A dialdehyde chitosan manganese dioxide nanozyme and its preparation and application

Abstract

A dialdehyde chitosan manganese dioxide nanozyme and its preparation and application relate to the technical field of nanozyme preparation, and include the following steps: (1) Prepare an aqueous solution of sodium periodate, then slowly pour the prepared sodium periodate solution into the chitosan solution, stir the reaction mixture in the dark at 37-50 °C, then add absolute ethanol to terminate the reaction and precipitate the crude product. Subsequently, the crude product is dialyzed successively in a sodium chloride solution and deionized water to purify the product; (2) Configure manganese acetate and the dialdehyde chitosan prepared in step (1) into an aqueous solution and magnetically stir and mix them. After magnetic stirring at room temperature, add an aqueous solution of sodium hydroxide, continue to carry out vigorous magnetic stirring at room temperature, and then rinse three times and disperse it in ultrapure water. The synthesized DAC / MnO2 nanozyme of the present invention has excellent catalase-like catalytic activity and oxidase-like catalytic activity, enriches the types of nanozymes, and can be used for the quantitative detection of hydrogen peroxide, and has good selectivity for hydrogen peroxide.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozyme preparation, and particularly relates to a dialdehyde chitosan manganese dioxide nanozyme and its preparation and application. Background Art

[0002] Hydrogen peroxide (H2O2), commonly known as hydrogen peroxide solution, is a colorless and transparent liquid with strong oxidizing and corrosive properties. As an important chemical product, hydrogen peroxide is widely used not only in the fields of medicine, food, national defense, textiles, pulp bleaching, synthesis of chemical products, and environmental protection, but also plays a crucial role in human metabolism. However, excessive production and accumulation of reactive free radicals generated by the decomposition of excessive H2O2 in the human body can cause harm to the human body, leading to serious cell damage and even triggering some dangerous diseases. Therefore, it is of great significance and necessity to establish a simple and accurate method for rapid detection of H2O2.

[0003] Currently, a variety of analytical techniques for the determination of H2O2 have been developed, such as conventional titration, spectrophotometry, high-performance liquid chromatography, colorimetry, electrochemistry, etc. The iodometric method in conventional titration is the most widely used method in factory practice for detecting the content of hydrogen peroxide in the bleaching process. This method is simple to operate, but has low sensitivity, many interfering factors, and an unclear color change at the end point. The application of electrochemistry methods and chromatography methods requires special equipment and is difficult to promote. Among them, the colorimetric detection based on horseradish peroxidase (HRP) has good application prospects in actual sample diagnosis due to its advantages such as simple operation, fast reaction, low cost, and high sensitivity, which has attracted wide attention. However, as a natural enzyme, the inherent disadvantages of HRP limit its application in some aspects. Therefore, in order to overcome these problems, researchers have made a lot of efforts in developing nanozymes with properties similar to HRP.

[0004] Nanozymes refer to nanomaterials with natural enzyme activity. As a new type of mimetic enzyme, nanozymes not only have the unique properties of nanomaterials but also can mimic the efficient catalytic function of natural enzymes. Compared with natural enzymes, nanozymes have low cost, strong tolerance, high stability, are easy to store for a long time, and can be mass-produced.

[0005] So far, a variety of nanomaterials such as metals, metal oxides, metal sulfides, metal-organic frameworks, and carbon-based nanomaterials have been reported to possess the catalytic activity of natural enzymes. Among them, manganese dioxide, as an important transition metal oxide, not only has excellent optical properties and enzyme-like activity, but more importantly, manganese dioxide has good oxidation activity towards reducing biomolecules such as glutathione and hydrogen peroxide. However, although nanoscale manganese dioxide has special advantages, its insolubility in almost all solvents limits its applications. Therefore, it is necessary to develop new manganese dioxide nanozyme materials to enrich the types of nanozymes and provide more options for the detection of hydrogen peroxide. Summary of the Invention

[0006] Based on the above deficiencies, the present invention proposes a dialdehyde chitosan manganese dioxide nanozyme.

[0007] The specific technical solution is as follows: The dialdehyde chitosan manganese dioxide nanozyme is prepared from manganese acetate and dialdehyde chitosan as raw materials.

[0008] Another technical solution: A method for preparing a dialdehyde chitosan manganese dioxide nanozyme, comprising the following steps:

[0009] (1) Preparation of dialdehyde chitosan: Weigh chitosan and dissolve it in an acetate buffer solution to prepare a chitosan acetate solution. Prepare an aqueous sodium periodate solution, and then slowly pour the prepared sodium periodate solution into the chitosan solution. Stir the reaction mixture in the dark at 37-50 °C, then add absolute ethanol to terminate the reaction and precipitate the crude product. Subsequently, the crude product is dialyzed successively in a sodium chloride solution and deionized water to purify the product;

[0010] (2) Preparation of DAC / MnO2 nanozyme: Mix manganese acetate and the dialdehyde chitosan prepared in step (1) into an aqueous solution for the first magnetic stirring. After magnetic stirring at room temperature, add an aqueous sodium hydroxide solution, and continue the second intense magnetic stirring at room temperature. Then rinse three times and disperse it into ultrapure water.

[0011] 3. According to the method for preparing a dialdehyde chitosan manganese dioxide nanozyme as described in claim 2, wherein the pH of the acetate buffer solution in step (1) is 4.4-4.6; the concentration of the prepared chitosan acetate solution is 15 mg / mL; the concentration of the prepared aqueous sodium periodate solution is 0.05 g / mL; the dark stirring time is 24-45 h; the volume of absolute ethanol is 800-950 mL, the reaction termination time is 2-3 h; the dialysis time is 36-48 h.

[0012] Preferably, in step (2), the concentration of manganese acetate is 100 mM, and the volume is 1-1.5 mL; the volume of dialdehyde chitosan is 5 mL, and the concentration is 1 mg / mL; the concentration of sodium hydroxide is 70 mM, and 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.

[0013] Another technical solution: Application of dialdehyde chitosan manganese dioxide nanozyme. The hydrogen peroxide is detected by the prepared DAC / MnO2 nanozyme: Take the DAC / MnO2 nanozyme, add PBS buffer solution, hydrogen peroxide and chromogenic substrate. After color development, add sulfuric acid solution to terminate the reaction, and use an ultraviolet spectrophotometer to read the absorbance value at a wavelength of 450 nm.

[0014] Preferably, the concentration of DAC / MnO2 nanozyme 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',5,5'-tetramethylbenzidine, and the concentration of the chromogenic substrate is 0.5-2.5 mg / mL.

[0015] Preferably, the catalytic reaction temperature of the DAC / MnO2 nanozyme is 25-60 °C, and the pH of the PBS buffer solution is 5-7.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1): The preparation method of the present invention has a simple process, mild reaction conditions, and is convenient and fast;

[0018] (2): The DAC / MnO2 prepared by the method of the present invention has excellent catalase-like catalytic activity and oxidase-like catalytic activity, enriching the types of nanozymes;

[0019] (3): The DAC / MnO2 prepared by the method of the present invention can be used for the quantitative detection of hydrogen peroxide. The synthesized nanomaterial has good sensitivity and low detection limit in the detection of hydrogen peroxide, has good selectivity for hydrogen peroxide, and can realize low-cost, rapid and simple detection;

[0020] (4): The DAC / MnO2 prepared by the method of the present invention, as a new type of nanozyme, has great application prospects. For example, it can be applied to the quantitative measurement of glucose and ascorbic acid in blood; the quantitative measurement of glutathione in human serum; the application of DAC / MnO2 nanozyme conjugated with antibodies, DNA and other biological probes. Description of the Drawings

[0021] Figure 1 It shows the influence of the amount of DAC added during the preparation of DAC / MnO2 on the peroxidase activity.

[0022] Figure 2 Prepare a comparison chart of DAC / MnO2 for low and high concentrations of DAC addition;

[0023] Figure 3 Effect of reaction conditions on the enzyme-like catalytic activity of DAC / MnO2: (a) pH of the buffer; (b) temperature; (c) concentration of H2O2; (d) concentration of TMB;

[0024] Figure 4 Time absorbance curves of DAC / MnO2 for (a) different concentrations of TMB and (b) H2O2; (c) Michaelis-Menten plots of TMB and (d) H2O2;

[0025] Figure 5 Standard curve for H2O2 detection. Detailed implementation mode

[0026] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] Example 1: A dialdehyde chitosan manganese dioxide nanozyme, and its preparation method includes the following steps:

[0028] (1) Prepare dialdehyde chitosan: Weigh chitosan and dissolve it in an acetate buffer solution with a pH of 4.4 - 4.6 to form a chitosan acetate solution with a concentration of 15 mg / mL; Prepare an aqueous sodium periodate solution with a concentration of 0.05 g / mL, then slowly pour the prepared sodium periodate solution into the chitosan solution, stir the reaction mixture in the dark at 37 - 50 °C for 24 - 45 h, then add 800 - 950 mL of absolute ethanol to terminate the reaction for 2 - 3 h and precipitate the crude product. Subsequently, the crude product is dialyzed in a sodium chloride solution (0.2 mol / L) and deionized water for 36 - 48 h to purify the product;

[0029] (2) Prepare DAC / MnO2 nanozyme: Mix manganese acetate with a concentration of 100 mM and a volume of 1 - 1.5 mL and the dialdehyde chitosan prepared in step (1) to form an aqueous solution, and perform the first magnetic stirring for 30 - 45 min. After magnetic stirring at room temperature, add an aqueous sodium hydroxide solution with a concentration of 70 mM and a volume of 4.6 - 5.2 mL, and continue to perform the second intense magnetic stirring at room temperature for 12 - 16 h. Then rinse three times and disperse it in ultrapure water, where the volume of dialdehyde chitosan is 5 mL and the concentration is 0.1 - 1 mg / mL.

[0030] Detection of hydrogen peroxide by DAC / MnO₂ nanozyme: Take DAC / MnO₂ nanozyme, add PBS buffer, hydrogen peroxide and chromogenic substrate. After color development, add sulfuric acid solution to terminate the reaction, and use an ultraviolet spectrophotometer to read the absorbance value at a wavelength of 450 nm. Among them, the concentration of DAC / MnO₂ nanozyme in the reaction system is 0.5 - 2.5 mg / mL; the concentration of hydrogen peroxide is 2 - 10 mg / mL; the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB), and the concentration of the chromogenic substrate is 0.5 - 2.5 mg / mL; the catalytic reaction temperature of DAC / MnO₂ nanozyme is 25 - 60 °C, and the pH of PBS buffer is 5 - 7.

[0031] Example 2: In this example, the preparation method of dialdehyde chitosan (DAC) is the same as that of DAC in Example 1.

[0032] Preparation of DAC / MnO₂ nanozyme: Mix manganese acetate with a concentration of 100 mM and a volume of 1 - 1.5 mL and the dialdehyde chitosan prepared in step (1) to form an aqueous solution and perform the first magnetic stirring for 30 min. After magnetic stirring at room temperature, add an aqueous sodium hydroxide solution with a concentration of 70 mM and a volume of 5 mL and react for 5 min. Continue to perform the second intense magnetic stirring at room temperature for 12 h, and then rinse three times and disperse it into ultrapure water. Among them, three groups of controls were set. In the three groups of control experiments, the volume of dialdehyde chitosan was 5 mL, and the concentrations were (1 mg / mL, 0.5 mg / mL, 0.1 mg / mL) respectively.

[0033] Experiment on the peroxidase activity of nanozyme: Take 10 μL of the nanozyme prepared in the three groups of control experiments, 350 μL of PBS buffer (pH = 6), 20 μL of H₂O₂ (concentration of 10 mM), and 2 μL of TMB (concentration of 2 mM) respectively. After color development for 2 min, add 100 μL of H₂SO₄ (concentration of 2 mol / L) to terminate the reaction, and use an ultraviolet spectrophotometer to read the absorbance value at a wavelength of 450 nm.

[0034] As Figure 1 shown, in the low concentration range, with the increase of dialdehyde chitosan, its peroxidase-like activity gradually increases. The reason may be that low-concentration dialdehyde chitosan can be strongly adsorbed on bulk manganese dioxide in the form of monomers, thus effectively exfoliating manganese dioxide nanosheets. However, high-concentration dialdehyde chitosan aggregates and cannot be strongly adsorbed on bulk manganese dioxide, resulting in ineffective exfoliation of manganese dioxide nanosheets, as Figure 2 shown. Therefore, 1 mg / mL dialdehyde chitosan is selected when preparing the material.

[0035] Example 3: Effects of different pH values on the peroxidase-like catalytic activity of DAC / MnO2: Phosphate buffer (PBS buffer) and acetic acid-sodium acetate buffer were prepared, and the pH values were set to (4, 5, 6, 7, 8). At the same time, other conditions were fixed: the concentration of DAC / MnO2 was 1 mg / mL, the catalytic reaction temperature was 35 °C, the concentration of H2O2 was 10 mM, and the concentration of TMB was 2 mM. The peroxidase-catalyzed oxidation reaction was carried out. During the reaction, 10 μL of DAC / MnO2 solution, 350 μL of PBS buffer, 20 μL of H2O2, and 20 μL of TMB were added. After color development for 2 min, 100 μL of H2SO4 (2 mol / L) was added to terminate the reaction, and the absorbance was measured successively.

[0036] As Figure 3 (a) shows, an appropriate buffer pH value can improve the catalytic activity of the nanozyme. The catalytic activity of DAC / MnO2 is active under weakly acidic conditions and has the maximum catalytic activity at pH = 6. Higher temperatures will cause the peroxidase-like activity to inactivate.

[0037] Effects of different temperatures on the peroxidase-like catalytic activity of DAC / MnO2: The temperatures were designed as (25, 30, 35, 40, 45, 50 °C). The pH value of the PBS buffer was fixed at 6, and the others were the same as in Example 3. The peroxidase-catalyzed oxidation reaction was carried out.

[0038] As Figure 3 (b) shows, with the increase of temperature, the peroxidase-like activity of DAC / MnO2 first increases and then decreases sharply, and the catalytic performance is the best at 35 °C.

[0039] Effects of different hydrogen peroxide concentrations on the peroxidase-like catalytic activity of DAC / MnO2: The hydrogen peroxide concentrations were set as (2, 4, 6, 8, 10 mM). The pH value of the PBS buffer was fixed at 6, and the others were the same as in Example 3. The peroxidase-catalyzed oxidation reaction was carried out.

[0040] As Figure 3 (c) shows, the optimal concentration of H2O2 is 4 mM.

[0041] Effects of different TMB concentrations on the peroxidase-like catalytic activity of DAC / MnO2: The TMB concentrations were set as (0.5, 1, 1.5, 2, 2.5 mM). The pH value of the PBS buffer was fixed at 6, and the others were the same as in Example 3. The peroxidase-catalyzed oxidation reaction was carried out.

[0042] As Figure 3 (d) shows, the optimal concentration of TMB is 1.5 mM.

[0043] Example 4: The concentration of H2O2 was fixed at 1.5 mM, and a series of different concentrations of TMB (0.04 mM - 2 mM) were prepared. During the reaction, 10 μL of DAC / MnO2 (1 mg / mL) solution, 20 μL of H2O2 solution, 20 μL of TMB, and 450 μL of PBS buffer (pH 6) were taken. The reaction was sampled at the same time intervals, and the absorbance was measured at 652 nm by a UV-Vis spectrophotometer.

[0044] Under the condition that other experimental conditions remained unchanged, the concentration of TMB was fixed at 4 mM, and the concentration of H2O2 was increased from 0.004 mM to 0.4 mM in sequence, and the same experimental steps were repeated.

[0045] As Figure 4 (a) shows that when the concentration of H2O2 is fixed, the reaction rate of the DAC / MnO2 peroxidase-like enzyme gradually increases with the increase of the TMB concentration. However, when the TMB concentration increases to a certain extent, the increase rate of the reaction rate slows down and gradually levels off. Secondly, as Figure 4 (b) shows that when the TMB concentration is fixed, the reaction rate of the DAC / MnO2 peroxidase-like enzyme gradually increases with the increase of H2O2. Similarly, when the concentration of H2O2 increases to a certain extent, the reaction rate also begins to gradually slow down and finally levels off. By fitting the data in the curve using the Lineweaver-Bur double reciprocal model, straight lines can be obtained, as Figure 4 (c) and 4(d) show.

[0046] The kinetic parameters Km of DAC / MnO2 oxidizing TMB and H2O2 were calculated from the intercept and slope on the straight line, which were 0.0776 mmol / L and 7.722 μmol / L respectively. The Km value is one of the characteristic constants of the enzyme, and it is only related to the nature of the enzyme. Generally, different enzymes have different Km values. The Km value represents the affinity between the enzyme and the substrate. The larger the Km value, the smaller the affinity between the enzyme and the substrate, and the smaller the Km value, the greater the affinity. Compared with the Km of natural horseradish peroxidase (the Km for oxidizing TMB is 0.434), these results indicate that the DAC / MnO2 material can be used for the study of artificial peroxidase and has significant enzyme activity.

[0047] Example 5: 350 μL of PBS buffer (pH = 6), 20 μL of TMB solution (2 mM), and 20 μL of H2O2 with different concentrations increasing sequentially were added to 10 μL of 1 mg / mL DAC / MnO2 solution. After 2 min of color development, 10 μL of H2SO4 (2 mol / L) was added to terminate the reaction. The resulting solution was measured for its absorption spectrum at 450 nm using a UV-visible spectrophotometer.

[0048] As Figure 5 shown, the absorbance at 450 nm increased with the H2O2 concentration from 5 μM to 500 μM. The absorbance intensity had a good linear relationship with the H2O2 concentration in the range of 4 μM to 400 μM, with a correlation coefficient of 0.997 and a detection limit of 11 μM. The detection limit was determined by LOD = KS0 / S, where K is a numerical factor selected according to the required confidence level, S0 is the standard deviation of blank measurements (n = 11, K = 3), and S is the slope of the calibration curve.

[0049] Table 1 compares the detection of hydrogen peroxide in the prior art with that of the present application

[0050] Material Method Linear range (μmol / L) Detection limit (μmol / L) <![CDATA[MoS2-PPy]]> Colorimetry 50-2000 45.0 <![CDATA[MoS2-rGO]]> Fluorometry 60-700 25.0 DA-AgNPs Colorimetry 30-70 3000 CNP Colorimetry 1-40 20 CoS Colorimetry 50-800 20 <![CDATA[Meso-CeO2 / C]]> Electrochemical method 250-5000 1300 NiS / MMT / GO Colorimetry 10-100 9.73 This method Colorimetry 4-400 11

[0051] According to the results in Table 1, the nanozyme prepared by this method has good sensitivity and a low detection limit in the detection of hydrogen peroxide.

[0052] In summary, the preparation method of the present invention has a simple process, mild reaction conditions, and is convenient and fast; the prepared DAC / MnO2 has excellent catalase-like catalytic activity and oxidase-like catalytic activity, enriching the types of nanozymes; and DAC / MnO2 can be used for the quantitative detection of hydrogen peroxide. The synthesized nanomaterial has good sensitivity and a low detection limit in the detection of hydrogen peroxide, has good selectivity for hydrogen peroxide, and can achieve low-cost, rapid, and simple detection.

[0053] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

Claims

1. A dialdehyde chitosan manganese dioxide nanozyme, characterized in that, It is prepared from manganese acetate and dialdehyde chitosan; A preparation method of dialdehyde chitosan manganese dioxide nanozyme, comprising the following steps: (1) Preparation of dialdehyde chitosan: Weigh chitosan and dissolve it in acetate buffer solution to prepare a chitosan acetate solution. Prepare an aqueous sodium periodate solution, and then slowly pour the prepared sodium periodate solution into the chitosan solution. Stir the reaction mixture in the dark at 37-50 °C, then add anhydrous ethanol to terminate the reaction and precipitate the crude product. Subsequently, the crude product is dialyzed in sodium chloride solution and deionized water in sequence to purify the product; (2) Preparation of DAC / MnO2 nanozyme: Prepare an aqueous solution of manganese acetate and the dialdehyde chitosan prepared in step (1) for the first magnetic stirring and mixing. After magnetic stirring at room temperature, add an aqueous sodium hydroxide solution, and continue the second intense magnetic stirring at room temperature. Then rinse three times and disperse it in ultrapure water; In step (1), the pH of the acetate buffer solution is 4.4-4.6, and the dark stirring time is 24-45 h; In step (2), the first magnetic stirring time is 30-45 min, and the second magnetic stirring time is 12-16 h.

2. A dialdehyde chitosan manganese dioxide nanozyme according to claim 1, characterized in that, In step (1), the concentration of the prepared chitosan acetate solution is 15 mg / mL; the concentration of the prepared aqueous sodium periodate solution is 0.05 g / mL; the volume of anhydrous ethanol is 800-950 mL, the reaction termination time is 2-3 h; the dialysis time is 36-48 h.

3. A dialdehyde chitosan manganese dioxide nanozyme according to claim 1, characterized in that, In step (2), the concentration of the manganese acetate is 100 mM, and the volume is 1-1.5 mL; the volume of dialdehyde chitosan is 5 mL, and the concentration is 0.1-1 mg / mL; the concentration of sodium hydroxide is 70 mM, and the volume is 4.6-5.2 mL.

4. Use of the dialdehyde chitosan manganese dioxide nanozyme according to claim 1, characterized in that, Detect hydrogen peroxide with the prepared DAC / MnO2 nanozyme: Take the DAC / MnO2 nanozyme, add PBS buffer solution, hydrogen peroxide and a chromogenic substrate. After color development, add sulfuric acid solution to terminate the reaction, and use an ultraviolet spectrophotometer to read the absorbance value at a wavelength of 450 nm.

5. The application of the dialdehyde chitosan manganese dioxide nanozyme according to claim 4, wherein The concentration of the DAC / MnO2 nanozyme is 0.5-2.5 mg / mL; the concentration of the hydrogen peroxide is 2-10 mg / mL; the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, and the concentration of the chromogenic substrate is 0.5-2.5 mg / mL.

6. The application of the dialdehyde chitosan manganese dioxide nanozyme according to claim 4, characterized in that, The catalytic reaction temperature of the DAC / MnO2 nanozyme is 25-60 °C, and the pH of the PBS buffer solution is 5-7.

7. Use of the dialdehyde chitosan manganese dioxide nanozyme according to claim 1, characterized in that, The DAC / MnO2 nanozyme is applied to the quantitative measurement of glucose and ascorbic acid in blood.

8. Use of the dialdehyde chitosan manganese dioxide nanozyme according to claim 1, characterized in that, The DAC / MnO2 nanozyme is applied to the quantitative measurement of glutathione in human serum.

9. Use of the dialdehyde chitosan manganese dioxide nanozyme according to claim 1, characterized in that, The DAC / MnO2 nanozyme is coupled with antibodies and DNA bioprobes and applied in the fields of electrochemistry and biosensors.

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