A preparation method of a nano-composite enzyme for detecting tannic acid

Abstract

The application discloses a preparation method of a nano-composite enzyme for detecting tannic acid, and the method comprises the following steps: step one, adopting alkaline amino acids and polypeptides with imidazole groups to simulate the primary structure of natural oxidase, and preparing amino acid and polypeptide modified cerium oxide with enhanced oxidase and peroxidase performance; step two, in-situ compounding the polypeptide modified cerium oxide and zeolite imidazolate framework material with large specific surface area, good stability, hydrophilicity and hydrophobicity, and specific electron distribution to obtain nano-composite enzyme with enhanced oxidase and peroxidase performance; and step three, after etching treatment of the nano-composite enzyme in a polar medium, the nano-composite enzyme with enhanced oxidase and peroxidase performance for detecting tannic acid is obtained. By utilizing the competition between tannic acid and a substrate, the tannic acid is detected by using a colorimetric or fluorescent method, and the operation process is simple and rapid, the selectivity is high, the detection limit is low, the detection range is wide, and the nano-composite enzyme shows a good application prospect for sensitive detection of tannic acid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of material preparation, and particularly relates to a preparation method of a nano-composite enzyme for detecting tannic acid. BACKGROUND

[0002] As an antioxidant, tannic acid is often used in the fields of bone regeneration, skin adhesive, wound dressing, food oxidation prevention, etc. Oral administration of low-concentration tannic acid is beneficial to maintaining the level of intestinal flora. However, high-concentration tannic acid can reduce the nutritional value of food and has cytotoxicity, which is harmful to human health. Therefore, it is of great significance to determine the content of tannic acid in food. At present, the methods for detecting tannic acid mainly include high-performance liquid chromatography-tandem mass spectrometry, electrochemical method, chemiluminescence method and fluorescence method, etc. However, these methods are expensive in instrument price, complicated in pretreatment and low in sensitivity. Therefore, it is necessary to develop a sensitive and rapid method for detecting tannic acid.

[0003] Oxidase and peroxidase can oxidize substrates 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, o-phenylenediamine or fluorescent red dye to generate colored products in the presence of oxygen and hydrogen peroxide, respectively. The antioxidant effect of tannic acid can reduce the absorbance or fluorescence signal of the colored products by reducing the colored products in the oxidized state to colorless. Therefore, tannic acid can be tested by colorimetric or fluorescence method under the action of oxidase or peroxidase, and the analysis speed is fast and the operation process is simple and rapid. However, natural enzymes are low in stability, high in cost and difficult to store, and although they have high catalytic activity, they are limited in practical application. Therefore, it has become a current trend to replace natural enzymes with nano-enzymes, which have the characteristics of high catalytic stability, easy functionalization and low cost. SUMMARY

[0004] Technical problem: The purpose of the present application is to provide a preparation method of a nano-composite enzyme for detecting tannic acid, which can catalyze substrates without the participation of exogenous hydrogen peroxide, construct a sensor with simple stability and high detection sensitivity, and realize the detection of tannic acid in food.

[0005] Technical solution: The preparation method of the nano-composite enzyme for detecting tannic acid comprises the following steps:

[0006] Step one: The basic amino acid and the polypeptide with imidazole group are used to simulate the primary structure of natural oxidase to prepare amino acid and polypeptide modified cerium oxide with enhanced oxidase and peroxidase performance;

[0007] Step two, the polypeptide modified cerium oxide and the zeolite imidazolate framework material with large specific surface area, good stability, hydrophilic and hydrophobic properties and specific electronic distribution are in-situ compounded to obtain a nano-composite enzyme with enhanced oxidase and peroxidase properties;

[0008] Step three, the nano-composite enzyme is etched in a polar medium for post-processing to obtain a nano-composite enzyme with enhanced oxidase and peroxidase properties for detecting tannic acid.

[0009] In step one, the polypeptide modified cerium oxide is synthesized by using a cerium salt, an amino acid and a polypeptide.

[0010] The cerium salt is cerium nitrate, cerium chloride, cerium acetate or cerium sulfate; the amino acid is arginine, lysine or histidine; and the polypeptide is carnosine, anserine or copper peptide.

[0011] The mass ratio of the amino acid and the polypeptide is 10:3-5:9, and the mass concentration of the cerium salt is 10-50 mg / mL.

[0012] In step two, the zeolite imidazolate framework material is synthesized by using a zinc salt, a cobalt salt, an organic ligand and a solvent.

[0013] The zinc salt is zinc nitrate, zinc chloride or zinc sulfate; the cobalt salt is cobalt nitrate, cobalt chloride or cobalt sulfate; the organic ligand is 2-methylimidazole, 2-ethylimidazole, benzimidazole or imidazole-2-carboxaldehyde; and the solvent is methanol, water or N,N-dimethylformamide.

[0014] The molar ratio of the zinc salt or the cobalt salt to the organic ligand is 1:1-1:20, and the molar ratio of the cobalt salt to the zinc salt is 1:0-1:2.

[0015] The volume ratio of the polypeptide modified cerium oxide to the solvent in which the organic ligand of the synthesized zeolite imidazolate framework material is dissolved is 1:8-1:40.

[0016] In step three, the nano-composite enzyme is etched in a polar medium, and the polar medium used is glycerol, ultrapure water or methanol.

[0017] The mass ratio of the nano-composite enzyme material to the polar medium is 1:100-1:500, and the pH value of the polar medium is 3-9.

[0018] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0019] (1) The catalytic performance on the surface of the nano-enzyme is related to the surface characteristics of the nano-enzyme and the catalytic environment. Appropriate surface modification adjusts the surface charge, acidity, active site and adsorption or desorption performance of the reactants or products of the nano-enzyme, thereby changing the catalytic activity of the nano-enzyme. Thus, the amino acid and polypeptide co-modified cerium oxide nano-enzyme is prepared. The amino acid provides an alkaline environment to promote the generation of cerium oxide. The distal imidazole of the polypeptide simulates the primary structure of the natural oxidase. The hydrogen bond and the delocalized pi electron of the imidazole are connected through the hydrogen bond between the nitrogen atom of the distal imidazole and the substrate, which is beneficial to the electron transfer and promotes the enrichment of the substrate on the surface of the nano-enzyme.

[0020] (2) The modified cerium oxide is synthesized by a green one-pot synthesis method. The truncated octahedral cerium oxide with uniform morphology and an average particle size of 5-10 nm is obtained. The truncated octahedral cerium oxide is in-situ synthesized with zeolite imidazolate framework-67. The excellent oxidase-like activity, microporous structure and large specific surface area of the cobalt-based and zinc-based zeolite imidazolate framework enhance the oxidase-like activity of the nano-enzyme. The morphology of the zeolite imidazolate framework material after compounding does not change significantly.

[0021] (3) The synthesized nano-composite enzyme increases the mesoporous structure after etching, improves the transport efficiency of the reactants and products in the catalytic process, and further improves the oxidase-like activity of the material.

[0022] (4) The synthesized nano-composite enzyme not only has oxidase-like activity, but also has peroxidase-like activity. The oxidase-like activity of the nano-composite enzyme can catalyze 3,3',5,5'-tetramethylbenzidine or fluorescent red dye to generate hydrogen peroxide in the presence of oxygen. Hydrogen peroxide generates hydroxyl radicals and superoxide anion radicals under the action of peroxidase-like activity. The high-activity hydroxyl radicals and superoxide anion radicals can further oxidize 3,3',5,5'-tetramethylbenzidine or fluorescent red dye, thereby improving the sensitivity of the reaction. The cascade reaction catalyzed by the nano-composite enzyme also occurs on the surface of the nano-composite enzyme, which shortens the mass transfer distance and improves the mass transfer efficiency. The catalytic process does not require the addition of unstable exogenous hydrogen peroxide, so that the sensing system is simpler and more stable.

[0023] (5) The sensor constructed based on the nano-composite enzyme has high selectivity and detection performance. The detection limit is 11 nM, which is higher than that of other reported sensors for detecting tannic acid. The recovery of nine food samples is detected, and the recovery rate ranges from 80.87% to 113.8%. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1are transmission electron microscope images of modified cerium oxide, zeolitic imidazolate framework-67 and nanocomposite enzyme, wherein A is a transmission electron microscope image of modified cerium oxide, B is a transmission electron microscope image of zeolitic imidazolate framework-67, and C is a transmission electron microscope image of nanocomposite enzyme before etching;

[0025] Figure 2 are transmission electron microscope images of nanocomposite enzyme after etching, wherein A is a transmission electron microscope image of nanocomposite enzyme just etched in water, B is a transmission electron microscope image of nanocomposite enzyme etched in water for three days, and C is a transmission electron microscope image of nanocomposite enzyme etched in water for six days;

[0026] Figure 3 are X-ray diffraction and Fourier infrared images of nanocomposite enzyme, wherein A is an X-ray diffraction image of zeolitic imidazolate framework-67 and nanocomposite enzyme before etching, B is an X-ray diffraction image of nanocomposite enzyme after etching, and C is a Fourier infrared image of nanocomposite enzyme before and after etching;

[0027] Figure 4 are ultraviolet-visible absorption spectrum images of zeolitic imidazolate framework-67, modified cerium oxide and different nanocomposite mimetic enzymes;

[0028] Figure 5 are fluorescence spectrum images of zeolitic imidazolate framework-67, modified cerium oxide and different nanocomposite mimetic enzymes;

[0029] Figure 6 are ultraviolet absorption intensity changes with tannic acid concentration and linear range of detection, wherein A is an ultraviolet absorption spectrum image before and after 1 mM tannic acid is added, B is an ultraviolet absorption spectrum image of different concentrations of tannic acid added, C is a tannic acid concentration dependent reaction curve image of nanocomposite enzyme colorimetric sensor, and D is a linear calibration image of tannic acid concentration of nanocomposite enzyme colorimetric sensor;

[0030] Figure 7 are fluorescence intensity changes with tannic acid concentration and linear range of detection, wherein A is a fluorescence intensity image before and after 1 mM tannic acid is added, B is a fluorescence intensity image of different concentrations of tannic acid added, C is a tannic acid concentration dependent reaction curve image of nanocomposite enzyme fluorescence sensor, and D is a linear calibration image of tannic acid concentration of nanocomposite enzyme fluorescence sensor;

[0031] Figure 8 is a specificity image of the sensor constructed by the nanocomposite enzyme. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further illustrated below by examples.

[0033] Reagents and instrumentations

[0034] 2-methylimidazole, Ce(NO3)3·6H2O, L-arginine were purchased from Aladdin. 3,3',5,5'-tetramethylbenzidine (TMB), Fluorescent red dye (AR), L-carnosine, Tannic acid (TA) were purchased from Macklin. All chemicals were of analytical grade and used without further purification. The solutions used in the experiment were prepared with ultrapure water (resistance ≥ 18.2 MΩ / cm).

[0035] UV-Vis absorption spectra and absorbance values were measured using a UV-Vis spectrophotometer (Cary 100, Agilent, Singapore). X-ray diffraction (XRD) patterns were obtained on an X-ray diffractometer (Ultima IV, Rigaku, Japan) using a graphite monochromator Cu K α radiation. Fourier transform infrared spectroscopy (FT-IR) was measured using a FT-IR spectrometer (NICOLET iS10, Thermo Fisher, USA).

[0036] The preparation method of the nanocomposite enzyme for detecting tannic acid comprises the following steps:

[0037] Step one, using basic amino acids and polypeptides with imidazole groups to simulate the primary structure of natural oxidase, preparing amino acid and polypeptide modified cerium oxide with enhanced oxidase and peroxidase performance;

[0038] Step two, in-situ compounding the polypeptide modified cerium oxide and zeolite imidazolate framework material with large specific surface area, good stability, hydrophilic and hydrophobic properties, and specific electronic distribution to obtain nanocomposite enzyme with enhanced oxidase and peroxidase performance;

[0039] Step three, etching and treating the nanocomposite enzyme in a polar medium to obtain nanocomposite enzyme with enhanced oxidase and peroxidase performance for detecting tannic acid.

[0040] Example 1 synthesis of nanocomposite enzyme

[0041] (1) 100 mg of L-arginine and 130 mg of L-carnosine were dissolved in 30 mL of ultrapure water, 10 mL of a solution containing 300 mg of Ce(NO3)3·6H2O was added dropwise, stirred at room temperature for 5 h, and then dialyzed against water using a dialysis bag (Mw 1 / 4 3500 Da) for 24 h, and the dialysis water was replaced at 2 h, 8 h, and 16 h, respectively. After dialysis, it was stored in a brown volumetric flask.

[0042] (2) 2.453g of cobalt nitrate hexahydrate was added to 50mL of methanol, and then 2mL of modified cerium oxide was added. The mixture was magnetically stirred at room temperature for 1h. Then, 50mL of methanol solution containing 5.419g of 2-methylimidazole was added. After stirring for 2h, the mixture was centrifuged (centrifuged at 13000 rpm for 3min), washed three times with methanol, centrifuged again, and then placed in a vacuum desiccator to dry for 6h.

[0043] (3) Add 2 mg of sample to 1 mL of deionized water for six days to etch, centrifuge (3 min at 13000 rpm), and freeze dry for 6 h.

[0044] Transmission electron microscopy image of the modified cerium oxide and nanocomposite enzyme in Example 1 is shown below. Figure 1 A, C and Figure 2 As shown, Figure 1 Transmission electron microscopy images of the modified cerium oxide, zeolite imidazole ester-67, and nanocomposite enzyme before etching. Figure 2 These are transmission electron microscope images of the nanocomposite enzyme in aqueous solution immediately after etching, after 3 days of etching, and after 6 days of etching. Figure 2 It can be seen that the nanocomposite enzyme underwent etching in the aqueous solution. The X-ray diffraction pattern and Fourier transform infrared image of the nanocomposite enzyme are shown below. Figure 3 As shown in the figure, the X-ray diffraction pattern of the nanocomposite enzyme in Figure A is consistent with that of zeolite imidazolium ester-67 in the range of 5–40°, and the peak shape of the nanocomposite enzyme in the Fourier transform infrared image in Figure C is also similar to that of zeolite imidazolium ester-67. This indicates that the in-situ synthesized nanocomposite enzyme has well maintained the high crystallinity of zeolite imidazolium ester-67. This shows that the synthesis of the nanocomposite material did not change the crystal form of the material. The carnosine-modified cerium oxide particles are small in size and highly dispersed in zeolite imidazolium ester-67. However, the X-ray diffraction pattern of the nanocomposite enzyme in Figure B shows the formation of Co-OH bonds after etching, indicating that the ligand network was continuously destroyed during the etching process, and cobalt ions were hydrolyzed to form cobalt hydroxide.

[0045] Example 2 Synthesis of Nanocomposite Enzymes

[0046] (1) Dissolve 90 mg L-lysine and 90 mg anserine in 30 mL of ultrapure water, then add 10 mL of a solution containing 300 mg cerium chloride heptahydrate. Stir at room temperature for 6 h, and then use a dialysis bag (Mw 1 / 4 The dialysis solution was 3500 Da and water was dialyzed for 24 hours. The dialysate was changed at 2 hours, 8 hours and 16 hours. After dialysis, the solution was stored in a brown volumetric flask.

[0047] (2) 3.421 g of zinc nitrate hexahydrate was added to 50 mL of water, 6.25 mL of modified cerium oxide was added, and the mixture was stirred magnetically at room temperature for 1 h. A solution containing 9.43 g of 2-ethylimidazole in 50 mL of water was added, and the mixture was stirred for 3 h. The mixture was centrifuged (at 13000 rpm for 3 min), washed with water 3 times, and centrifuged again. The mixture was then dried in a vacuum desiccator for 8 h.

[0048] (3) 6 mg of the sample was etched in 1 mL of glycerol for 6 days, centrifuged (at 13000 rpm for 3 min), and dried in a freeze dryer for 6 h.

[0049] Example 3 Synthesis of nanocomposite enzyme

[0050] (1) 80 mg of L-histidine and 30 mg of copper peptide were dissolved in 30 mL of ultrapure water, and 10 mL of a solution containing 100 mg of cerium sulfate tetrahydrate was added dropwise. The mixture was stirred at room temperature for 4 h, dialyzed against water in a dialysis bag (Mw 1 / 4 3500 Da) for 24 h, and the dialysis water was changed at 2 h, 8 h, and 16 h, respectively. After dialysis, the mixture was stored in a brown volumetric flask.

[0051] (2) 1.223 g of cobalt nitrate hydrate and 1.238 g of zinc nitrate hexahydrate were added to 50 mL of N,N-dimethylformamide, 1.25 mL of modified cerium oxide was added, and the mixture was stirred magnetically at room temperature for 1 h. A solution containing 0.6913 g of benzimidazole in 50 mL of N,N-dimethylformamide was added, and the mixture was stirred for 5 h. The mixture was centrifuged (at 13000 rpm for 3 min), washed with N,N-dimethylformamide 4 times, and centrifuged again. The mixture was then dried in a vacuum desiccator for 7 h.

[0052] (3) 10 mg of the sample was etched in 1 mL of methanol for 6 days, centrifuged (at 13000 rpm for 3 min), and dried in a freeze dryer for 6 h.

[0053] Example 4 Synthesis of nanocomposite enzyme

[0054] (1) 60 mg of L-histidine and 90 mg of carnosine were dissolved in 30 mL of ultrapure water, and 10 mL of a solution containing 200 mg of cerium acetate was added dropwise. The mixture was stirred at room temperature for 3.5 h, dialyzed against water in a dialysis bag (Mw 1 / 4 3500 Da) for 24 h, and the dialysis water was changed at 2 h, 8 h, and 16 h, respectively. After dialysis, the mixture was stored in a brown volumetric flask.

[0055] (2) 1.361 g of zinc chloride and 2.512 g of cobalt chloride hexahydrate were added to 50 mL of methanol, 3 mL of modified cerium oxide was added, and magnetic stirring was performed at room temperature for 1 h. A solution containing 4.282 g of imidazole-2-formaldehyde in 50 mL of methanol was added, and stirring was performed for 3 h. Centrifugation was performed (13000 rpm for 3 min), and washing was performed three times with methanol. Centrifugation was continuously performed, and then drying was performed in a vacuum desiccator for 6 h.

[0056] (3) 3 mg of the sample was etched in 1 mL of deionized water for six days, centrifugation was performed (13000 rpm for 3 min), and drying was performed in a freeze dryer for 6 h.

[0057] Example 5 Synthesis of a nano-composite enzyme

[0058] (1) 50 mg of L-lysine and 90 mg of copper peptide were dissolved in 30 mL of ultrapure water, 10 mL of a solution containing 400 mg of cerium nitrate hexahydrate was added dropwise, stirring was performed at room temperature for 3 h, and dialysis was performed against water in a dialysis bag (Mw 1 / 4 3500 Da) for 24 h, and the dialysis water was replaced at 2 h, 8 h, and 16 h, respectively. After dialysis, it was stored in a brown volumetric flask.

[0059] (2) 0.2876 g of zinc sulfate heptahydrate and 2.121 g of cobalt sulfate were added to 50 mL of N,N-dimethylformamide, 3 mL of modified cerium oxide was added, magnetic stirring was performed at room temperature for 1.5 h, a solution containing 3.325 g of benzimidazole in 50 mL of N,N-dimethylformamide was added, stirring was performed for 3 h, centrifugation was performed (13000 rpm for 3 min), washing was performed three times with N,N-dimethylformamide, centrifugation was continuously performed, and then drying was performed in a vacuum desiccator for 6 h.

[0060] (3) 5 mg of the sample was etched in 1 mL of glycerol for six days, centrifugation was performed (13000 rpm for 3 min), and drying was performed in a freeze dryer for 6 h.

[0061] Example 6 Feasibility study of a sensor established based on a nano-composite enzyme

[0062] 800 μL of an acetic acid-sodium acetate buffer solution (pH = 3.5) was added to a 2 mL centrifuge tube, 50 μL of a 2 mg / mL nano-enzyme solution, 50 μL of 3,3',5,5'-tetramethylbenzidine (4 mM) were added, and an absorption spectrum was collected using a UV-visible spectrophotometer after reaction at room temperature for 10 min.

[0063] 440 μL of an acetic acid-sodium acetate buffer solution (pH = 5.5) was added to a 2 mL centrifuge tube, 50 μL of a 2 mg / mL nano-enzyme solution, 10 μL of a fluorescent red dye (0.5 mM) were added, and detection was performed using a fluorescence spectrometer after reaction at room temperature for 10 min.

[0064] Figure 4 and Figure 5 UV-Vis absorption spectrum peaks and fluorescence intensity peaks for the feasibility study of colorimetric and fluorescent sensors based on nano-composite enzymes. Among them, Figure 4 UV-Vis absorption spectrum peak diagram of nano-composite enzyme, zeolite imidazolate framework-67, modified cerium oxide; Figure 5 Fluorescence intensity peak diagram of nano-composite enzyme, zeolite imidazolate framework-67, modified cerium oxide. From Figure 4 and Figure 5 It can be seen that the oxidase-like performance of the nano-composite enzyme is the best.

[0065] Example 7 Detection of different concentrations of tannic acid

[0066] The specific steps for tannic acid detection are as follows:

[0067] Add 750 μL of acetic acid-sodium acetate buffer solution with pH of 3.5, add 50 μL of nano-enzyme composite enzyme prepared in Example 1, 50 μL of 3,3',5,5'-tetramethylbenzidine with a concentration of 4 mM, and 50 μL of tannic acid with a concentration of 0 μM, 20 μM, 30 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 180 μM, 200 μM, 250 μM, 260 μM, and 270 μM, respectively. React at room temperature for 10 min, filter, and measure the UV absorbance, as shown in Figure 6 B-D, the UV absorbance value is linearly related to the tannic acid concentration in the range of 20-270 μM, and the detection limit is 1.45 μM.

[0068] Add 390 μL of acetic acid-sodium acetate buffer solution with pH of 5.5, add 50 μL of nano-composite enzyme prepared in Example 1, 10 μL of fluorescent red dye with a concentration of 0.5 mM, and 50 μL of tannic acid with a concentration of 0 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM, 7 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM, respectively. React at room temperature for 10 min, filter, and measure the fluorescence intensity. As shown in Figure 7 B-D, the fluorescence intensity is linearly related to the tannic acid concentration in the range of 0.05-50 μM, and the detection limit is 0.011 μM.

[0069] Example 8 Detection of tannic acid specificity

[0070] Specificity of tannic acid detection

[0071] Add 750 μL of acetate-sodium acetate buffer solution at pH 3.5, 50 μL of the nanocomposite enzyme prepared in Example 1, 50 μL of 4 mM 3,3',5,5'-tetramethylbenzidine, and 1 mM of different interfering substances (cation selection: Na+). + K + Ca 2 + Mg 2+ The sugars used were glucose, and the antioxidants were gallic acid, oxalic acid, and citric acid, replacing 100 μM tannic acid. The reaction was carried out at room temperature for 10 min, filtered, and the UV absorbance was measured. For the fluorescence method, 390 μL of acetate-sodium acetate buffer solution (pH 5.5), 50 μL of the nanocomposite enzyme prepared in Example 1, 10 μL of 0.5 mM fluorescent red dye, and 1 mM of the aforementioned different interfering substances replacing 100 μM tannic acid were added. The reaction was carried out at room temperature for 10 min, filtered, and the fluorescence intensity was measured. Figure 8 As shown, the ultraviolet absorbance and fluorescence intensity are highest at tannic acid, indicating that the detection of tannic acid is specific.

[0072] Example 9

[0073] The specific steps for detecting tannic acid in food are as follows:

[0074] For food and beverages, choose coffee and green tea; for fruits, choose apples, grapes, persimmons, and citrus fruits; for spices, choose fennel and cinnamon; and for herbs, choose gallnuts and dried tangerine peel. Steep 1.2g of coffee and green tea in 50mL of boiling water. Juice the fruits. Steep 2g each of fennel, cinnamon, and gallnuts in 50mL of boiling water, and steep 1g of dried tangerine peel in 50mL of boiling water. After cooling to room temperature, filter using a needle filter.

[0075] Add 700 μL of acetate-sodium acetate buffer solution at pH 3.5, 50 μL of the nanocomposite enzyme prepared in Example 1, 50 μL of the sample diluted 20-fold with buffer, 50 μL of 4 mM 3,3',5,5'-tetramethylbenzidine, and 50 μL of tannic acid at concentrations of 0 μM, 50 μM, 150 μM, and 230 μM. React at room temperature for 10 min, filter, and measure UV absorbance.

[0076] Add 340 μL of acetate-sodium acetate buffer solution at pH 5.5, 50 μL of the nanocomposite enzyme prepared in Example 1, 50 μL of sample diluted 600 times with buffer, 10 μL of 0.5 mM fluorescent red dye, and 50 μL of tannic acid at concentrations of 0 μM, 0.5 μM, 5 μM, and 10 μM. React at room temperature for 10 min, filter, and measure fluorescence intensity.

[0077] As shown in Tables 1, 2, the low, medium and high concentrations in the linear range were used to determine the recovery rate. Compared with the standard curve, the recovery rate of colorimetric method was 80.87% to 113.2%, and the recovery rate of fluorescence method was 89.02% to 113.8%. The relative standard deviation was 0.2% to 7%, which was within the allowable error range. This indicated that the nano-composite enzyme was feasible for the detection of actual samples.

[0078] Table 1 Recovery rate of tannic acid in food

[0079]

[0080]

[0081] Table 2 Recovery rate of tannic acid in food

[0082]

[0083]

Claims

1. A method for preparing a nanocomposite enzyme for detecting tannic acid, characterized by, The method comprises the following steps: Step one, using basic amino acids and polypeptides with imidazole groups to simulate the primary structure of natural oxidase, preparing amino acid and polypeptide modified cerium oxide with enhanced oxidase and peroxidase performance; the amino acid is arginine, lysine or histidine; the polypeptide is camosin or cupric peptide; Step two, in-situ compounding the polypeptide modified cerium oxide and zeolite imidazolate framework material with large specific surface area, good stability, hydrophilic and hydrophobic properties and specific electronic distribution to obtain a nano-composite enzyme with enhanced oxidase and peroxidase performance; the polypeptide modified cerium oxide is synthesized by using cerium salt, amino acid and polypeptide; the zeolite imidazolate framework material is synthesized by using zinc salt, cobalt salt, organic ligand and solvent; Step three, etching the nano-composite enzyme in a polar medium for post-treatment to obtain a nano-composite enzyme with enhanced oxidase and peroxidase performance for detecting tannic acid; the nano-composite enzyme is etched in a polar medium, and the polar medium used is glycerol, ultrapure water or methanol.

2. The method for preparing a nanohybrid enzyme for detecting tannin according to claim 1, wherein, The cerium salt is cerium nitrate, cerium chloride, cerium acetate or cerium sulfate.

3. The method for preparing a nanocomposite enzyme for detecting tannin according to claim 1, wherein, The mass ratio of the amino acid and the polypeptide is 10:3-5:9; the mass concentration of the cerium salt is 10-50 mg / mL.

4. The method for preparing a nanocomposite enzyme for detecting tannin according to claim 1, characterized by: The zinc salt is zinc nitrate, zinc chloride or zinc sulfate; the cobalt salt is cobalt nitrate, cobalt chloride or cobalt sulfate; the organic ligand is 2-methyl imidazole, 2-ethyl imidazole, benzimidazole or imidazole-2-carboxaldehyde; and the solvent is methanol, water or N,N-dimethylformamide.

5. The method for preparing a nanocomposite enzyme for detecting tannin according to claim 4, characterized by: The molar ratio of the zinc salt or the cobalt salt to the organic ligand is 1:1-1:20; the molar ratio of the cobalt salt to the zinc salt is 1:0-1:

2.

6. The method for preparing a nanohybrid enzyme for detecting tannin according to claim 1, wherein, The volume ratio of the polypeptide modified cerium oxide to the solvent in which the organic ligand of the synthesized zeolite imidazolate framework material is dissolved is 1:8-1:

40.

7. The method for preparing a nanohybrid enzyme for detecting tannin according to claim 1, wherein, The mass ratio of the nano-composite enzyme material to the polar medium is 1:100-1:500; and the pH value of the polar medium is 3-9.

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