Simulated enzyme material as well as preparation method and application thereof

Abstract

The invention discloses a mimic enzyme material as well as a preparation method and application thereof, and belongs to the technical field of mimic enzymes. The Cu2V4O11 with the rod-like structure is prepared through a hydrothermal synthesis method, and the material has good peroxidase catalytic simulation performance, can be used for rapidly detecting H2O2 and glucose, and has potential application prospects in the fields of immunoassay, environment detection and the like. Meanwhile, the material has the characteristics of simple and feasible preparation method, low price, good repeatability and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of mimetic enzymes, and particularly relates to a mimetic enzyme material, a preparation method thereof and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Peroxidase (Horseradish peroxidase, abbreviated as HRP) is a class of redox enzymes produced by microorganisms or plants. It is a natural enzyme that catalyzes the oxidation of substrates with H2O2 as an electron acceptor and is often used for H2O2 detection. However, as a kind of natural enzyme, peroxidase is essentially a protein with a supramolecular structure. Although peroxidase can catalyze specific substrates under mild conditions, its catalytic activity is easily affected by conditions such as temperature and pH, and it is prone to inactivation or denaturation under extreme conditions such as high temperature, strong acid and strong base. Moreover, the extraction process of peroxidase is very complex, with high costs and low yields (Wei et al., Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev. 42 (2013) 6060-6093). Therefore, the development of materials with stable catalytic performance and good catalytic activity to replace natural peroxidase has received extensive attention from researchers.

[0004] Among many artificial enzyme materials, the properties of nanomaterials in terms of size, shape, surface charge, etc. are very similar to those of natural enzymes. Therefore, nanomaterials with a large specific surface area and numerous active sites on the surface provide the possibility for studying highly efficient artificial enzyme activity. Moreover, nanomaterials have a low synthesis cost, are easy to store, and have excellent heat resistance, acid and alkali resistance. These advantages have greatly promoted the wide application of nanomaterial-based artificial enzymes. Especially in 2007, after Gao et al. reported that Fe3O4 nanoparticles (Gao et al., Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2 (2007) 577-583) could act as peroxidase mimics, researchers were more certain that the existence of nanomaterials would change the research process of artificial enzymes. In recent years, vanadate nanomaterials have shown significant advantages among artificial enzyme materials. Their unique electronic structure and surface properties enable them to efficiently catalyze a variety of reactions, similar to the active centers of natural enzymes. Vanadate nanomaterials have excellent stability and tunability, and can maintain high catalytic activity under harsh conditions. Their nanoscale effect and large specific surface area provide abundant active sites, enhancing substrate adsorption and reaction rate. In addition, vanadate nanomaterials are easy to functionalize and modify, which can further optimize their catalytic performance and expand their application potential in fields such as biosensing and environmental treatment. So far, only a few vanadate nanomaterials, such as Co2V2O7, FeVO4, and AgVO3, have been reported as artificial enzyme nanomaterials for detecting hydrogen peroxide. However, these materials may have drawbacks, including low catalytic efficiency, insufficient stability, sensitivity to environmental conditions, and they may tend to aggregate during the reaction, affecting their activity and reusability.

[0005] Therefore, how to provide an artificial enzyme material with higher catalytic activity, better stability, and lower toxicity is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] To solve the deficiencies of the prior art, the purpose of the present invention is to provide an artificial enzyme material, its preparation method, and its application. The present invention prepares Cu2V4O with a rod-like structure through a hydrothermal synthesis method 11 , which has good peroxidase mimic catalytic performance, can rapidly detect H2O2 and glucose, and has potential application prospects in fields such as immunoassay. At the same time, this material has the characteristics of simple and easy preparation method, low price, and good repeatability.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a mimetic enzyme material, which is a rod-shaped Cu2V4O 11 ;

[0009] The mimetic enzyme material is a substance with peroxidase-like activity.

[0010] In one or more embodiments, the width of the rod-shaped structure is 500 - 1000 nm, and the length is 2000 - 8000 m.

[0011] In a second aspect, the present invention provides a method for preparing the above mimetic enzyme material, comprising the following steps: hydrothermal reaction of ammonium metavanadate and copper nitrate to obtain it.

[0012] In one or more embodiments, the molar ratio of ammonium metavanadate (NH4VO3) to copper nitrate (Cu(NO3)2) is 1:1 - 4, preferably 1:1 - 2.

[0013] In one or more embodiments, the specific steps are: ammonium metavanadate and copper nitrate are respectively dissolved in an organic solvent to obtain a mixed solution, the pH is adjusted to 1 - 7, and hydrothermal treatment is carried out.

[0014] Further, ammonium metavanadate is dissolved in an organic solvent, denoted as solution A; copper nitrate is dissolved in an organic solvent, denoted as solution B; solution A is slowly added dropwise to solution B to obtain a mixed solution.

[0015] Further, the organic solvent is methanol or ethanol.

[0016] Further, when ammonium metavanadate is dissolved in methanol, the ratio of ammonium metavanadate to methanol is 0.5 - 2 mmol: 20 - 40 ml; when copper nitrate is dissolved in methanol, the ratio of copper nitrate to methanol is 0.5 - 2 mmol: 20 - 40 ml;

[0017] Further, the pH of the mixed solution is adjusted to 1 - 7 with HCl solution or NaOH solution, preferably 3 - 5. The color development is the best at pH = 4, and there is no color development in an alkaline environment, such as pH = 9.

[0018] The concentration of the HCl solution or NaOH solution is 1.0 - 5.0 M.

[0019] Further, the temperature of the hydrothermal treatment is 120 - 200 °C, preferably 170 - 190 °C, and the time is 12 - 48 h, preferably 20 - 26 h.

[0020] Further, it also includes centrifugation, washing, and drying. Drying is carried out at 40 - 80 °C for 6 - 36 h.

[0021] In a preferred embodiment, 0.5 - 2 mmol of ammonium metavanadate (NH4VO3) is dissolved in 30 ml of methanol, denoted as solution A. Meanwhile, 0.5 - 2 mmol of copper nitrate (Cu(NO3)2) is dissolved in 30 ml of methanol, denoted as solution B. Under stirring conditions, A is slowly added dropwise to solution A to obtain a mixed solution. Then, the pH of the mixed solution is adjusted to 1 - 7 using HCl solution or NaOH solution. After stirring the mixed solution evenly, it is transferred to a polytetrafluoroethylene high-pressure reaction kettle and heat-treated in a forced-air drying oven. After the reaction stops, the reaction kettle is naturally cooled to room temperature, and after centrifugation, washing, and drying in a forced-air drying oven at 40 - 80 °C for 6 - 36 h, Cu2V4O 11 simulated enzyme material.

[0022] In a third aspect, the present invention provides the use of the above-mentioned simulated enzyme material or the simulated enzyme material obtained by the above-mentioned preparation method as a simulated peroxidase.

[0023] Preferably, biological immunoassay and environmental detection are carried out using the simulated peroxidase.

[0024] Preferably, it includes colorimetric detection of H2O2 and glucose.

[0025] In a fourth aspect, the present invention provides a method for colorimetric detection of H2O2, which uses the above-mentioned simulated enzyme material or the simulated enzyme material obtained by the above-mentioned preparation method;

[0026] Phosphate buffer solution (PBS), the H2O2 solution to be detected, an alcohol solution of TMB (3,3’,5’,5’-tetramethylbenzidine), water, and a dispersion of the above-mentioned simulated enzyme material are added in sequence, and the reaction is carried out at 20 - 60 °C for 5 - 20 minutes, and the absorbance at 400 - 800 nm is measured using an ultraviolet spectrophotometer.

[0027] Further, the pH value of the phosphate buffer solution (PBS) is 4 - 7. The concentration of the phosphate buffer solution is 40 - 60 mM.

[0028] Further, the concentration of the H2O2 solution is 10 - 30 mM.

[0029] Further, the concentration of the alcohol solution of TMB (3,3’,5’,5’-tetramethylbenzidine) is 0.5 - 10 mM. The alcohol is ethanol.

[0030] Further, the concentration of the dispersion of the simulated enzyme material is 10 - 15 μg / mL.

[0031] Further, the volume ratio of the phosphate buffer solution, the H2O2 solution to be detected, the alcohol solution of TMB (3,3’,5’,5’-tetramethylbenzidine), water, and the dispersion of the above-mentioned mimetic enzyme material is 300 - 500 μL: 50 - 150 μL: 50 - 150 μL: 200 - 400 μL: 50 - 150 μL.

[0032] Further, react for 5 - 10 min.

[0033] In a fifth aspect, the present invention provides a method for detecting glucose by colorimetry, which uses the above-mentioned mimetic enzyme material or the mimetic enzyme material obtained by the above-mentioned preparation method;

[0034] Add a glucose solution and a glucose oxidase (GOx) solution. After incubation, add PBS (phosphate buffer solution), TMB alcohol solution, water, and the dispersion of the above-mentioned mimetic enzyme material to the incubation solution, react at 20 - 60 °C for 5 - 20 minutes, and measure the absorbance at 650 - 660 nm with a UV spectrophotometer.

[0035] Further, react for 5 - 10 min.

[0036] The absorbance wavelength is 652 nm.

[0037] The final concentration of the glucose solution is 0 - 2 mM, preferably 0.01 - 2 mM, and more preferably 0.25 - 2 mM.

[0038] The concentration of the glucose oxidase (GOx) solution is 40 - 60 mg / mL.

[0039] The concentration of the TMB alcohol solution is 0.5 - 10 mg / mL.

[0040] The concentration of the dispersion of the mimetic enzyme material is 130 - 150 μg / mL.

[0041] The pH of the phosphate buffer solution is 4 - 7, and the concentration is 40 - 60 mM.

[0042] The volume ratio of the glucose solution, the glucose oxidase (GOx) solution, the TMB alcohol solution, the dispersion of the mimetic enzyme material, water, and the phosphate buffer solution is 50 - 150 μL: 10 - 30 μL: 50 - 150 μL: 50 - 150 μL: 250 - 350 μL: 300 - 500 μL.

[0043] In a sixth aspect, the present invention provides a visual test strip, which includes the above-mentioned mimetic enzyme material or the mimetic enzyme material obtained by the above-mentioned preparation method.

[0044] In a seventh aspect, the present invention provides the application of the above-mentioned visual test strip in detecting H2O2 or glucose.

[0045] One or some of the above technical solutions have the following advantages or beneficial effects:

[0046] (1) The present invention prepares the Cu2V4O 11 simulated enzyme material by a simple hydrothermal synthesis method, and the preparation process is simple, easy to control, and low in cost.

[0047] (2) The Cu2V4O 11 simulated enzyme material prepared by the present invention has a rod-like structure, a relatively large specific surface area, and is easy to recycle and reuse.

[0048] (3) The Cu2V4O 11 simulated enzyme material prepared by the present invention has good peroxidase-like catalytic performance, can rapidly detect H2O2 and glucose by colorimetry, and has good stability and reusability, and has potential application prospects in the fields of immunoassay and the like.

[0049] (4) Compared with the problems of low catalytic activity, poor stability, and potential biological toxicity existing in the simulated enzyme materials in the prior art, the Cu2V4O 11 simulated enzyme material provided by the present invention has higher catalytic efficiency and better chemical stability. Its unique crystal structure enables it to exhibit excellent performance in catalytic reactions. At the same time, its detection limit for hydrogen peroxide is lower and its application range is wider. In addition, the 11 biocompatibility and environmental friendliness of Cu2V4O also make it have potential application value in the fields of biosensing and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0051] Figure 1 The X-ray diffraction (XRD) pattern of the Cu2V4O 11 simulated enzyme material prepared in Example 1 of the present invention;

[0052] Figure 2 The scanning electron microscope (SEM) photograph of the Cu2V4O 11 simulated enzyme material prepared in Example 1 of the present invention;

[0053] Figure 3 The ultraviolet-visible absorption spectrum of the reaction system for the Cu2V4O 11 simulated enzyme material prepared in Example 1 of the present invention to simulate the peroxidase-like detection of H2O2;

[0054] Figure 4Cu2V4O prepared in Example 1 of the present invention 11 Absorbance value of the mimic enzyme material after repeating the mimic enzyme reaction 10 times;

[0055] Figure 5 Cu2V4O prepared in Example 1 of the present invention 11 Reaction kinetic curve of the mimic enzyme material for detecting glucose in the peroxidase mimic reaction system;

[0056] Figure 6 Cu2V4O prepared in Example 1 of the present invention 11 Standard curve of the mimic enzyme material for detecting glucose by peroxidase mimic. Detailed implementation manners

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0058] Example 1 Preparation of rod-shaped Cu2V4O 11 Preparation

[0059] Prepared by hydrothermal synthesis method. Dissolve 1 mmol ammonium metavanadate (NH4VO3) in 30 ml methanol, denoted as solution A. At the same time, dissolve 1 mmol copper nitrate (Cu(NO3)2) in 30 ml methanol, denoted as solution B. Under stirring conditions, slowly drop solution A into solution B to obtain a mixed solution. Then, adjust the pH of the mixed solution to 4 with 2.0 M HCl solution or NaOH solution. After stirring the mixed solution evenly, transfer it to a polytetrafluoroethylene high-pressure reaction kettle and place it in a blast drying oven for heat treatment at 180 °C for 24 h. After the reaction stops, naturally cool the reaction kettle to room temperature, and after centrifugation, washing, and drying in a blast drying oven at 60 °C for 24 h, Cu2V4O 11 Mimic enzyme material can be obtained.

[0060] Figure 1 XRD pattern of the sample prepared in Example 1. As can be seen from the figure, the positions of all diffraction peaks are completely consistent with the standard card JCPDS No. 54-1241 of Cu2V4O 11 and no impurity phase appears, so it can be determined that the sample prepared in Example 1 is pure Cu2V4O 11 . In addition, as can be seen from the figure, the diffraction peak intensity of the sample is large and the diffraction peak is sharp, indicating that the prepared Cu2V4O 11 has good crystallinity.

[0061] Figure 2 SEM photograph of the sample prepared in Example 1. As can be seen from the figure, the prepared Cu2V4O 11It is in a rod-like structure, with a width of about 700 nm and a length of about 2000 - 8000 nm. This structure has a large specific surface area.

[0062] Example 2 Cu2V4O 11 Detection of H2O2 by mimicking peroxidase

[0063] Take a 1.5 mL centrifuge tube, add 400 μL of 50 mM phosphate buffer (PBS, pH = 4.0), 100 μL of 20 mM H2O2 solution, 100 μL of 8 mM TMB ethanol solution and 300 μL of ultrapure water, and then add 100 μL of the Cu2V4O 11 dispersion (14 μg / mL) prepared in Example 1. After reacting for 7 min, observe the change of the solution and record the ultraviolet-visible absorption spectrum of the reaction system at 400 - 800 nm (see Figure 3 ).

[0064] In the figure, the characteristic absorption peak of oxidized TMB is at 652 nm. As can be seen from the figure, there are almost no absorption peaks in the three systems of H2O2 + Cu2V4O 11 , TMB + Cu2V4O 11 and TMB + H2O2, while the system of TMB + H2O2 + Cu2V4O 11 has an obvious absorption peak at 652 nm and the peak shape is good. In addition, by observing the colors of the three systems of H2O2 + Cu2V4O 11 , TMB + Cu2V4O 11 and TMB + H2O2, it is found that the colors of the three systems are all colorless and transparent without obvious changes, while the solution in the system of TMB + H2O2 + Cu2V4O 11 appears obvious blue, indicating that Cu2V4O 11 catalyzes the oxidation of TMB to generate blue oxide in the presence of H2O2.

[0065] The results show that the Cu2V4O 11 material prepared by the present invention has good peroxidase-like catalytic activity and is a peroxidase-like material. It can catalyze the oxidation of TMB to generate blue oxide in the presence of H2O2. Through this color reaction, rapid detection of H2O2 can be realized, and it has potential application prospects in the fields of bio-immunoassay and environmental detection.

[0066] Example 3 Cu2V4O 11 Repeated application for detection of H2O2 by mimicking peroxidase

[0067] The Cu2V4O used in Example 2 11The material was centrifugally recovered, washed several times with ultrapure water and absolute ethanol respectively, dried at 60 °C, and then the next simulated enzymatic reaction was carried out according to the steps in Example 2. This was continuously carried out 10 times while keeping other conditions unchanged.

[0068] The results are as Figure 4 shown. After continuously carrying out the simulated enzymatic experiment 10 times, the absorbance value of the reaction system did not decrease significantly, and the color of the solution still showed an obvious blue, indicating the good 11 reusability and stability of the Cu2V4O mimetic enzyme material.

[0069] Example 4 Detection of glucose by Cu2V4O 11 mimetic peroxidase

[0070] Similarly, 100 μL of glucose solutions with different final concentrations (0.01 mM, 0.025 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM) and 20 μL of 50 mg / mL GOx were added to a centrifuge tube. Subsequently, 100 μL of 8.0 mM TMB, 100 μL of 140 μg / mL Cu2V4O 11 , 280 μL of ultrapure water and 400 μL of 50.0 mM PBS (pH = 4.0) were added to the above solutions respectively. Finally, the absorbance changes of all solutions were recorded at 652 nm using a UV-visible spectrophotometer within 7 min.

[0071] The results are as Figure 5 shown. As the glucose concentration increased, the absorbance of the reaction system at 652 nm gradually increased, and at the same time, the blue color of the reaction system gradually deepened. GOx can react with glucose at different concentrations to generate H2O2 at different concentrations. The Cu2V4O 11 mimetic enzyme material can catalyze the oxidation of TMB to form a blue oxide in the presence of H2O2. Through this color reaction, the rapid detection of H2O2 can be achieved, and the concentration of H2O2 has a linear relationship with the concentration of glucose. Therefore, the rapid detection of glucose can be realized.

[0072] Figure 6 The linear equation for glucose detection is shown: A 652nm = 0.1117C + 0.0243 (mM), and its linear range is 10 - 75 μM (R 2 = 0.9971), and the detection limit for glucose is 2.24 μM (S / N = 3).

[0073] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A mimetic enzyme material, characterized in that: The enzyme-mimicking material is a rod-shaped Cu2V4O 11 ; The enzyme-mimicking material is a substance having peroxidase-like activity.

2. The enzyme-mimicking material according to claim 1, characterized in that: The rod-like structure has a width of 500-1000 nm and a length of 2000-8000 nm.

3. A method for preparing the enzyme mimicking material according to claim 1 or 2, characterized in that: The following steps are involved: It is obtained by hydrothermal reaction of ammonium metavanadate and copper nitrate.

4. The preparation method according to claim 3, characterized in that: The molar ratio of ammonium metavanadate to copper nitrate is 1:1 to 4, preferably 1:1 to 2; Preferably, the temperature of the hydrothermal treatment is 120-200° C., preferably 170-190° C., and the time is 12-48 h, preferably 20-26 h.

5. The preparation method according to claim 3, characterized in that: The specific steps are: dissolving ammonium metavanadate and copper nitrate in organic solvents respectively to obtain a mixed solution, adjusting the pH to 1-7, and performing hydrothermal treatment; Preferably, ammonium metavanadate is dissolved in an organic solvent, which is referred to as solution A; copper nitrate is dissolved in an organic solvent, which is referred to as solution B; solution A is slowly added dropwise to solution B to obtain a mixed solution; Preferably, the organic solvent is methanol or ethanol; Preferably, when ammonium metavanadate is dissolved in methanol, the ratio of ammonium metavanadate to methanol is 0.5-2mmol:20-40ml; when copper nitrate is dissolved in methanol, the ratio of copper nitrate to methanol is 0.5-2mmol:20-40ml; Preferably, the pH of the mixed solution is adjusted to 1-7 with HCl solution or NaOH solution; Preferably, the concentration of the HCl solution or the NaOH solution is 1.0-5.0 M; Preferably, the method further comprises centrifugation, washing and drying; and drying at 40 to 80° C. for 6 to 36 hours.

6. Use of the mimetic enzyme material according to claim 1 or 2 or the mimetic enzyme material obtained by the preparation method according to any one of claims 3 to 5 as a mimetic peroxidase; Preferably, simulated peroxidase is used for biological immunoassay and environmental testing; Preferably, colorimetric detection of H2O2 and glucose is included.

7. A method for detecting H2O2 by colorimetry, characterized in that: The method uses the simulated enzyme material according to claim 1 or 2 or the simulated enzyme material obtained by the preparation method according to any one of claims 3 to 5 as simulated peroxidase, comprising the following steps: Add phosphate buffer, H2O2 solution to be detected, alcohol solution of 3,3',5',5'-tetramethylbenzidine, water and dispersion of mimic enzyme material in sequence, react at 20-60°C for 5-20 minutes, and measure the absorbance at 400-800nm ​​with a UV spectrophotometer; Preferably, the pH value of the phosphate buffer is 4-7; the concentration of the phosphate buffer is 40-60 mM; Preferably, the concentration of the H2O2 solution is 10-30 mM; Preferably, the concentration of the alcohol solution of 3,3',5',5'-tetramethylbenzidine is 0.5-10 mM; Preferably, the concentration of the dispersion of the enzyme-mimicking material is 10-15 μg / mL; Preferably, the volume ratio of phosphate buffer, H2O2 solution to be detected, alcohol solution of 3,3',5',5'-tetramethylbenzidine, water and dispersion of the mimetic enzyme material is 300-500 μL: 50-150 μL: 50-150 μL: 200-400 μL: 50-150 μL.

8. A method for detecting glucose by colorimetry, characterized in that: The method uses the simulated enzyme material according to claim 1 or 2 or the simulated enzyme material obtained by the preparation method according to any one of claims 3 to 5 as simulated peroxidase, comprising the following steps: Adding glucose solution and glucose oxidase solution, after incubation, adding phosphate buffer, alcohol solution of 3,3',5',5'-tetramethylbenzidine, water and dispersion of mimic enzyme material to the incubation solution, reacting at 20-60° C. for 5-20 minutes, and measuring the absorbance at 650-660 nm with a UV spectrophotometer; Preferably, the final concentration of the glucose solution is 0-2 mM, preferably 0.01-2 mM; Preferably, the concentration of the glucose oxidase solution is 40-60 mg / mL; Preferably, the concentration of the alcohol solution of 3,3',5',5'-tetramethylbenzidine is 0.5-10 mg / mL; Preferably, the concentration of the dispersion of the enzyme-mimicking material is 130-150 μg / mL; Preferably, the pH of the phosphate buffer is 4-7 and the concentration is 40-60 mM; Preferably, the volume ratio of glucose solution, glucose oxidase solution, alcohol solution of 3,3',5',5'-tetramethylbenzidine, dispersion of mimetic enzyme material, water and phosphate buffer is 50-150 μL: 10-30 μL: 50-150 μL: 50-150 μL: 250-350 μL: 300-500 μL.

9. A visual test paper, characterized in that: The invention comprises the enzyme-mimicking material according to claim 1 or 2 or the enzyme-mimicking material obtained by the preparation method according to any one of claims 3 to 5.

10. Use of the visual test paper according to claim 9 in detecting H2O2 or glucose.

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