Application of Zn3 (OH) 2V2O7. 2H2O nano material as mimetic peroxidase

Abstract

The invention relates to the field of chemical detection of biological materials, in particular to application of a Zn3 (OH) 2V2O7. 2H2O nano material as mimetic peroxidase. The Zn3 (OH) 2V2O7. 2H2O nano material with the banded structure is provided for the first time and can be used as a mimetic peroxidase, the enzyme catalytic activity of the nano material can be comparable with that of natural horse radish peroxidase (HRP), and the nano material can be used for detecting the concentration of hydrogen peroxide in a solution sample.

Description

Technical Field

[0001] The present invention relates to the field of chemical detection of biological materials, and particularly to the application of Zn3(OH)2V2O7·2H2O nanomaterials as peroxidase mimics. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background 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 an enzyme that catalyzes the oxidation of substrates with H2O2 as the electron acceptor and is often used for the detection of H2O2. However, due to its high selectivity and high-efficiency catalytic activity, natural enzymes have important applications in the fields of biology, chemistry, agriculture, and food processing. However, natural enzymes are susceptible to experimental conditions such as temperature and pH value, which can cause denaturation and inactivation, resulting in reduced stability, high price, difficult purification, and high storage and use costs. These disadvantages greatly limit the application scope of enzymes. Therefore, it is necessary to develop mimetic enzymes with the catalytic characteristics of enzymes and more stable structures.

[0004] Compared with natural enzymes, nanomaterials have the advantages of low cost, adjustable catalytic activity, high stability, easy handling and storage, etc. In recent years, various inorganic material mimetic enzymes with peroxidase characteristics have been developed successively. Currently, the nanomaterial peroxidase mimics mainly include three categories: metals, such as platinum, gold, and iron-cobalt alloys, etc.; metal oxides, such as magnetite, cobalt tetroxide, and copper oxide, etc.; carbon-based materials, such as graphene oxide, carbon quantum dots, and carbon nanotubes with helical structures, etc. These nanomaterial mimetic enzymes generally have the disadvantages of low catalytic efficiency, poor tolerance, poor stability, and non-recyclability. Summary of the Invention

[0005] In order to overcome the above problems, the present invention provides the application of Zn3(OH)2V2O7·2H2O nanomaterials as peroxidase mimics.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the application of Zn3(OH)2V2O7·2H2O nanomaterials as peroxidase mimics in the determination of hydrogen peroxide (H2O2) concentration.

[0008] The second aspect of the present invention provides a peroxidase mimetic enzyme, which comprises Zn3(OH)2V2O7·2H2O nanomaterials; the Zn3(OH)2V2O7·2H2O nanomaterials are in a strip structure, with a length of 5-10 μm and a width of 300-500 nm.

[0009] The third aspect of the present invention provides a preparation method of the Zn3(OH)2V2O7·2H2O nanomaterials described in the first aspect, comprising the following steps:

[0010] Disperse a surfactant, a zinc source and a vanadium source in water, adjust the pH of the mixed solution to 4-7, and obtain the Zn3(OH)2V2O7·2H2O nanomaterials through a hydrothermal reaction.

[0011] The fourth aspect of the present invention provides a method for determining the concentration of hydrogen peroxide by using Zn3(OH)2V2O7·2H2O nanomaterials, comprising the following steps:

[0012] (1) Disperse the Zn3(OH)2V2O7·2H2O nanomaterials in a solvent to obtain a Zn3(OH)2V2O7·2H2O nanomaterial dispersion;

[0013] (2) Add a test solution containing hydrogen peroxide, an ethanol solution of 3,3’,5,5’-tetramethylbenzidine (TMB) and the Zn3(OH)2V2O7·2H2O nanomaterial dispersion prepared in step (1) into a centrifuge tube containing a buffer solution to obtain a colorimetric detection system, and perform ultraviolet-visible absorption signal detection after 6-8 minutes.

[0014] The beneficial effects of the present invention are as follows:

[0015] (1) The present invention for the first time provides that the strip-structured Zn3(OH)2V2O7·2H2O nanomaterials can be used as peroxidase mimetic enzymes, and their enzymatic catalytic activity can be comparable to that of natural horseradish peroxidase (HRP), and can be used to detect the concentration of hydrogen peroxide in solution samples.

[0016] (2) Compared with natural enzymes, the strip-structured Zn3(OH)2V2O7·2H2O nanomaterials have higher stability in extreme environments such as high temperature, strong acid or strong alkali, and can be reused while maintaining high catalytic activity.

[0017] (3) The preparation process of the strip-structured Zn3(OH)2V2O7·2H2O nanomaterials in the present invention is simple, has good repeatability, high stability and low cost. As a novel peroxidase mimetic enzyme, it can replace peroxidase and be applied in fields such as immunoassay, biological detection, and clinical diagnosis. Description of the Drawings

[0018] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0019] Figure 1 XRD pattern of the strip-structured Zn3(OH)2V2O7·2H2O nanomaterial prepared in Example 1;

[0020] Figure 2 SEM photograph of the strip-structured Zn3(OH)2V2O7·2H2O nanomaterial prepared in Example 1;

[0021] Figure 3 Ultraviolet-visible absorption spectra corresponding to four reaction systems, where a is H2O2 + Zn3(OH)2V2O7·2H2O, b is TMB + Zn3(OH)2V2O7·2H2O, c is TMB + H2O2, and d is TMB + H2O2 + Zn3(OH)2V2O7·2H2O;

[0022] Figure 4 Effect of pH value on the catalytic activity of the reaction system;

[0023] Figure 5 Effect of temperature on the catalytic activity of the reaction system;

[0024] Figure 6 Relationship between absorbance and hydrogen peroxide concentration;

[0025] Figure 7 Absorbance values after the strip-structured Zn3(OH)2V2O7·2H2O nanomaterial prepared in Example 1 was repeatedly subjected to 10 simulated enzyme reactions. Detailed Description of the Invention

[0026] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] The first typical embodiment of the present invention provides the application of Zn3(OH)2V2O7·2H2O nanomaterials as peroxidase mimics in the determination of hydrogen peroxide (H2O2) concentration.

[0029] In one or more embodiments, the Zn3(OH)2V2O7·2H2O nanomaterials have a ribbon structure, with a length of 5 - 10 μm and a width of 300 - 500 nm.

[0030] The second typical embodiment of the present invention provides a peroxidase mimic, which comprises Zn3(OH)2V2O7·2H2O nanomaterials; the Zn3(OH)2V2O7·2H2O nanomaterials have a ribbon structure, with a length of 5 - 10 μm and a width of 300 - 500 nm.

[0031] The third typical embodiment of the present invention provides a preparation method of the Zn3(OH)2V2O7·2H2O nanomaterials described in the first aspect, comprising the following steps:

[0032] Disperse a surfactant, a zinc source, and a vanadium source in water, adjust the pH of the mixed solution to 4 - 7, and obtain the Zn3(OH)2V2O7·2H2O nanomaterials through hydrothermal reaction.

[0033] In one or more embodiments, the surfactant is selected from sodium dodecyl sulfate (SDS). The surfactant adsorbs on specific crystal planes, affects the crystal growth direction, and thus controls the morphology of the material. By adjusting the concentration and type, the surfactant can limit the particle growth and control the material size.

[0034] In one or more embodiments, the concentration of the surfactant is 0.05 - 0.15 g / mL, preferably 0.1 g / mL.

[0035] In one or more embodiments, the zinc source is selected from zinc acetate (Zn(CH3COO)2).

[0036] In one or more embodiments, the vanadium source is selected from sodium orthovanadate (Na3VO4).

[0037] In one or more embodiments, the molar ratio of the vanadium source to the zinc source is 1:(1 - 2).

[0038] In one or more embodiments, the concentration of the zinc source is 0.016 - 0.033 mol / L.

[0039] In one or more embodiments, the temperature of the hydrothermal reaction is 120 - 200 °C, preferably 180 °C; the time of the hydrothermal reaction is 12 - 48 h, preferably 24 h.

[0040] The fourth typical embodiment of the present invention provides a method for determining the concentration of hydrogen peroxide by using Zn3(OH)2V2O7·2H2O nanomaterials, which includes the following steps:

[0041] (1) Disperse Zn3(OH)2V2O7·2H2O nanomaterials in a solvent to obtain a dispersion of Zn3(OH)2V2O7·2H2O nanomaterials;

[0042] (2) Add the test solution containing hydrogen peroxide, an ethanol solution of 3,3’,5,5’-tetramethylbenzidine (TMB), and the dispersion of Zn3(OH)2V2O7·2H2O nanomaterials prepared in step (1) into a centrifuge tube containing a buffer solution to obtain a colorimetric detection system, and perform ultraviolet-visible absorption signal detection after 6-8 minutes.

[0043] In one or more embodiments, the solvent in step (1) is selected from water.

[0044] In one or more embodiments, the pH value of the colorimetric detection system is 2-9.

[0045] The buffer solution in step (2) includes phosphate buffer solution.

[0046] In one or more embodiments, the final concentration of the Zn3(OH)2V2O7·2H2O nanomaterials is 8-10 μg / mL, preferably 9 μg / mL.

[0047] In one or more embodiments, the final concentration of the 3,3’,5,5’-tetramethylbenzidine is 0.7-0.9 mmol / L, preferably 0.8 mmol / L.

[0048] In one or more embodiments, in step (2), ultraviolet-visible absorption signals at wavelengths of 400-800 nm are used for detection.

[0049] In one or more embodiments, a mixed colorimetric detection system is prepared using hydrogen peroxide solutions with standard gradient concentrations, ultraviolet-visible absorption signal detection is performed, a standard curve is constructed, and the detection value of the test solution is brought into the calculation to quantitatively analyze the hydrogen peroxide in the sample to be tested.

[0050] Preferably, the concentration range of the hydrogen peroxide with gradient concentrations is 1-25 μM.

[0051] Preferably, the standard curve is constructed using ultraviolet-visible absorption signals at wavelengths of 400-800 nm.

[0052] 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.

[0053] Example 1

[0054] Preparation of ribbon-like structure Zn3(OH)2V2O7·2H2O nanomaterial:

[0055] Dissolve 0.6 g of sodium dodecyl sulfate (SDS), 1 mmol of zinc acetate (Zn(CH3COO)2), and 2 mmol of sodium orthovanadate (Na3VO4) in 60 mL of ultrapure water, and adjust the pH of the mixed solution to 5 with 2 M NaOH solution. Then, after stirring the mixed solution evenly, transfer it to a polytetrafluoroethylene high-pressure reaction kettle and place it in a forced-air drying oven for heat treatment at 180 °C for 24 h. After the reaction is completed, let the reaction kettle cool naturally to room temperature, carry out suction filtration, and wash the precipitate with water and absolute ethanol for many times. Finally, the obtained product is dried in a forced-air drying oven at 60 °C for 6 h.

[0056] Figure 1 This is the XRD pattern of the sample prepared in this example. From Figure 1 it can be seen that the positions of all diffraction peaks are completely consistent with the standard card of hexagonal phase Zn3(OH)2V2O7·2H2O (JCPDS No. 50-0570), and no impurity phase appears. It can be determined that the sample prepared in this example is pure Zn3(OH)2V2O7·2H2O. In addition, from Figure 1 it can be seen that the diffraction peak intensity of the sample is relatively large and the diffraction peak is relatively sharp, indicating that the prepared Zn3(OH)2V2O7·2H2O has good crystallinity.

[0057] Figure 2 This is the SEM photograph of the sample prepared in this example. As can be seen from the figure, the prepared Zn3(OH)2V2O7·2H2O is in a ribbon-like structure, with a width of about 335 nm and a length of 6 μM. This structure has a relatively large specific surface area.

[0058] Example 2

[0059] Ribbon-like structure Zn3(OH)2V2O7·2H2O nanomaterial mimicking peroxidase:

[0060] Take a 1.5 mL centrifuge tube, add 400 μL of 50 mM phosphate buffer solution (PBS, pH = 4.0), 100 μL of 20 mM H2O2 solution, 100 μL of 8 mM ethanol solution of TMB, and 310 μL of ultrapure water. Then add 90 μL of the Zn3(OH)2V2O7·2H2O dispersion prepared in Example 1 (the solvent is water, 100 μg / mL). After reacting for 7 min, observe the change of the solution and record the ultraviolet-visible absorption spectrum at 400 - 800 nm (see Figure 3 ).

[0061] Figure 3 are the ultraviolet-visible absorption spectra corresponding to the four reaction systems. 652 nm is the characteristic absorption peak of oxidized TMB. From Figure 3 it can be seen that there are almost no absorption peaks in the three systems of H2O2+Zn3(OH)2V2O7·2H2O, TMB+Zn3(OH)2V2O7·2H2O, and TMB+H2O2, while the TMB+H2O2+Zn3(OH)2V2O7·2H2O system has an obvious absorption peak at 652 nm and a good peak shape. In addition, by observing the color changes of the three systems of H2O2+Zn3(OH)2V2O7·2H2O, TMB+Zn3(OH)2V2O7·2H2O, and TMB+H2O2, it is found that the colors of the solutions in the three systems are colorless and transparent without obvious changes, while the solution in the TMB+H2O2+Zn3(OH)2V2O7·2H2O system shows an obvious blue color, indicating that Zn3(OH)2V2O7·2H2O catalyzes the oxidation of TMB to generate blue oxide in the presence of H2O2. The results show that the Zn3(OH)2V2O7·2H2O nanomaterial prepared in the present invention has good peroxidase-like catalytic activity, is a peroxidase mimetic material, can catalyze the oxidation of TMB to generate blue oxide in the presence of H2O2, and can realize the rapid detection of H2O2 through this color reaction, and has potential application prospects in the fields of bio-immunoassay and environmental detection.

[0062] Example 3

[0063] Figure 4 reflects the influence of pH value on the catalytic activity of the reaction system. It can be clearly seen from the experimental results that when pH = 4, the absorbance of the reaction mixture reaches the maximum value and the catalytic activity is the best.

[0064] Example 4

[0065] Figure 5It reflects the influence of temperature on the catalytic activity of the reaction system. As the temperature increases, the absorbance of the reaction system at 652 nm gradually increases, indicating an improvement in catalytic activity. Considering a series of factors such as ease of implementation and observation, 25 °C is taken as the standard temperature for the entire experiment.

[0066] Example 5

[0067] The belt-like structure Zn3(OH)2V2O7·2H2O nanomaterial can ingeniously act as a peroxidase mimetic to catalytically promote the oxidation of TMB in the presence of hydrogen peroxide. Therefore, a simple method for detecting hydrogen peroxide was established based on the unique colorimetric change exhibited by TMB after oxidation. Figure 6 It reflects the relationship between absorbance and hydrogen peroxide concentration. The results show that the absorbance in the reaction system gradually increases with the increase in hydrogen peroxide concentration and shows a positive correlation within the linear range of 1 - 25 μM. The linear equation is A 652 nm = 0.01006 + 3.98612C (mM), and the correlation coefficient is 0.9902. The lowest detection limit of this detection method is 0.71 μM.

[0068] Example 6

[0069] The belt-like structure Zn3(OH)2V2O7·2H2O nanomaterial was repeatedly applied to detect H2O2 as a mimetic peroxidase

[0070] The belt-like structure Zn3(OH)2V2O7·2H2O nanomaterial used in Example 2 was centrifugally recovered, washed repeatedly with ultrapure water and absolute ethanol, dried at 60 °C, and then the next mimetic enzyme performance test was carried out according to the steps in Example 2 for 10 consecutive times while keeping other conditions unchanged. The results are as Figure 7 shown. After 10 consecutive mimetic enzyme tests, the absorbance value of the reaction system did not decrease significantly, and the solution color still showed an obvious blue, indicating good reusability and stability of the belt-like structure Zn3(OH)2V2O7·2H2O nanomaterial as a mimetic enzyme material.

[0071] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. 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. Application of Zn3(OH)2V2O7·2H2O nanomaterial as peroxide mimetic enzyme in the determination of hydrogen peroxide (H2O2) concentration.

2. The use according to claim 1, characterized in that The Zn3(OH)2V2O7·2H2O nano material is a strip structure with a length of 5 to 10 μm and a width of 300 to 500 nm.

3. A peroxidase mimetic enzyme, characterized in that The invention comprises a Zn3(OH)2V2O7·2H2O nano material; the Zn3(OH)2V2O7·2H2O nano material is a band structure with a length of 5-10 μm and a width of 300-500 nm.

4. The method for preparing the Zn3(OH)2V2O7·2H2O nanomaterial according to claim 1 or 2, characterized in that: The steps include: The surfactant, the zinc source and the vanadium source are dispersed in water, the pH value of the mixed solution is adjusted to 4-7, and the Zn3(OH)2V2O7·2H2O nanomaterial is obtained by hydrothermal reaction.

5. The preparation method according to claim 4, characterized in that: The surfactant is selected from sodium lauryl sulfate; Or, the zinc source is selected from zinc acetate; Or, the vanadium source is selected from sodium orthovanadate; Or, the molar ratio of the vanadium source to the zinc source is 1:(1-2); Alternatively, the concentration of the zinc source is 0.016 to 0.033 mol / L.

6. The preparation method according to claim 4, characterized in that: The temperature of the hydrothermal reaction is 120-200° C., preferably 180° C.; the time of the hydrothermal reaction is 12-48 hours, preferably 24 hours.

7. A method for measuring the concentration of hydrogen peroxide using Zn3(OH)2V2O7·2H2O nanomaterials, characterized in that: The steps include: (1) dispersing Zn3(OH)2V2O7·2H2O nanomaterial in a solvent to obtain a Zn3(OH)2V2O7·2H2O nanomaterial dispersion; (2) Adding a test solution containing hydrogen peroxide, an ethanol solution of 3,3',5,5'-tetramethylbenzidine (TMB), and the Zn3(OH)2V2O7·2H2O nanomaterial dispersion prepared in step (1) to a centrifuge tube containing a buffer solution to obtain a colorimetric detection system, and performing UV-visible absorption signal detection after 6 to 8 minutes.

8. The method according to claim 7, characterized in that The solvent in step (1) is selected from water; Or, the pH value of the colorimetric detection system is 4 to 10; preferably, the buffer solution in step (2) comprises a phosphate buffer; Or, the final concentration of the Zn3(OH)2V2O7·2H2O nanomaterial is 8-10 μg / mL, preferably 9 μg / mL; Alternatively, the final concentration of the 3,3',5,5'-tetramethylbenzidine is 0.7-0.9 mmol / L, preferably 0.8 mmol / L.

9. The method according to claim 7, characterized in that In step (2), ultraviolet-visible absorption signals at a wavelength of 400 to 800 nm are used for detection.

10. The method according to claim 7, characterized in that A mixed colorimetric detection system is prepared using hydrogen peroxide solutions of standard gradient concentrations, UV-visible absorption signal detection is performed, a standard curve is constructed, the detection value of the solution to be tested is brought into the calculation, and the hydrogen peroxide in the sample to be tested is quantitatively analyzed; Preferably, the concentration range of the gradient concentration of hydrogen peroxide is 1 to 25 μM; Preferably, the standard curve is constructed using ultraviolet-visible absorption signals at a wavelength of 400 to 800 nm.

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