Manganese dioxide nano-enzyme loaded with gold and silver nano-clusters as well as preparation method and application thereof

Abstract

The invention provides manganese dioxide nano-enzyme loaded with gold and silver nano-clusters as well as a preparation method and application of the manganese dioxide nano-enzyme, and relates to the technical field of biomedical detection. The manganese dioxide nano-enzyme loaded with the gold and silver nano-clusters has enzyme activity similar to that of peroxidase. The manganese dioxide nano-enzyme loaded with the gold and silver nano-clusters is combined with glucose oxidase to realize high-sensitivity and selective detection on glucose, wherein the detection linear range is 0.025-0.2 mM, and the detection limit is 0.012 mM. The detection method is simple and convenient to operate, does not need a complicated free radical oxidation process, and avoids color development interference and uncertainty caused by free radical reaction in a general method. Meanwhile, the method does not need complex instruments and equipment, the glucose concentration can be preliminarily judged only through naked eyes, the method is suitable for rapid detection and field application, and the detection efficiency and convenience are greatly improved. Compared with a natural enzyme, the nano-enzyme provided by the invention has better stability and durability, and can keep activity in a wider temperature and pH range.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomedical detection, in particular to a manganese dioxide nanometer enzyme loaded with gold and silver nanometer clusters and a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a global metabolic disease that can cause a variety of serious complications, such as kidney failure, cardiovascular disease and vision loss, and has a great negative impact on the mental health and quality of life of patients. Therefore, the detection and control of blood glucose become a crucial issue. At present, there are various methods for monitoring blood glucose levels, including photometry, colorimetry, electrochemistry and chemiluminescence, etc. Among them, the colorimetric detection method is concerned due to its simplicity, low cost and practicability.

[0003] The mainstream blood glucose monitoring instrument in the market at present is based on electrochemistry, and glucose oxidase / glucose dehydrogenase (GOD / GDH) and a conductive medium are fixed on the electrode surface of the blood glucose test paper. The blood glucose in the blood reacts with the GOD / GDH on the electrode of the reaction area of the test paper to generate electrons, and the electrons are transmitted to the electrode by the conductive medium. Under the driving of a certain voltage, the current change flowing through the electrode is linearly related to the glucose concentration, and then the content of glucose in the blood is calculated. This method has fast detection speed, small blood volume, and the instrument is easy to carry, and can realize bedside instant detection, but the oxygen concentration in the blood sample, pH, endogenous and exogenous drug interference, as well as the temperature, humidity and altitude during testing, and the operation proficiency of the operator will affect the measurement result.

[0004] The colorimetric method is to use glucose to react with certain reagents to produce colored products, and the glucose content is determined by measuring the absorbance of the products. Common colorimetric reagents include phenol red and bromocresol green. This method is relatively simple to operate, but the sensitivity and specificity are relatively low, and there are many interference factors. It is generally used for qualitative or semi-quantitative detection of glucose in some systems with low precision requirements, such as preliminary detection of glucose content in honey, fruit juice and other food in the food industry.

[0005] The titration method is to add a standard solution with a known concentration drop by drop until the glucose in the solution is completely consumed by the reactant in the titrant, so as to determine the content of glucose. Common titrants include sodium hydroxide solution and copper sulfate solution. This method is a classic chemical analysis method, which is suitable for the determination of glucose content in some simple systems, but the operation is relatively complicated, time-consuming, and there may be interference for complex samples. The accuracy is not as good as enzyme method and electrochemistry, and the application in clinical and scientific research is gradually reduced.

[0006] SERS and near-infrared spectroscopy are also included in the spectral analysis method. Li Yang's team used a new type of silver nanosheet prepared by scratching the silver plate with a nano-structured surface to scratch the smooth silver sheet. The capture of glucose molecules and SERS signal response is particularly sensitive, with a detection limit as low as 0.5 amole per liter, and can show the structure and composition of glucose. It is expected to be used for the development of wearable and portable detection equipment to monitor glucose SERS signals at any time through blood, urine, sweat, tears, etc. However, this technology is still in the research stage and has not been widely used in clinical practice. Many biological tissues have unique light absorption in the near-infrared region, and a small part of the reflected or absorbed infrared rays representing blood glucose characteristics can be used for blood glucose value determination. However, due to the interference of organic molecules, it is difficult to obtain blood glucose information, and the test results need to be corrected with minimally invasive test results. The physiological conditions of different human bodies also affect light wave absorption, and the current monitoring results are not ideal. However, with the advancement of technology, there is certain development potential.

[0007] Enzymatic method is the most commonly used method for glucose detection, which can be further divided into glucose oxidase method and hexokinase method. Glucose oxidase catalyzes the oxidation of glucose to release hydrogen peroxide, which is condensed with a chromogenic oxygen acceptor to form a blue or red compound under the catalysis of peroxidase, and its absorbance value is proportional to the amount of glucose. This method meets the clinical requirements in terms of accuracy and precision, and is simple to operate. It is a routine test method for blood glucose determination and can also be used for cerebrospinal fluid glucose concentration determination. Hexokinase method refers to the reaction of glucose with adenosine triphosphate (ATP) in the presence of hexokinase and magnesium ions. The product, glucose-6-phosphate, is reduced to NADPH by glucose-6-phosphate dehydrogenase under the catalysis of glucose-6-phosphate dehydrogenase. The glucose content is determined by measuring the amount of NADPH generated at 340 nm. This method is recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) as the reference method. The results are not affected by factors such as mild hemolysis, jaundice, and heparin. It can be used for urine glucose quantitative detection, but the operation is relatively complex and the experimental conditions are relatively high. +

[0008] Nanoparticle enzyme detection of glucose is a new detection technology with unique advantages and characteristics compared to traditional enzymes. The detection principle is to use the enzyme-like catalytic activity of nanoparticle enzymes to simulate biological enzymes for specific recognition and catalytic reaction of glucose. For example, some nanomaterials have glucose oxidase-like activity and can directly catalyze the oxidation of glucose to generate corresponding products; some nanomaterials have peroxidase-like activity and can further catalyze the reaction of hydrogen peroxide with specific substrates to generate color or other detectable reactions based on the catalysis of glucose oxidase to generate hydrogen peroxide from glucose. The amount of reaction product is used to determine the content of glucose.

[0009] ​Common nanoscale enzyme materials include metal nanomaterials, such as gold nanoclusters / palladium nanocube heterostructures (AuNCs / PdNCs), which have glucose oxidase-like activity and can be used to construct self-powered biosensors for detecting glucose. As an anode catalyst, combined with a cathode catalyst, it can convert the chemical energy generated by the oxidation of glucose into electrical energy, and through the detection of current signals, it can achieve high sensitivity, high selectivity, and long-term stability for detecting glucose; metal oxide nanomaterials, such as ferroferric oxide nanoparticles (Fe3O4 NPs) with peroxidase-like activity, which can catalyze the generation of hydrogen peroxide from glucose in detection, and Fe3O4 NPs can catalyze the oxidation of substrate 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue products, and the absorbance or color change of the blue products can be used to quantify glucose; other nanomaterials, such as tannic acid-modified gold nanoflowers (TA@AuNFs), which have both peroxidase-like and glucose oxidase activities, can catalyze the oxidation of glucose to generate a colorimetric product, enabling visual detection of serum glucose.

[0010] The nanoscale enzyme glucose detection technology has the following advantages:

[0011] (1) Low cost: The preparation of nanoscale enzymes is relatively simple and can be mass-produced, which significantly reduces the cost compared to natural enzymes, making it suitable for widespread application; good stability: nanoscale enzymes are not easily affected by environmental factors such as temperature and pH, and can maintain good catalytic activity and stability under different conditions, allowing for long-term storage and use. For example, Fe3O4 NPs can maintain high catalytic efficiency after being recycled 5 times and stored at room temperature for 11 days.

[0012] (2) Simple operation: The glucose detection method based on nanoscale enzymes usually has a simple operation process, such as nanoscale enzyme colorimetric method, which does not require complex instruments and professional operation skills, making it easy to promote.

[0013] (3) Rapid detection: It can complete the detection in a short time, such as the nanoscale enzyme colorimetric method based on the smart phone sensing platform, which can complete the quantitative detection of glucose within 30 minutes, meeting the demand for on-site rapid detection.

[0014] The nanoscale enzyme glucose detection technology also has the following limitations:

[0015] (1) Sensitivity needs to be improved: Although some nanoscale enzyme detection methods have high sensitivity, compared with some advanced instrument analysis methods, the overall sensitivity still needs to be improved, and there may be some difficulties in detecting low concentration glucose.

[0016] (2) The selectivity is not ideal: In complex biological samples, other substances may exist that interfere with the reaction between nanozymes and glucose, affecting the accuracy of the detection results. Although some studies have improved the selectivity through optimization methods, further improvements are still needed.

[0017] (3) Standardization issues: The preparation and detection methods of nanozymes have not been fully standardized. The performance of nanozymes prepared by different laboratories may vary, which may affect the comparability and repeatability of the detection results.

[0018] Nanozyme technology for glucose detection shows promising prospects. With the continuous advancement of nanomaterials science and analytical techniques, nanozyme technology for glucose detection is expected to achieve breakthroughs in sensitivity, selectivity, and standardization, providing a more efficient, convenient, and accurate method for glucose detection and playing an important role in clinical diagnosis, food safety testing, and biomedical research.

[0019] However, the natural enzymes used in traditional colorimetric methods have limitations such as poor stability and long reaction times. With the rapid development of nanozyme technology, researchers are dedicated to developing nanomaterials with similar enzyme activities to replace natural enzymes. Among them, manganese dioxide (MnO2) nanozymes have attracted much attention due to their unique properties. They typically possess multiple enzyme activities, including oxidase-like, catalase-like, and peroxidase-like activities, and can undergo a colorimetric reaction with 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of glucose with the assistance of natural enzymes. Furthermore, the colorimetric behavior changes with different glucose concentrations, thus enabling the quantitative detection of glucose.

[0020] Nevertheless, the multiple enzyme activities of manganese dioxide nanozymes also present new challenges. Their complex reaction mechanism involves multiple reaction processes, resulting in TMB exhibiting different colors. This complexity not only makes it difficult to explore the nature of the color changes but also increases the difficulty of precise control and optimization, thereby increasing the complexity of detection and potentially adversely affecting the accuracy and reliability of the results. Summary of the Invention

[0021] To address the technical problems existing in the prior art, this invention provides a manganese dioxide nanozyme loaded with gold and silver nanoclusters, its preparation method, and its application. The technical solution is as follows:

[0022] A manganese dioxide nanozyme loaded with gold and silver nanoclusters, wherein the manganese dioxide nanozyme loaded with gold and silver nanoclusters has peroxidase-like enzyme activity.

[0023] Optionally, the manganese dioxide nanozyme loaded with gold and silver nanoclusters is prepared by the following method:

[0024] Dendritic MnO2 nanorods were synthesized using a hydrothermal method to prepare manganese dioxide nanozymes. The structure of the nanozymes was optimized by calcination, and Au and Ag nanoparticles were grown in situ on the surface of the manganese dioxide nanozymes to prepare the manganese dioxide nanozymes loaded with gold and silver nanoclusters.

[0025] The method for preparing manganese dioxide nanozymes loaded with gold and silver nanoclusters includes the following steps:

[0026] (1) Synthesis of manganese dioxide nanorods: A solution containing KMnO4 and MnSO4 is transferred to a high-pressure reactor and reacted at 100 ℃-150 ℃ to obtain manganese dioxide nanorods, wherein the molar ratio of KMnO4 and MnSO4 is 4:1-8:1, preferably 6:1;

[0027] (2) The manganese dioxide nanorods are calcined at 500-600℃ to obtain a defect-rich MnO2 material;

[0028] (3) Dissolve the MnO2 material in water to obtain a MnO2 solution, wherein the concentration of the MnO2 solution is 0.5-5 mg / mL, preferably 2 mg / mL;

[0029] (4) Add chloroauric acid solution, silver nitrate solution and sodium borohydride solution to the MnO2 solution to obtain the solution of manganese dioxide nanozyme loaded with gold and silver nanoclusters, wherein the molar ratio of chloroauric acid solution, silver nitrate solution, sodium borohydride solution and MnO2 solution is 1:1:10:(0.01-0.1). Centrifuge the solution of manganese dioxide nanozyme loaded with gold and silver nanoclusters to obtain the manganese dioxide nanozyme loaded with gold and silver nanoclusters.

[0030] Optionally, in step (2), the calcination is carried out in a muffle furnace, and the heating parameters are adjusted to make the heating rate 15 °C / min.

[0031] The application of the manganese dioxide nanozyme loaded with gold and silver nanoclusters in glucose detection methods or in the preparation of glucose test strips or test kits.

[0032] A method for detecting glucose, the method comprising the following steps:

[0033] 1) React the solution of the manganese dioxide nanozyme loaded with gold and silver nanoclusters, glucose oxidase, glucose-containing solution, or the sample to be tested with a solution of TMB.

[0034] 2) The change in absorbance of the reaction solution at 652 nm was measured using a UV-Vis absorption spectrometer to achieve quantitative detection of glucose;

[0035] Alternatively, the method may include the following steps:

[0036] i) The filter paper is soaked in a solution containing manganese dioxide nanozyme loaded with gold and silver nanoclusters and glucose oxidase, and then dried to obtain glucose test paper.

[0037] ii) The concentration of glucose in the solution is identified by RGB photography by immersing the glucose test strip in a solution containing glucose or a mixture of the sample to be tested and TMB.

[0038] A glucose test strip, the glucose test strip comprising:

[0039] (1) The manganese dioxide nanozyme loaded with gold and silver nanoclusters; and

[0040] (2) Glucose oxidase.

[0041] Optionally, the glucose test strip is prepared by immersing filter paper in a solution containing manganese dioxide nanozyme loaded with gold and silver nanoclusters and glucose oxidase, and then drying it to obtain the glucose test strip.

[0042] And / or, the mass ratio of the glucose oxidase to the manganese dioxide nanozyme loaded with gold and silver nanoclusters is 1:(4-8), preferably 1:6.

[0043] A glucose detection kit, the kit comprising: the manganese dioxide nanozyme loaded with gold and silver nanoclusters.

[0044] Optionally, the kit may further include: glucose oxidase and / or TMB solution;

[0045] And / or, the manganese dioxide nanozyme loaded with gold and silver nanoclusters is present in the kit in the form of a solution or test strip.

[0046] When the manganese dioxide nanozyme loaded with gold and silver nanoclusters is present in the kit in solution form, the concentration of the solution is 0.05-0.5 mg / mL, preferably 0.2 mg / mL.

[0047] When the test strip is present in the kit, it is a glucose detection test strip.

[0048] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0049] 1. High sensitivity and selectivity: By optimizing the structure of MnO2 and growing Au and Ag nanoparticles in situ, followed by the combination with glucose oxidase, the manganese dioxide nanozyme loaded with gold and silver nanoclusters exhibits more sensitive and specific detection of glucose. The linear detection range of the manganese dioxide nanozyme loaded with gold and silver nanoclusters of this invention is 0.025 to 0.2 mM, and the detection limit is 0.012 mM.

[0050] 2. Simple and efficient: This detection method is easy to operate, requiring no complex free radical oxidation process, thus avoiding colorimetric interference and uncertainties caused by free radical reactions in general methods. Furthermore, this method requires no complex instruments; glucose concentration can be preliminarily determined by visual inspection alone, making it suitable for rapid detection and on-site applications, greatly improving detection efficiency and convenience.

[0051] 3. High Stability: Compared with natural enzymes, the nanozymes of this invention exhibit better stability and durability, maintaining activity over a wider temperature and pH range. In particular, by optimizing the structure of the MnO2 nanozyme and introducing Au and Ag nanoparticles, the detection sensitivity is further enhanced. Furthermore, the nanozyme itself catalyzes the decomposition of H2O2, thereby improving the reliability and repeatability of the detection.

[0052] 4. Low cost: The materials and preparation methods used are relatively economical, reducing the testing cost.

[0053] 5. Environmentally friendly: The detection method of this invention does not involve toxic or harmful reagents and is environmentally friendly.

[0054] The manganese dioxide nanozyme loaded with gold and silver nanoclusters of the present invention differs from the manganese dioxide nanozyme of the prior art in the following aspects:

[0055] (1) The new MnO2-Au / Ag nanomaterials prepared by loading gold and silver nanoclusters on the surface of MnO2 effectively improved its peroxidase-like activity, thereby improving the sensitivity of detection;

[0056] (2) By mixing manganese dioxide nanozymes loaded with gold and silver nanoclusters with peroxidase-like activity with glucose oxidase, glucose can be qualitatively detected.

[0057] (3) Manganese dioxide nanozymes loaded with gold and silver nanoclusters and glucose oxidase can be used for glucose detection. This is also due to the fact that glucose oxidase can react with glucose, which provides a preparation scheme for detection reagents for the detection of other disease biomarkers. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0059] Figure 1 This is a SEM image of MnO2 provided in Example 1 of the present invention, showing that its nanorods have an average diameter of approximately 20-50 nm;

[0060] Figure 2 This is a TEM image of MnO2-Au / Ag provided in Example 1 of the present invention, showing that Au and Ag are deposited on the MnO2 surface with a particle size of 5 nm;

[0061] Figure 3 This is the XRD pattern of MnO2-Au / Ag provided in Embodiment 1 of the present invention. It shows that MnO2@Au-Ag has obvious characteristic peaks at 2θ angles of 37.44°, 38.14° and 44.34°, which correspond to the crystal structures of MnO2 and Au(Ag), respectively.

[0062] Figure 4 This is a selective detection diagram of glucose by manganese dioxide nanozyme loaded with gold and silver nanoclusters provided in Example 1 of the present invention.

[0063] Figure 5 This is a linear calibration graph of the colorimetric detection of glucose by manganese dioxide nanozyme loaded with gold and silver nanoclusters provided in Example 1 of this invention, where R... 2 The value of 0.99 indicates that the absorbance has a good linear relationship with the glucose concentration, and its linear range for glucose detection is 0.025 mM to 0.2 mM, with a detection limit of 0.012 mM.

[0064] Figure 6 These are visualization images of glucose detection using manganese dioxide nanozymes loaded with gold and silver nanoclusters provided in Example 1 of this invention, demonstrating that it can perform rapid colorimetric detection of glucose in the range of 0-12 mM. Detailed Implementation

[0065] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0066] This invention aims to provide a manganese dioxide nanozyme loaded with gold and silver nanoclusters and exhibiting peroxidase-like activity, and to apply it to glucose detection. Currently, nanozymes have significant application potential in the field of glucose detection; however, a pressing issue in this field is how to optimize and adjust the properties of nanozymes to synthesize a nanozyme that can efficiently promote the decomposition of H₂O₂, thereby simplifying the complex free radical generation process.

[0067] Given the multiple enzymatic activities of manganese dioxide nanozymes, researchers have begun exploring other nanomaterials to improve detection performance. Gold (Au) and silver (Ag) nanomaterials have been extensively studied due to their unique physicochemical properties and excellent oxidation performance. They not only possess excellent optical properties but also exhibit certain enzyme-mimicking activities, particularly demonstrating good catalytic effects in redox reactions. Therefore, the inventors believe that Au and Ag nanomaterials can be used to adjust and optimize the properties of manganese dioxide nanozymes, thereby making glucose concentration detection simpler, more efficient, and more accurate.

[0068] Therefore, this invention relies on the characteristic of traditional manganese dioxide nanozymes, which not only possess peroxidase-like activity but also catalyze the decomposition of H2O2. It transforms the process of H2O2 generating free radicals into the decomposition of H2O2, and converts the colorimetric reaction of free radical oxidation of TMB into a reaction of direct oxidation of TMB by MnO2. Through this transformation, a simple, efficient, low-cost, and highly sensitive and selective colorimetric method for glucose detection has been developed.

[0069] The core of this invention is the synthesis of manganese dioxide nanozymes via a hydrothermal method, with optimized structure and performance. Calcination enriches the surface of manganese dioxide with defects, enhancing its catalytic activity. Furthermore, in-situ growth of Au and Ag nanoparticles further enhances its oxidation capacity. This modified manganese dioxide nanozyme exhibits strong peroxidase-like activity and catalytic properties. When the material encounters glucose, glucose is oxidized to H₂O₂ by glucose oxidase. Under the catalysis of the nanozyme, H₂O₂ generates hydroxyl radicals, which efficiently catalyze the oxidation of TMB to the blue oxidation product oxTMB. Based on this unique reaction mechanism, this invention establishes a simple and efficient colorimetric detection method for glucose.

[0070] Example 1

[0071] The specific steps are as follows:

[0072] 1. Synthesis of manganese dioxide nanozymes:

[0073] ① Dissolve 6 mM KMnO4 and 1 mM MnSO4 in 70 mL of deionized water, and transfer the resulting solution to a 100 mL Teflon-lined stainless steel autoclave. Seal the autoclave and place it in an oven at 120°C for 2 hours, then allow it to cool naturally to room temperature. During this process, KMnO4 acts as a strong oxidizing agent, reducing the manganese oxidation state from +7 to +4, generating MnO2 nanorods. Simultaneously, the manganese in MnSO4 is oxidized from +2 to +4, generating manganese dioxide nanorods.

[0074] ②After the reactor cools down to room temperature, collect the product by vacuum filtration, wash it 6 times with deionized water, and dry the product in a 60℃ oven.

[0075] MnO2 was photographed using a scanning electron microscope.

[0076] 2. Adjusting and optimizing the performance of manganese dioxide nanozymes:

[0077] ① The product was poured into a crucible and transferred to a muffle furnace for calcination at 600℃. The heating rate of the muffle furnace was adjusted to 15℃ / min. This resulted in a defect-rich MnO2 material.

[0078] ② Prepare 0.01 M chloroauric acid solution, 0.01 M silver nitrate solution, and 0.1 M sodium borohydride solution respectively. Sodium borohydride must be dissolved in ice water. Simultaneously, dissolve 6 mg of MnO2 material in 3 mL of deionized water and sonicate to ensure uniform dispersion.

[0079] ③ Add 7 mL of chloroauric acid solution, 10 mL of silver nitrate solution, and 4 mL of sodium borohydride solution to the MnO2 solution. Stir on a vortex mixer for 1 min to obtain the MnO2-Au / Ag solution;

[0080] The MnO2-Au / Ag solution was centrifuged at 6000 rpm for 50 min, and the MnO2-Au / Ag precipitate was retained. The sample was then washed with deionized water at 8000 rpm, repeated three times. After each centrifugation, the supernatant was removed, and the precipitate was retained. The precipitate was then lyophilized to obtain the manganese dioxide nanozyme MnO2-Au / Ag loaded with gold and silver nanoclusters.

[0081] Transmission electron microscopy (TEM) and powder X-ray diffraction (XRD) analysis were performed on the manganese dioxide nanozyme MnO2-Au / Ag loaded with gold and silver nanoclusters. The XRD analysis procedure can be found in the paper "A Versatile Sunscreen with Minimal ROS Damage and Low Permeability".

[0082] ④ Mix GOx (glucose oxidase, purchased from Aladdin Reagent (Shanghai) Co., Ltd.) with MnO2-Au / Ag at a mass ratio of 1:6, and add an appropriate amount of deionized water to make the concentration of MnO2-Au / Ag 100 µg / mL.

[0083] ⑤ Place the size 1 1cm 2 The filter paper was soaked in a mixed solution of 20 mg / mL glucose oxidase and MnO2-Au / Ag nanozyme for two minutes, and then dried in an oven at 40 °C to obtain glucose test strips.

[0084] 3. Glucose detection:

[0085] ① Add 150 µL of the mixed solution from step ④ above, and 10 µL of glucose solutions of different concentrations (0, 0.01, 0.025, 0.05, 0.075, 0.10, 0.15, 0.20, and 0.25 mM) to 765 µL of deionized water. Then, add 75 µL of 1 mg / mL TMB solution to each reaction system, react under specific conditions for a period of time, and observe the color intensity of the solution to preliminarily determine the glucose concentration.

[0086] ②Quantitative detection of glucose was achieved by measuring the change in absorbance of the reaction solution at 652 nm using a UV-Vis absorption spectrometer.

[0087] Alternatively, glucose can be detected using the following methods:

[0088] The test strip from step ⑤ above is immersed in a solution containing glucose (or the sample to be tested) and a mixed solution of TMB, and the concentration of glucose in the solution is identified by RGB photography.

[0089] Experimental results:

[0090] 1) Figure 1 This is a SEM image of MnO2. The results are from observations using a scanning electron microscope, that is... Figure 1 As can be seen, MnO2 has a dendritic morphology with a diameter of 20-50 nm;

[0091] 2) Figure 2This is a TEM image of MnO2-Au / Ag, showing Au and Ag deposited on the MnO2 surface with a particle size of 5 nm. This is from a transmission electron microscope image, i.e. Figure 2 It can be seen that distinct spherical particles with a diameter of about 5 nm appeared on the surface of manganese dioxide nanorods, but the overall dendritic structure still exists;

[0092] 3) To investigate the crystal structure of the prepared MnO2-Au / Ag, we performed powder X-ray diffraction (XRD) analysis. The results are as follows: Figure 3 As shown, besides MnO2's 2θ = In addition to the characteristic peaks at 12.75°, 18.04°, 25.62°, 28.66°, 36.48°, 37.46°, 41.88°, 49.70°, 56.16°, 60.14°, 65.13°, 69.44° and 72.72° of α-MnO2, corresponding to the (110), (200), (220), (310), (400), (211), (301), (411), (600), (521), (002), (541) and (321) crystal planes respectively, new diffraction peaks at 2θ=38.11°, 44.30°, 65.14° and 77.50° were observed, corresponding to the (111), (220), (220) and (311) crystal planes of Au / Ag respectively. Therefore, we confirm the successful preparation of MnO2-Au / Ag;

[0093] 4) Figure 4 This is a graph showing the selective detection of glucose by manganese dioxide nanozymes loaded with gold and silver nanoclusters. (Example:) Figure 4 As shown, at a wavelength of 652 nm, the absorbance of the control group was significantly lower than that of the glucose group. Compared with the glucose group, the solution of the control group was almost colorless, while the glucose group showed a distinct blue color. Therefore, the above results clearly demonstrate that the manganese dioxide nanozyme loaded with gold and silver nanoclusters still has excellent specificity and can be used for reliable glucose detection in real samples.

[0094] 5) Figure 5 This is a linear calibration graph of colorimetric detection of glucose by manganese dioxide nanozymes loaded with gold and silver nanoclusters. Figure 5 The results showed that the absorbance of the solution increased within the glucose concentration range of 0.025–0.20 mM, and the color gradually deepened with increasing glucose concentration. The linear regression equation was y = 3.2559x + 0.01768, and the regression coefficient R0 was [value missing]. 2 The limit of concentration (LOC) was 0.9904, and the detection limit (LOD) was 0.012 mM. Therefore, the detection system using manganese dioxide nanozymes loaded with gold and silver nanoclusters can be used for selective and sensitive glucose detection.

[0095] 6)Figure 6 These are visual images of a glucose test strip containing manganese dioxide nanozymes loaded with gold and silver nanoclusters, demonstrating its ability to detect different concentrations of glucose. (Example:) Figure 6 As shown, when the glucose concentration changes within the range of 0-12 mM, the color of the test strip becomes significantly darker, exhibiting a more pronounced blue hue.

[0096] Example 2

[0097] 1. Synthesis of manganese dioxide nanozymes:

[0098] ① Dissolve 4 mM KMnO4 and 1 mM MnSO4 in 70 mL of deionized water, and transfer the resulting solution to a 100 mL Teflon-lined stainless steel autoclave. Seal the autoclave and place it in an oven at 150°C for 2 hours, then allow it to cool naturally to room temperature. During this process, KMnO4 acts as a strong oxidizing agent, reducing the manganese oxidation state from +7 to +4, generating MnO2 nanorods. Simultaneously, the manganese in MnSO4 is oxidized from +2 to +4, generating manganese dioxide nanorods.

[0099] ②After the reactor cools down to room temperature, collect the product by vacuum filtration, wash it 6 times with deionized water, and dry the product in a 60℃ oven.

[0100] MnO2 was photographed using a scanning electron microscope.

[0101] 2. Adjusting and optimizing the performance of manganese dioxide nanozymes:

[0102] ① The product was poured into a crucible and transferred to a muffle furnace for calcination at 500℃. The heating rate of the muffle furnace was adjusted to 15℃ / min. This resulted in a defect-rich MnO2 material.

[0103] ② Prepare 0.01 M chloroauric acid solution, 0.01 M silver nitrate solution, and 0.1 M sodium borohydride solution respectively. Sodium borohydride must be dissolved in ice water. Simultaneously, dissolve 3 mg of MnO2 material in 3 mL of deionized water and sonicate to ensure uniform dispersion.

[0104] ③ Add 7 mL of chloroauric acid solution, 10 mL of silver nitrate solution, and 4 mL of sodium borohydride solution to the MnO2 solution. Stir on a vortex mixer for 1 min to obtain the MnO2-Au / Ag solution;

[0105] The MnO2-Au / Ag solution was centrifuged at 6000 rpm for 50 min, and the MnO2-Au / Ag precipitate was retained. The sample was then washed with deionized water at 8000 rpm, repeated three times. After each centrifugation, the supernatant was removed, and the precipitate was retained. The precipitate was then lyophilized to obtain the manganese dioxide nanozyme MnO2-Au / Ag loaded with gold and silver nanoclusters.

[0106] Transmission electron microscopy (TEM) and powder X-ray diffraction (XRD) analysis were performed on the manganese dioxide nanozyme MnO2-Au / Ag loaded with gold and silver nanoclusters. The XRD analysis procedure can be found in the paper "A Versatile Sunscreen with Minimal ROS Damage and Low Permeability".

[0107] ④ Mix GOx (glucose oxidase, purchased from Aladdin Reagent (Shanghai) Co., Ltd.) with MnO2-Au / Ag at a mass ratio of 1:4, and add an appropriate amount of deionized water to make the concentration of MnO2-Au / Ag 100 µg / mL.

[0108] ⑤ Place the size 1 1cm 2 The filter paper was soaked in a mixed solution of 20 mg / mL glucose oxidase and manganese dioxide nanozyme loaded with gold and silver nanoclusters for two minutes, and then dried in an oven at 40°C to obtain glucose test strips.

[0109] 3. Glucose detection:

[0110] ① Add 150 µL of the mixed solution from step ④ above, and 10 µL of glucose solutions of different concentrations (0, 0.01, 0.025, 0.05, 0.075, 0.10, 0.15, 0.20, and 0.25 mM) to 765 µL of deionized water. Then, add 75 µL of 1 mg / mL TMB solution to each reaction system, react under specific conditions for a period of time, and observe the color intensity of the solution to preliminarily determine the glucose concentration.

[0111] ②Quantitative detection of glucose was achieved by measuring the change in absorbance of the reaction solution at 652 nm using a UV-Vis absorption spectrometer.

[0112] Alternatively, glucose can be detected using the following methods:

[0113] The test strip from step ⑤ above is immersed in a solution containing glucose (or the sample to be tested) and a mixed solution of TMB, and the concentration of glucose in the solution is identified by RGB photography.

[0114] Experimental results:

[0115] The experimental results were the same as in Example 1, and will not be repeated here.

[0116] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A manganese dioxide nanozyme loaded with gold and silver nanoclusters, characterized in that, The manganese dioxide nanozyme loaded with gold and silver nanoclusters has peroxidase-like enzyme activity.

2. The manganese dioxide nanozyme loaded with gold and silver nanoclusters according to claim 1, characterized in that, The manganese dioxide nanozyme loaded with gold and silver nanoclusters was prepared by the following method: Dendritic MnO2 nanorods were synthesized using a hydrothermal method to prepare manganese dioxide nanozymes. The structure of the nanozymes was optimized by calcination, and Au and Ag nanoparticles were grown in situ on the surface of the manganese dioxide nanozymes to prepare the manganese dioxide nanozymes loaded with gold and silver nanoclusters.

3. The method for preparing manganese dioxide nanozymes loaded with gold and silver nanoclusters according to any one of claims 1-2, characterized in that, The method includes the following steps: (1) Synthesis of manganese dioxide nanorods: A solution containing KMnO4 and MnSO4 is transferred to a high-pressure reactor and reacted at 100℃-150℃ to obtain manganese dioxide nanorods, wherein the molar ratio of KMnO4 and MnSO4 is 4:1-8:1, preferably 6:1; (2) The manganese dioxide nanorods are calcined at 500-600℃ to obtain a defect-rich MnO2 material; (3) Dissolve the MnO2 material in water to obtain a MnO2 solution, wherein the concentration of the MnO2 solution is 0.5-5 mg / mL, preferably 2 mg / mL; (4) Add chloroauric acid solution, silver nitrate solution and sodium borohydride solution to the MnO2 solution to obtain the solution of manganese dioxide nanozyme loaded with gold and silver nanoclusters, wherein the molar ratio of chloroauric acid solution, silver nitrate solution, sodium borohydride solution and MnO2 solution is 1:1:10:(0.01-0.1). Centrifuge the solution of manganese dioxide nanozyme loaded with gold and silver nanoclusters to obtain the manganese dioxide nanozyme loaded with gold and silver nanoclusters.

4. The preparation method according to claim 3, characterized in that, In step (2), the calcination is carried out in a muffle furnace, and the heating rate is adjusted to 15 °C / min by adjusting the heating parameters.

5. The application of manganese dioxide nanozymes loaded with gold and silver nanoclusters according to any one of claims 1-2 in methods for detecting glucose or in the preparation of glucose test strips or test kits.

6. A method for detecting glucose, characterized in that, The method includes the following steps: 1) React a solution of manganese dioxide nanozyme loaded with gold and silver nanoclusters according to any one of claims 1-2, glucose oxidase, a solution containing glucose, or the sample to be tested with a solution of TMB. 2) The change in absorbance of the reaction solution at 652 nm was measured using a UV-Vis absorption spectrometer to achieve quantitative detection of glucose; Alternatively, the method may include the following steps: i) Immerse filter paper in a solution containing manganese dioxide nanozyme loaded with gold and silver nanoclusters according to any one of claims 1-2 and glucose oxidase, and then dry it to obtain glucose test paper; ii) The concentration of glucose in the solution is identified by RGB photography by immersing the glucose test strip in a solution containing glucose or a mixture of the sample to be tested and TMB.

7. A glucose test strip, characterized in that, The glucose test strip contains: (1) The manganese dioxide nanozyme loaded with gold and silver nanoclusters according to any one of claims 1-2; and (2) Glucose oxidase.

8. The glucose test strip according to claim 7, characterized in that, The glucose test strip is prepared by the following method: filter paper is soaked in a solution containing manganese dioxide nanozyme loaded with gold and silver nanoclusters according to any one of claims 1-2 and glucose oxidase, and then dried to obtain the glucose test strip. And / or, the mass ratio of the glucose oxidase to the manganese dioxide nanozyme loaded with gold and silver nanoclusters is 1:(4-8), preferably 1:

6.

9. A glucose detection kit, characterized in that, The kit comprises: manganese dioxide nanozymes loaded with gold and silver nanoclusters according to any one of claims 1-2.

10. The glucose detection kit according to claim 9, characterized in that, The kit also includes: glucose oxidase and / or TMB solution; And / or, the manganese dioxide nanozyme loaded with gold and silver nanoclusters is present in the kit in the form of a solution or test strip. When the manganese dioxide nanozyme loaded with gold and silver nanoclusters is present in the kit in solution form, the concentration of the solution is 0.05-0.5 mg / mL, preferably 0.2 mg / mL. When the test strip is present in the kit in the form of a test strip, the test strip is a glucose detection test strip according to any one of claims 7-8.

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