Aluminum-doped Prussian blue nano-enzyme, preparation method thereof and GSH content detection method
By developing aluminum-doped Prussian blue nanoenzyme, the problems of poor stability and high cost of horseradish peroxidase in the existing technology are solved, and the rapid, accurate and low-cost detection of glutathione is achieved, and there are wide application prospects.
Patent Information
- Application Number
- CN202510061889.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-05-06
AI Technical Summary
When existing horseradish peroxidase is used for glutathione detection, it has poor stability and high cost, resulting in inaccurate detection results and excessive cost, making it difficult to meet the needs of on-site detection.
An aluminum-doped Prussian blue nanoenzyme has high peroxidase activity that can catalyze colorless TMB to produce blue ox-TMB and is reduced to colorless TMB in the presence of glutathione, thereby achieving detection of glutathione. The nanoenzyme is synthesized by hydrothermal method and has high catalytic activity and stability.
Fast, accurate and low-cost detection of glutathione is achieved, reducing detection costs, improving detection sensitivity and specificity, and can be used to assist in the diagnosis of related diseases and assessing antioxidant properties of food and environmental pollution.
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Figure CN119929841A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of synthesis of doped nanomaterials mimicking enzymes. More specifically, the present invention relates to an aluminum-doped Prussian blue nanozyme and a preparation method thereof and a GSH content detection method. Background Art
[0002] Glutathione (GSH) is a thiol-containing tripeptide composed of glutamic acid, cysteine and glycine, which has the effects of anti-aging, anti-oxidation and maintaining normal immune function. Under physiological and biochemical conditions, abnormal glutathione levels can lead to the occurrence of related diseases such as Alzheimer's disease, cancer, cardiovascular disease, etc. Glutathione-derived vegetables and fruits exist in various plant-based foods as a well-known antioxidant and dietary supplement, which has attracted widespread attention. So far, a variety of methods for detecting GSH have been established, such as high-performance liquid chromatography, electrochemical method, enzymatic method, colorimetric method, etc. Among them, the colorimetric sensing platform has attracted much attention due to its simplicity, low cost and convenience, and is widely used in the detection of proteins, viruses, pathogenic microorganisms, heavy metal ions, etc. For example, when detecting the concentration of glutathione, the property of glutathione that it can interact with the HRP-H2O2-chromogenic substrate reaction system is used. First, glutathione acts as a reducing agent to reduce the substrate. Secondly, glutathione can compete with hydrogen peroxide for the active site of HRP, thereby inhibiting the catalytic effect of HRP on the oxidation reaction of the substrate. By detecting the degree of inhibition of the substrate oxidation reaction, the concentration of glutathione can be indirectly determined. However, when using horseradish peroxidase to detect glutathione, on the one hand, the stability of horseradish peroxidase will affect the test results, and on the other hand, the high price of horseradish peroxidase will undoubtedly increase the detection cost of glutathione. In order to meet the requirements of simple, rapid, accurate and low-cost on-site detection of glutathione, it is of great significance to develop a nanozyme that can replace horseradish peroxidase. Summary of the invention
[0003] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.
[0004] Another object of the present invention is to provide an aluminum-doped Prussian blue nanozyme with high peroxidase-like activity, which can catalyze colorless TMB to generate blue ox-TMB. When glutathione is present, ox-TMB can be reduced to colorless TMB. The nanozyme can be used to detect glutathione.
[0005] In order to achieve these purposes and other advantages according to the present invention, a method for preparing an aluminum-doped Prussian blue nanozyme with high peroxidase-like activity is provided, comprising the following steps: 1) Stir and mix polyvinyl pyrrolidone, aluminum nitrate, potassium ferrocyanide and hydrochloric acid to obtain a mixed solution; 2) The mixed solution is heated for reaction, centrifuged, washed with ultrapure water, and dried to obtain the aluminum-doped Prussian blue nanozyme with high peroxidase-like activity.
[0006] Preferably, the mass ratio of polyvinyl pyrrolidone, aluminum nitrate, potassium ferrocyanide and brine is 10-100:0.5-5:10-50:100-500.
[0007] Preferably, the heating reaction temperature is 50-100°C and the reaction time is 20-30 h.
[0008] Preferably, the drying temperature is 40-50°C.
[0009] Preferably, it is prepared by the method for preparing aluminum-doped Prussian blue nanozyme with high peroxidase-like activity.
[0010] The method for detecting glutathione comprises the following steps: Step 1, adding TMB solution to disodium hydrogen phosphate-citrate buffer, the nanozyme, H2O2 and the test solution, incubating at 30-40°C for 5-30 min to obtain an incubation product; Step 2: Draw a standard curve based on the spectrum-glutathione concentration relationship or the RGB value-glutathione concentration relationship, measure the absorption spectrum or RGB value of the incubation product, and calculate the glutathione concentration based on the absorption spectrum or RGB value through the corresponding standard curve.
[0011] Preferably, the incubation product is dropped onto the paper substrate with the hydrophobic barrier printed thereon, and then photographed using a handheld device, and the RGB value of the incubation product at the paper substrate position is read using the color recognition software in the handheld device. The handheld device may be a smart phone or a tablet.
[0012] The present invention has at least the following beneficial effects: First, the aluminum-doped Prussian blue nanozyme (AlPB) prepared by the present invention has high peroxidase activity, and its active center can react with hydrogen peroxide (H2O2) to produce an intermediate with strong oxidizing properties (highly similar to the high-valent active oxygen species formed in the catalytic process of natural peroxidase). This intermediate can undergo a redox reaction with tetramethylbenzidine (TMB) to oxidize the colorless TMB into blue oxidized TMB (ox-TMB). In this process, the AlPB nanozyme plays a role in catalyzing and accelerating the reaction, reducing the activation energy of the reaction, so that TMB is oxidized and colored under relatively mild conditions. Glutathione (GSH) is a strong reducing agent, and the sulfhydryl group (-SH) in its molecular structure has high reactivity. In the detection system, when GSH is present, it will preferentially react with the intermediate in the AlPB-H2O2 reaction system and reduce it, thereby preventing TMB from being oxidized to ox-TMB. Because the reaction rate of GSH with the intermediate product is much higher than the reaction rate of TMB with the intermediate product, GSH produces a competitive inhibitory effect on the oxidation reaction of TMB. In addition, due to the reduction effect of GSH, ox-TMB will also be reduced to TMB. As the concentration of GSH increases, more intermediate products are consumed by GSH, the degree of TMB oxidation decreases, and the color of the reaction system gradually becomes lighter. There is a quantitative relationship between this color change and the concentration of GSH, which serves as the basis for colorimetric detection of GSH and realizes the colorimetric detection of GSH. Second, the present invention can be used to assist in the detection of diseases related to abnormal glutathione levels, such as Alzheimer's disease, cancer, cardiovascular disease, etc. By quickly and accurately detecting the glutathione content in biological samples (such as blood, tissue fluid, etc.), it provides an important reference for early diagnosis of diseases, disease monitoring and treatment effect evaluation. In biomedical research, it helps to gain a deeper understanding of the intracellular redox state, antioxidant defense mechanism, and the effect of drugs on glutathione metabolism. Third, the present invention can be used to detect the glutathione content in food and evaluate the antioxidant properties and quality of food. For example, detect changes in glutathione in fruits, vegetables and other agricultural products during picking, storage and processing to determine their freshness and nutritional value; monitor the effects of food additives, preservatives, etc. on glutathione levels in food to ensure food safety. Fourth, the present invention can be used to evaluate the effects of environmental pollutants (such as heavy metals, organic pollutants, etc.) on glutathione levels in organisms, and as a biomarker to reflect the degree of environmental pollution and biological toxicity effects. In toxicological research, it helps researchers understand the interference mechanism of toxins on the antioxidant system of organisms, and provides data support for the formulation of environmental quality standards and toxic risk assessments. Fifth, the present invention successfully synthesized AlPB nanozymes with high peroxidase-like activity using a hydrothermal method.The nanozyme prepared can be used to construct a colorimetric sensor array based on the competitive inhibition of glutathione on the nanozyme-TMB reaction system. Under the multi-hole parallel acquisition strategy, the RGB value of the colorimetric image is adaptively and accurately read, and the sensor array realizes the colorimetric-smartphone dual mode for quantitative analysis of glutathione in biological samples and food samples. Sixth, the method of the present invention is simple, fast, and low-cost, and provides a new detection method for on-site detection of various glutathiones.
[0013] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 A flow chart of portable detection of glutathione content provided by the present invention; Figure 2 Characterization results of AlPB; A: scanning electron microscopy; B: transmission electron microscopy; CX-ray diffraction pattern; D: Fourier transform infrared spectrum; Figure 3 It is the result diagram of enzyme activity determination and feasibility analysis of AlPB; Figure 4 This is the result of enzyme kinetic investigation of AlPB; Figure 5 This is the result diagram of condition optimization of the test system; Figure 6 is the absorbance standard curve for glutathione detection; Figure 7 RGB standard curve for mobile phone photography of glutathione detection; Figure 8 This is a graph showing the interference test results for common compounds. DETAILED DESCRIPTION
[0015] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.
[0016] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.
[0017] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.
[0018] 1. Synthesis and characterization of nanozyme (AlPB) Polyvinylpyrrolidone PVP (5.0 g), aluminum nitrate Al (NO3)3 (0.098 g), potassium ferrocyanide K3[Fe(CN)6] (0.395 g) and 1 M hydrochloric acid (40 mL) were mixed under magnetic stirring to obtain a clear solution. Subsequently, the obtained solution was transferred to a Teflon-lined stainless steel autoclave and reacted in an oven at 80 °C for 24 h. Then centrifuged, the precipitate was washed three times with ultrapure water to obtain the product, and dried at 40 °C to obtain aluminum-doped Prussian blue nanozyme.
[0019] Scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared were used for observation. The results are as follows Figure 2 shown.
[0020] 2. Enzyme activity determination and feasibility test (1) Add 10 μg / mL AlPB solution (15 μL), 50 mM H2O2 (40 μL), and 10 mM TMB (35 μL) to sodium dihydrogen phosphate-citrate buffer (240 μL), respectively, make up to 400 μL with water, incubate at 30 °C for 15 min, and scan the absorption spectrum using a UV-visible spectrophotometer.
[0021] (2) To sodium hydrogen phosphate-citrate buffer (240 μL), 10 μg / mL AlPB solution (15 μL), 50 mM H2O2 (40 μL), 10 mM TMB (35 μL), and 20 μL glutathione solution (final concentration 5 μM) were added, respectively, and the volume was made up to 400 μL with water. The solution was incubated at 30 °C for 15 min, and the absorption spectrum was scanned using a UV-visible spectrophotometer.
[0022] (3) Add 50 mM H2O2 (40 μL) and 10 mM TMB (35 μL) to sodium hydrogen phosphate-citrate buffer (240 μL), make up to 400 μL with water, incubate at 30 °C for 15 min, and scan the absorption spectrum using a UV-visible spectrophotometer.
[0023] (4) Add 50 mM H2O2 (40 μL), 10 mM TMB (35 μL), and 20 μL of glutathione solution (final concentration: 5 μM) to disodium hydrogen phosphate-citrate buffer (240 μL), make up to 400 μL with water, incubate at 30 °C for 15 min, and scan the absorption spectrum using a UV-visible spectrophotometer.
[0024] (5) Add 10 μg / mL AlPB solution (15 μL), 10 mM TMB (35 μL), and 20 μL glutathione solution (final concentration 5 μM) to disodium hydrogen phosphate-citrate buffer (240 μL), respectively, make up to 400 μL with water, incubate at 30 °C for 15 min, and scan the absorption spectrum using a UV-visible spectrophotometer.
[0025] (6) Add 10 mM TMB (35 μL) and 20 μL of glutathione solution (final concentration of 5 μM) to sodium hydrogen phosphate-citrate buffer (240 μL), make up to 400 μL with water, incubate at 30 °C for 15 min, and scan the absorption spectrum using a UV-visible spectrophotometer.
[0026] The results are as follows Figure 3 As shown. When only TMB+H2O2, TMB+GSH, TMB+H2O2+GSH, TMB+GSH+AlPB exist in the system, the absorption peak at 652nm is very weak. After adding AlPB, TMB+H2O2+AlPB can be observed to have a typical absorption peak of oxidized TMB at 652nm, indicating that when AlPB or H2O2 exists alone in the solution, TMB cannot be oxidized, and other substances in the solution will not affect the absorbance of TMB. When AlPB and H2O2 exist at the same time, they can catalyze the oxidation of TMB to generate a blue product. It is proved that AlPB can catalyze the oxidation of TMB by H2O2. When GSH is added to the solution, the absorbance at 652 nm decreases, indicating that GSH can reduce ox-TMB to TMB.
[0027] 3. Steady-state kinetics of nanozymes (1) To 240 μL of sodium dihydrogen phosphate-citrate buffer (pH 5.2), different volumes (0-35 μL) of 10 mM TMB solution, 20 μL of AlPB (20 μg / mL), and 40 μL of H2O2 (50 mM) were added, and the total volume was made up to 400 μL. The mixture was incubated at 30 °C for 15 min, and the absorption spectrum was scanned using a UV-visible spectrophotometer.
[0028] (2) Different volumes (0 to 40 μL) of 50 mM hydrogen peroxide solution, 10 mM TMB (35 μL), and 20 μL AlPB (20 μg / mL) were added to 240 μL of sodium dihydrogen phosphate-citrate buffer (pH 5.2). The total volume was filled to 400 μL and incubated at 30 °C for 15 min. The absorption spectrum was scanned using a UV-visible spectrophotometer.
[0029] The initial absorption of TMB and hydrogen peroxide at different concentrations at 652 nm was recorded by UV-visible spectrometer. Figure 4 A and 4B.
[0030] The kinetic parameters were calculated according to the Michaelis-Menten equation (1) and the Lineweaver-Burk plot equation (2): V = V max × [C] / (K m +[C]) (1) 1 / V = (K m / V max ) (1 / [C]) + (1 / V max ) (2) Where V is the initial velocity, V max is the maximum reaction rate, [C] is the concentration of substrate (TMB or H2O2), K m is the Michaelis constant.
[0031] The Michaelis-Menten equation and Lineweaver-Burk equation were used to calculate the kinetic parameters and gain a deeper understanding of the catalytic properties of the nanozyme, such as the maximum reaction rate (Vmax), Michaelis constant (Km), etc., to provide a basis for optimizing the detection conditions and understanding the reaction mechanism. Figure 4 From the results of C and 4D, it can be seen that the nanozyme prepared by the present invention has good catalytic activity and stability.
[0032] 4. Experimental Condition Optimization Test (1) 35 μL of TMB solution (10 mM), 20 μL of AlPB (20 μg / mL) and 40 μL of H2O2 (50 mM) were added to 240 μL of sodium dihydrogen phosphate-citrate buffer with different pH values (2.2-7.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for 15 min and the absorption spectrum was scanned using a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 5 As shown in A.
[0033] (2) 10 mM TMB solution (10 mM), 20 μL AlPB (20 μg / mL) and 40 μL H2O2 (50 mM) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2) respectively, and the total volume was made up to 400 μL. The mixture was incubated at 30°C for different time periods (0-30 min) and the absorption spectrum was scanned using a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 5 As shown in B.
[0034] (3) 10 mM TMB solution (10 mM), 20 μL AlPB (20 μg / mL) and 40 μL H2O2 (50 mM) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2) respectively, and the total volume was made up to 400 μL. The mixture was incubated at different temperatures (25-45°C) for different times of 7 min, and the absorption spectrum was scanned with a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 5 As shown in C.
[0035] (4) 10 mM, different volumes of TMB solution (10 mM), 20 μL AlPB (20 μg / mL) and 40 μL H2O2 (50 mM) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for different times of 7 min, and the absorption spectrum was scanned using a UV-visible spectrophotometer. The results are shown in Figure 4. Figure 5 As shown in D.
[0036] (5) 10 mM, 35 μL TMB solution (10 mM), 20 μL AlPB (20 μg / mL) and different volumes of H2O2 (50 mM) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for different time periods of 7 min, and the absorption spectrum was scanned using a UV-visible spectrophotometer. The results are shown in Figure 5. Figure 5 As shown in E.
[0037] (6) 10 mM, 35 μL TMB solution (10 mM), different volumes of AlPB (20 μg / mL) and 40 μL H2O2 (50 mM) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for different times of 7 min and the absorption spectrum was scanned using a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 5 As shown in F.
[0038] When the amount of H2O2 is 40 μL and the amount of AlPB is 20 μL, when the color intensity of ox-TMB meets the detection requirements, the absorbance range is between 0.2 and 0.8, and the absorbance meets the requirements. And as the amount increases, the absorbance increases, and the error also increases. Considering the cost, sensitivity and reducing the absorbance transmittance error, the present invention selects a 40 μL H2O2 solution of 50 mM. Therefore, the optimal reaction conditions are pH 5.2, 35 μL of TMB solution (10 mM), 20 μL AlPB (20 μg / mL) and 40 μL H2O2 (50 mM), replenishing water to a total volume of 400 μL, and incubating at 30 ° C for 7 minutes. By optimizing the reaction conditions, the detection system achieves the best response difference, thereby improving the accuracy and sensitivity of the detection.
[0039] V. Methodological Investigation Test (i) 10 mM, 35 μL TMB solution (10 mM), 20 μL AlPB (20 μg / mL), 40 μL H2O2 (50 mM) and different concentrations of glutathione were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for different time periods of 7 min, and the absorbance at 652 nm was measured using a UV-visible spectrophotometer. The results are shown in Figure 1. Figure 6 shown.
[0040] (ii) 10 mM, 35 μL TMB solution (10 mM), 20 μL AlPB (20 μg / mL), 40 μL H2O2 (50 mM) and different concentrations of glutathione were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for different times for 7 min, and then dropped on the paper substrate with a hydrophobic barrier printed (prepared by the method provided by LIX, TIAN J, GARNIER G, et al. Fabrication of paper-based microfluidicsensorsby printing). A mobile phone was used to take a photo and extract the RGB value of the system. For example, the software Quick Viewer on the mobile phone APP, an Android application developed based on Java language, uses the OpenCV library to adaptively extract RGB values from the colorimetric image. The application can directly use the camera function to obtain the colorimetric image. The RGB value is obtained and output by colorimetric image analysis. In this detection method, after the incubation mixture changes color on the paper substrate printed with the hydrophobic barrier, a smartphone takes a picture of it through the App "Quick Viewer". Specifically, first, the app converts the color image (including RGB channels) into a grayscale image (displaying a single brightness channel from 0 to 255). Subsequently, a binary segmentation threshold is performed on the image according to the OTSU method using the foreground (all pixels with grayscale values greater than or equal to the threshold) and background (all pixels with grayscale values less than the threshold) with the maximum inter-class variance, resulting in a binary image that only presents two colors, black and white, 0 or 255. To further optimize the image, OpeningMorphology was applied to eliminate small miscellaneous points. Then, the OTSU The method identifies and isolates the contours of the foreground image. Subsequently, it determines the radius and center position of the smallest enclosing circle of these contour lines. After that, the application selects circles with suitable radius to obtain ROIs (i.e., the areas where the incubation mixture produces color changes on the paper substrate), and then the positions of the selected circles are sorted row by row. Within the ROI, RGB values are extracted based on the color information of the image pixels. Since the depth of color is related to the GSH concentration, and the RGB value can quantify the color information, a standard curve of RGB value and GSH concentration is established (such as Figure 7 ), the concentration of GSH can be quantitatively detected based on the RGB values obtained by the smartphone through this standard curve.
[0041] (III) The effects of common compounds in biological samples (glycine, tryptophan, threonine, valine, leucine, isoamino acids, phenylalanine, alanine, arginine, serine, citrulline, glutamine, proline, histidine, proline, lysine, methionine, aspartic acid, glutamic acid, cystine, and cysteine) on the colorimetric response were investigated. Interference solutions were added to the reaction system, incubated and detected under the same conditions, and the selectivity of the sensor was evaluated to ensure accurate detection of glutathione in complex biological samples. 10 mM, 35 μL TMB solution (10 mM), 20 μL AlPB (20 μg / mL), 40 μL H2O2 (50 mM) and 20 μL of the above compounds (final concentration 5 μM) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled to 400 μL. The mixture was incubated at 30 °C for 7 min at different times, and the absorbance at 652 nm was measured using a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 8 shown.
[0042] (IV) After 50 g of garlic was squeezed into juice with 50 mL of water, the garlic juice was diluted with sodium dihydrogen phosphate-citric acid buffer at a ratio of 1:500. 10 mM, 35 μL of TMB solution (10 mM), 20 μL of AlPB (20 μg / mL), 40 μL of H2O2 (50 mM), 20 μL of the above diluted garlic juice and different concentrations of glutathione (final concentrations of 15 μM, 25 μM, and 35 μM, respectively) were added to 240 uL of sodium dihydrogen phosphate-citric acid buffer (pH 5.2), and the total volume was filled with water to 400 μL. The mixture was incubated at 30 °C for different times of 7 min, and the absorbance at 652 nm was measured with a UV-visible spectrophotometer. The recovery rate ranged from 93.6% to 108.1%, and the RSD ranged from 0.7% to 4.1%. This indicates that the method of the present invention is not easily interfered by interfering substances and has high reliability.
[0043] (V) Healthy human serum was diluted at a ratio of 1:100. 10 mM, 35 μL TMB solution (10 mM), 20 μL AlPB (20 μg / mL), 40 μL H2O2 (50 mM), 20 μL of the above diluted serum and different concentrations of glutathione (final concentrations of 15 μM, 25 μM, and 35 μM, respectively) were added to 240 μL sodium dihydrogen phosphate-citrate buffer (pH 5.2), and the total volume was filled with water to 400 μL. The samples were incubated at 30 °C for 7 min at different times, and the absorbance at 652 nm was measured by UV-visible spectrophotometer. The recovery rate ranged from 93.3% to 115.9%, and the RSD ranged from 1.56% to 6.56%. This indicates that the method of the present invention is not easily interfered by interfering substances and has high reliability.
[0044] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.
Claims
1. A method for preparing aluminum-doped Prussian blue nanozyme with high peroxidase-like activity, characterized in that: The following steps are involved: 1) Stir and mix polyvinyl pyrrolidone, aluminum nitrate, potassium ferrocyanide and hydrochloric acid to obtain a mixed solution; 2) The mixed solution of step 1) is heated for reaction, centrifuged, washed with ultrapure water, and dried to obtain the aluminum-doped Prussian blue nanozyme with high peroxidase-like activity.
2. The aluminum-doped Prussian blue nanozyme with high peroxidase-like activity according to claim 1, characterized in that: The mass ratio of polyvinyl pyrrolidone, aluminum nitrate, potassium ferrocyanide and brine is 10-100:0.5-5:10-50:100-500.
3. The aluminum-doped Prussian blue nanozyme with high peroxidase-like activity according to claim 1, characterized in that: The heating reaction temperature is 50-100 ℃ and the reaction time is 20-30 h.
4. The method for preparing aluminum-doped Prussian blue nanozyme according to claim 1, characterized in that: The drying temperature is 40-50 ℃.
5. Aluminum-doped Prussian blue nanozyme with high peroxidase-like activity, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 3.
6. A method for detecting glutathione, characterized in that: The following steps are involved: Step 1, adding TMB solution, the nanozyme according to claim 1, H2O2 and the test solution to disodium hydrogen phosphate-citrate buffer, incubating at 30-40°C for 5-30 min to obtain an incubation product; Step 2: Draw a standard curve based on the spectrum-glutathione concentration relationship or the RGB value-glutathione concentration relationship, measure the absorption spectrum or RGB value of the incubation product, and calculate the glutathione concentration based on the absorption spectrum or RGB value through the corresponding standard curve.
7. The method for detecting glutathione according to claim 6, characterized in that: The incubation product is dropped onto the paper base with the hydrophobic barrier printed on it, and then a handheld device is used to take a photo. The color recognition software in the handheld device is used to read the RGB value of the incubation product at the position of the paper base.