework material, denoted as DA-PEG-DA / GOx@aZIF-7;

[0016] (3) Preparation of polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase: The mesoporous metal-organic framework immobilized glucose oxidase was mixed with a dopamine solution and the mixture was shaken at a pH of 7.0 to 10.0. The dopamine self-polymerized to form polydopamine, which then attached to the mesoporous metal-organic framework immobilized glucose oxidase (DA-PEG-DA / GOx@aZIF-7). The mixture was centrifuged, washed, and freeze-dried to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0017] As a further improvement to the technical solution, the above-described method for preparing a polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase includes nitrogen-containing heterocyclic organic ligands such as benzimidazole, 2-methylimidazole, and 2,5-dihydroxy-1,4-benzylcarboxylic acid.

[0018] As a further improvement to the technical solution, the preparation method of the polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase described above, wherein the hydrophilic polymer modified with dopamine at both ends is one of polyethylene glycol modified with dopamine at both ends, polypyrrolidone modified with dopamine at both ends, or polyvinylimidazole modified with dopamine at both ends.

[0019] As a further improvement to the technical solution, the above-described method for preparing a polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase uses zinc ions, cobalt ions, and copper ions as divalent metal ions.

[0020] As a further improvement to the technical solution, in the above-described method for preparing a polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, the concentration of the nitrogen-containing heterocyclic organic ligand aqueous solution is 0.01-0.1 mol / L; the concentration of the divalent metal ion aqueous solution is 0.01-0.1 mol / L; in the mixed solution of the dopamine-modified hydrophilic polymer and glucose oxidase, the concentration of the dopamine-modified hydrophilic polymer is 0.01-0.08 mmol / L, the concentration of glucose oxidase is 3-8 mg / mL, and the concentration of the dopamine solution is 1-3 mg / mL.

[0021] As a further improvement to the technical solution, in the above-described method for preparing a polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, the molar ratio of the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, dopamine-modified hydrophilic polymer, and dopamine is 3250-49998:1934-25220:1.23-10.08:6.5-52:2637.5-15825.

[0022] As a further improvement to the technical solution, in the above-described method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, the biomimetic mineralization reaction time is 0.5-10 h, and the oscillation reaction time is 2-10 h.

[0023] As a further improvement to the technical solution, in the above-described method for preparing a polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, the hydrophilic polymer in the dopamine-modified hydrophilic polymer has a molecular weight of 2000-12000.

[0024] A method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase as described above, characterized in that: the enzyme is a black amorphous cluster of spherical particles smaller than 100 nm, with a particle size of approximately 461 nm; a specific surface area of ​​74.70 m² / g; a pore size of 17.31 nm, classifying it as a mesoporous material; and a Km value of 10.35 ± 0.80 mM and a Vmax value of 2.86 ± 0.13 μM·min. -1 The Kcat value is 3.77 ± 0.17 s. -1 The Kcat / Km value is 0.36 ± 0.01 s. -1 mM -1 .

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) In this invention, glucose oxidase solution is mixed with DA-PEG-DA, and then mixed with Bim and divalent metal ions in sequence. Mesoporous metal-organic framework material immobilized glucose oxidase (DA-PEG-DA / GOx@aZIF-7) is successfully prepared under mild aqueous conditions. The immobilized enzyme is then mixed with dopamine solution to successfully prepare polydopamine-coated mesoporous metal-organic framework material immobilized glucose oxidase (DA-PEG-DA / GOx@aZIF-7 / PDA). Compared with free glucose oxidase, its stability is improved and enzyme leaching is prevented during the reaction.

[0027] (2) This invention optimizes the immobilization conditions by adjusting the amounts of DA-PEG-DA, Bim, divalent metal ions, and enzyme, thereby maximizing the immobilized enzyme with high catalytic activity. When the enzyme concentration is 5 mg / mL, the enzyme is dissolved in Tris-HCl buffer at pH 7.0, the molecular weight of DA-PEG-DA is 10000, the concentration is 0.04 mmol / L, the concentration of Bim is 38.46 mmol / L, the concentration of divalent metal ions is 4.31 mmol / L, the immobilization time is 40 min, and the immobilization temperature is 35 °C, the enzyme encapsulation efficiency and enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized by mesoporous metal-organic framework material reach the optimal values, which are 99.93 ± 3.01% and 106.46 ± 1.65%, respectively.

[0028] (3) In this invention, glucose oxidase (DA-PEG-DA / GOx@aZIF-7) immobilized with mesoporous metal-organic framework material was mixed with dopamine solution. When the reaction time was adjusted to 10 h and the dopamine concentration was 2.0 mg / mL, the leakage rate of the immobilized enzyme was effectively reduced from 14.53% to 0.11%. At this time, the enzyme activity recovery rate of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized with polydopamine-coated mesoporous metal-organic framework material was 99.02 ± 1.03%.

[0029] (4) The present invention investigated the enzymatic properties of the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA and found that it has significant improvements in stability and other aspects. The three-dimensional pore structure of the DA-PEG-DA / GOx@aZIF-7 / PDA material provides a rigid shielding environment for glucose oxidase, thereby effectively reducing the impact of adverse external environments on enzyme activity and improving its thermal stability, acid and alkali resistance, storage stability, and reusability. Attached Figure Description

[0030] Figure 1The diagram shows the synthesis principle of DA-PEG-DA / GOx@aZIF-7 / PDA according to the present invention (a) and the appearance of DA-PEG-DA / aZIF-7 (b), DA-PEG-DA / GOx@aZIF-7 (c), and DA-PEG-DA / GOx@aZIF-7 / PDA (d).

[0031] Figure 2 These are SEM images of DA-PEG-DA / aZIF-7(a), DA-PEG-DA / GOx@aZIF-7(b), and DA-PEG-DA / GOx@aZIF-7 / PDA(c) prepared according to the present invention.

[0032] Figure 3 These are TEM images of DA-PEG-DA / aZIF-7(a), DA-PEG-DA / GOx@aZIF-7(b), and DA-PEG-DA / GOx@aZIF-7 / PDA(c) prepared according to the present invention.

[0033] Figure 4 These are particle size analysis diagrams of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA prepared in this invention.

[0034] Figure 5 This is the infrared spectrum of the biocomposite material prepared in this invention.

[0035] Figure 6 These are sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) images of free GOx (lane 1), washed DA-PEG-DA / GOx@aZIF-7 (lane 2), and washed GOx-on-DA-PEG-DA / aZIF-7 (lane 3).

[0036] Figure 7 This is a thermogravimetric analysis diagram of the biocomposite material prepared in this invention.

[0037] Figure 8 This is the X-ray diffraction (XRD) pattern of the biocomposite material prepared in this invention.

[0038] Figure 9 These are the XPS full spectrum (a) and high-resolution XPS C 1s (b), XPS N 1s (c), and XPS Zn 2p (d) spectra of the DA-PEG-DA / GOx@aZIF-7 / PDA prepared according to the present invention.

[0039] Figure 10The images show the Ar adsorption-desorption isotherm (a) and pore size distribution curve (b) of the biocomposite material prepared in this invention.

[0040] Figure 11 The figure shows the effects of DA-PEG-DA molecular weight (a), DA-PEG-DA concentration (b), and dopamine concentration (c) on enzyme immobilization efficiency and enzyme activity recovery.

[0041] Figure 12 The figure shows the effect of pH on the catalytic activity of free GOx and its immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0042] Figure 13 The figure shows the effect of temperature on the catalytic activity of free GOx and its immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0043] Figure 14 The figures are Michaelis-Menten curves (a) and Lineweaver-Burk double reciprocal curves (b) for free GOx and its immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0044] Figure 15 This is a graph showing the results of pH stability verification of free GOx and its immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0045] Figure 16 This is a graph showing the results of the thermal stability verification of free GOx and its immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0046] Figure 17 This is a graph showing the results of the reusability stability verification of the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA prepared in this invention.

[0047] Figure 18 This is a graph showing the results of the storage stability verification of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0048] Figure 19 This is a graph showing the results of verifying the stability of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA against adverse environments.

[0049] Figure 20 This is a graph showing the results of substrate selectivity verification for the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. These embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0051] The properties of the immobilized enzyme obtained in this invention were verified in the following manner:

[0052] I. Raw material preparation:

[0053] Preparation of aqueous solution of nitrogen-containing heterocyclic organic ligand: Dissolve the nitrogen-containing heterocyclic organic ligand in deionized water to prepare an aqueous solution of nitrogen-containing heterocyclic organic ligand; the concentration is 0.01-0.1 mol / L, and the solution is prepared according to a range of 0.01 mol / L.

[0054] Preparation of zinc nitrate hexahydrate with divalent metal ions: Dissolve divalent metal ions in deionized water to prepare an aqueous solution of divalent metal ions; the concentration is 0.01-0.1 mol / L, and the solution is prepared according to a range of 0.01 mol / L.

[0055] Preparation of a mixed solution of dopamine-modified hydrophilic polymer and glucose oxidase: Place the dopamine-modified hydrophilic polymer and glucose oxidase lyophilized powder in centrifuge tubes, and add Tris-HCl buffer to prepare a mixed solution of dopamine-modified hydrophilic polymer and glucose oxidase; the concentration of the dopamine-modified hydrophilic polymer is 0.01-0.08 mmol / L, prepared according to a range of 0.02 mmol / L; the concentration of glucose oxidase is 3-8 mg / mL, prepared according to a range of 1 mg / mL.

[0056] Preparation of dopamine solution: Dissolve dopamine in Tris-HCl buffer and mix well to obtain a dopamine solution; the concentration is 1-3 mg / mL, and it is prepared according to a range of 0.5 mol / L.

[0057] II. Measurement methods used in the experiment:

[0058] (1) Determination of glucose oxidase (GOx) activity:

[0059] Enzyme activity was measured using a colorimetric method. Glucose was used as the substrate, and the product of catalytic hydrolysis was hydrogen peroxide (H₂O₂). ABTS was then catalytically oxidized in the presence of horseradish peroxidase (HRP). 2- Formation of ABTS -· It has a characteristic absorption peak at 415 nm, which can be directly measured by colorimetry.

[0060] Preparation of 10mM pH 7.4 PBS buffer: Weigh 1.36g potassium dihydrogen phosphate and add 1000mL of deionized water to a final volume of 10mmol / L potassium dihydrogen phosphate solution. Weigh 3.58g disodium hydrogen phosphate dodecahydrate and add 1000mL of deionized water to a final volume of 10mmol / L disodium hydrogen phosphate dodecahydrate solution. Adjust both solutions to a pH of 7.4 using a pH meter.

[0061] Preparation of 1M glucose solution: Weigh 4.955g of glucose into a beaker, dissolve it with an appropriate amount of PBS, and then bring the volume to 25mL.

[0062] Preparation of 50mM ABTS solution: Weigh 0.2743g of ABTS into a beaker, dissolve it with an appropriate amount of PBS, and then bring the volume to 10mL.

[0063] Preparation of 100 μg / mL HRP solution: Weigh 5 mg HRP into a 10 mL centrifuge tube and add 5 mL PBS to prepare a 1 mg / mL HRP solution. Dilute the 1 mg / mL HRP solution 10 times to prepare a 100 μg / mL HRP solution.

[0064] Enzyme activity assay: Add 995 μL of working solution (including 785 μL of 10 mM pH 7.4 PBS buffer, 100 μL of 1 M glucose solution, 10 μL of 50 mM ABTS solution, and 100 μL of 100 μg / mL HRP solution) and 5 μL of enzyme solution or supernatant and washing solution after centrifugation to a 1 mL slit cuvette. After inverting and mixing well, quickly place the cuvette into the sample cell of a spectrophotometer and detect the change in absorbance at 415 nm over time within 1 min.

[0065] (2) Calculation of enzyme activity:

[0066] The activity of glucose oxidase is defined as the amount of enzyme that catalyzes the production of H2O2 from glucose per unit time (min) at 25°C.

[0067] (3) Enzyme encapsulation efficiency determination:

[0068] Add 995 μL of working solution (including 785 μL of 10 mM pH 7.4 PBS buffer, 100 μL of 1 M glucose solution, 10 μL of 50 mM ABTS solution, and 100 μL of 100 μg / mL HRP solution) and 5 μL of enzyme solution or supernatant and washing solution after centrifugation to a 1 mL slit cuvette. After inverting and mixing well, quickly place the cuvette into the sample cell of a spectrophotometer and detect the change in absorbance at a wavelength of 415 nm over time within 1 min.

[0069] Enzyme immobilization efficiency (%) = (Enzyme activity of added enzyme - Enzyme activity in supernatant - Enzyme activity in washing solution) / Enzyme activity of added enzyme × 100%................................................................................................(1)

[0070] (4) Calculation of the relative enzyme activity of immobilized enzymes:

[0071] Relative enzyme activity of immobilized enzyme (%) = Immobilized enzyme activity / Encapsulated free enzyme activity × 100% ... (2)

[0072] (5) Calculation of enzyme leakage rate

[0073] Enzyme leakage rate (%) = Enzyme activity leaked from the supernatant / Enzyme activity encapsulated × 100% .......................(3)

[0074] (6) Determination of DA-PEG-DA / GOx@aZIF-7 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0075] First, 25 mg of GOx was added to 1 mg of DA-PEG-DA / aZIF-7 powder for adsorption for 40 min. The mixture was then washed three times with deionized water to elute unfixed GOx, yielding GOx-on-DA-PEG-DA / aZIF-7. 1 mg of DA-PEG-DA / GOx@aZIF-7 and GOx-on-DA-PEG-DA / aZIF-7 were dissolved separately in 2 mL of 1M hydrochloric acid. After complete dissolution, the solutions were concentrated by centrifugation (7000 rpm) using a 10 kDa filter. Deionized water was added to the concentrated enzyme solution to prepare a dilute solution with an enzyme concentration of 1 mg / mL. 7.5 μL of the prepared enzyme solution was mixed with the prepared Laemmli sample buffer and heated in water at 95 °C for 5 min. The mixture was then electrophoresed on an SDS-PAGE gel (12% acrylamide) at 80 V, and the gel was stained with Coomassie Brilliant Blue. Excess Coomassie Brilliant Blue was rinsed off the gel with deionized water.

[0076] III. Examples

[0077] Example 1:

[0078] A 0.01 mmol / L DA-PEG-DA (where PEG is polyethylene glycol) with a molecular weight of 2000 was added to a 3 mg / mL glucose oxidase (GOx) solution at pH 3.0 and mixed thoroughly. Then, a 0.01 mol / L Bim (where Bim is benzimidazole) solution and a 0.03 mol / L zinc nitrate solution were added and mixed thoroughly to achieve a molar ratio of 3250:1934:1.23:6.5 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 20 °C for 0.5 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework material, designated DA-PEG-DA / GOx@aZIF-7. The enzyme encapsulation efficiency of the supernatant obtained after centrifugation was determined.

[0079] The glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized in the prepared mesoporous metal-organic framework was reacted with a 1 mg / mL dopamine solution. The molar ratio of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 (based on the amount of glucose oxidase) to dopamine was adjusted to 1.23:2637.5. The reaction was carried out under shaking conditions at pH 7.0 for 2 h. After the reaction was completed, the mixture was centrifuged, washed with deionized water, and the precipitate was collected and freeze-dried under vacuum to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0080] Then, the encapsulation efficiency, relative enzyme activity of the immobilized enzyme, and enzyme leakage rate of glucose oxidase were calculated using the determination methods (1), (2), and (3) used in the experiment. In this embodiment, the encapsulation efficiency of glucose oxidase immobilized by mesoporous metal-organic framework material was 68.08±2.21%, the enzyme activity recovery rate of DA-PEG-DA / GOx@aZIF-7 was 40.13±2.65%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized by polydopamine-coated mesoporous metal-organic framework material was 41.05±2.13%, and the enzyme leakage rate was 11±2.40%.

[0081] Example 2:

[0082] A 0.02 mmol / L DA-PEG-DA (where PEG is polyethylene glycol) with a molecular weight of 10,000 was added to a 4 mg / mL glucose oxidase (GOx) solution at pH 7.0 and mixed thoroughly. Then, a 0.04 mol / L Bim (where Bim is benzimidazole) solution and a 0.04 mol / L zinc nitrate solution were added and mixed thoroughly to achieve a molar ratio of 10,000:5,000:1.42:10 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 30 °C for 2 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework, designated DA-PEG-DA / GOx@aZIF-7. The enzyme encapsulation efficiency of the supernatant obtained after centrifugation was determined.

[0083] The glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized in the prepared mesoporous metal-organic framework was reacted with a 2 mg / mL dopamine solution. The molar ratio of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 (based on the amount of glucose oxidase) to dopamine was adjusted to 1.42:5000. The reaction was carried out under shaking conditions at pH 8.0 for 4 h. After the reaction was completed, the mixture was centrifuged, washed with deionized water, and the precipitate was collected and freeze-dried under vacuum to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0084] Then, the encapsulation efficiency of glucose oxidase, the relative enzyme activity of the immobilized enzyme, and the enzyme leakage rate of glucose oxidase were calculated using the determination methods (1), (2), and (3) used in the experiment. In this embodiment, the encapsulation efficiency of glucose oxidase immobilized by mesoporous metal-organic framework material was 85.83±3.21%, the enzyme activity recovery rate of DA-PEG-DA / GOx@aZIF-7 was 86.46±2.7%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized by polydopamine-coated mesoporous metal-organic framework material was 87.12±1.07%, and the enzyme leakage rate was 5.15±1.30%.

[0085] Example 3

[0086] A 0.04 mmol / L DA-PEG-DA (where PEG is polypyrrolidone) with a molecular weight of 6000 was added to a 5 mg / mL glucose oxidase (GOx) solution at pH 5.0 and mixed thoroughly. Then, a 0.05 mol / L Bim (where Bim is benzimidazole) solution and a 0.056 mol / L cobalt nitrate solution were added and mixed thoroughly to achieve a molar ratio of 20000:10000:1.62:15 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 35 °C for 3 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework, designated DA-PEG-DA / GOx@aZIF-7. The enzyme encapsulation efficiency of the supernatant obtained after centrifugation was determined.

[0087] The glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized in the prepared mesoporous metal-organic framework was reacted with a 3 mg / mL dopamine solution. The molar ratio of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 (based on the amount of glucose oxidase) to dopamine was adjusted to 1.62:10000. The reaction was carried out under shaking conditions at pH 8.5 for 6 h. After the reaction was completed, the mixture was centrifuged, washed with deionized water, and the precipitate was collected and freeze-dried under vacuum to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0088] Then, the encapsulation efficiency of glucose oxidase, the relative enzyme activity of the immobilized enzyme, and the enzyme leakage rate of glucose oxidase were calculated using the determination methods (1), (2), and (3) used in the experiment. In this embodiment, the encapsulation efficiency of the immobilized glucose oxidase was 86.93±3.31%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized with mesoporous metal-organic framework material was 84.46±2.54%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized with polydopamine-coated mesoporous metal-organic framework material was 58.12±2.03%, and the enzyme leakage rate was 0.51±1.00%.

[0089] Example 4

[0090] A 0.04 mmol / L DA-PEG-DA (where PEG is polyethylene glycol) with a molecular weight of 8000 was added to a 5 mg / mL glucose oxidase (GOx) solution at pH 7.0 and mixed thoroughly. Then, a 0.005 mol / L Bim (where Bim is benzimidazole) solution and a 0.056 mol / L zinc nitrate solution were added and mixed thoroughly to achieve a molar ratio of 24999:2801:6.3:26 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 35 °C for 0.7 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework, designated DA-PEG-DA / GOx@aZIF-7. The enzyme encapsulation efficiency of the supernatant obtained after centrifugation was determined.

[0091] The glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized in the prepared mesoporous metal-organic framework was reacted with a 2 mg / mL dopamine solution. The molar ratio of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 (based on the amount of glucose oxidase) to dopamine was adjusted to 6.3:10550. The reaction was carried out under shaking conditions at pH 8.5 for 10 h. After the reaction was completed, the mixture was centrifuged, washed with deionized water, and the precipitate was collected and freeze-dried under vacuum to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0092] Then, the encapsulation efficiency of glucose oxidase, the relative enzyme activity of the immobilized enzyme, and the enzyme leakage rate of glucose oxidase were calculated using the determination methods (1), (2), and (3) used in the experiment. In this embodiment, the encapsulation efficiency of glucose oxidase immobilized by mesoporous metal-organic framework material was 99.93±3.01%, the enzyme activity recovery rate of DA-PEG-DA / GOx@aZIF-7 was 106.46±1.65%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized by polydopamine-coated mesoporous metal-organic framework material was 99.02±1.03%, and the enzyme leakage rate was 0.11±1.00%.

[0093] Example 5

[0094] A 0.04 mmol / L DA-PEG-DA (where PEG is polyvinylimidazole) with a molecular weight of 10,000 was added to a 6 mg / mL glucose oxidase (GOx) solution at pH 6.0 and mixed thoroughly. Then, a 0.08 mol / L Bim (where Bim is 2-methylimidazole) solution and a 0.08 mol / L copper nitrate solution were added and mixed thoroughly to achieve a molar ratio of 35,000:20,000:2.0:35 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 50 °C for 6 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework material, designated DA-PEG-DA / GOx@aZIF-7. The enzyme encapsulation efficiency of the supernatant obtained after centrifugation was determined.

[0095] The glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized in the prepared mesoporous metal-organic framework was reacted with a dopamine solution at a concentration of 2 mg / mL. The molar ratio of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 (based on the amount of glucose oxidase) to dopamine was adjusted to 2.0:12000. The reaction was carried out under shaking conditions at pH 9.0 for 8 h. After the reaction was completed, the mixture was centrifuged, washed with deionized water, and the precipitate was collected and freeze-dried under vacuum to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0096] Then, the encapsulation efficiency of glucose oxidase, the relative enzyme activity of the immobilized enzyme, and the enzyme leakage rate of glucose oxidase were calculated using the determination methods (1), (2), and (3) used in the experiment. In this embodiment, the encapsulation efficiency of the immobilized glucose oxidase was 96.93±2.01%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized with mesoporous metal-organic framework material was 68.46±2.45%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized with polydopamine-coated mesoporous metal-organic framework material was 75.12±3.13%, and the enzyme leakage rate was 1.15±1.40%.

[0097] Example 6

[0098] A 0.08 mmol / L DA-PEG-DA (where PEG is polyethylene glycol) with a molecular weight of 10000 was added to an 8 mg / mL glucose oxidase (GOx) solution at pH 8.0 and mixed thoroughly. Then, a 0.1 mol / L Bim (2,5-dihydroxy-1,4-benzylcarboxylic acid) solution and a 0.1 mol / L zinc nitrate solution were added and thoroughly mixed to achieve a molar ratio of 49998:25220:10.08:52 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 60 °C for 10 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework material, designated DA-PEG-DA / GOx@aZIF-7. The enzyme encapsulation efficiency of the supernatant obtained after centrifugation was determined.

[0099] The glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized in the prepared mesoporous metal-organic framework was reacted with a 2.5 mg / mL dopamine solution. The molar ratio of the glucose oxidase DA-PEG-DA / GOx@aZIF-7 (based on the amount of glucose oxidase) to dopamine was adjusted to 10.08:15825. The reaction was carried out under shaking conditions at pH ~ 10.0 for 10 h. After the reaction was completed, the mixture was centrifuged, washed with deionized water, and the precipitate was collected and freeze-dried under vacuum to obtain polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase, denoted as DA-PEG-DA / GOx@aZIF-7 / PDA.

[0100] Then, the encapsulation efficiency of glucose oxidase, the relative enzyme activity of the immobilized enzyme, and the enzyme leakage rate of glucose oxidase were calculated using the determination methods (1), (2), and (3) used in the experiment. In this embodiment, the encapsulation efficiency of the immobilized glucose oxidase was 99.70±2.01%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 immobilized with mesoporous metal-organic framework material was 56.56±2.15%, the enzyme activity recovery rate of glucose oxidase DA-PEG-DA / GOx@aZIF-7 / PDA immobilized with polydopamine-coated mesoporous metal-organic framework material was 59±2.13%, and the enzyme leakage rate was 0.15±0.60%.

[0101] Comparative Example:

[0102] Comparative Example 1

[0103] A 0.04 mmol / L DA-PEG-DA (where PEG is polyethylene glycol) with a molecular weight of 2000 was added to a 3 mg / mL glucose oxidase (GOx) solution at pH 3.0 and mixed thoroughly. Then, a 0.01 mol / L Bim (where Bim is benzimidazole) solution and a 0.01 mol / L zinc nitrate solution were added and mixed thoroughly to achieve a molar ratio of 3250:1934:1.23:6.5 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 20 °C for 0.5 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework material, designated DA-PEG-DA / GOx@aZIF-7.

[0104] Comparative Example 2

[0105] A 0.02 mmol / L DA-PEG-DA (where PEG is polyethylene glycol) with a molecular weight of 10,000 was added to a 4 mg / mL glucose oxidase (GOx) solution at pH 7.0 and mixed thoroughly. Then, a 0.04 mol / L Bim (where Bim is benzimidazole) solution and a 0.04 mol / L zinc nitrate solution were added and mixed thoroughly to achieve a molar ratio of 10,000:5,000:1.42:10 for the nitrogen-containing heterocyclic organic ligand, divalent metal ions, glucose oxidase, and dopamine-modified hydrophilic polymer. The mixture was subjected to a biomimetic mineralization reaction at 30 °C for 2 h. After the reaction, the mixture was centrifuged, washed with deionized water, and the collected precipitate was freeze-dried under vacuum to obtain glucose oxidase immobilized in a mesoporous metal-organic framework material, designated DA-PEG-DA / GOx@aZIF-7.

[0106] IV. Characterization of Mesoporous Metal-Organic Framework Materials

[0107] Figure 1 The diagram shows the synthesis principle of DA-PEG-DA / GOx@aZIF-7 / PDA (a) and the appearance of DA-PEG-DA / aZIF-7 (b), DA-PEG-DA / GOx@aZIF-7 (c), and DA-PEG-DA / GOx@aZIF-7 / PDA (d).

[0108] As shown in the figure, DA-PEG-DA / aZIF-7 is a white granular sample; DA-PEG-DA / GOx@aZIF-7 is a yellow granular sample; and DA-PEG-DA / GOx@aZIF-7 / PDA is a black granular sample.

[0109] Figure 2 The images show SEM images of the prepared DA-PEG-DA / aZIF-7(a), DA-PEG-DA / GOx@aZIF-7(b), and DA-PEG-DA / GOx@aZIF-7 / PDA(c).

[0110] Figure 3 These are TEM images of DA-PEG-DA / aZIF-7(a), DA-PEG-DA / GOx@aZIF-7(b), and DA-PEG-DA / GOx@aZIF-7 / PDA(c). Comparison shows that the SEM and TEM images of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are basically consistent, all showing clusters of irregular spherical particles smaller than 100 nm, indicating that the introduction of glucose oxidase and polydopamine did not disrupt the morphology of aZIF-7. The TEM images show that the particle size of DA-PEG-DA / GOx@aZIF-7 increases after the addition of GOx, which may be because the introduction of GOx accelerates the synthesis of aZIF-7. TEM images of the DA-PEG-DA / GOx@aZIF-7 / PDA sample show that PDA forms on the outer surface of DA-PEG-DA / GOx@aZIF-7 and tethers the nanoparticles into larger aggregates.

[0111] Figure 4 The figures show particle size analysis results for the prepared DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA. As shown in the figures, the cluster particles of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are 270.7 nm, 356.9 nm, and 461 nm, respectively. The particle size increases sequentially, consistent with TEM results.

[0112] Figure 5 This is the infrared spectrum of the biocomposite material. The characteristic peak of DA-PEG-DA / aZIF-7 appears at 1461 cm⁻¹. -1 742cm -1 (C=C and CH of the benzene functional group), 1241cm -1 (CC stretching vibration of the imidazole ring peak), 428 cm⁻¹ -1 (Zn-N bond), 1114cm -1 (C=O stretching mode in DA-PEG-DA) and 2889cm -1(Stretching mode of the -CH2 group in DA-PEG-DA); the characteristic peak of GOx appears at 1635 cm⁻¹. -1 (C=O stretching mode of amide I), 1511cm -1 and 1540cm -1 (Amide II produced by the combined bending of NH and stretching of CN), DA-PEG-DA / GOx@aZIF-7 contains characteristic peaks of DA-PEG-DA / aZIF-7 and GOx, and also at 1652 cm⁻¹. -1 The characteristic peak at that location corresponds to Zn 2+ The coordination with the carboxyl group on the enzyme indicates that GOx is not simply adsorbed, but rather intercalated into DA-PEG-DA / GOx@aZIF-7; DA-PEG-DA / GOx@aZIF-7 / PDA contains the characteristic peaks of DA-PEG-DA / GOx@aZIF-7, and furthermore, in the 3150-3700 cm⁻¹ range... -1 The broad infrared spectral bands correspond to the extension of alcohol, catechol, and NH bonds in the PDA structure. These results indicate, from a functional group perspective, the successful preparation of the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA.

[0113] Figure 6 This is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of free GOx (lane 1), washed DA-PEG-DA / GOx@aZIF-7 (lane 2), and washed GOx-on-DA-PEG-DA / aZIF-7 (lane 3). Clear protein bands appear in lanes 1 and 2, while no protein bands appear in lane 3. This result clearly demonstrates that GOx is indeed embedded in DA-PEG-DA / GOx@aZIF-7, and that the embedded GOx molecules are not easily removed from the biocomposite material. Conversely, GOx molecules on the surface of GOx-on-DA-PEG-DA / aZIF-7 are easily removed because they are only physically adsorbed onto the DA-PEG-DA / aZIF-7 surface.

[0114] Figure 7This is a thermogravimetric analysis diagram of a biocomposite material. From 35℃ to 275℃, DA-PEG-DA / aZIF-7 lost approximately 5.95% of its mass due to the evaporation of water molecules in the sample; from 275℃ to 800℃, DA-PEG-DA / aZIF-7 decomposed, resulting in a loss of approximately 76.42% of its mass. From 35℃ to 113℃, DA-PEG-DA / GOx@aZIF-7 lost approximately 1.51% of its mass due to the evaporation of water molecules in the sample; from 113℃ to 800℃, DA-PEG-DA / aZIF-7 decomposed, resulting in a loss of approximately 82.58% of its mass. From 35℃ to 132.3℃, DA-PEG-DA / GOx@aZIF-7 / PDA lost approximately 2.57% of its mass due to the evaporation of water molecules in the sample; from 132.3℃ to 800℃, DA-PEG-DA / GOx@aZIF-7 / PDA decomposed, resulting in a loss of approximately 81.83% of its mass. Analysis of the three weight loss curves for DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA shows that GOx was successfully immobilized in DA-PEG-DA / aZIF-7. The weight of GOx in DA-PEG-DA / GOx@aZIF-7 accounts for approximately 6.16% of the total weight of the material, while the weight of GOx in DA-PEG-DA / GOx@aZIF-7 / PDA accounts for approximately 5.41% of the total weight of the material.

[0115] Figure 8 The figures show the X-ray diffraction (XRD) patterns of the biocomposite materials. As can be seen, ZIF-7 obtained without the addition of DA-PEG-DA during the in-situ aqueous synthesis exhibits a partially crystalline structure, with characteristic peak positions similar to ZIF-7-Ⅲ. However, the X-ray diffraction patterns of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA obtained with the addition of DA-PEG-DA indicate the presence of more amorphous structures. This suggests that the introduction of DA-PEG-DA can cause the loss of long-range order in ZIF-7, inducing coordination defects in the DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA materials.

[0116] Figure 9XPS full spectrum (a) and high-resolution XPS C 1s (b), XPS N 1s (c), and XPS Zn 2p (d) spectra of the prepared DA-PEG-DA / GOx@aZIF-7 / PDA were obtained to detect the chemical structure of DA-PEG-DA / GOx@aZIF-7 / PDA. The XPS full spectrum of DA-PEG-DA / GOx@aZIF-7 / PDA shows that the biocomposite material contains C, N, O, and Zn elements, consistent with the elements added during synthesis. The XPS C 1s spectrum can be fitted as CC (284.74 eV), CO / CN (286.22 eV), and O=CO (288.11 eV). The XPS N 1s spectrum can be fitted as N-Zn (399.21 eV), imidazole C=N (400.34 eV), and amine CN (398.65 eV). The XPS Zn 2p spectrum shows that Zn 2p... 3 / 2 and Zn 2p 1 / 2 The peak binding energies were 1022.03 and 1045.08, respectively, consistent with the previously reported Zn 2p of ZIF-7, indicating that the amorphous DA-PEG-DA / GOx@aZIF-7 / PDA exhibits Zn and N coordination similar to that of ZIF-7. This demonstrates the successful synthesis of the biocomposite material.

[0117] Figure 10 Figure (a) shows the Ar adsorption-desorption isotherm and pore size distribution curve (b) of the biocomposite materials. As can be seen from Figure (a), the intersection points of the adsorption and desorption isotherms of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are all between 0.6 and 0.8, indicating that DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are mesoporous materials. Combining this with the pore size distribution diagram in Figure (b), it can be seen that the pores in DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are mainly mesopores, classifying them as mesoporous materials. The presence of some mesopores may be due to material aggregation. Meanwhile, the specific surface areas of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are 103.77 m², respectively. 2 / g、73.20m 2 / g and 74.70m 2 / g. The pore sizes of DA-PEG-DA / aZIF-7, DA-PEG-DA / GOx@aZIF-7, and DA-PEG-DA / GOx@aZIF-7 / PDA are 23.51 nm, 18.64 nm, and 17.31 nm, respectively. The decrease in specific surface area and pore size indicates that the added GOx and PDA blocked some of the pores in DA-PEG-DA / aZIF-7, further demonstrating the successful synthesis of DA-PEG-DA / GOx@aZIF-7 / PDA.

[0118] V. Enzymatic properties of immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA

[0119] (1) Optimal catalytic pH values ​​for free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA:

[0120] pH is a crucial factor affecting enzyme activity. Enzyme conformation is easily altered by pH changes, leading to a loss of enzyme activity. For example... Figure 12 As shown, the optimal reaction pH for both the free and immobilized enzymes is 6.0, indicating that the conformation of GOx in DA-PEG-DA / GOx@aZIF-7 / PDA did not change significantly. Compared to free GOx, DA-PEG-DA / GOx@aZIF-7 / PDA exhibited higher relative activity at pH values ​​of 5.0–8.0.

[0121] (2) Optimal catalytic reaction temperature of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA:

[0122] Temperature is another important factor affecting the catalytic activity of enzymes. This invention investigated the catalytic activity of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDAA in different temperature systems. Figure 13 As shown, the optimal reaction temperature for free GOx is 40℃, and the optimal reaction temperature for immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA is 45℃.

[0123] (3) Kinetic parameters of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA:

[0124] Glucose substrates at concentrations of 0-100 mM were prepared, and a catalytic reaction was carried out over a short period. The enzyme activities of free GOx and the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA were measured. The Km, kcat, and kcat / Km values ​​of both were determined using the Lineweaver-Burk method. The results are as follows: Figure 14The Mie curve (a) and the Lineweaver-Burk double reciprocal curve (b) are shown in Table 1.

[0125] Table 1. Kinetic parameters of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA

[0126]

[0127] As shown in Table 1, the Km value of DA-PEG-DA / GOx@aZIF-7 / PDA is significantly lower than that of free GOx, indicating that DA-PEG-DA / GOx@aZIF-7 / PDA has better affinity for the substrate. Furthermore, the catalytic constant (kcat) and catalytic efficiency (Kcat / Km) of DA-PEG-DA / GOx@aZIF-7 / PDA are both improved compared to free GOx, indicating that DA-PEG-DA / GOx@aZIF-7 / PDA has higher catalytic efficiency than free GOx.

[0128] (4) pH stability of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA

[0129] GOx is a protein whose catalytic performance is easily affected by the pH value of the environment. Too high or too low pH values ​​can destroy the three-dimensional structure of the enzyme or prevent the substrate from binding to the enzyme, thereby reducing the activity of the enzyme molecules or even inactivating them. Figure 15 The stability of free enzyme and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA after treatment at different pH values ​​for 30 min was investigated. As shown in the figure, pH value has a greater impact on the catalytic performance of the free enzyme than on DA-PEG-DA / GOx@aZIF-7 / PDA. DA-PEG-DA / GOx@aZIF-7 / PDA maintained high relative activity (>75%) within a pH range of 4.5–8.5, but for free GOx, the relative activity was only 69.50 ± 3.00% after treatment in a buffer solution at pH 8.5 for 30 min. Therefore, the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA exhibits good stability over a relatively wide pH range.

[0130] (5) Temperature stability of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA

[0131] Figure 16 The temperature stability of the free enzyme GOx and the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA was evaluated. Figure 16It is evident that below 30℃, the relative activities of both the free enzyme GOx and the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA decrease slowly. However, above 30℃, the decreasing trend in relative activity of the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA is less pronounced compared to the free enzyme. At 70℃, the relative activity of the free enzyme GOx is only 17.69±3.76%, while DA-PEG-DA / GOx@aZIF-7 / PDA still retains a relative activity of 54.83±2.41%. This indicates that the thermostability of the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA is significantly higher than that of the free enzyme. This is because high temperatures disrupt the spatial structure of GOx, leading to irreversible inactivation, while DA-PEG-DA / GOx@aZIF-7 / PDA provides a rigid protective layer for GOx, thus improving its thermostability.

[0132] (6) Reusability stability of immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA:

[0133] like Figure 17 As shown, excluding factors that could impair enzyme activity, such as centrifugation, DA-PEG-DA / GOx@aZIF-7 / PDA maintained a relative activity of 96.68 ± 2.33% after 10 cycles, even with 100% enzyme activity in the first degradation cycle. The immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA is simple to use, can be effectively recovered, and is reusable, meeting the requirements of economic efficiency and green chemistry.

[0134] (7) Storage stability of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA:

[0135] Storage conditions and stability of enzymes are crucial issues that must be addressed in the industrial application of enzymes. In this example, free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA were stored at 4°C, and their relative enzyme activities were measured over 10 days. Figure 18 As shown, under 4℃ conditions, the relative activity of the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA remained at 98.02±3.41% after 10 days of storage, while the free GOx only maintained a relative activity of 62.21±3.26%. This demonstrates that the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA can be successfully preserved without losing activity within a certain temperature and time range, which may be due to the protective effect of the material.

[0136] (8) Stability of free GOx and immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA against adverse environments:

[0137] Enzymes exposed to adverse environments, such as polar organic solvents, urea, and trypsin, typically experience reduced activity or even inactivation. This example investigated the tolerance of free GOx and the immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA to an aqueous solution containing 1 mg / mL trypsin, 6 M urea, 50% dimethyl sulfoxide, 50% methanol, and 50% acetone. Figure 19 As shown, after treatment, the residual activity of DA-PEG-DA / GOx@aZIF-7 / PDA was higher than that of free GOx. This indicates that DA-PEG-DA / GOx@aZIF-7 / PDA has good protective and shielding effects, which may be due to the improved enzyme molecular stability resulting from the interaction between the enzyme and the carrier.

[0138] (9) Substrate selectivity of immobilized enzyme DA-PEG-DA / GOx@aZIF-7 / PDA:

[0139] Due to its safety, high efficiency, and high specificity, GOx is often used as a bio-element in glucose detection biosensors. To avoid interference from other substances during the detection process, this embodiment tested the selectivity of DA-PEG-DA / GOx@aZIF-7 / PDA for glucose in the presence of different analytes (including glucose, sucrose, fructose, galactose, arabinose, xylose, rhamnose, and maltose) at the same concentration. The relative enzyme activity of DA-PEG-DA / GOx@aZIF-7 / PDA catalyzing the glucose reaction was calculated as 100%, and the relative enzyme activity of DA-PEG-DA / GOx@aZIF-7 / PDA catalyzing the reaction with interfering substances was also calculated. Figure 20 As shown, DA-PEG-DA / GOx@aZIF-7 / PDA showed almost no response to the catalytic interferences sucrose, fructose, galactose, arabinose, xylose, and rhamnose, and its relative activity in catalyzing maltose was extremely low, below 6%. Furthermore, visual observation of the reaction solution at the same time point revealed that, at the same concentration, only the reaction solution containing glucose exhibited a distinct blue color. These results demonstrate that DA-PEG-DA / GOx@aZIF-7 / PDA exhibits a sensitive and specific color response to glucose, demonstrating good substrate selectivity.

[0140] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing glucose oxidase immobilized on a polydopamine-coated mesoporous metal-organic framework, characterized in that: Specifically, the steps include the following: (1) Raw material preparation: Preparation of aqueous solutions of nitrogen-containing heterocyclic organic ligands: Dissolve the nitrogen-containing heterocyclic organic ligands in deionized water to prepare an aqueous solution of nitrogen-containing heterocyclic organic ligands; Preparation of divalent metal ion aqueous solution: Dissolve divalent metal ions in deionized water to prepare divalent metal ion aqueous solution; Preparation of a mixed solution of a dopamine-modified hydrophilic polymer and glucose oxidase: Place the dopamine-modified hydrophilic polymer and glucose oxidase lyophilized powder in a centrifuge tube, and add Tris-HCl buffer to prepare a mixed solution of the dopamine-modified hydrophilic polymer and glucose oxidase. Preparation of dopamine solution: Dissolve dopamine in Tris-HCl buffer and mix well to obtain dopamine solution; (2) Preparation of glucose oxidase immobilized by mesoporous metal-organic framework: A hydrophilic polymer modified with dopamine at both ends was mixed with glucose oxidase, and an aqueous solution of nitrogen-containing heterocyclic organic ligand and divalent metal ion was added. A biomimetic mineralization reaction was carried out under the conditions of pH 3.0-8.0 and temperature 20-60℃ to coordinate the polymerization of nitrogen-containing heterocyclic organic ligand and divalent metal ion and embed glucose oxidase in situ. Then, the mixture was centrifuged, washed, and the precipitate was collected to obtain glucose oxidase immobilized by mesoporous metal-organic framework. (3) Preparation of polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase: The mesoporous metal-organic framework immobilized glucose oxidase was mixed with dopamine solution and the reaction was shaken at pH 7.0-10.0; the dopamine self-polymerized to form polydopamine, and the polydopamine attached to the mesoporous metal-organic framework immobilized glucose oxidase. After centrifugation, washing, and freeze drying, polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase was obtained. The nitrogen-containing heterocyclic organic ligand is benzimidazole; The dopamine-modified hydrophilic polymer is polyethylene glycol modified with dopamine at both ends; The divalent metal ion mentioned is a zinc ion.

2. The method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase according to claim 1, characterized in that: The concentration of the nitrogen-containing heterocyclic organic ligand aqueous solution is 0.01-0.1 mol / L; the concentration of the divalent metal ion aqueous solution is 0.01-0.1 mol / L; in the mixed solution of the dopamine-modified hydrophilic polymer and glucose oxidase, the concentration of the dopamine-modified hydrophilic polymer is 0.01-0.08 mmol / L, the concentration of glucose oxidase is 3-8 mg / mL; and the concentration of the dopamine solution is 1-3 mg / mL.

3. The method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase according to claim 1, characterized in that: The molar ratio of the nitrogen-containing heterocyclic organic ligand, divalent metal ion, glucose oxidase, dopamine-modified hydrophilic polymer, and dopamine is 3250-49998:1934-25220:1.23-2.52:6.5-52:2637.5-15825.

4. The method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase according to claim 3, characterized in that: The biomimetic mineralization reaction time is 0.5-10 h, and the oscillation reaction time is 2-10 h.

5. The method for preparing polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase according to claim 1, characterized in that: The hydrophilic polymer in the dopamine-modified hydrophilic polymer has a molecular weight of 2000 to 12000.

6. A polydopamine-coated mesoporous metal-organic framework immobilized glucose oxidase prepared by a method according to any one of claims 1-5.

Images