A bimetallic MOF nanozyme and a preparation method and application thereof

Abstract

The application relates to the field of analytical detection technology, and discloses a bimetallic MOF nano-enzyme as well as a preparation method and application thereof. The bimetallic MOF nano-enzyme comprises iron, cerium and an organic skeleton, the organic skeleton is 1,3-benzenedicarboxylic acid, and the iron and the cerium replace H atoms in carboxyl groups of the organic skeleton. The preparation method comprises the following steps: dissolving FeCl3.6H2O and Ce(SO4)2.4H2O in a solvent to obtain solution A; dissolving 1,3-benzenedicarboxylic acid in a DMF solution to obtain solution B; and mixing and reacting the solution A with the solution B to obtain the bimetallic MOF nano-enzyme. The bimetallic MOF nano-enzyme is used for quantitative detection of total antioxidant capacity, glutathione, ascorbic acid and cysteine, has the advantages of high stability, high reliability, high sensitivity, good selectivity, simple operation and the like, and can be widely applied in the fields of biomedicine, food safety, environmental monitoring and the like.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a bimetallic MOF nanozyme, its preparation method, and its application. Background Technology

[0002] Vegetables and fruits are an important part of our daily diet, containing abundant antioxidants such as vitamin C, vitamin E, carotenoids, and polyphenols. These antioxidants possess strong antioxidant capabilities, scavenging free radicals, reducing oxidative stress, and thus protecting cells from oxidative damage. They also have health benefits such as anti-tumor effects, lowering blood pressure, lowering blood sugar, and lowering blood lipids. Total antioxidant capacity refers to the overall antioxidant capacity of all antioxidants in food, and it can serve as an important quality indicator in vegetables and fruits. The types and amounts of antioxidants in vegetables and fruits vary depending on the variety, growing environment, and factors such as harvesting, storage, and processing. Furthermore, detecting total antioxidant capacity provides an important monitoring method for potential decreases in antioxidant capacity during production and processing, as well as for nutrient loss during food storage, ensuring consumer health and safety. Therefore, detecting the total antioxidant capacity of vegetables and fruits is of great significance for understanding their nutritional value and their contribution to human health. Total antioxidant capacity (TAC) refers to the ability of all antioxidants in a sample to eliminate free radicals, and is usually characterized by the reducing power of antioxidants against standard oxidants.

[0003] In recent years, MOF-mimicking enzymes have been termed artificial enzymes with catalytic activity designed and prepared using metal-organic framework (MOF) materials. Their active centers consist of metal ions and organic ligands within the MOF material. MOF nanozymes exhibit high stability, controllability, and catalytic efficiency. Furthermore, due to their large specific surface area, tunable physicochemical properties, porous structure, and diverse compositions, they have been explored as mimics for various enzymes. Their catalytic activity is closely related to the metal active center. However, in single-metal MOF catalysts, the coordination structure formed by the metal ions is unstable, exhibiting low thermal and chemical stability, and thus cannot maintain stable catalytic activity during catalytic reactions. Summary of the Invention

[0004] This invention provides a bimetallic MOF nanozyme, its preparation method, and its application, which solves the problem that existing single-metal MOF catalysts have unstable coordination structures formed by metal ions, low thermal and chemical stability, and cannot maintain stable catalytic activity in catalytic reactions.

[0005] A bimetallic MOF nanozyme comprising iron, cerium, and an organic framework, wherein the organic framework is 1,3-phthalic acid, and the iron and cerium replace the H atoms in the carboxyl groups of the organic framework.

[0006] A second objective of this invention is to protect the method for preparing the bimetallic MOF nanozyme, comprising the following steps:

[0007] S1. Dissolve FeCl3·6H2O and Ce(SO4)2·4H2O in a solvent to obtain solution A; wherein, FeCl3·6H2O:Ce(SO4)2·4H2O:solvent = (0.5~1.5)mM:1mM:10mL;

[0008] S2. Dissolve 1,3-phthalic acid in N,N-dimethylformamide solution to obtain solution B; wherein, 1,3-phthalic acid:N,N-dimethylformamide = 1 mM:10 mL;

[0009] S3. Mix solution A and solution B to react. After the reaction is complete, centrifuge, wash, and dry to obtain bimetallic MOF nanozymes; wherein the volume ratio of solution A to solution B is 1:1.

[0010] Preferably, the solvent in S1 is N,N-dimethylformamide or anhydrous ethanol.

[0011] Preferably, the reaction in S3 is carried out using a DBD microplasma device, wherein the voltage applied to the reaction tube in the DBD microplasma device is 70-90V and the reaction time is 30-50min.

[0012] Preferably, the drying temperature in step S3 is 60–70°C, and the drying time is 8–12 hours.

[0013] The third objective of this invention is to protect the application of bimetallic MOF nanozymes in the detection of total antioxidant capacity.

[0014] The fourth objective of this invention is to protect bimetallic MOF nanozymes for the detection of glutathione.

[0015] The fifth objective of this invention is to protect bimetallic MOF nanozymes for the detection of ascorbic acid.

[0016] The sixth objective of this invention is to protect bimetallic MOF nanozymes for the detection of cysteine.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] (1) The Ce / Fe-MOF nanoenzyme sensing platform of bimetallic MOF material has the advantages of high stability, high reliability, high sensitivity, good selectivity and simple operation for the quantitative detection of ascorbic acid, glutathione, cysteine ​​and total antioxidant capacity. It is widely used in the fields of biomedicine, food safety and environmental monitoring.

[0019] (2) The present invention uses micro-plasma to synthesize the bimetallic MOF material Ce / Fe-MOF in one step. Compared with monometallic MOF, Ce / Fe-MOF has better catalytic performance and three types of enzyme activity, and has better application prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Ce / Fe-MOF bimetallic MOF synthesis apparatus provided in Embodiment 1 of the present invention;

[0021] Figure 2 XPS spectra of Fe-MOF, Ce-MOF, Ce / Fe-MOF and physically mixed Fe-MOF / Ce-MOF provided in Example 2 of this invention;

[0022] Figure 3 The FT-IR spectra of Fe-MOF, Ce-MOF, and Ce / Fe-MOF provided in Example 2 of this invention;

[0023] Figure 4 Scanning electron microscope images (a, b) of Ce-MOF provided in Embodiment 2 of the present invention; Scanning electron microscope images (c, d) of Fe-MOF;

[0024] Figure 5 Scanning electron microscope (a, b), transmission electron microscope (c), and elemental analysis diagram (dg) of Ce / Fe-MOF provided in Embodiment 2 of the present invention;

[0025] Figure 6 The high-resolution XPS spectra of Ce / Fe-MOF, Fe-MOF, and Ce-MOF provided in Embodiment 2 of the present invention;

[0026] Figure 7 High-resolution XPS spectra of Ce / Fe-MOF, Fe-MOF, and Ce-MOF provided in Embodiment 2 of the present invention: O1s;

[0027] Figure 8 The electron paramagnetic resonance spectrum of Ce / Fe-MOF provided in Embodiment 2 of the present invention;

[0028] Figure 9High-resolution XPS spectra of Ce-MOF (9(a)), Fe-MOF (9(b)), Ce / Fe-MOF before catalytic reaction (9(c)), and Ce / Fe-MOF after catalytic reaction (9(d)) provided in Example 2 of the present invention: O 1s;

[0029] Figure 10 Here are the high-resolution XPS spectra of Ce / Fe-MOF before and after the reaction with TMB, as provided in Example 2 of this invention: Ce 3d;

[0030] Figure 11 Here are the high-resolution XPS spectra of Fe-MOF before and after the reaction of Ce / Fe-MOF with TMB, as provided in Example 2 of this invention: Fe 2p;

[0031] Figure 12 Comparative analysis of the oxidase-like properties of Ce / Fe-MOF, Ce-MOF, and Fe-MOF provided in Example 2 of this invention;

[0032] Figure 13 Steady-state kinetic analysis of Ce / Fe-MOF-like oxidases provided in Example 2 of the present invention: kinetic curve (a) and double reciprocal plot (b) of TMB;

[0033] Figure 14 Steady-state kinetic analysis of Fe-MOF-like oxidases provided in Example 2 of the present invention: kinetic curve (a) and double reciprocal plot (b) of TMB;

[0034] Figure 15 Cyclic voltammograms of Ce / Fe-MOF, Ce-MOF, and Fe-MOF modified GCEs provided in Embodiment 2 of the present invention;

[0035] Figure 16 This is a gas-dependent experimental analysis diagram of an oxidase-like enzyme provided in Example 2 of the present invention;

[0036] Figure 17 The free radical types of Ce / Fe-MOF oxidases provided in Example 2 of this invention;

[0037] Figure 18 The following are examples of the steady-state kinetic analysis of Ce / Fe-MOF-like oxidases provided in Example 2 of this invention: kinetic curves and double reciprocal plots of TMB (a, b), and kinetic curves and double reciprocal plots of H2O2 (c, d).

[0038] Figure 19 Comparative analysis of the peroxidase-like performance of Ce / Fe-MOF provided in Example 2 of this invention;

[0039] Figure 20This study investigates the types of free radicals in Ce / Fe-MOF peroxidases provided in Example 2 of the present invention.

[0040] Figure 21 Verification of superoxide dismutase activity using the pyrogallol auto-oxidation method;

[0041] Figure 22 To validate superoxide dismutase activity using an SOD activity assay kit;

[0042] Figure 23 The percentage of superoxide dismutase inhibition;

[0043] Figure 24 This is a comparison of the Ce / Fe-MOF synthesis conditions provided in Example 2 of the present invention;

[0044] Figure 25 pH comparison of the oxidase-like activity of Ce / Fe-MOF provided in Example 2 of this invention;

[0045] Figure 26 This is a comparison of the concentrations of oxidase-like activities of Ce / Fe-MOF provided in Example 2 of the present invention;

[0046] Figure 27 This is a comparison of the reaction time for the oxidase-like activity of Ce / Fe-MOF provided in Example 2 of the present invention;

[0047] Figure 28 The linear relationship graph of AA;

[0048] Figure 29 A linear relationship graph for GSH;

[0049] Figure 30 A linear relationship graph of cys;

[0050] Figure 31 For interference testing;

[0051] Figure 32 Stability analysis of Ce / Fe-MOF;

[0052] Figure 33 The TAC content of three samples was analyzed using AA concentration as a standard. Detailed Implementation

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood in the field to which the subject matter described herein belongs.

[0054] When this application refers to a range of values, such as a concentration range, percentage range, or proportion range, unless the context clearly specifies otherwise, it includes one-tenth of the lower limit unit and refers to all intermediate values ​​within the upper and lower limits of the range. This application also includes independent sub-ranges composed of any smaller upper and lower limits within the range, and these embodiments are also included in the patent, subject to any specific exclusion restrictions within the specified range. Ranges that include one or two upper (lower) limits or exclude one or two upper (lower) limits are also included in this application.

[0055] Throughout the description of various embodiments, the word "comprising" is used. However, in certain specific circumstances, it will be understood in the art that the language of "substantially consisting of" or "consisting of" may be used to describe embodiments instead.

[0056] To better understand this application and without limiting its scope, all figures representing quantities, percentages, proportions, and other numerical values ​​used in the specification and claims, unless otherwise stated, should be understood to be modified by the term "about". Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximations that may vary depending on the desired properties obtained, and at least each numerical parameter should be interpreted based on the number of significant figures reported and by applying general rounding rules.

[0057] The plasma in this invention is a low-temperature plasma confined to a limited spatial range at the millimeter scale or even lower. Compared with traditional gas discharge plasma, micro-plasma exhibits unique properties and application potential in terms of size, morphology, and chemical reactions. Micro-plasma possesses characteristics such as high electric field strength, high reactivity and high energy density, low energy consumption, and controllable operation. Due to its low-temperature plasma state, micro-plasma can realize a variety of unconventional chemical reactions, such as electrochemical reactions, redox reactions, pyrolysis reactions, and ion exchange reactions. These reactions are typically difficult to achieve at room temperature and pressure, but can proceed efficiently in micro-plasma. Therefore, micro-plasma has broad prospects for application in chemistry, materials, and environmental fields, such as surface modification, material synthesis and modification, biomedicine, and environmental remediation.

[0058] In this invention, the redox behavior of Ce / Fe-MOF was verified using cyclic voltammetry (CV) and electrochemical impedance spectroscopy. Cyclic voltammetry (CV) was performed using an electrochemical analyzer (CHI 760E workstation), with an Ag / AgCl electrode and a Pt sheet as the reference and auxiliary electrodes, respectively. Ce / Fe-MOF, Ce-MOF, and Fe-MOF-modified GCE were used as working electrodes, and cyclic voltammetry tests were conducted within a potential range of -0.3 to 1.1 V.

[0059] This invention employs the Michaleis-Menten equation and the Lineweaver-Burk equation to analyze the relationship between enzyme-catalyzed reaction rate and substrate concentration. The Michaleis-Menten equation describes the relationship between enzyme-catalyzed reaction rate and substrate concentration, while the Lineweaver-Burk equation is commonly used for the analysis and comparison of enzyme kinetic experimental data. The Michaleis-Menten equation is shown below:

[0060] V = Vmax * [S] / (Km + [S])

[0061] Where V represents the reaction rate, Vmax represents the maximum reaction rate at infinitely high substrate concentration, [S] represents the substrate concentration, and Km represents the enzyme's substrate affinity constant. The Michaelis-Menten equation shows that when the substrate concentration is very low, the reaction rate is linearly related to the substrate concentration; while when the substrate concentration approaches Km, the reaction rate tends to its maximum value Vmax. The Lineweaver-Burk equation is a linearization of the Michaelis-Menten equation, and its form is as follows:

[0062] 1 / V=(Km / Vmax)×(1 / [S]+1 / Km)

[0063] Taking the reciprocal of the Michaelis-Menten equation and plotting it yields a straight line with an intercept of 1 / Vmax and a slope of Km / Vmax. From this, the values ​​of Km and Vmax can be obtained from experimental data, allowing for the calculation of enzyme kinetic parameters. This enables the evaluation of the catalytic activity and substrate affinity of Ce / Fe-MOF and Fe-MOF.

[0064] Example 1

[0065] Synthesis of Ce / Fe-MOF

[0066] Ce / Fe-MOF was synthesized in one step via DBD microplasma method, with 1,3-phthalic acid (H2BDC) as the organic framework and Ce and Fe as the metal centers, with iron and cerium replacing the H atoms in the carboxyl groups of the organic framework.

[0067] The specific preparation steps are as follows:

[0068] S1. Dissolve 0.271 g (1 mM) FeCl3·6H2O and 0.4043 g (1 mM) Ce(SO4)2·4H2O in 10 mL of N,N-dimethylformamide (DMF) solution to obtain solution A.

[0069] S2. Dissolve 0.254 g (1 mM) H2BDC in 10 mL of DMF solution to obtain solution B.

[0070] S3. Add solution A to solution B and transfer it to the reaction tube in the microplasma device. React for 30 minutes using the DBD microplasma device at 80V. The DBD microplasma device used in this embodiment consists of an open concentric glass tube (outer tube containing reactants), a copper rod, and copper wire. The copper wire is wrapped around the glass tube, and the copper rod is located at the bottom inside the glass tube and extends outwards. Both the copper rod and the copper wire are connected to a power source (e.g.,...). Figure 1 Then, the precipitate was separated from the reaction mixture by centrifugation at 5000 rpm for 5 min. It was washed three times with ultrapure water, then three times with ethanol, and finally dried in an oven at 60°C for 12 hours.

[0071] The specific steps for synthesizing Ce-MOF are as follows: 0.4043 g (1 mmol) of Ce(SO4)2·4H2O was dissolved in 10 mL of DMF solution to obtain solution C. Solution C was transferred to a reaction tube in a microplasma apparatus, and the reaction was carried out at 80 V for 30 min using a DBD microplasma apparatus. The precipitate was then separated from the reaction mixture by centrifugation at 5000 rpm for 5 min. The precipitate was washed three times with ultrapure water, then three times with ethanol, and finally dried in an oven at 60 °C for 12 h to obtain Ce-MOF.

[0072] The specific steps for synthesizing Fe-MOF are as follows: 0.271 g (1 mmol) of FeCl3·6H2O was dissolved in 10 mL of DMF solution to obtain solution D; solution D was transferred to a reaction tube in a micro-plasma apparatus, and the reaction was carried out at 80 V for 30 minutes using a DBD micro-plasma apparatus. The precipitate was then separated from the reaction mixture by centrifugation at 5000 rpm for 5 min. The precipitate was washed three times with ultrapure water, then three times with ethanol, and finally dried in an oven at 60 °C for 12 hours to obtain Fe-MOF.

[0073] refer to Figure 2 The crystal structures of Fe-MOF, Ce-MOF, Ce / Fe-MOF, and physically mixed Fe-BDC and Ce-BDC were determined by powder XRD. The chemical composition and crystal structure of each sample were accurately determined. The strong and narrow peaks indicate good crystallinity of the prepared samples. The XRD patterns of Fe-MOF and Ce-MOF were consistent with previous reports. The main diffraction peaks exhibited by the bimetallic MOF were also consistent with those of the single metal, and the crystal structure differed from that of the physically mixed materials. This indicates that the materials synthesized using microplasma equipment are not simple physical mixtures, but rather bimetallic MOFs with a certain crystal structure, demonstrating the successful synthesis of this material.

[0074] refer to Figure 3The types of chemical bonds in the prepared material were determined using infrared spectroscopy. From the FT-IR spectrum of Ce-MOF, it can be seen that at 3430 cm⁻¹... -1 The peak is related to the stretching vibration of OH, and is located in the range of 1610-1560 cm⁻¹. -1 and 1480-1370cm -1 The characteristic peaks at 500-700 cm⁻¹ belong to the asymmetric and symmetric stretching of the carboxyl group, respectively. -1 The infrared peak at 549 cm⁻¹ is due to the Ce-O stretching vibration, confirming the presence of the Ce-O bond and the dicarboxylic acid linker, and thus demonstrating the successful synthesis of Ce-MOF. In the FT-IR spectrum of Fe-MOF, the peak at 549 cm⁻¹ is due to the Ce-O stretching vibration, proving the presence of the Ce-O bond and the dicarboxylic acid linker, and also confirming the successful synthesis of Ce-MOF -1 The peak at 3430 cm⁻¹ can be attributed to the stretching vibration of the Fe-O bond, indicating that the Fe-O cluster exists between the carboxyl group and the inorganic metal. -1 The peak is related to the stretching vibration of OH, at 1391 cm⁻¹. -1 1535-1667cm -1 The strong characteristic peaks at the positions are attributed to the asymmetric and symmetric stretching of the carboxyl groups, respectively, proving the existence of the dicarboxylic acid linker and indicating the successful synthesis of Fe-MOF. The characteristic peaks of Ce / Fe-MOF correspond to those of single-metal MOF, exhibiting similar absorption peaks, indicating the successful synthesis of Ce / Fe-MOF.

[0075] refer to Figures 4 to 5 The surface morphology and microstructure of Ce-MOF, Fe-MOF, and Ce / Fe-MOF were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM images of Ce-MOF showed its rod-like structure with a rough surface, consistent with previous reports. Fe-MOF, on the other hand, consisted of three-dimensional peony-shaped microspheres composed of two-dimensional nanosheets. With the addition of iron, Ce / Fe-MOF also exhibited a rod-like shape, but with a much smaller particle size than Ce-MOF and more pores and structural defects, providing a larger surface area and more catalytic active sites, resulting in better catalytic performance. EDS elemental analysis mapping images further demonstrated that Fe, Ce, C, and O were uniformly distributed throughout the Ce / Fe-MOF.

[0076] refer to Figures 6 to 11The surface composition and chemical state of Ce / Fe-MOF were determined using XPS spectroscopy. Peaks for C, O, Fe, and Ce were observed in the measured spectrum of Ce / Fe-MOF, indicating the simultaneous presence of these elements in the material and confirming its successful synthesis. Analysis of the O1s spectra of Ce / Fe-MOF, Ce-MOF, and Fe-MOF revealed that, compared to individually synthesized monometallic MOFs, the binding energy position of the oxygen peak in Ce / Fe-MOF shifted significantly to a higher binding energy position, indicating the presence of oxygen deficiency and a change in the electronic structure of oxygen, potentially leading to the creation of oxygen vacancies. The oxygen peak position in the reacted material shifted significantly to a lower binding energy position, suggesting that oxygen vacancies participated in the catalytic process. The presence of oxygen vacancies was further confirmed by electron paramagnetic resonance (EPR) testing. Simultaneously, by analyzing the characteristic peaks of oxygen, it was found that the O(1s) spectrum conforms to three characteristic peaks, with binding energies corresponding to lattice oxygen (Olat), oxygen vacancies (Ov), and carboxylic acid organic oxygen (Oorg), respectively. The lattice oxygen intensity of Ce / Fe-MOF is greater than that of its monometallic MOF, indicating that Ce / Fe-MOF has a better ability to bind oxygen and is more stable. Oxygen vacancies are an indicator of the degree of lattice defects, which are generated by the reduction of high-valence metal ions to low-valence metal ions. Through the characteristic peak analysis of the Ce 3d and Fe 2p energy levels of the material, it was found that Ce / Fe-MOF contains both +3 and +4 valence states of Ce and +2 and +3 valence states of Fe, and the presence of mixed valence states further illustrates the existence of oxygen vacancies. In the Fe 2p XPS spectrum, there are two main peaks at 711.1 and 725.1 eV, belonging to the 2p3 / 2 and 2p1 / 2 of Fe, respectively. The binding energies of Fe(III) at Fe 2p1 / 2 and Fe 2p3 / 2 are 707.9 and 724.8 eV, respectively, while those of Fe(II) are 710.8 and 722.6 eV, corresponding to Fe 2p1 / 2. Furthermore, characteristic satellite peaks at 715.6, 719.2, and 726.7 eV are observed, further confirming the binding energies of Fe. 3+ The presence of species. High-resolution XPS spectra of Ce 3d show the presence of mixed valence states (Ce) in the material. 3+ / Ce 4+ ), with Ce 4+ The relevant peaks were 883.2, 887.8, 901.4, and 907.8 eV, respectively, and were associated with Ce. 3+ The relevant peaks are 886.1 and 904.9 eV, respectively, and it has a characteristic satellite peak of 917.4 eV, further confirming Ce 3+ The presence of species. Fe before the reaction of Ce / Fe-MOF with TMB. 3 + / Fe 2+ The ratio is approximately 1.49, Ce4+ / Ce 3+ The ratio is approximately 1.18, and the Fe after the reaction 3+ / Fe 2+ The ratio is 0.94, Fe 3+ / Fe 2+ The ratio is approximately 1.27, indicating that both Fe and Ce elements act as active reaction sites in this catalytic process, synergistically promoting catalytic activity.

[0077] Example 2: Multienzyme mimicry activity of Ce / Fe-MOF

[0078] 2.1. Oxidase-like activity of Ce / Fe-MOF.

[0079] Evaluation of oxidase activity using TMB (3,3',5,5'-tetramethylbenzidine) as a substrate is a commonly used method, assessing catalytic activity by measuring changes in absorbance produced during the catalytic reaction. For example... Figures 12 to 17 As shown, in a mixture of (50 μL, 1 mM) TMB and (0.9 mL, pH 4) NaAc / HAc buffer, (50 μL, 5 mg / mL) of TMB and (0.9 mL, pH 4) NaAc / HAc buffer were added respectively. -1 Ce / Fe-BDC, Ce-MOF, and Ce-MOF suspensions were reacted at room temperature for 6 minutes, and absorbance was measured at 652 nm. The absorption at 652 nm was almost negligible with the addition of Ce-MOF, and Fe-MOF showed low absorbance at 652 nm. The bimetallic MOF, however, exhibited a strong absorption peak, indicating that Ce / Fe-MOF possesses inherent oxidation-like activity, and its enzyme-like activity is far superior to that of single-metal MOF nanomaterials. This may be due to the synergistic effect of the two metal ions in the bimetallic MOF, which enhances the efficiency of the catalytic reaction. Furthermore, due to the larger specific surface area and more abundant catalytic active sites, the bimetallic MOF exhibits higher catalytic activity in the reaction system. In addition, the catalytic efficiency and affinity of Ce / Fe-MOF were evaluated by measuring its kinetic constant. The catalytic rate constant reflects the rate of substrate-to-product conversion, while the Michaelis constant reflects the affinity between the enzyme and the substrate, i.e., the degree of enzyme affinity for the substrate. The results show that the Km and Vmax values ​​of Ce / Fe-MOF are 0.62 mM and 49.1 × 10⁻⁶ mM, respectively. -7 Ms -1 Ce-MOF does not possess oxidase-like activity, while Fe-MOF has Km and Vmax values ​​of 1.79 mM and 34.5 × 10⁻⁶ mM, respectively. -7 Ms -1This indicates that Ce / Fe-MOF has a stronger affinity for TMB, and the higher V.max value indicates a higher conversion rate from substrate to product, exhibiting enhanced oxidase-like activity. Cyclic voltammetry (CV) was used to explore the redox behavior of Ce / Fe-MOF. Using Ce / Fe-MOF, Ce-MOF, and Fe-MOF-modified GCE as working electrodes, cyclic voltammetry was performed in the potential range of -0.3 to 1.1 V. It was observed that when TMB was present in the buffer solution, Ce / Fe-MOF and Fe-MOF showed a strong redox peak, while Fe-MOF did not show a redox peak, indicating that the electron transfer efficiency in the redox couple of Ce / Fe-MOF was superior to that of Fe-MOF and Ce-MOF. To determine whether the oxidase-mimicking activity of Ce / Fe-MOF is oxygen-dependent, a series of gas-dependent experiments were conducted, including oxidation experiments in air, oxygen, and nitrogen. The results showed that the absorbance was highest under oxygen-enriched conditions, demonstrating that oxygen is one of the main factors affecting the activity of Ce / Fe-MOF oxidases. Further investigation of free radicals in the reaction system was carried out using free radical capture experiments, which showed that ·O2... - ·OH and 1O2 all play important roles in the catalytic process.

[0080] 2.2. Peroxidase-like activity of Ce / Fe-MOF.

[0081] refer to Figures 18 to 20 The catalytic activity of Ce / Fe-MOF with hydrogen peroxide and TMB substrates was assessed by detecting changes in absorbance in the reaction system. The results showed that the bimetallic MOF exhibited higher absorbance values ​​in the presence of hydrogen peroxide, indicating that it possessed good peroxidase-like activity for hydrogen peroxide. Similarly, the catalytic efficiency and affinity of Ce / Fe-MOF were evaluated by measuring its kinetic constants. The results showed that when the TMB concentration was fixed at 0.5 mM, the reaction rate (v) gradually increased with increasing hydrogen peroxide concentration. According to the double reciprocal plot, the Km value of Ce / Fe-MOF was 1.67 mM, and the Vmax value was 17.7 × 10⁻⁶. -7 Ms -1 Similarly, the relevant enzyme kinetic parameters of TMB's Ce / Fe-MOF were determined at a hydrogen peroxide concentration of 4 mM. The Km value of Ce / Fe-MOF was 2.03 mM, and the Vmax value was 79.4 × 10⁻⁶. -7 Ms -1 In free radical capture experiments, ·OH and ·O2 were found to be effective. - Both 1O2 and 1O2 play important roles in the catalytic process, with ·OH playing the most significant role.

[0082] 2.3. Superoxide dismutase-like activity of Ce / Fe-MOF.

[0083] refer to Figures 21 to 23 First, the superoxide scavenging capacity of Ce / Fe-MOF was determined using the pyrogallol autoxidation method. Then, the superoxide dismutase (SOD) activity assay kit was used to verify the superoxide dismutase-like activity. Using the superoxide dismutase (SOD) activity assay kit (Sangon Biotecch, Shanghai), superoxide anions (·O2) were generated through a xanthine and xanthine oxidase reaction system. - ), O2 - It can reduce nitroblue tetrazolium to form blue formazan, which has an absorption at 560 nm; SOD can scavenge O2. - This inhibits the formation of formazan; the darker the color of the reaction solution, the lower the SOD activity, and vice versa. The SOD activity was quantified by calculating the decrease in absorbance compared to the case without Ce / Fe-MOF (ΔA blank), and expressed as the percentage inhibition of formazan formation.

[0084] Inhibition percentage = (ΔA blank - ΔA determination) ÷ ΔA blank × 100%

[0085] The results showed that Ce / Fe-MOF had a good inhibitory effect on superoxide radicals and could effectively scavenge O2. ·- .

[0086] Example 3: Comparison of synthesis conditions and oxide-like activity

[0087] 3.1 Comparison of Synthesis Conditions

[0088] First, the catalytic activity of different molar ratios of iron and cerium on chromogenic substrates was compared, see [reference needed]. Figure 24 Experimental results show that the material exhibits the highest catalytic activity when the content ratio is 1:1. Therefore, the following experiments all use catalysts with a cerium to iron molar ratio of 1.

[0089] 3.2. Comparison of Oxide-like Activity Conditions

[0090] refer to Figures 25 to 27To compare the activity conditions of oxidases, it is usually necessary to study multiple experimental conditions, including pH, catalytic reaction time, catalyst dosage, and TMB concentration. Experimental results show that these conditions can significantly affect the catalytic activity and efficiency of oxidases. First, pH plays a crucial role in oxidase activity. By conducting catalytic reactions at a series of different pH values, we observed that oxidase activity may vary at different pH values. Experimental results showed that Ce / Fe-MOF exhibited the best catalytic effect at a buffer solution pH of 4; therefore, subsequent experiments were conducted under NaAc-HAc buffer (0.1M pH 4). Second, the duration of the catalytic reaction directly affects the evaluation results of oxidase activity. Shorter reaction times may not fully demonstrate the catalytic ability of oxidases, while longer reaction times may lead to over-reaction, affecting the accuracy of the measurement results. Experiments were conducted at different catalytic reaction times. The results showed that after 6 minutes, the solution began to change from blue to green and then yellow. This is likely because with increasing reaction time, oxTMB further catalyzes the oxidation to form a diamine (yellow product). Therefore, 6 minutes is the optimal reaction time for this catalytic reaction system. Furthermore, the amount of catalyst is also a key factor affecting the activity of oxidase-like enzymes. Too little catalyst may lead to a low reaction rate, while too much catalyst may lead to overactivation, thus interfering with the reaction products or impairing the catalytic ability of the nanozyme. Therefore, the catalyst concentration was investigated. The experimental results showed that the catalytic effect was optimal when the catalyst concentration reached 0.5 mg / mL. As the catalyst concentration increased, the solution color gradually changed from blue to green and then yellow. This is likely because the increasing catalyst concentration leads to the further catalytic oxidation of oxTMB to form a diamine (yellow product). Therefore, in subsequent experiments, the catalyst concentration was maintained at 0.5 mg / mL.

[0091] Example 4: GSH, cys, AA detection and TAC detection

[0092] refer to Figures 28 to 30 AA (ascorbic acid), GSH (glutathione), and cys (cysteine) are common antioxidants that can be detected colorimetrically by reducing TMB and causing changes in absorbance. This colorimetric detection method is commonly used to assess the antioxidant capacity of antioxidants. Based on the oxidase-like properties of Ce / Fe-MOF, a simple method was developed as a sensing platform for the quantitative detection of GSH, AA, and cys.

[0093] 4.1. Detection of AA

[0094] Antioxidants (AA), as a general antioxidant, play an important physiological role in organisms. Their main function is to react with reactive oxygen species (ROS) through redox reactions, thereby reducing intracellular oxidative stress and protecting cells from oxidative damage. Ce / Fe-MOF oxidizes the substrate TMB to generate the colored product oxTMB. When AA is present in the system, it acts as an antioxidant, reducing the oxidation of TMB and leading to a decrease in absorbance. By measuring the change in absorbance, the concentration of AA can be reflected, thus enabling quantitative detection of AA. Within the range of 2–60 μM, the absorbance of TMB at 652 nm shows a good linear relationship with the concentration of AA, with a linear relationship of y = 0.0123x - 0.0010 and a correlation coefficient (R²). 2 The LOD was 0.9978. Using the formula LOD = 3σ / k (σ is the standard deviation of 11 blank signals and k is the slope of the standard curve), the LOD of AA was calculated to be 1.3 μM. The detection limit of ascorbic acid by colorimetric method is in the range of 0.3-8 μM.

[0095] 4.2. GSH Detection

[0096] GSH, as an important antioxidant in living organisms, plays a crucial role in maintaining homeostasis. Traditional GSH detection methods primarily rely on specialized instruments such as high-performance liquid chromatography (HPLC), which are complex and costly, limiting their widespread application. However, nanozyme-based GSH detection methods offer advantages such as simplicity, speed, high sensitivity, and strong specificity, enabling rapid quantitative detection of GSH. Based on the activity of bimetallic MOF oxidases, TMB can be used for the quantitative detection of GSH. Similar to the AA detection method described above, Ce / Fe-MOF catalyzes the oxidation of TMB, turning colorless TMB into blue oxTMB. Upon addition of GSH, the solution color and absorbance change, showing a linear relationship between absorbance and GSH concentration. By measuring the absorbance of the solution after the reaction, the concentration of GSH in the sample can be calculated. Experimental results show that in the colorimetric detection system for GSH, the linear relationship within the concentration range of 2-50 μM GSH is y = 0.0108x + 0.0198, with a correlation coefficient (R²). 2 The limit of detection (LOD) for GSH is 0.9992. The LOD for GSH is 1.2 μM, and the detection limit for GSH by colorimetric method ranges from 0.1 to 5.6 μM.

[0097] 4.3. Detection of Cys

[0098] Cysteine ​​is an important amino acid that plays a variety of biological functions in the human body, such as participating in protein synthesis, cell proliferation and differentiation, and regulating the immune system. In addition, cysteine ​​is also an important antioxidant, which can protect the body from oxidative stress by working synergistically with other antioxidants such as glutathione. Similar to the detection of AA and GSH mentioned above, in the colorimetric detection system of cys, the linear relationship between absorbance and cys concentration is y = 0.0178x + 0.0099, with a linear range of 2-30 μM, a correlation coefficient (R²) of 0.9957, a limit of detection (LOD) of cys of 1.4 μM, and a limit of detection (LOD) of 0.9-4.0 μM for colorimetric detection of GSH.

[0099] Example 5 Interference Test

[0100] refer to Figure 31 To test the interference resistance of the colorimetric sensor, potential interfering substances in real samples were tested, including 100 times the amount of K. + Na + Ca 2+ Cl - Glucose (Glu), fructose (Fru), lactose (Fru), glycine (Gly), glutamic acid (Glu), histidine (His), tryptophan (L-Tr), glutamine (L-Glu), tyrosine (L-Ty), from Figure 31 As can be seen, except for cys and GSH, the interference effect was almost negligible compared to AA. Considering that the concentration of amino acids in food is much lower than that of AA, the experimental results indicate that the colorimetric method based on the Ce / Fe-MOF-based oxidase system has high selectivity for AA.

[0101] Example 6 Stability Analysis

[0102] refer to Figure 32 Good stability is crucial for the practical application of colorimetric sensing methods. The analytical performance of the same Ce / Fe-MOF catalyst was compared after storage for different numbers of days. The results showed that the material's performance remained almost unchanged after 30 days, indicating the stability of the Ce / Fe-MOF catalyst and its applicability to the detection of real samples.

[0103] Example 7: Detection of TAC

[0104] Total antioxidant capacity (TAC) is a key indicator of the effectiveness of several antioxidants in food and pharmaceuticals. Based on the above investigation, we established a traditional determination method. In the following experiments, TAC was expressed as micromoles of AA / L to assess the nutritional value of food. The practicality of this colorimetric method was further tested in real samples. First, sample solutions diluted 10-fold were measured, and then real samples with known concentrations of AA were added to confirm the reliability of TAC assessment in fresh apples, yellow watermelons, and cucumbers. The results in Table 1 show that the recovery rate of AA ranged from 93.2% to 101.97%, with a relative standard deviation (RSD) of less than 1.54%. Among the tested samples, apples had a higher TAC content than other samples, indicating that this method is reliable for the detection of TAC in real samples. Figure 33 .

[0105] Table 1. Determination results of TAC in real samples (n=3)

[0106]

[0107] The embodiments described in this invention can be combined in various ways. Those skilled in the art will recognize that certain aspects or features described for a particular embodiment can be combined with another embodiment presented or suggested in this disclosure. Although various novel features of the principles of the invention have been shown, described, and pointed out as specific embodiments, it should be understood that various omissions, substitutions, and modifications can be made by those skilled in the art without departing from the spirit of this disclosure. Those skilled in the art will recognize that the principles of the invention can be implemented in other embodiments besides those described, which are illustrative and not restrictive.

Claims

1. A bimetallic MOF nanozyme, characterized in that, The bimetallic MOF nanozyme comprises iron, cerium, and an organic framework, wherein the organic framework is 1,3-phthalic acid, and the iron and cerium replace the H atoms in the carboxyl groups of the organic framework.

2. The method for preparing a bimetallic MOF nanozyme according to claim 1, characterized in that, Includes the following steps: S1. Dissolve FeCl3·6H2O and Ce(SO4)2·4H2O in a solvent to obtain solution A; wherein, FeCl3·6H2O:Ce(SO4)2·4H2O:solvent = (0.5~1.5)mM:1mM:10mL; S2. Dissolve 1,3-phthalic acid in N,N-dimethylformamide solution to obtain solution B; wherein, 1,3-phthalic acid:N,N-dimethylformamide = 1 mM:10 mL; S3. Mix solution A and solution B to react. After the reaction is complete, centrifuge, wash, and dry to obtain bimetallic MOF nanozymes; wherein the volume ratio of solution A to solution B is 1:

1.

3. The method for preparing a bimetallic MOF nanozyme according to claim 2, characterized in that, The solvent mentioned in S1 is N,N-dimethylformamide or anhydrous ethanol.

4. The method for preparing a bimetallic MOF nanozyme according to claim 2, characterized in that, The reaction in S3 uses a DBD micro-plasma device, where the voltage applied to the reaction tube is 70-90V and the reaction time is 30-50min.

5. The method for preparing a bimetallic MOF nanozyme according to claim 4, characterized in that, The drying temperature described in S3 is 60–70°C, and the drying time is 8–12 hours.

6. The application of the bimetallic MOF nanozyme of claim 1 in the detection of total antioxidant capacity.

7. An application as described in claim 6, characterized in that, Bimetallic MOF nanozymes are used to detect glutathione.

8. An application as described in claim 6, characterized in that, Bimetallic MOF nanozymes are used to detect ascorbic acid.

9. An application as described in claim 6, characterized in that, Bimetallic MOF nanozymes are used to detect cysteine.

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