Preparation method and application of iron-based covalent organic framework enzyme-carrying biological cascade catalytic material

Abstract

This invention discloses a method for preparing and applying an iron-based covalent organic framework-loaded enzyme biocatalytic cascade material, belonging to the field of materials science. This invention employs a one-pot method to prepare an iron-based covalent organic framework material, which is then further loaded with glucose oxidase to obtain a biocatalytic cascade material suitable for glucose detection. The biocatalytic cascade material prepared by this invention combines the advantages of nanozymes and natural enzymes, exhibiting excellent catalytic efficiency and lifetime. Furthermore, the Fe-COFs prepared by this invention possess strong peroxidase-like activity and high enzyme loading capacity, making them adaptable to various enzyme-loaded cascade systems, thus broadening the application prospects of biocatalytic cascade systems based on COFs-loaded natural enzymes.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying an iron-based covalent organic framework-loaded enzyme biocatalytic cascade material, belonging to the field of materials. Background Technology

[0002] In recent years, the combination of biocatalysis and chemocatalysis has attracted widespread attention due to its excellent substrate selectivity, high catalytic efficiency, and sustained stability. However, the practical application of this combination is limited by the inherent instability and insufficient support capacity of natural enzymes, as well as the low catalytic activity of the catalysts. Constructing cascade catalytic systems by co-immobilizing nanozymes and natural enzymes on porous materials is considered an effective strategy to overcome these limitations. Various materials have been explored as supports for immobilizing natural enzymes, including silica, carbon nanotubes, nanofibers, hydrogels, and metal-organic frameworks (MOFs). However, these support materials face some obstacles in practical applications, such as low enzyme loading capacity, poor enzyme compatibility, and unclear mass transfer channel structures. Further exploration of universal methods to overcome these challenges is still needed.

[0003] Covalent organic frameworks (COFs) are crystalline porous networks composed of organic molecules, possessing tunable physicochemical properties, high crystallinity, large specific surface area, superior adsorption capacity, and significant stability. Due to their tunable structure, flexible functionality, and well-defined channels, COFs can immobilize specific natural enzymes through covalent bonds or other interactions, allowing for high-density natural enzyme loading and efficient substrate diffusion. They serve as ideal host materials for natural enzymes, suitable for complex, sequential reactions necessary in biotransformation or cascade processes, effectively improving enzyme stability and reusability. Furthermore, COFs are typically composed of non-toxic, light elements such as C, H, O, N, and B, conforming to green chemistry principles. Their selective encapsulation capabilities and strong customizability make COFs ideal platforms for biocatalysis, biosensors, and environmental remediation applications. Therefore, further research is urgently needed to develop suitable COFs and effectively composite them with natural enzymes to construct bio-cascade catalysts for green chemical production.

[0004] Glucose oxidase (GOx) and horseradish peroxidase (HRP) are commonly used in enzyme cascade catalytic reactions, particularly for blood glucose monitoring in humans. The principle is that glucose oxidase catalyzes the oxidation of glucose to produce hydrogen peroxide (H₂O₂), while horseradish peroxidase catalyzes the oxidation of the colorimetric substrate TMB, thus detecting glucose. Existing technology CN117645992A, a cascade enzyme catalytic nanocomposite material and its preparation method and application, discloses a material using COFs to load glucose oxidase and horseradish peroxidase as a cascade catalytic agent. However, simultaneously loading two enzymes onto a single carrier to construct a cascade system increases the risk of enzyme inactivation and leaching. In the cascade reaction, if the spatial distribution of the two enzymes within the COFs is unreasonable, the diffusion efficiency of the intermediate product (H₂O₂) may become the rate-limiting step. Therefore, a more stable and efficient bio-cascade catalytic material for glucose detection is needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, an iron-based covalent organic framework material was prepared using a one-pot method, and then glucose oxidase was loaded onto it to prepare a bio-cascade catalytic material that can be used for glucose detection.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a method for preparing an iron-based covalent organic framework-loaded enzyme-based biocatalytic cascade material, comprising the following steps:

[0008] (1) Select iron-containing metal salts and COFs monomers containing polyaldehyde groups and COFs monomers containing polyamino groups, and obtain iron-based covalent organic frameworks by solvothermal reaction under vacuum.

[0009] (2) The iron-based covalent organic framework obtained in step (1) is mixed with glucose oxidase in an acidic buffer solution, and then centrifuged and dried to obtain the iron-based covalent organic framework enzyme-loaded biocatalytic cascade material.

[0010] In one embodiment of the present invention, the iron-containing metal salt in step (1) is FeCl3·6H2O.

[0011] In one embodiment of the present invention, the polyaldehyde-containing COFs monomer in step (1) is 2,2′-bipyridine-5,5′-dicarboxaldehyde.

[0012] In one embodiment of the present invention, the polyamino COFs monomer in step (1) is one or both of 1,3,6,8-tetra-(p-aminophenyl)pyrene and tetra-(4-aminophenyl)ethylene.

[0013] In one embodiment of the present invention, the solvothermal reaction in step (1) involves reacting the iron-containing metal salt with the polyaldehyde-containing COFs monomer and the polyamino-containing COFs monomer in a mixed solvent, wherein the mixed solvent is a mixture of 1,4-dioxane and mesitylene in a volume ratio of 1:0.5 to 2.

[0014] In one embodiment of the present invention, the mass ratio of the iron-containing metal salt, the polyamino-containing COFs monomer, and the polyaldehyde-containing COFs monomer in step (1) is 15-20:15-25:10-30. More preferably, it is 15-20:16-24:12-26, and even more preferably, it is 15:17:12.8 and 20:23.55:25.6.

[0015] In one embodiment of the present invention, the mass-to-volume ratio of the iron-containing metal salt and the mixed solvent in the mixed solution in step (1) is 10-15 mg / mL.

[0016] In one embodiment of the present invention, the reaction in step (1) uses an acetic acid solution as a catalyst.

[0017] In one embodiment of the present invention, the concentration of the acetic acid solution is 2M to 4M.

[0018] In one embodiment of the present invention, the amount of catalyst added in step (1) is 5% to 15% of the volume of the mixed solvent.

[0019] In one embodiment of the present invention, the mass-to-volume ratio of amino-based COFs monomers to mixed solvents in the mixed solution of step (1) is 5-15 mg / mL.

[0020] In one embodiment of the present invention, the mass-to-volume ratio of aldehyde-containing COFs monomers to the mixed solvent in the mixed solution of step (1) is 5-15 mg / mL.

[0021] In one embodiment of the present invention, the reaction described in step (1) is carried out at 90-130°C for 60-80 hours.

[0022] In one embodiment of the present invention, step (1) is as follows:

[0023] Add polyaldehyde and polyamino COF monomers to a container, along with a mixed solvent, and ultrasonically mix until homogeneous to obtain a mixed solution of the two monomers. Dissolve an iron-containing metal salt in the mixed solvent to obtain a mixed solution containing iron ions. Then, add the mixed solution containing iron ions to the mixed solution of the two monomers, ultrasonically mix until homogeneous, transfer to a Pyrex tube, and add an appropriate amount of acetic acid to carry out the Fe-COF synthesis reaction. After the reaction is complete, wash, centrifuge, and dry to obtain the Fe-COF material.

[0024] In one embodiment of the present invention, the acidic buffer solution in step (2) is an acetate / sodium acetate buffer solution with a pH of 3.0-5.0.

[0025] In one embodiment of the present invention, the mass ratio of the iron-based covalent organic framework to glucose oxidase in step (2) is 1:0.5-2, and the mass-volume ratio of the iron-based covalent organic framework and glucose oxidase to the acidic buffer is 0.2-0.6 mg / mL.

[0026] In one embodiment of the present invention, the iron-based covalent organic framework and glucose oxidase are stirred in an acidic buffer solution in a water bath at a temperature of 20-65°C.

[0027] In one embodiment of the present invention, the iron-based covalent organic framework and glucose oxidase are stirred in an acidic buffer solution in a water bath for 3-6 hours.

[0028] In one embodiment of the present invention, the drying in step (2) is vacuum drying, with specific parameters: temperature of 45-55℃ and drying time of 10-12h.

[0029] In one embodiment of the present invention, step (2) specifically comprises:

[0030] The Fe-COFs obtained in step (1) were mixed with glucose oxidase in an acidic buffer solution and stirred in a water bath. After washing, centrifugation and drying, iron-based covalent organic framework enzyme-loaded biocatalytic cascade material was obtained.

[0031] The second objective of this invention is to prepare an iron-based covalent organic framework-loaded enzyme biocatalytic cascade material by means of the method described in this invention.

[0032] The third objective of this invention is to apply the iron-based covalent organic framework enzyme-loaded biocatalytic cascade material described herein to the fields of bioanalysis, biocatalysis, enzyme loading, medical diagnostics, and drug delivery.

[0033] The beneficial effects of this invention are:

[0034] (1) This invention adopts a one-pot synthesis method: selecting a metal salt containing iron ions and synthesizing it with COFs monomers in one pot, eliminating the cumbersome steps of the post-coordination method, and the preparation method is simple and easy to operate. The post-coordination method requires first synthesizing pure organic COFs without metals, and then introducing metals through impregnation, ion exchange, etc., which is cumbersome and time-consuming; in the post-coordination method, metal ions are loaded onto the pores or surface of the pre-synthesized COFs through adsorption or coordination, which is prone to uneven metal distribution due to diffusion limitation; the metal loading in the post-coordination method is limited by the adsorption capacity of COFs and the diffusion efficiency of metal precursors, which is prone to saturation bottleneck; the metal introduced later in the post-coordination method will to some extent destroy the periodic arrangement of the original framework, resulting in a decrease in the crystallinity of COFs.

[0035] (2) The Fe-COFs prepared in this invention have excellent peroxidase-like activity and do not have oxidase activity interference. The maximum reaction rate and affinity with the substrate are superior to those of natural peroxidase.

[0036] (3) The iron-based covalent organic framework material prepared in this invention has an enzyme loading efficiency of up to 93% (w / w). After 10 cycles, it can still maintain 90% of the catalytic activity of the initial cascade catalytic material, which has excellent catalytic efficiency and catalytic life compared with natural enzymes. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a scanning electron microscope image of Fe-COF1 from Example 1;

[0039] Figure 2 This is a scanning electron microscope image of Fe-COF2 from Example 2;

[0040] Figure 3 Image of Fe-COF1@GOx loaded with glucose oxidase from Example 1;

[0041] Figure 4 Image of Fe-COF2@GOx loaded with glucose oxidase from Example 2;

[0042] Figure 5 This is a bar chart comparing the activity levels of Fe-COF1 peroxidase-like activity under optimal catalytic temperature conditions in Example 1.

[0043] Figure 6This is a bar chart comparing the activity levels of Fe-COF2 peroxidase-like enzymes under optimal catalytic temperature conditions in Example 2.

[0044] Figure 7 This is a bar chart comparing the activity levels of Fe-COF1 peroxidase-like activity under optimal catalytic pH conditions in Example 1.

[0045] Figure 8 This is a bar chart comparing the activity levels of Fe-COF2 peroxidase-like activity under optimal catalytic pH conditions in Example 2.

[0046] Figure 9 This is a bar chart comparing the activity levels of the glucose oxidase cascade catalytic activity supported on iron-based COFs in Example 1 under the optimal catalytic temperature conditions.

[0047] Figure 10 This is a bar chart comparing the activity levels of the glucose oxidase cascade catalytic activity supported on iron-based COFs in Example 2 under the optimal catalytic temperature conditions.

[0048] Figure 11 This is a bar chart comparing the activity levels of the glucose oxidase cascade supported on iron-based COFs in Example 1 under optimal catalytic pH conditions.

[0049] Figure 12 This is a bar chart comparing the activity levels of the glucose oxidase cascade catalytic activity supported on iron-based COFs in Example 2 under optimal catalytic pH conditions.

[0050] Figure 13 A comparison of the catalytic effects of Fe-COF1, Fe-COF2, and GOx under acidic and neutral incubation conditions;

[0051] Figure 14 Enzymatic kinetics of Fe-COFs over a given time period;

[0052] Figure 15 Steady-state kinetic analysis of Fe-COFs under different H2O2 and TMB concentrations;

[0053] Figure 16 The relative activity of Fe-COFs@GOx over 10 reaction cycles;

[0054] Figure 17 Enzymatic kinetics of Fe-COFs@GOx over a given time period;

[0055] Figure 18 Steady-state kinetic analysis of Fe-COFs@GOx under different glucose and TMB concentrations. Detailed Implementation

[0056] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0057] Source of raw materials

[0058] 2,2′-Bipyridine-5,5′-dicarboxaldehyde (Bpy), 1,3,6,8-tetra-(p-aminophenyl)pyrene (Tpy), and Tetri-(4-aminophenylphenyl)ethylene (TE) were purchased from Jilin Yanshen Technology Co., Ltd., Chinese Academy of Sciences. 1,4-Dioxane, trimethylene, acetic acid, acetone, tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO) were purchased from Shanghai Siwei Chemical Technology Co., Ltd. FeCl3·6H2O was purchased from Beijing Bailingwei Technology Co., Ltd. Glucose oxidase (GOx), horseradish peroxidase (HRP), glucose, and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Aladdin Company. H2O2 was purchased from Sinopharm Chemical Reagent Co., Ltd. Anhydrous sodium acetate was purchased from Maclean's Reagent Company. All chemicals and reagents were purchased from commercial suppliers and used without further purification; all solutions were prepared with ultrapure water.

[0059] Test method:

[0060] Enzyme loading determination conditions: The absorbance of blank sample, standard sample and test sample at 562 nm was measured by ultraviolet absorption spectroscopy using a protein content determination kit-spectrophotometry (BCA method) to calculate the protein content.

[0061] Ultraviolet absorption spectroscopy test conditions: A UV-Vis spectrophotometer (UV-2700i, Japan) was used. The wavelength for protein content determination was 562 nm, and the wavelength for enzyme activity determination was 652 nm.

[0062] Protein content determination - spectrophotometry (BCA method).

[0063] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0064] Example 1:

[0065] A method for preparing a biocatalytic cascade material of glucose oxidase supported on an iron-based covalent organic framework includes the following steps:

[0066] (1) 1,3,6,8-tetra-(p-aminophenyl)pyrene (17 mg, 0.03 mM), 2,2′-bipyridine-5,5′-dicarboxaldehyde (12.8 mg, 0.06 mM), and FeCl3·6H2O (15 mg) were mixed in a Pyrex tube with a solvent mixture of 1,4-dioxane / trimethylbenzene (3 ml, 1:1, v / v) and 3M acetic acid (300 μL). The resulting mixture was sonicated to obtain a homogeneous solution and subjected to three freeze-thaw cycles for degassing. The mixture was then reacted in a 120 °C oven for 3 days. After cooling to room temperature, the mixture was washed several times with tetrahydrofuran and acetone, and the sample was collected by centrifugation. Finally, the product was vacuum dried at 60 °C for 12 h to obtain Fe-COF1 material, the morphology of which is characterized as follows: Figure 1 .

[0067] (2) The 2 mg Fe-COF1 powder obtained in step (1) was ultrasonically dispersed in 5 ml of HAc-NaAc buffer (0.1 M, pH = 4.0), 2 mg of glucose oxidase was added, the mixture was stirred, and incubated in a water bath at 37 °C for 4 h. The composite material was collected, centrifuged, and washed three times with ultrapure water (8000 rpm, 5 min). Finally, the product was vacuum dried at 50 °C for 12 h to obtain the iron-based COF material loaded with glucose oxidase (Fe-COF1@GOx), whose morphology is characterized as follows: Figure 3 .

[0068] Example 2:

[0069] A method for preparing a biocatalytic cascade material of glucose oxidase supported on an iron-based covalent organic framework includes the following steps:

[0070] (1) Tetra-(4-aminophenyl)ethylene (0.06 mM, 23.55 mg), 2,2′-bipyridine-5,5′-dicarboxaldehyde (25.6 mg, 0.12 mM), and FeCl3·6H2O (20 mg) were mixed in a Pyrex tube with a solvent mixture of 1,4-dioxane / trimethylbenzene (3 ml, 1:1, v / v) and 3M acetic acid (300 μL). The resulting mixture was sonicated to obtain a homogeneous solution and subjected to three freeze-thaw cycles for degassing. The mixture was then reacted in a 120 °C oven for 3 days. After cooling to room temperature, the mixture was washed several times with tetrahydrofuran and acetone, and the sample was collected by centrifugation. Finally, the product was vacuum dried at 60 °C for 12 h to obtain Fe-COF2 material, the morphology of which is characterized as follows: Figure 2 .

[0071] (2) The 2 mg Fe-COF2 powder obtained in step (1) was ultrasonically dispersed in 5 ml of HAc-NaAc buffer (0.1 M, pH = 4.0), 2 mg glucose oxidase was added, the mixture was stirred, and incubated in a water bath at 37 °C for 4 h. The composite material was collected, centrifuged, and washed three times with ultrapure water (8000 rpm, 5 min). Finally, the product was vacuum dried at 50 °C for 12 h to obtain the iron-based COF material loaded with glucose oxidase (Fe-COF2@GOx), whose morphology is characterized as follows: Figure 4 .

[0072] Example 3:

[0073] Optimal catalytic temperature conditions for iron-based COFs peroxidase activity regulation

[0074] The optimal catalytic temperature for the peroxidase-like activity of Fe-COFs for the substrate H2O2 in step (1) of Examples 1 and 2 was adjusted within the temperature range of 20-65℃. At 5℃ intervals, the absorbance of the ·OH generated from H2O2 catalyzed by Fe-COFs at 652nm was measured using a UV-Vis spectrophotometer. The test results of Fe-COF1 obtained in step (1) of Example 1 are as follows... Figure 5 The Fe-COF2 test results obtained in step (1) of Example 2 are as follows: Figure 6 .

[0075] Example 4:

[0076] Optimal catalytic pH conditions for iron-based COFs peroxidase activity

[0077] The optimal catalytic pH for the peroxidase-like activity of Fe-COFs on the substrate H2O2 was determined by adjusting the pH in step (1) of Examples 1 and 2. The pH range was set between 3 and 7, with intervals of 0.5 pH. The absorbance of the ·OH generated from H2O2 catalyzed by Fe-COFs was measured at 652 nm using a UV-Vis spectrophotometer. The test results for Fe-COF1 obtained in step (1) of Example 1 are as follows... Figure 7 The Fe-COF2 test results obtained in step (1) of Example 2 are as follows: Figure 8 .

[0078] Example 5:

[0079] Optimal catalytic temperature conditions for glucose oxidase cascade catalytic activity supported by iron-based COFs

[0080] The optimal catalytic temperature for the cascade catalytic activity of Fe-COFs-loaded glucose oxidase for the substrate glucose in step (2) of Examples 1 and 2 was determined by adjusting the temperature range of 20-65℃. At 5℃ intervals, the absorbance of the ·OH generated in the cascade catalytic system at 652nm was measured using a UV-Vis spectrophotometer. The test results of Fe-COF1@GOx obtained in step (2) of Example 1 are as follows: Figure 9 The Fe-COF2@GOx test results obtained in step (2) of Example 2 are as follows: Figure 10 .

[0081] Example 6:

[0082] Optimal catalytic temperature conditions for glucose oxidase cascade catalytic activity supported by iron-based COFs

[0083] The optimal catalytic pH for the cascade catalytic activity of Fe-COFs-loaded glucose oxidase for the substrate glucose was determined by adjusting the pH in step (2) of Examples 1 and 2. The pH range was set between 3 and 7, with intervals of 0.5 pH. The absorbance of the ·OH generated in the cascade catalytic system at 652 nm was measured using a UV-Vis spectrophotometer. The test results for Fe-COF1@GOx obtained in step (2) of Example 1 are as follows: Figure 11 The Fe-COF2@GOx test results obtained in step (2) of Example 2 are as follows: Figure 12 .

[0084] Comparative Example 1:

[0085] Fe-COF1 material was prepared according to step (1) in Example 1. 2 mg of Fe-COF1 powder was ultrasonically dispersed in 5 ml of HAc-NaAc buffer (pH = 7.0), 2 mg of glucose oxidase was added, the mixture was stirred, and incubated in a water bath at 37°C for 4 h. The composite material was collected, centrifuged, and washed three times with ultrapure water (8000 rpm, 5 min). Finally, the product was vacuum dried at 50°C for 12 h to obtain the iron-based COF material loaded with glucose oxidase.

[0086] Fe-COF2 material was prepared according to step (1) in Example 2. 2 mg of Fe-COF2 powder was ultrasonically dispersed in 5 ml of HAc-NaAc buffer (pH = 7.0), 2 mg of glucose oxidase was added, the mixture was stirred, and incubated in a water bath at 37°C for 4 h. The composite material was collected, centrifuged, and washed three times with ultrapure water (8000 rpm, 5 min). Finally, the product was vacuum dried at 50°C for 12 h to obtain the iron-based COF material loaded with glucose oxidase.

[0087] Using TMB as a probe, the catalytic activity of the two materials in Examples 1, 2, and Comparative Example 1 was determined. In 4 mL of sodium acetate buffer containing TMB (0.25 mM) or the different materials mentioned above (10 μg / mL), 1 mM glucose was added, and the mixture was reacted at 50 °C for 15 min. The absorbance at 652 nm was recorded using a UV-Vis spectrophotometer. The results are as follows: Figure 13 As shown in the figure, the comparison shows that both Fe-COFs and GOx exhibit higher absorbance under acidic conditions than under neutral conditions, indicating that acidic incubation is more conducive to the binding of GOx and Fe-COFs and their participation in the cascade reaction.

[0088] Comparative Example 2:

[0089] The catalytic activities of Fe-COFs with natural horseradish peroxidase (HRP) and other enzyme-like materials are shown in Table 1. In the table, Feporphyrin COF is a material with porphyrin as one of the COF building blocks and Fe as a coordination element; MIL-100(Fe) is a metal-organic framework material centered on Fe ions; Fe3O4@MIL-100(Fe) is the above-mentioned metal-organic framework material further loaded with Fe3O4; and Fe3O4@COF@Os is a COF material loaded with Fe3O4 and Os. All of these materials exhibit peroxidase-like activities.

[0090] Using H₂O₂ or TMB as substrates, the catalytic performance of peroxidase-like enzymes was determined using a steady-state kinetic method, and kinetic parameters were obtained. Kinetic experiments were conducted at 50 °C in 4.0 mL of HAc-NaAc buffer (0.1 M, pH = 3.8) with different materials, different concentrations of H₂O₂ (0.1–0.5 mM), or different concentrations of TMB (0.05–0.25 mM) at 10 μg / mL. The absorbance of the separated supernatant was then recorded at 652 nm. The Michaelis-Menten constant was calculated using the Lineweaver-Burk double reciprocal plot: 1 / V = K. m / V m (1 / [S]+1 / K m ), where K m Here, [S] represents the Michaelis-Menten constant, V is the initial velocity, and [S] corresponds to the substrate concentration (TMB or H₂O₂). max This represents the maximum reaction rate.

[0091] The results are shown in Table 1. The results indicate that Fe-COF1 and Fe-COF2 in this invention exhibit a larger V than natural HRP and other enzyme-like materials. max and lower K m This indicates that Fe-COFs possess excellent peroxidase-like activity and better substrate affinity.

[0092] Table 1 Comparison of catalytic activity parameters of different types of peroxidases

[0093]

[0094] Test example:

[0095] The bio-cascade catalytic material prepared by the method of the present invention combines the advantages of nanozymes and natural enzymes, exhibiting excellent catalytic efficiency and catalytic lifetime; moreover, the Fe-COFs prepared by the present invention have strong peroxidase-like activity and high enzyme loading capacity.

[0096] (1) Catalytic efficiency was determined by comparing the steady-state kinetics of Fe-COFs peroxidase activity with the Michaelis-Menten equation to calculate the maximum reaction rate V. max and the Michaelis-Menten constant K m Value. Where V max A higher K value indicates a faster reaction rate and higher reactivity. m The smaller the value, the higher the substrate affinity.

[0097] Test conditions: The catalytic activity of Fe-COFs was determined using a TMB probe. In 4 mL of sodium acetate buffer containing TMB (0.25 mM) or different materials (10 μg / mL), with or without 0.1 mM H2O2, the reaction was carried out at 50 °C for 15 min. Photos were taken, and the absorbance at 652 nm was recorded using a UV-Vis spectrophotometer.

[0098] Furthermore, the peroxidase-like catalytic performance of Fe-COFs was determined using steady-state kinetics with H2O2 or TMB as substrates, and kinetic parameters were obtained. Kinetic experiments were conducted at 50 °C in 4.0 mL of HAc-NaAc buffer (0.1 M, pH = 3.8) with 10 μg / mL Fe-COFs, different concentrations of H2O2 (0.1–0.5 mM), or different concentrations of TMB (0.05–0.25 mM). The absorbance of the separated supernatant was then recorded at 652 nm. The Michaelis-Menten constant was calculated using the Lineweaver-Burk double reciprocal plot: 1 / V = K. m / V m (1 / [S]+1 / K m ), where K m Here, [S] represents the Michaelis-Menten constant, V is the initial velocity, and [S] corresponds to the substrate concentration (TMB or H₂O₂). max This represents the maximum reaction rate.

[0099] The results are as follows Figure 14 , Figure 15 As shown, both Fe-COFs exhibited superior K+ compared to natural horseradish peroxidase (HRP). m and V m Values. Among them, the V values ​​of HRP for H2O2 and TMB. m They are 0.0871 mM s -1 and 0.1000mM s -1 Fe-COF1 were 2.412 mM s -1 and 1.3976mM s -1 Fe-COF2 were 1.812 mM s -1 and 0.8913mM s -1 Fe-COF1 and Fe-COF2 on the K of H2O2 m The values ​​were 0.2322 mM and 0.2078 mM, respectively, for the K values ​​of the two Fe-COFs. m The values ​​are very close, both far lower than HRP (3.7000mM).

[0100] (2) Cyclic stability test: 1 mM glucose was added to 4 mL of sodium acetate buffer containing TMB (0.25 mM) and different materials (10 μg / mL), and the mixture was reacted at 50 °C for 15 min. After each reaction, Fe-COF1@GOx was washed with deionized water and centrifuged to proceed to the next catalytic cycle. The same doses of glucose and TMB were added to react with the recovered Fe-COF1@GOx. The relative intensities were recorded to evaluate the repeatability of Fe-COF1@GOx in detecting glucose. The results are shown below. Figure 16 As shown, after 10 cycles of testing, the catalytic activity of the initial cascade catalytic material is still maintained at 90%.

[0101] (3) Characterization of loading effect: Elemental distribution imaging was performed using energy dispersive spectroscopy (EDS), which showed that the complex enzyme contained S element belonging to glucose oxidase (GOx); amide I and amide II bonds belonging to GOx were observed using infrared Fourier transform spectroscopy; the overall decomposition temperature of natural glucose oxidase is 350℃, and thermogravimetric analysis showed that after loading GOx, GOx lost weight along with the Fe-COFs material at 450℃; Zeta potential test showed that the Fe-COFs material was positively charged due to the coordination of Fe ions, and after loading the enzyme, GOx neutralized the positive charge, and the complex enzyme turned negative, showing a stable state close to electroneutrality.

[0102] Loading capacity determination: The loading capacity of GOx in Fe-COFs was determined using a BCA protein (spectrophotometric) assay kit. The test solution was the supernatant of 1 mg Fe-COFs and 1 mg GOx, incubated in 2.5 ml HAc-NaAc buffer for 4 h, and centrifuged (8000 rpm, 5 min). Specifically, the working solution was first preheated in a 60°C water bath for at least 30 minutes. Three 2 ml centrifuge tubes were used, each containing 20 μL of ultrapure water, standard, and test solution, respectively. Then, 1000 μL of preheated working solution was added to each tube, mixed thoroughly, and incubated in a 60°C water bath for 30 minutes. The tubes were then cooled with tap water for 10 minutes, and the absorbance was measured at 562 nm and recorded as A. blank A standard A determination Protein content Cpr (mg / ml) = (C0 - C determination ) / C0, where C standard =0.05mg / ml, C0=0.4mg / ml. The final loadings of Fe-COF1 and Fe-COF2 were determined to be 92.5% (w / w) and 93.6% (w / w), respectively, which are superior to most support materials.

[0103] Catalytic activity assay: The catalytic activity of Fe-COFs@GOx was determined using a TMB probe. 1 mM glucose was added to 4 mL of sodium acetate buffer containing TMB (0.25 mM) and different materials (10 μg / mL). The mixture was reacted at 50 °C for 15 min, photographed, and the absorbance at 652 nm was recorded over a given time using a UV-Vis spectrophotometer. The results are shown below. Figure 17 As shown.

[0104] Steady-state kinetics test: Using glucose or TMB as substrates, the catalytic performance of Fe-COFs@GOx was determined using steady-state kinetics, and kinetic parameters were obtained. Different materials, glucose concentrations (0.5-4 mM), or TMB concentrations (0.05-0.35 mM) were added to 4.0 mL of HAc-NaAc buffer (0.1 M, pH = 4.0), and kinetic experiments were conducted at 50 °C. Subsequently, the absorbance of the separated supernatant at 652 nm was recorded. The Michaelis-Menten constant was calculated using the Lineweaver-Burk double reciprocal plot: 1 / V = K. m / V m (1 / [S]+1 / K m ), where K m Here, [S] represents the Michaelis-Menten constant, V is the initial velocity, and [S] corresponds to the substrate concentration (TMB or glucose). max This represents the maximum reaction rate. Steady-state kinetics studies can be found in [link to study]. Figure 18Because cascade enzymes include nanozymes and biological enzymes, the number of active sites is unknown, and steady-state kinetic data do not possess significant characteristics.

[0105] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing an iron-based covalent organic framework-supported enzyme biocatalytic cascade material, characterized in that, Includes the following steps: (1) Select iron-containing metal salts and mix them with polyaldehyde-containing COFs monomers and polyamino-containing COFs monomers, and obtain iron-based covalent organic frameworks by solvothermal reaction under vacuum. (2) The iron-based covalent organic framework obtained in step (1) is mixed with glucose oxidase in an acidic buffer solution, and then centrifuged and dried to obtain the iron-based covalent organic framework enzyme-loaded biocatalytic cascade material. The iron-containing metal salt mentioned in step (1) is FeCl3·6H2O; The polyaldehyde-containing COFs monomer mentioned in step (1) is 2,2′-bipyridine-5,5′-dicarboxaldehyde; the polyamino-containing COFs monomer is 1,3,6,8-tetra-(p-aminophenyl)-pyrene; The solvothermal reaction in step (1) involves placing the iron-containing metal salt, polyaldehyde-containing COFs monomers, and polyamino-containing COFs monomers in a mixed solvent and reacting at 90-130℃ for 60-80 hours. The mixed solvent is a mixture of 1,4-dioxane and mesitylene in a volume ratio of 1:0.5~2. In step (1), the mass ratio of the iron-containing metal salt, the polyamino COF monomer, and the polyaldehyde COF monomer is 15-20:15-25:10-30. The reaction in step (1) uses acetic acid solution as a catalyst; the concentration of acetic acid solution is 2 M to 4 M; the amount added is 5% to 15% of the volume of the mixed solvent; The pH of the acidic buffer solution in step (2) is 3.0-5.

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2. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the iron-based covalent organic framework to glucose oxidase is 1:0.5~2, and the mass-volume ratio of the iron-based covalent organic framework and glucose oxidase to the acidic buffer is 0.2~0.6 mg / mL.

3. An iron-based covalent organic framework enzyme-carrying biocatalytic cascade material prepared by the method described in any one of claims 1 to 2.

4. The application of the iron-based covalent organic framework enzyme-supported biocatalytic cascade material as described in claim 3 in enzyme loading.

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