CN116586112B - A dialdehyde chitosan manganese dioxide nanozyme and its preparation and application - Google Patents
A dialdehyde chitosan manganese dioxide nanozyme and its preparation and application Download PDFInfo
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Abstract
Preparing sodium periodate aqueous solution, slowly pouring the prepared sodium periodate solution into chitosan solution, stirring a reaction mixture at 37-50 ℃ in a dark place, adding absolute ethyl alcohol to terminate the reaction and precipitate a crude product, sequentially dialyzing the crude product in sodium chloride solution and deionized water to purify the product, preparing manganese acetate and the dialdehyde chitosan prepared in the step (1) into aqueous solution, magnetically stirring and mixing the aqueous solution, adding sodium hydroxide aqueous solution after magnetically stirring at room temperature, continuously and severely magnetically stirring at room temperature, flushing for three times, and dispersing into ultrapure water. The synthesized DAC/MnO2 nano-enzyme has excellent catalase-like catalytic activity and oxidase-like catalytic activity, enriches the variety of nano-enzymes, can be used for quantitative detection of hydrogen peroxide, and has good selectivity to hydrogen peroxide.
Description
Technical Field
The invention relates to the technical field of nano-enzyme preparation, in particular to dialdehyde chitosan manganese dioxide nano-enzyme and preparation and application thereof.
Background
Hydrogen peroxide (H 2O2) is commonly called hydrogen peroxide, and is colorless transparent liquid with strong oxidizing property and corrosiveness. Hydrogen peroxide is an important chemical product, and is widely applied to the fields of medicine, food, national defense, textile, pulp bleaching, synthesis of chemical products, environmental protection and the like, and plays a vital role in human metabolism. However, excessive production and accumulation of active radicals generated by the decomposition of H 2O2 in human body is harmful to human body, resulting in serious cell damage and even some dangerous diseases. Therefore, the establishment of a simple and accurate H 2O2 rapid detection method has important significance and necessity.
Currently, a variety of analytical techniques for determining H 2O2 have been developed, such as conventional titration, spectrophotometry, high performance liquid chromatography, colorimetry, electrochemistry and the like. The iodine method in the conventional titration method is the most widely used method for detecting the hydrogen peroxide content in the bleaching process in factory practice, and the method is simple to operate, low in sensitivity, multiple in interference factors and less obvious in end-point color change. The application of electrochemical methods and chromatography requires special equipment and is difficult to popularize and use. The detection based on horseradish peroxidase (HRP) colorimetric method has good application prospect in practical sample diagnosis due to the advantages of simple operation, quick reaction, low cost, high sensitivity and the like, and is widely focused by people. However, HRP is a natural enzyme, and its inherent disadvantages have limited its use in some applications. Thus, in order to overcome these problems, researchers have made much effort to develop nanoenzymes having similar properties to HRP.
Nano-enzymes refer to nano-materials having natural enzymatic activity. As a novel mimic enzyme, the nano-enzyme not only has the unique property of nano-materials, but also can mimic the high-efficiency catalytic function of natural enzymes. Compared with natural enzymes, the nano-enzyme has low cost, strong tolerance, high stability, easy long-term storage, large-scale production and the like.
Up to now, various nanomaterials such as metals, metal oxides, metal sulfides, metal organic frameworks and carbon-based nanomaterials have been reported to have catalytic activity of natural enzymes. Among them, manganese dioxide has not only excellent optical properties and enzyme-like activity as an important transition metal oxide, but also, more importantly, manganese dioxide has good oxidation activity to reducing biomolecules such as glutathione, hydrogen peroxide and the like. However, nano manganese dioxide, while having particular advantages, has limited application due to its insolubility in nearly all solvents. Therefore, it is necessary to develop a novel manganese dioxide nano enzyme material, enrich the variety of nano enzymes and provide more choices for hydrogen peroxide detection.
Disclosure of Invention
Based on the defects, the invention provides dialdehyde chitosan manganese dioxide nano-enzyme.
The specific technical scheme is that the dialdehyde chitosan manganese dioxide nano enzyme is prepared from manganese acetate and dialdehyde chitosan serving as raw materials.
The preparation method of the dialdehyde chitosan manganese dioxide nano-enzyme comprises the following steps:
(1) Preparing dialdehyde chitosan, namely weighing chitosan, dissolving the chitosan in an acetate buffer solution, preparing a chitosan acetic acid solution, preparing a sodium periodate aqueous solution, slowly pouring the prepared sodium periodate solution into the chitosan solution, stirring a reaction mixture at 37-50 ℃ in a dark place, adding absolute ethyl alcohol to terminate the reaction and precipitate a crude product, and sequentially dialyzing the crude product in sodium chloride solution and deionized water to purify the product;
(2) Preparing DAC/MnO 2 nano enzyme, namely preparing manganese acetate and the dialdehyde chitosan prepared in the step (1) into an aqueous solution, carrying out first magnetic stirring and mixing, adding a sodium hydroxide aqueous solution after room temperature magnetic stirring, continuously carrying out second magnetic stirring which is intense continuously at room temperature, and then flushing for three times and dispersing into ultrapure water.
The preparation method of the dialdehyde chitosan manganese dioxide nano-enzyme according to claim 2 is characterized in that in the step (1), the pH of an acetate buffer solution is 4.4-4.6, the concentration of a prepared chitosan acetic acid solution is 15mg/mL, the concentration of a prepared sodium periodate aqueous solution is 0.05g/mL, the light-shielding stirring time is 24-45 h, the volume of absolute ethyl alcohol is 800-950 mL, the termination reaction time is 2-3 h, and the dialysis time is 36-48 h.
Preferably, in the step (2), the concentration of the manganese acetate is 100mM, the volume is 1-1.5 mL, the volume of the dialdehyde chitosan is 5mL, the concentration is 1mg/mL, the concentration of the sodium hydroxide is 70mM, the volume is 4.6-5.2 mL, the first magnetic stirring time is 30-45 min, and the second magnetic stirring time is 12-16 h.
According to another technical scheme, the application of the dialdehyde chitosan manganese dioxide nano-enzyme is that the prepared DAC/MnO 2 nano-enzyme is used for detecting hydrogen peroxide, namely DAC/MnO 2 nano-enzyme is taken, PBS buffer solution, hydrogen peroxide and a chromogenic substrate are added, after chromogenic reaction is carried out, sulfuric acid solution is added for stopping the reaction, and an ultraviolet spectrophotometer is used for reading absorbance value under the wavelength of 450 nm.
Preferably, the concentration of the nano DAC/MnO 2 nano enzyme in the reaction system is 0.5-2.5 mg/mL, the concentration of the hydrogen peroxide is 2-10 mg/mL, the chromogenic substrate is 3,3', 5' -tetramethylbenzidine, and the concentration of the chromogenic substrate is 0.5-2.5 mg/mL.
Preferably, the catalytic reaction temperature of the DAC/MnO 2 nano-enzyme is 25-60 ℃, and the pH of the PBS buffer solution is 5-7.
Compared with the prior art, the invention has the beneficial effects that:
(1) The preparation method has simple process, mild reaction condition, convenience and rapidness;
(2) DAC/MnO 2 prepared by the method has excellent catalase-like catalytic activity and oxidase-like catalytic activity, and enriches the variety of nano enzymes;
(3) DAC/MnO 2 prepared by the method can be used for quantitative detection of hydrogen peroxide, and the synthesized nano material has good sensitivity and lower detection limit in detecting hydrogen peroxide, has good selectivity to hydrogen peroxide, and can realize low-cost, rapid and simple detection;
(4) The DAC/MnO 2 prepared by the method has a great application prospect as a novel nano-enzyme, and can be applied to quantitative measurement of glucose and ascorbic acid in blood, quantitative measurement of glutathione in human serum and biological probes such as DAC/MnO 2 nano-enzyme coupled antibodies and DNA.
Drawings
FIG. 1 is a graph showing the effect of the amount of DAC added during the preparation of DAC/MnO 2 on peroxidase activity;
FIG. 2 is a graph comparing DAC/MnO 2 prepared with low concentration and high concentration DAC additions;
FIG. 3 shows the effect of reaction conditions on the catalytic activity of DAC/MnO 2 class enzyme (a) pH of buffer, (b) temperature, (c) concentration of H 2O2, and (d) concentration of TMB;
FIG. 4 is a graph of Michaelis-Menten plot of TMB and (d) H 2O2 versus time absorbance of (a) TMB at different concentrations and (b) H 2O2 for DAC/MnO 2;
fig. 5 is a standard graph of H 2O2 detection.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1A dialdehyde chitosan manganese dioxide nano-enzyme, its preparation method includes the following steps:
(1) Preparing dialdehyde chitosan, namely weighing chitosan, dissolving chitosan in an acetate buffer solution with the pH of 4.4-4.6 to prepare a chitosan acetic acid solution with the concentration of 15mg/mL, preparing a sodium periodate aqueous solution with the concentration of 0.05g/mL, slowly pouring the prepared sodium periodate solution into the chitosan solution, stirring the reaction mixture at 37-50 ℃ for 24-45 hours in a dark place, adding 800-950 mL of absolute ethyl alcohol to terminate the reaction for 2-3 hours and precipitate a crude product, and then sequentially dialyzing the crude product in sodium chloride solution (0.2 mol/L) and deionized water for 36-48 hours to purify the product;
(2) Preparing DAC/MnO 2 nano enzyme, namely preparing manganese acetate with the concentration of 100mM and the volume of 1-1.5 mL and dialdehyde chitosan prepared in the step (1) into an aqueous solution, carrying out first magnetic stirring for 30-45 min, adding a sodium hydroxide aqueous solution with the concentration of 70mM and the volume of 4.6-5.2 mL after magnetic stirring at room temperature, continuously carrying out second magnetic stirring for 12-16 h at room temperature, and flushing for three times to disperse in ultrapure water, wherein the volume of dialdehyde chitosan is 5mL and the concentration is 0.1-1 mg/mL.
The DAC/MnO 2 nano enzyme is used for detecting hydrogen peroxide, namely DAC/MnO 2 nano enzyme is taken, PBS buffer solution, hydrogen peroxide and chromogenic substrate are added, sulfuric acid solution is added to stop the reaction after chromogenic reaction, and an ultraviolet spectrophotometer is used for reading absorbance value under the wavelength of 450 nm. The nano DAC/MnO 2 nano enzyme concentration in the reaction system is 0.5-2.5 mg/mL, the hydrogen peroxide concentration is 2-10 mg/mL, the chromogenic substrate is 3,3', 5' -tetramethyl benzidine (TMB), the chromogenic substrate concentration is 0.5-2.5 mg/mL, the DAC/MnO 2 nano enzyme catalytic reaction temperature is 25-60 ℃, and the pH of PBS buffer solution is 5-7.
Example 2 the preparation of dialdehyde chitosan (DAC) in this example was performed in the same manner as the DAC in example 1.
Preparing DAC/MnO 2 nano enzyme, namely preparing manganese acetate with the concentration of 100mM and the volume of 1-1.5 mL and dialdehyde chitosan prepared in the step (1) into an aqueous solution, carrying out first magnetic stirring for 30min, adding a sodium hydroxide aqueous solution with the concentration of 70mM and the volume of 5mL for reaction for 5min after magnetic stirring at room temperature, continuing to carry out second magnetic stirring for 12h at room temperature, and flushing three times for dispersion into ultrapure water. Three groups of controls were made, and in the three groups of control experiments, the volume of the dialdehyde chitosan was 5mL, and the concentrations were respectively (1 mg/mL, 0.5mg/mL, 0.1 mg/mL).
Nano enzyme exploration peroxidase activity experiment 10. Mu.L of PBS buffer solution (pH 6), 20. Mu.L of H 2O2 (concentration 10 mM) and 2. Mu.L of TMB (concentration 2 mM) prepared in three groups of control experiments are respectively taken, after color development for 2min, 100. Mu.L of H 2SO4 (concentration 2 mol/L) is added to terminate the reaction, and an absorbance value at a wavelength of 450nm is read by using an ultraviolet spectrophotometer.
As shown in fig. 1, in the low concentration range, the activity of the peroxygenase gradually increases as the dialdehyde chitosan increases, probably because the dialdehyde chitosan with low concentration can be strongly adsorbed on the bulk manganese dioxide in the form of a monomer to effectively peel off the manganese dioxide nano-sheets, whereas the dialdehyde chitosan with high concentration is aggregated and cannot be strongly adsorbed on the bulk manganese dioxide to cause ineffective peeling of the manganese dioxide nano-sheets, as shown in fig. 2. Thus 1mg/mL dialdehyde chitosan was optionally added during the preparation of the material.
Example 3 effect of different pH on the catalytic activity of the peroxidase-like DAC/MnO 2 phosphate buffer (PBS buffer) and acetate-sodium acetate buffer were formulated and the pH values were set to (4, 5, 6, 7, 8), respectively. While other conditions were fixed such that the concentration of DAC/MnO 2 was 1mg/mL, the catalytic reaction temperature was 35 ℃, the concentration of H 2O2 was 10mM, and the concentration of TMB was 2mM, and the peroxidase catalytic oxidation reaction was performed. In the reaction, 10. Mu.L of DAC/MnO 2 solution, 350. Mu.L of PBS buffer, 20. Mu.L of H 2O2 and 20. Mu.L of TMB were added, and after development of color for 2min, 100. Mu.L of H 2SO4 (2 mol/L) was added to terminate the reaction, and the absorbance was measured in that order.
As shown in fig. 3 (a), the appropriate pH of the buffer can increase the catalytic activity of the nano-enzyme, and the catalytic activity of DAC/MnO 2 is active under weak acidic conditions, and the catalytic activity is the greatest at ph=6. Higher temperatures can lead to deactivation of peroxide-like activity.
The effect of different temperatures on the catalytic activity of DAC/MnO 2 type peroxidases were carried out by fixing the pH of PBS buffer to 6 at temperatures of 25,30,35,40,45,50℃respectively, and otherwise carrying out the catalytic oxidation reaction of the peroxidases in accordance with example 3.
As shown in FIG. 3 (b), DAC/MnO2 peroxide-like activity increases and then decreases sharply with increasing temperature, and the catalytic performance at 35 ℃ is optimal.
Effect of different hydrogen peroxide concentrations on catalytic activity of DAC/MnO 2 class peroxidase the hydrogen peroxide concentrations were set at (2, 4, 6, 8, 10 mM) and the PBS buffer was fixed at pH 6, otherwise the peroxidase catalytic oxidation reaction was performed in accordance with example 3.
As shown in FIG. 3 (c), the optimal concentration of H 2O2 was 4mM, respectively.
Effect of different TMB concentrations on catalytic activity of DAC/MnO 2 class peroxidase the catalytic oxidation reaction of peroxidase was performed in the same manner as in example 3 except that TMB concentrations were set at (0.5, 1, 1.5, 2, 2.5 mM) and PBS buffer was fixed at pH 6.
As shown in FIG. 3 (d), the optimal concentration of TMB was 1.5mM.
Example 4H 2O2 concentration was fixed at 1.5mM and a series of different concentrations of TMB (0.04 mM-2 mM) were prepared. During the reaction, 10. Mu.L of DAC/MnO 2 (1 mg/mL) solution, 20. Mu.L of H 2O2 solution and 20. Mu.L of TMB, 450. Mu.L of PBS buffer (pH 6) were taken. The reaction was sampled at equal time intervals and absorbance was measured at 652nm by a UV-Vis spectrophotometer.
The same experimental procedure was repeated with the other experimental conditions unchanged, fixing the concentration of TMB to 4mM, increasing the concentration of H 2O2 from 0.004mM to 0.4mM in sequence.
As shown in fig. 4 (a), when the concentration of H 2O2 was fixed, the reaction rate of DAC/MnO 2 type peroxidase gradually increased with an increase in the concentration of TMB, but when the concentration of TMB was increased to some extent, the rate of increase in the reaction rate became slow and gradually smoothed. Next, as shown in FIG. 4 (b), when TMB concentration is fixed, the reaction rate of DAC/MnO 2 type peroxidases gradually increases with the increase of H 2O2. Also, as the concentration of H 2O2 increases to some extent, the reaction rate also begins to slow down gradually and eventually stabilizes. By fitting the data in the curve using the Lineweaver-Bur double reciprocal model, straight lines can be obtained, as shown in FIGS. 4 (c) and 4 (d).
DAC/MnO 2 oxidation TMB and H 2O2 kinetic parameters Km were calculated from the intercept and slope on the straight line, 0.0776mmol/L and 7.722. Mu. Mol/L, respectively. The Km value is one of the characteristic constants of enzymes, and is only related to the properties of the enzymes, and in general, km values of different enzymes are different. The Km value indicates the degree of affinity between the enzyme and the substrate, and the larger the Km value, the smaller the affinity between the enzyme and the substrate, and the larger the affinity. These results indicate that DAC/MnO 2 material can be used as an artificial peroxidase study and has intentional enzymatic activity, as compared to the Km of natural horseradish peroxidase (Km of oxidized TMB is 0.434).
Example 5. Mu.L of PBS buffer (pH=6), 20. Mu.L of TMB solution (2 mM), 20. Mu.L of H 2O2 at different concentrations and increasing in sequence at different concentrations were added to 10. Mu.L of 1mg/mL DAC/MnO 2 solution. After 2min of color development, 10. Mu.L of H 2SO4 (2 mol/L) was added to terminate the reaction. The resulting solution was subjected to measurement of absorption spectrum at 450nm by an ultraviolet-visible spectrophotometer.
As shown in FIG. 5, the absorbance at 450nm increases from 5. Mu.M to 500. Mu.M with H 2O2 concentration, the absorbance intensity and H 2O2 concentration are in good linear relationship in the range of 4. Mu.M to 400. Mu.M, the correlation coefficient is 0.997, and the detection limit is 11. Mu.M. The limit of detection is determined by lod=ks 0/S, where K is a numerical factor selected according to the required confidence level, S 0 is the standard deviation of the blank measurement (n=11, k=3), and S is the slope of the calibration curve.
Table 1 shows the comparison of the hydrogen peroxide detected in the prior art with the hydrogen peroxide detected in the present application
| Material | Method of | Linear range (mu mol/L) | Detection limit (mu mol/L) |
| MoS2-PPy | Colorimetric method | 50-2000 | 45.0 |
| MoS2-rGO | Fluorescence method | 60-700 | 25.0 |
| DA-AgNPs | Colorimetric method | 30-70 | 3000 |
| CNP | Colorimetric method | 1-40 | 20 |
| CoS | Colorimetric method | 50-800 | 20 |
| Meso-CeO2/C | Electrochemical process | 250-5000 | 1300 |
| NiS/MMT/GO | Colorimetric method | 10-100 | 9.73 |
| The method | Colorimetric method | 4-400 | 11 |
From the results in Table 1, it can be seen that the nano-enzyme prepared by the method has good sensitivity and low detection limit in detecting hydrogen peroxide.
In conclusion, the preparation method is simple in process, mild in reaction condition, convenient and quick, the prepared DAC/MnO 2 has excellent catalase-like catalytic activity and oxidase-like catalytic activity, the variety of nano enzymes is enriched, the DAC/MnO 2 can be used for quantitatively detecting hydrogen peroxide, and the synthesized nano material has good sensitivity and lower detection limit in detecting hydrogen peroxide, has good selectivity on hydrogen peroxide, and can realize low-cost, rapid and simple detection.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications according to the technical scheme and the inventive concept thereof within the scope of the present invention.
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| KR20230008624A (en) * | 2021-07-07 | 2023-01-16 | 서울대학교산학협력단 | Metal complex nanozyme, manufacturing method thereof and blood glucose measuring apparatus using the same |
| CN114163662A (en) * | 2021-12-16 | 2022-03-11 | 江南大学 | A kind of nanozyme functionalized hydrogel and preparation method thereof |
-
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- 2023-05-24 CN CN202310593226.6A patent/CN116586112B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109354634A (en) * | 2018-11-17 | 2019-02-19 | 赵兵 | A kind of Chitosan-Thiolated Polymers and preparation method thereof |
| CN115651965A (en) * | 2022-10-24 | 2023-01-31 | 遵义医科大学 | Nano composite catalyst and method for determining glycated albumin by one-step method |
Non-Patent Citations (1)
| Title |
|---|
| "MnO2基类氧化酶活性材料制备及双模检测研究";张志伟;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20230215(第2期);第5页第2段，第6页第2段，第7页第2段，第35页第3段至第36段第1段 * |
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