Multi-enzyme-activity-like silver-nanoparticle-loaded silver-based metal organic framework nano-enzyme as well as preparation and application thereof

Abstract

The invention discloses a multi-enzyme-like activity silver-nanoparticle-loaded silver-based metal organic framework nano-enzyme as well as preparation and application of the multi-enzyme-like activity silver-nanoparticle-loaded silver-based metal organic framework nano-enzyme. 2-methylimidazole serves as an organic ligand, triethylamine serves as a deprotonation reagent, an alkaline environment is provided, methyl alcohol serves as a solvent and a reducing agent of silver ions, and the silver-based metal organic framework is obtained through the coordination effect of the deprotonated 2-methylimidazole and the silver ions; meanwhile, under the alkaline condition, silver ions adsorbed on the surface of the silver-based metal organic framework are reduced into silver nanoparticles by utilizing methanol, and the silver-nanoparticle-loaded silver-based metal organic framework nano-enzyme is prepared. The nano-enzyme can show multiple enzyme activities such as oxidoid enzyme activity, peroxidase activity and laccase activity under different conditions, and colorimetric detection of multiple microanalysis objects in different types of complex samples can be realized by utilizing different enzyme activities under different detection conditions.

Description

Technical Field

[0001] The invention belongs to the field of synthesis and application of nanomaterials, and specifically relates to a silver-based metal organic framework nanozyme loaded with silver nanoparticles with multi-enzyme activity and the preparation and application of the nanozyme. Background Art

[0002] Metal-organic frameworks (MOFs) are materials formed by self-assembly of organic ligands and metal ions as secondary structural units. They are crystalline porous inorganic-organic hybrid materials with a periodic network structure. Due to their outstanding design flexibility, excellent surface area and diversity of topological structures, they have shown a wide range of potential applications in gas adsorption / separation, catalysis, sensing, chromatographic separation and energy storage. At present, the research focus of MOFs has shifted from early synthesis to performance optimization and application scenario expansion. In the field of nanozyme research, MOFs also show unparalleled advantages over other traditional materials due to their unique properties. Compared with natural enzymes, MOFs materials can not only provide more active sites for nanozymes, giving them excellent enzyme-like catalytic activity, but also have the advantages of low cost, simple preparation, high stability and durability.

[0003] Most of the MOF nanozymes reported so far only have single enzyme mimicking activity, while there are few reports of MOF materials with multi-enzyme mimicking activity, and their preparation methods are complex and require high conditions. Compared with nanozymes with single enzyme activity, nanozymes with multi-enzyme activity have many excellent properties, including synergistic effects, cascade reactions, and different response activities in different environments, which make them more widely used in practice. Based on this, the present invention uses 2-methylimidazole as an organic ligand, triethylamine as a deprotonating agent and provides an alkaline environment, methanol as a solvent and a reducing agent for silver ions, and utilizes the coordination of deprotonated 2-methylimidazole with silver ions to obtain a silver-based metal organic framework; at the same time, under alkaline conditions, methanol is cleverly used to reduce the silver ions adsorbed on the surface of the silver-based metal organic framework into silver nanoparticles, and a silver-based metal organic framework nanozyme loaded with silver nanoparticles is prepared. The preparation method of the present invention is simple and easy, the synthesis conditions are mild, and it can be directly synthesized at room temperature without high temperature and high pressure, without expensive instruments. This nanozyme can simultaneously exhibit three different simulated enzyme activities: oxidase-like, peroxidase-like, and laccase-like, and can achieve simple, fast, and low-cost multi-target colorimetric detection in different response scenarios. It has very important application value in the fields of bioanalysis, colorimetric sensing, etc. Summary of the invention

[0004] The object of the present invention is to provide a silver-based metal organic framework (AgNPs@Ag-MOF) nanozyme loaded with silver nanoparticles having multi-enzyme-like activity, and to realize colorimetric detection applications in different application scenarios. The present invention adopts a deprotonation method to increase the coordination bond strength between silver ions and nitrogen atoms on the metal organic framework, and generates silver nanoparticles in situ on the silver-based metal organic framework by reducing silver ions with a reducing agent methanol under alkaline conditions, thereby realizing the preparation of AgNPs@Ag-MOF nanozymes. On the one hand, the imidazole ring of 2-methylimidazole has deprotonable ammonia nitrogen, and the protons on the ammonia nitrogen are removed by the proton extraction reaction of triethylamine and 2-methylimidazole to make it negatively charged, thereby enhancing the coordination ability of silver ions and ammonia nitrogen. At the same time, 2-methylimidazole is a molecule with a π-π conjugated system, and changing the electron distribution in the conjugated system can enhance the coordination ability of nitrogen atoms. The present invention utilizes a weakly polar solvent (methanol) to reduce the solvent's attraction to electrons around nitrogen atoms, thereby increasing the electron density of nitrogen atoms on the imidazole ring, making it easier for nitrogen atoms to coordinate with silver ions in Lewis base-Lewis acid, enhancing the coordination ability between 2-methylimidazole and silver ions, and improving the enzyme-like activity of the material. On the other hand, methanol will generate alcohol salts under alkaline conditions, and as a reducing agent for silver ions, the silver ions are reduced in situ on the silver-based metal organic framework to generate silver nanoparticles; the silver nanoparticles generated in situ on the silver-based metal organic framework further improve the enzyme-like activity of the material. AgNPs@Ag-MOF nanozyme can directly oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the blue oxidation product oxTMB, showing oxidase-like activity; in the presence of hydrogen peroxide (H2O2), it can quickly oxidize the substrate 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to the green product ABTS·+, showing peroxidase activity; it can also catalyze the phenolic substance 2,4-dichlorophenol (2,4-DCP) into 2,4-dichlorobenzoquinone, and form a pink coupling product with 4-aminoantipyrine (4-AAP), showing laccase-like activity.

[0005] The present invention proposes a mild preparation method for a silver-based metal organic framework nanozyme loaded with silver nanoparticles, and demonstrates the multi-enzyme activity of the new nanozyme, further expanding the application of metal organic framework materials in the fields of biological analysis and colorimetric detection.

[0006] To achieve the above object, the present invention adopts the following technical solution: The preparation method of the silver-based metal organic framework nanozyme loaded with silver nanoparticles comprises the following steps: (1) Using 2-methylimidazole as an organic ligand, methanol as a solvent (the solvent methanol also serves as a reducing agent for silver ions in the next step), and triethylamine as a deprotonating agent and providing an alkaline environment required for the reduction reaction, a deprotonated 2-methylimidazole methanol solution is prepared.

[0007] (2) Prepare an aqueous silver nitrate solution with the same concentration as the deprotonated 2-methylimidazole methanol solution, then slowly drip an equal amount of the aqueous silver nitrate solution into the deprotonated 2-methylimidazole methanol solution, stirring continuously until the addition is complete, and react at room temperature in the dark for 2-24 hours to obtain a uniform milky white suspension; then centrifuge, wash the obtained solid product with water and ethanol three times each, and dry it in a vacuum drying oven at 60°C for 24 hours to obtain AgNPs@Ag-MOF nanozyme.

[0008] Multi-enzyme activity demonstration of silver-based metal-organic framework nanozymes loaded with silver nanoparticles: (1) Oxidase-like activity: AgNPs@Ag-MOF was used as nanozyme, and the color-developing substrate TMB was added. The solution changed from colorless to blue, and its oxidase-like activity was demonstrated by combining the UV-visible absorption spectrum. The specific steps are as follows: 50 μL AgNPs@Ag-MOF dispersion (final concentration of 25 μg / mL) and 50 μL TMB solution (final concentration of 0.5 mM) were added to 1900 μL acetic acid-sodium acetate (HAc-NaAc) solution (12 mM, pH 5.0), mixed evenly, and reacted at 20 °C for 20 minutes. At the same time, TMB solution and AgNPs@Ag-MOF dispersion were used as control groups, and the absorbance value at a wavelength of 650 nm (the characteristic peak position of oxidized TMB (oxTMB)) was recorded using a UV-visible absorption spectrometer. The results showed that the solutions in the control group did not turn blue. Only when AgNPs@Ag-MOF and TMB were present at the same time did the solution turn blue, and a characteristic absorption peak appeared at 650nm, proving that AgNPs@Ag-MOF nanozyme has oxidase-like activity.

[0009] (2) Peroxidase-like activity: AgNPs@Ag-MOF was used as nanozyme. When the chromogenic substrate ABTS was added in the presence of H2O2, the solution changed from colorless to green. Combined with the UV-visible absorption spectrum, its peroxidase-like activity was demonstrated. The specific steps are as follows: add 100 μL AgNPs@Ag-MOF dispersion (final concentration is 20 μg / mL), 50 μL ABTS solution (final concentration is 0.5 mM) and 50 μL H2O2 solution (final concentration is 0.1 mM) to 1800 μL HAc-NaAc solution (10 mM, pH 3.0), mix well, and react at 37.5°C for 40 min. At the same time, ABTS solution, H2O2 solution + ABTS solution, AgNPs@Ag-MOF dispersion + ABTS solution, and H2O2 solution + AgNPs@Ag-MOF dispersion are used as control groups, and the absorbance value at a wavelength of 414 nm (the characteristic peak position of ABTS·+) is recorded using a UV-visible absorption spectrometer. The results showed that the solutions in the control group did not turn green. Only when AgNPs@Ag-MOF, H2O2 and ABTS were present at the same time did the solutions turn green, and a characteristic absorption peak appeared at 414nm, proving that AgNPs@Ag-MOF nanozyme has peroxidase-like activity.

[0010] (3) Laccase-like activity: When AgNPs@Ag-MOF was used as nanozyme and the phenolic substance 2,4-DCP and the chromogenic substrate 4-AAP were added, the solution changed from colorless to pink, and combined with the UV-visible absorption spectrum, it showed its laccase-like activity. The specific steps are as follows: add 100 μL AgNPs@Ag-MOF dispersion (final concentration of 250 μg / mL), 200 μL 2,4-DCP solution (final concentration of 2.5 mM) and 200 μL 4-AAP solution (final concentration of 0.6 mM) to 1500 μL 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (20 mM, pH 6.0), mix well, react at 40 ° C for 30 minutes, centrifuge and take the supernatant. At the same time, 2,4-DCP solution, 2,4-DCP solution + AgNPs@Ag-MOF dispersion, 4-AAP solution, and 4-AAP solution + AgNPs@Ag-MOF dispersion are used as control groups, and the absorbance value at a wavelength of 510 nm (the characteristic peak position of the pink coupling product) is recorded using a UV-visible absorption spectrometer. The results showed that the solutions in the control group did not turn pink. Only when AgNPs@Ag-MOF, 2,4-DCP and 4-AAP existed at the same time did the solutions turn pink and a characteristic absorption peak appeared at 510 nm, proving that the AgNPs@Ag-MOF nanozyme had laccase-like activity.

[0011] The significant advantages of the present invention are: ① Compared with traditional nanocomposite materials, the silver-based metal organic framework nanozyme loaded with silver nanoparticles provided by the present invention is simple to prepare, low-cost, does not require expensive instruments, and is easy to promote; ② The silver-based metal organic framework nanozyme loaded with silver nanoparticles provided by the present invention can exhibit different enzyme-like activities in different usage scenarios under different conditions through different substrates, so that the actual usage scenario of the nanozyme is no longer single; ③ The silver-based metal organic framework nanozyme loaded with silver nanoparticles provided by the present invention can be combined with colorimetric detection and other methods to achieve its wide application in the fields of biological analysis, colorimetric sensing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a transmission electron microscopy morphology image of the silver-based metal organic framework nanozyme loaded with silver nanoparticles prepared by the present invention.

[0013] Figure 2 The transmission electron microscope high-angle annular dark field (HAADF) scanning image and particle size distribution diagram of the silver-based metal organic framework nanozyme loaded with silver nanoparticles prepared by the present invention.

[0014] Figure 3 This is an X-ray photoelectron spectroscopy characterization diagram of the silver-based metal organic framework nanozyme loaded with silver nanoparticles prepared by the present invention.

[0015] Figure 4 This is an X-ray powder diffraction characterization diagram of the silver-based metal organic framework nanozyme loaded with silver nanoparticles prepared in the present invention.

[0016] Figure 5 This is a diagram of the oxidase-like activity of the silver-based metal-organic framework nanozyme loaded with silver nanoparticles prepared in the present invention.

[0017] Figure 6 This is a diagram of the peroxidase-like activity of the silver-based metal-organic framework nanozyme loaded with silver nanoparticles prepared in the present invention.

[0018] Figure 7 This is a diagram of the laccase-like activity of the silver-based metal-organic framework nanozyme loaded with silver nanoparticles prepared in the present invention.

[0019] Figure 8 The present invention is a working curve for colorimetric detection of ascorbic acid (AA) standard solutions of different concentrations using the silver-based metal organic framework loaded with silver nanoparticles prepared by the present invention as a nanozyme.

[0020] Fig. 9 The working curve is a colorimetric detection curve of H2O2 standard solutions of different concentrations by the silver-based metal organic framework seat nanozyme loaded with silver nanoparticles prepared by the present invention.

[0021] Fig.10 The working curve is a colorimetric detection of adrenaline standard solutions of different concentrations using the silver-based metal organic framework loaded with silver nanoparticles prepared by the present invention as a nanozyme. DETAILED DESCRIPTION

[0022] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0023] Embodiment 1: The preparation of silver-based metal organic framework nanozymes loaded with silver nanoparticles comprises the following steps: (1) Preparation of a deprotonated organic ligand solution: 1 mmol of triethylamine and 2 mmol of 2-methylimidazole were added to 10 mL of methanol and dissolved by ultrasonication at room temperature to obtain a deprotonated organic ligand solution.

[0024] (2) Preparation of silver-based metal-organic framework nanozymes loaded with silver nanoparticles: 2 mmol of silver nitrate was dissolved in 10 mL of secondary water, and the resulting silver nitrate aqueous solution was slowly dripped into the deprotonated organic ligand solution of step (1) and stirred continuously until the addition was complete. The mixture was reacted in the dark at room temperature for 12 hours to obtain a uniform milky white suspension. The solid product was collected by centrifugation, and the solid product was washed three times with water and three times with ethanol, and then dried in a vacuum drying oven at 60°C for 24 hours to obtain silver-based metal-organic framework (AgNPs@Ag-MOF) nanozymes loaded with silver nanoparticles.

[0025] The silver-based metal-organic framework (AgNPs@Ag-MOF) nanozyme loaded with silver nanoparticles prepared according to the above steps was characterized by transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Figure 1 This is the TEM morphology of the nanozyme, from which we can see that there are many silver nanoparticles distributed in the nanozyme material. Figure 2 Shown Figure 1 High-angle annular dark field (HAADF) scanning image of the morphology image, from which the morphology of silver nanoparticles can be observed more clearly. Figure 2 The particle size distribution diagram of silver nanoparticles is also shown, and it can be found that the average particle size is 5.9nm. The above results prove the successful synthesis of AgNPs@Ag-MOF nanozyme. Figure 3 This is the full-band XPS energy spectrum of the nanozyme material, from which the four element signals of C, N, O, and Ag can be clearly seen, which also proves the successful synthesis of AgNPs@Ag-MOF nanozyme.

[0026] Embodiment 2: The preparation of silver-based metal organic framework nanozymes loaded with silver nanoparticles comprises the following steps: (1) Preparation of a deprotonated organic ligand solution: 2 mmol of triethylamine and 2 mmol of 2-methylimidazole were added to 10 mL of methanol and dissolved by ultrasonication at room temperature to obtain a deprotonated organic ligand solution.

[0027] (2) Preparation of silver-based metal-organic framework nanozymes loaded with silver nanoparticles: 2 mmol of silver nitrate was dissolved in 10 mL of secondary water, and the resulting silver nitrate aqueous solution was slowly dripped into the deprotonated organic ligand solution of step (1) and stirred continuously until the addition was complete. The mixture was reacted in the dark at room temperature for 16 hours to obtain a uniform milky white suspension. The solid product was collected by centrifugation, and the solid product was washed with water and ethanol for 3 times each, and then dried in a vacuum drying oven at 60°C for 24 hours to obtain silver-based metal-organic framework (AgNPs@Ag-MOF) nanozymes loaded with silver nanoparticles.

[0028] The silver-based metal organic framework (AgNPs@Ag-MOF) nanozyme loaded with silver nanoparticles prepared according to the above steps was characterized by X-ray powder diffraction (XRD). Figure 4 As shown, the main diffraction peaks show several very sharp peaks at 2θ = 16.90°, 22.45°, 28.00° and 28.70°, which are respectively related to (011), (200), It is related to the (013) crystal plane, indicating that the nanozyme has good crystallinity and is a typical polycrystalline material, which also proves the successful synthesis of AgNPs@Ag-MOF nanozyme.

[0029] Embodiment 3: (1) The AgNPs@Ag-MOF prepared in Example 1 was used as nanozyme to investigate the oxidase-like activity.

[0030] The experiment used a UV-visible absorption spectrometer as a detection instrument to prove that AgNPs@Ag-MOF has oxidase-like activity as a nanozyme. 50μL AgNPs@Ag-MOF dispersion (final concentration of 25μg / mL) and 50μL TMB solution (final concentration of 0.5mM) were added to 1900μL acetic acid-sodium acetate (HAc-NaAc) solution (12mM, pH5.0), mixed evenly, and reacted at 20°C for 20 minutes. At the same time, TMB solution and AgNPs@Ag-MOF dispersion were used as control groups, and the absorbance value at a wavelength of 650nm was recorded. The results are shown in Figure 2. Figure 5As shown, there is no characteristic absorption peak at 650nm in the control group. Only when AgNPs@Ag-MOF and TMB exist at the same time, a characteristic absorption peak appears at a wavelength of 650nm, which proves that AgNPs@Ag-MOF nanozyme has oxidase-like activity.

[0031] (2) The AgNPs@Ag-MOF prepared in Example 1 was used as nanozyme to investigate its peroxidase-like activity.

[0032] The experiment used a UV-visible absorption spectrometer as a detection instrument to prove that AgNPs@Ag-MOF as a nanozyme has peroxidase-like activity. 100μL AgNPs@Ag-MOF dispersion (final concentration of 20μg / mL), 50μLA ABTS solution (final concentration of 0.5mM) and 50μL H2O2 solution (final concentration of 0.1mM) were added to 1800μL HAc-NaAc solution (10mM, pH3.0), mixed evenly, and reacted at 37.5°C for 40 minutes. At the same time, ABTS solution, H2O2 solution + ABTS solution, AgNPs@Ag-MOF dispersion + ABTS solution, and H2O2 solution + AgNPs@Ag-MOF dispersion were used as control groups, and the absorbance value at a wavelength of 414nm was recorded. The results are as follows: Figure 6 As shown, the control group H2O2 solution + ABTS solution has a smaller peak at 414nm. The reason is that at 37.5℃, hydrogen peroxide decomposes under heat to produce ABTS·+; and only when AgNPs@Ag-MOF is added and AgNPs@Ag-MOF, H2O2, and ABTS exist at the same time, will an obvious characteristic absorption peak appear at a wavelength of 414nm, proving that the AgNPs@Ag-MOF nanozyme has oxidase-like activity.

[0033] (3) The AgNPs@Ag-MOF prepared in Example 1 was used as nanozyme to investigate the laccase-like activity.

[0034] The experiment used a UV-visible absorption spectrometer as a detection instrument to prove that AgNPs@Ag-MOF has laccase-like activity as a nanozyme. 100μL AgNPs@Ag-MOF dispersion (final concentration of 250μg / mL), 200μL 2,4-DCP solution (final concentration of 2.5mM) and 200μL 4-AAP solution (final concentration of 0.6mM) were added to 1500μL 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) solution (20mM, pH 6.0), mixed evenly, reacted at 40°C for 30 minutes, and centrifuged to obtain the supernatant. At the same time, 2,4-DCP solution, 2,4-DCP solution + AgNPs@Ag-MOF dispersion, 4-AAP solution, and 4-AAP solution + AgNPs@Ag-MOF dispersion were used as control groups, and the absorbance value at a wavelength of 510nm was recorded. The results are as follows Figure 7 As shown, there is no characteristic absorption peak at 510 nm in the control group. Only when AgNPs@Ag-MOF, 2,4-DCP and 4-AAP exist at the same time, a characteristic absorption peak appears at a wavelength of 510 nm, proving that AgNPs@Ag-MOF nanozyme has laccase-like activity.

[0035] Embodiment 4: The AgNPs@Ag-MOF prepared in Example 1 was used as a nanozyme, and ascorbic acid (AA) with reducing properties was added to the oxidase-like color development system, wherein the blue oxTMB was reduced to colorless TMB by AA, and the color changes produced by adding different concentrations of AA were compared, and the difference in absorbance change was recorded to achieve colorimetric detection of AA. The specific steps are as follows: 50μL AgNPs@Ag-MOF dispersion (final concentration of 25μg / mL) and 50μL TMB solution (final concentration of 0.5mM) were added to 1900μL HAc-NaAc solution (12mM, pH 5.0), mixed evenly, reacted at 20°C for 20 minutes, and the absorbance at 650nm was recorded; then 50μL ascorbic acid standard solutions of different concentrations (0.001-3.0mM) were added to the above solutions, allowed to react for 5 minutes, and the absorbance at 650nm was recorded. The colorimetric detection of AA was achieved by detecting the absorbance difference produced by AA solutions of different concentrations.

[0036] AgNPs@Ag-MOF, as a nanozyme with oxidase-like activity, has a high affinity for the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The amount of blue oxTMB produced by the oxidase-like reaction mediated by AgNPs@Ag-MOF decreases after the addition of AA solution. Combined with UV-visible spectrophotometer as a readout device, a simple and rapid AA colorimetric detection method was established. By comparing the absorbance difference produced by AA solutions of different concentrations, a working curve ( Figure 8 ). The linear equation of the curve is Δ 650nm =0.40433C AA +0.05249(R 2 =0.992), the linear range is 0.001-3 mM, and the detection limit is 0.3 μM. The above results show that the silver-based metal organic framework nanozyme (AgNPs@Ag-MOF) loaded with silver nanoparticles provided by the present invention can be used as an oxide-like nanozyme and has good application prospects in the field of colorimetric detection.

[0037] Embodiment 5: The AgNPs@Ag-MOF prepared in Example 2 was used as a peroxidase-like nanozyme. The concentration of the product ABTS·+ was changed by changing the concentration of H2O2, and the absorbance value was recorded by using the color change to achieve colorimetric detection of H2O2. The specific experiment is as follows: 100 μL AgNPs@Ag-MOF dispersion (final concentration of 20 μg / mL), 50 μL ABTS solution (final concentration of 0.5 mM) and 50 μL H2O2 solution of different concentrations (0.004-4.5 mM) were added to 1800 μL HAc-NaAc solution (10 mM, pH 3.0), mixed evenly, and reacted at 37.5°C for 40 minutes. The absorbance value at a wavelength of 414 nm was recorded using a UV-visible absorption spectrometer.

[0038] AgNPs@Ag-MOF, as a nanozyme with peroxidase-like activity, has a high affinity for the substrate 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) in the presence of hydrogen peroxide. The amount of green ABTS·+ produced by the peroxidase-like reaction mediated by AgNPs@Ag-MOF increases with the concentration of H2O2. Combined with a UV-visible spectrophotometer as a readout device, a simple and rapid H2O2 colorimetric detection method was established. Fig. 9 is the working curve for colorimetric detection of H2O2 solutions with different concentrations. The linear equation of the curve is Δ 650nm =0.19884+0.68586C H2O2 (R 2 =0.998), the linear range is 0.004-4.5 mM, and the detection limit is 2.0 μM. The above results show that the AgNPs@Ag-MOF provided by the present invention can be used as a peroxidase-like nanozyme and has good practical application prospects in the field of colorimetric detection.

[0039] Embodiment 6: The AgNPs@Ag-MOF prepared in Example 2 was used as a laccase-like nanozyme and adrenaline was used as the analysis object to investigate its colorimetric detection application. The specific experiment is as follows: 100 μL (final concentration of 0.25 mg / mL) of AgNPs@Ag-MOF dispersion and 200 μL of adrenaline solution of different concentrations (final concentration of 6-50 μM) were added to 1700 μL (20 mM, pH = 6.0) HEPES solution. After mixing and reacting at 40°C for 30 minutes, 1.5 mL of the supernatant was taken after centrifugation at 8000 rpm for 3 minutes, and the absorbance value at a wavelength of 510 nm was recorded using a UV-visible absorption spectrometer.

[0040] AgNPs@Ag-MOF, as a nanozyme with laccase-like activity, can directly catalyze adrenaline. The pink substance produced by the laccase-like activity mediated by AgNPs@Ag-MOF increases with the amount of adrenaline added. Combined with a UV-visible spectrophotometer as a readout device, a simple and rapid colorimetric detection method for adrenaline was established. Fig.10 This is the working curve for colorimetric detection of epinephrine solutions of different concentrations. The linear equation of this curve is Δ 510nm =0.13157+0.00242C EPi (R 2 =0.998), the linear range is 6-50 μM, and the detection limit is 2 μM. The above results show that the AgNPs@Ag-MOF provided by the present invention can be used as a laccase-like nanozyme and has good application prospects in the field of colorimetric detection.

[0041] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing a silver-based metal organic framework nanozyme loaded with silver nanoparticles with multi-enzyme activity, characterized in that: The following steps are involved: 1) Add triethylamine and 2-methylimidazole to methanol and dissolve them under ultrasonication at room temperature to obtain a deprotonated organic ligand solution; 2) Dissolve silver nitrate in secondary water, then slowly drip the obtained silver nitrate aqueous solution into the deprotonated organic ligand solution and keep stirring until the addition is complete, and react in the dark at room temperature for 2 to 24 hours to obtain a uniform milky white suspension; collect the solid product by centrifugation, wash the solid product with water and ethanol three times respectively, and then dry it in a vacuum drying oven at 60°C for 24 hours to obtain a silver-based metal organic framework nanozyme loaded with silver nanoparticles with multi-enzyme activity.

2. The preparation method according to claim 1, characterized in that: The molar ratio of the triethylamine, 2-methylimidazole and silver nitrate is 1-2:2:

2.

3. The preparation method according to claim 1, characterized in that: The multi-enzyme-like activity includes oxidase-like activity, peroxidase-like activity, and laccase-like activity.

4. A silver-based metal organic framework nanozyme loaded with silver nanoparticles with multi-enzyme activity, characterized in that: The method is prepared by any one of claims 1 to 3.

5. The silver-based metal-organic framework nanozyme according to claim 4, characterized in that: The multi-enzyme-like activity includes oxidase-like activity, peroxidase-like activity, and laccase-like activity.

6. Application of the silver-based metal organic framework nanozyme as described in any one of claims 4 to 5 in the field of bioanalysis and colorimetric detection.

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