Preparation method and application of a nano-enzyme with a heterostructure

Abstract

The application provides a preparation method of a heterostructure nano-enzyme and application of the heterostructure nano-enzyme, and the heterostructure nano-enzyme is FeMoO4@Fe7S8, and the preparation method comprises the following steps: dissolving Na2MoO4.2H2O, oxalic acid and thiourea in ultrapure water to form a solution A; mixing FeCl3.6H2O, NaAc and anhydrous ethylene glycol to form a solution B; reacting the mixed solution obtained by uniformly mixing the solution B and the solution A at 195-205 DEG C for 20-30 h; and drying the product after cleaning with anhydrous ethanol and ultrapure water. The heterostructure nano-enzyme solves the aggregation problem of FeMoO4 nanoparticles. The heterostructure nano-enzyme is a peroxidase-like enzyme, has high enzyme-like catalytic activity, has good affinity to H2O2 and TMB, and has high stability. The heterostructure nano-enzyme has universality in catalyzing color development of a color developing substrate, has a wide linear range in oxytetracycline detection, has a low detection limit, is simple to operate, and has low detection cost.

Description

Technical Field

[0001] This invention relates to the field of water quality testing technology, specifically to a method for preparing a heterogeneous nanozyme and its application. Background Technology

[0002] Oxytetracycline (OTC, C) 22 H 24 Nitrogen oxides (OTCs) are a typical new type of antibiotic pollutant, characterized by large-scale use and strong ecotoxicity, and their ecological risks have attracted widespread attention from the academic community. With the development of intensive and large-scale livestock farming, oxytetracycline has begun to be widely used in livestock and poultry farming, not only for the treatment and prevention of animal diseases, but also added to livestock and poultry feed at sub-therapeutic doses to stimulate animal growth and increase production. Since OTCs entering the animal body cannot be completely absorbed, they eventually return to the environment. Large amounts of OTC entering the environment can disrupt ecosystem functions, induce the production of resistance genes, and even threaten human health, leading to kidney disease and cancer. Therefore, countries around the world have regulations on limits for OTCs in food. In my country, the maximum residue limit for OTCs is 100 μg / kg in milk, 200 μg / kg in eggs, and 300 μg / kg and 600 μg / kg in the liver and kidneys of food animals, respectively. Currently, commonly used methods for OTC detection include liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay (ELISA), molecular imprinting, aptamer antibody detection, photochemical detection, fluorescence detection, and surface-enhanced Raman scattering (SERS). However, most of these methods rely on large-scale instruments and equipment, involve cumbersome sample pretreatment and experimental procedures, and require highly skilled personnel, thus limiting their application. Therefore, it is essential to establish a simple, low-cost, rapid, and sensitive method for detecting / monitoring OTC in environmental and food media to ensure environmental and food safety.

[0003] Colorimetric methods have gained widespread attention in the detection field due to their advantages such as low cost, fast detection speed, and direct visual observation. Colorimetric detection methods based on natural enzymes are simple and efficient; however, the preparation, purification, and storage costs of the natural enzymes used as catalysts are high, and their recycling is difficult. Furthermore, the activity of natural enzymes is more susceptible to interference from external conditions, such as temperature, pH, and inhibitors, which limits their application in colorimetric analysis. Nanozymes, as nanomaterials with similar natural enzyme activity, are stable, easy to store, and can withstand harsh conditions, showing great application potential. Therefore, developing nanozymes for the colorimetric detection of oxytetracycline is of significant practical importance for the accurate, rapid, and low-cost detection of oxytetracycline residues in food. Summary of the Invention

[0004] The problem to be solved by this invention is to provide a method for preparing heterostructured nanozymes FeMoO4@Fe7S8 and their application in the detection of oxytetracycline.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for preparing a heterostructured nanozyme, the method comprising: dissolving Na2MoO4·2H2O, oxalic acid and thiourea in ultrapure water to form solution A;

[0007] FeCl3·6H2O and NaAc were mixed with anhydrous ethylene glycol to form solution B;

[0008] The mixture of solution B and solution A was reacted at 195–205 °C for 20–30 h. The product was washed with anhydrous ethanol and ultrapure water and then dried to obtain the heterostructured nanozyme.

[0009] Preferably, in solution A, the molar ratio of Na2MoO4·2H2O, oxalic acid and thiourea is 1:(1~2):(4~6).

[0010] Preferably, in solution B, the molar ratio of FeCl3·6H2O to NaAc is 1:(1.5~2.5).

[0011] Preferably, in the mixture, the molar ratio of Na2MoO4·2H2O to FeCl3·6H2O is (1~1.3):1.

[0012] Preferably, in solution A, the molar concentration of Na2MoO4·2H2O is 2-4 mM, the molar concentration of oxalic acid is 3-5 mM, and the concentration of thiourea is 14-16 mM.

[0013] Preferably, in solution B, the molar concentration of FeCl3·6H2O is 4–6 mM, and the molar concentration of NaAc is 8–12 mM.

[0014] Preferably, the volume ratio of solution A to solution B is (1.5-2.5):1.

[0015] Preferably, the product is washed alternately with anhydrous ethanol and ultrapure water, and then dried in a vacuum oven at 55–65°C.

[0016] This invention also provides a heterostructured nanozyme prepared by the above-described method. The heterostructured nanozyme is composed of FeMoO4 nanoparticles anchored on the surface of Fe7S8 nanosheets. The heterostructured nanozyme exhibits a two-dimensional layered heterostructure, and its surface contains Fe... 2+ and Fe 3+Therefore, the heterostructured nanozyme in this invention is named FeMoO4@Fe7S8.

[0017] According to the embodiments, the Fe, Mo, O and S elements are evenly distributed in the heterostructured nanozyme, with Fe accounting for 49% to 50% of the mass, Mo accounting for 6% to 7% of the mass, O accounting for 32% to 33% of the mass and S accounting for 10% to 12% of the mass.

[0018] According to the embodiments, the specific surface area of ​​the heterostructured nanozyme is 6-7 m². 2 / g, the nitrogen adsorption-desorption curves show type II isothermal characteristics, indicating the presence of mesoporous structures with an average pore size of approximately 17–20 nm.

[0019] The present invention also provides a heterostructured nanozyme prepared by the above preparation method or the application of the above heterostructured nanozyme in the detection of oxytetracycline.

[0020] This invention also provides a method for detecting oxytetracycline, which is based on colorimetric detection. The sample to be tested is incubated with a heterostructured nanozyme prepared by the above preparation method or the above heterostructured nanozyme, H2O2 and a chromogenic substrate at 30-50°C. The chromogenic substrate is one or more of ABTS, DAB, TMB and OPD.

[0021] According to the examples, the reaction was carried out in an acetate-sodium acetate buffer solution with an initial pH of 3 to 4.

[0022] Preferably, the sample to be tested is a sample that has undergone protein removal treatment.

[0023] According to the embodiments, the color change before and after the reaction is observed visually, or the absorbance value of the reaction solution at 654nm is measured using an ultraviolet spectrophotometer, or the reaction solution is photographed and the gray value is calculated, and colorimetric data is processed using digital imaging colorimetric software.

[0024] The present invention also provides a kit for detecting oxytetracycline, comprising a heterostructured nanozyme prepared by the above preparation method or the above heterostructured nanozyme.

[0025] Preferably, the kit further includes hydrogen peroxide and a chromogenic substrate, wherein the chromogenic substrate is one or more of ABTS, DAB, TMB and OPD.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The heterostructured nanozyme FeMoO4@Fe7S8 prepared by the method of this invention exhibits a composite structure in which nanoparticles are anchored on nanosheets, thus solving the aggregation problem of FeMoO4 nanoparticles and containing Fe 2+ / Fe 3+ It has a complex valence state and mesoporous structure, many active sites, strong catalytic activity, and good magnetic properties, which facilitates separation.

[0028] FeMoO4@Fe7S8 is a peroxidase-like enzyme with high catalytic activity, good affinity for H2O2 and TMB, and high stability. It has universal applicability to catalytic colorimetric development of substrates (TMB, ABTS, DAB, OPD).

[0029] FeMoO4@Fe7S8 is used for the detection of oxytetracycline, which has a wide linear range, low detection limit, simple operation, and low detection cost. Attached Figure Description

[0030] Figure 1 SEM image of the heterostructured nanozyme FeMoO4@Fe7S8 prepared in Example 1;

[0031] Figure 2 TEM image of the heterostructured nanozyme FeMoO4@Fe7S8 prepared in Example 1;

[0032] Figure 3 Energy dispersive spectroscopy (EDS) and elemental analysis (mapping) of the heterostructured nanozyme FeMoO4@Fe7S8 prepared in Example 1;

[0033] Figure 4 XRD pattern of the heterostructured nanozyme FeMoO4@Fe7S8 prepared in Example 1;

[0034] Figure 5 The specific surface area (BET) and pore size of the heterostructured nanozyme FeMoO4@Fe7S8 prepared in Example 1;

[0035] Figure 6 Investigation of peroxidase-like catalytic conditions for FeMoO4@Fe7S8: UV-Vis absorption spectra of (AD) reacted with four colorimetric systems: ABTS, DAB, TMB, and OPD (insets show the corresponding solutions).

[0036] Figure 7 Steady-state kinetic curves for FeMoO4@Fe7S8: (A) TMB and H2O2 are Michaelis-Menten curves of the substrates, respectively; (C) TMB and H2O2 are Lineweaver-Burk plots of the substrates, respectively.

[0037] Figure 8To detect OTC using FeMoO4@Fe7S8: (A) UV-Vis absorption spectra of OTC at different concentrations, (B) Relationship between absorbance difference at 654 nm (ΔA654) and OTC concentration, (CD) Linearity graph of OTC in the range of 0.1–90 μM;

[0038] Figure 9 This is a schematic diagram of the detection based on a smartphone platform. Detailed Implementation

[0039] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually conditions in conventional experiments.

[0040] In the following examples, all chemicals were of analytical or chromatographic grade. Ultrapure water (>18.2 MΩ) was produced using a Milli-Q water purifier (Bedford, MA, USA). Scanning electron microscopy (SEM, Quanta 250; FEI, USA); transmission electron microscopy (Talos F200XG2; FEI, USA); X-ray diffraction (D8-Advance, Bruker, Germany); X-ray photoelectron spectroscopy (K-Alpha+ type; Thermo Scientific, USA). Enzyme kinetic data and UV-Vis spectra were obtained using a UV-8000 spectrophotometer (Shanghai, China).

[0041] Example 1

[0042] Preparation of FeMoO4@Fe7S8 heterostructure nanozymes:

[0043] First, solution A was prepared by dissolving 3 mM Na₂MoO₄·2H₂O, 4 mM oxalic acid, and 15 mM thiourea in 40 mL of ultrapure water. Simultaneously, 5 mM FeCl₃·6H₂O and 10 mM NaAc were added to 20 mL of anhydrous EG, and the mixture was stirred to form an orange-red mixed solution B. Solution B was then added dropwise to solution A, and stirring continued for 1 h. The mixed liquid was transferred to a high-temperature reactor and reacted at 200 °C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, washed three times alternately with anhydrous ethanol and ultrapure water, and the product was collected by centrifugation. The washed product was then dried in a vacuum oven at 60 °C for 4 h to obtain a heterostructured nanozyme.

[0044] Morphological and structural characteristics of heterogeneous nanozymes:

[0045] according to Figure 1 , Figure 2 and Figure 3 The heterostructured nanozyme is composed of a large number of FeMoO4 nanoparticles anchored on the surface of Fe7S8 nanosheets, forming a two-dimensional (2D) layered heterostructure. The nanozyme surface contains Fe... 2+ and Fe 3+ The overall distribution of Fe, Mo, O, and S elements is uniform, accounting for 49.9%, 6.6%, 32.4%, and 11.1%, respectively.

[0046] according to Figure 4 The XRD test results of FeMoO4@Fe7S8 correspond to FeMoO4 (JPCDS No. 22-0628) and Fe7S8 (JPCDS No. 71-0591), respectively.

[0047] according to Figure 5 The specific surface area of ​​this nanozyme is 6.3 m². 2 / g, with an average pore size of approximately 18.1nm, and nitrogen adsorption-desorption curves exhibiting type II isothermal characteristics, indicating the presence of mesoporous structures.

[0048] Based on the characterization results, the heterostructured nanozyme prepared in this embodiment is named FeMoO4@Fe7S8.

[0049] Example 2

[0050] Investigation on the enzyme activity of FeMoO4@Fe7S8 heterostructure nanoenzymes.

[0051] A dispersion of 75 μL FeMoO4@Fe7S8 nanozyme (solvent: water, concentration: 20 μg / mL) was prepared. -1 100 μL of TMB (6 mM DMSO) and 75 μL of H2O2 (50 mM water) were added to 1650 μL of HAc-NaAc buffer (0.2 M, pH 3.6). The mixture was incubated at 40 °C for 20 min, and the absorbance at specific wavelengths was measured using a UV-Vis spectrophotometer.

[0052] FeMoO4@Fe7S8 heterostructure nanozymes exhibit excellent peroxidase-like activity, and can catalyze the chromogenic substrates ABTS, DAB, TMB, and OPD to turn green in the presence of H2O2. Figure 6 (Figure A) Orange Figure 6 (Figure B in the middle) and blue (Figure B in the middle) Figure 6 (C diagram) and yellow ( Figure 6 (Figure D). The principle of catalytic color development is:

[0053] Fe 2+ +H₂O₂→Fe 3+ +·OH+OH - (1)

[0054] Fe 3+ +H₂O₂→Fe 2+ +HO·2+H + (2)

[0055] Fe 2+ +.OH→Fe 3+ +·OH - (3)

[0056] Mo 4+ +2Fe 3+ →Mo 6+ +2Fe 2+ (4).

[0057] Fe 2+ / Fe 3+ Contact with H2O2 will produce more reactive oxygen species (ROS), such as ·OH and ·O2. - ROS can oxidize the chromogenic substrate, causing it to become oxidized and exhibit different colors. The presence of Mo ions promotes electron transfer within the material and Fe... 2+ / Fe 3+ The cyclical transformation. For TMB, K m It is 0.09mM, V max 3.10*10 -8 M / s, ( Figure 7 (Figures A and C) For H2O2, K m It is 0.33mM, V max 3.24*10 -8 M / s( Figure 7 (Figures B and D in the middle).

[0058] Example 3

[0059] A method for detecting OTC using FeMoO4@Fe7S8 heterostructure nanozymes.

[0060] (1) Prepare a solution with a concentration of 0.01 mg / mL using water as the solvent. -1 FeMoO4@Fe7S8 heterostructure nanoenzyme dispersion;

[0061] (2) Prepare a 50 mM H2O2 solution using water as the solvent; prepare a 6 mM TMB solution using DMSO as the solvent;

[0062] (3) Prepare a 1 mM OTC stock solution using DMSO as the solvent. Then, use ultrapure water as the solvent to dilute the stock solution and prepare OTC working solutions with concentrations ranging from 0.1 to 120 μM.

[0063] (4) Pipette 75μL H2O2 (50mM), 100μL TMB (6mM), 75μL FeMoO4@Fe7S8 (0.01mg mL -1 Mix with 1650 μL of NaAc-HAc buffer solution (pH = 3.6, 0.2 M), then add 100 μL of different concentrations of OTC (0.1-120 μM) to maintain a total solution volume of 2 mL. Incubate at 40 °C for 20 min, and then measure A using a UV-Vis spectrophotometer. 654 Value. According to A 654 A standard curve for OTC detection using UV-Vis spectrophotometry was established to establish the relationship between the concentration and the OTC value, and the detection limit and linear range were calculated. The results are as follows: Figure 8 As shown, in the range of 0.1-20 μM, the linear equation is y = 0.0408x + 0.0358, R0 2 =0.988; in the 20-90 μM range, the linear equation is y = 0.0120x + 0.533, R 2 =0.996;

[0064] (5) Repeat step (4) of the experiment, transfer the reacted solution to a black 96-well plate, place it in a darkroom, and use a mobile phone to take a picture of the color development result; import the color development result image into the "Thing Identify" software, construct the relationship between the gray value of the color development result and the OTC concentration, establish a standard curve for mobile phone visualization detection of OTC, and calculate the detection limit and linear range. Figure 9 Within the range of 0.1–20 μM, a good linear relationship exists between the solution color intensity and the OTC concentration (R0). 2 =0.964), the regression equation is y = -1.30x + 238.76, and the LOD obtained through the smartphone APP is 0.03μM.

[0065] (6) Quantitative detection: 100 μL of the prepared sample was mixed with 75 μL of H2O2 (50 mM), 100 μL of TMB (6 mM), and 75 μL of FeMoO4@Fe7S8 (0.01 mg / mL). -1 Mix with 1650 μL NaAc-HAc buffer solution (pH = 3.6, 0.2 M), incubate at 40 °C for 20 min, and the OTC content in the sample can be obtained by the standard curve of step (4) or the standard curve of step (5).

[0066] (7) Actual Sample Detection: To evaluate the feasibility of detecting OTC drugs based on the FeMoO4@Fe7S8 nanozyme colorimetric method, milk was used as the actual sample. 1% trichloroacetic acid solution (10 mL) was added to 1 mL of milk sample to remove protein. After filtration and centrifugation, the supernatant was collected as the test sample. The results are shown in Table 1. Three spiking levels (1, 5, 15 μM) were selected for the experiment, and the spiked recoveries ranged from 93.0% to 107.87%, with RSD < 5.06%. Compared with UV-Vis spectrophotometry and HPLC-DAC, the spiked recoveries and RSD values ​​of this method are similar, indicating that the constructed FeMoO4@Fe7S8 nanozyme-based intelligent visualization detection method has significant application potential in the detection of antibiotics in complex food matrices.

[0067] Table 1 Comparison of Actual Sample Detection Results

[0068]

[0069] In this embodiment, the principle of OTC detection is: Fe in FeMoO4@Fe7S8 nanozyme 2+ Complexing with nitrogen-containing groups on OTC, promoting Fe 2+ and Fe 3+ Electron transfer between them catalyzes the decomposition of H2O2 to produce more ·OH, causing the blue color of the "FeMoO4@Fe7S8 nanozyme + H2O2 + TMB" color development system to deepen, the absorbance value at 654nm to increase, and the grayscale value of the resulting image to increase. Based on A... 654 The relationship between the grayscale value and OTC concentration, and the relationship between the grayscale value of the image and OTC concentration, enables rapid quantitative detection of OTC. After adding OTC, the light blue color of the solution gradually changes to dark blue.

[0070] The FeMoO4@Fe7S8 heterostructure nanozyme of this invention exhibits universality in catalytic colorimetric development of substrates (TMB, ABTS, DAB, OPD). Therefore, in other embodiments, ABTS, DAB, and OPD can also be selected as colorimetric substrates. The OTC detection method provided in this embodiment has a wide linear range, low detection limit, simple operation, and low detection cost. The mobile phone-based visual colorimetric detection method has good sensitivity and anti-interference ability.

[0071] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing a heterostructured nanozyme FeMoO4@Fe7S8, characterized in that, The preparation method includes: Dissolve Na2MoO4·2H2O, oxalic acid, and thiourea in ultrapure water to form solution A; FeCl3·6H2O and NaAc are mixed with anhydrous ethylene glycol to form solution B; The mixture of solution B and solution A was reacted at 195~205℃ for 20~30h. The product was washed with anhydrous ethanol and ultrapure water and then dried to obtain the heterostructured nanozyme FeMoO4@Fe7S8.

2. The preparation method according to claim 1, characterized in that, In solution A, the molar ratio of Na2MoO4·2H2O, oxalic acid, and thiourea is 1:(1~2):(4~6). In solution B, the molar ratio of FeCl3·6H2O to NaAc is 1:(1.5~2.5). In the mixture, the molar ratio of Na2MoO4·2H2O to FeCl3·6H2O is (1~1.3):

1.

3. The preparation method according to claim 2, characterized in that, In solution A, the molar concentration of Na2MoO4·2H2O is 2~4 mM, the molar concentration of oxalic acid is 3~5 mM, and the concentration of thiourea is 14~16 mM. In solution B, the molar concentration of FeCl3·6H2O is 4~6mM, and the molar concentration of NaAc is 8~12mM. The volume ratio of solution A to solution B is (1.5~2.5):

1.

4. The preparation method according to claim 1, characterized in that, The product was washed alternately with anhydrous ethanol and ultrapure water and then dried in a vacuum oven at 55-65°C.

5. A heterostructured nanozyme FeMoO4@Fe7S8 prepared by the preparation method according to any one of claims 1 to 4, characterized in that, The heterostructured nanoenzyme FeMoO4@Fe7S8 is composed of FeMoO4 nanoparticles anchored on the surface of Fe7S8 nanosheets. The FeMoO4@Fe7S8 heterostructured nanoenzyme exhibits a two-dimensional layered heterostructure, with Fe atoms present on its surface. 2+ and Fe 3+ .

6. The heterostructured nanoenzyme FeMoO4@Fe7S8 according to claim 5, characterized in that, The heterostructured nanozyme FeMoO4@Fe7S8 contains uniformly distributed Fe, Mo, O, and S elements, with Fe accounting for 49%~50% of the mass, Mo accounting for 6%~7% of the mass, O accounting for 32%~33% of the mass, and S accounting for 10%~12% of the mass.

7. The heterostructured nanoenzyme FeMoO4@Fe7S8 according to claim 5, characterized in that, The specific surface area of ​​the heterostructured nanozyme FeMoO4@Fe7S8 is 6~7 m². 2 / g, the nitrogen adsorption-desorption curves show type II isothermal characteristics, indicating the presence of mesoporous structures with an average pore size of 17~20 nm.

8. The application of a heterostructured nanozyme FeMoO4@Fe7S8 prepared by the preparation method according to any one of claims 1 to 4, or the heterostructured nanozyme FeMoO4@Fe7S8 according to any one of claims 5 to 7, in the detection of oxytetracycline.

9. A method for detecting oxytetracycline, characterized in that, The colorimetric method for detecting oxytetracycline involves incubating the sample to be tested with the heterostructured nanozyme FeMoO4@Fe7S8 prepared by the preparation method according to any one of claims 1 to 4 or the heterostructured nanozyme FeMoO4@Fe7S8 according to any one of claims 5 to 7, H2O2, and a chromogenic substrate at 30-50°C. The chromogenic substrate is one or more of ABTS, DAB, TMB, and OPD.

10. The detection method according to claim 9, characterized in that, The reaction was carried out in an acetate-sodium acetate buffer solution with an initial pH of 3-4; And / or, the sample to be tested is a sample that has undergone protein removal treatment; And / or, visually observe the color change before and after the reaction, or measure the absorbance of the reaction solution at 654 nm using a UV spectrophotometer, or photograph the reaction solution and calculate the grayscale value, and process the colorimetric data using digital imaging colorimetric software.

11. A kit for detecting oxytetracycline, characterized in that, It includes the heterostructured nanozyme FeMoO4@Fe7S8 prepared by the preparation method of any one of claims 1 to 4 or the heterostructured nanozyme FeMoO4@Fe7S8 prepared by any one of claims 5 to 7.

12. The kit according to claim 11, characterized in that, The kit also includes hydrogen peroxide and a chromogenic substrate, wherein the chromogenic substrate is one or more of ABTS, DAB, TMB and OPD.

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