B, n-mo se2-based bimetallic nanoszyme, preparation method and application thereof

Abstract

The present application relates to the field of nanometer enzyme catalysis and colorimetric sensing technology, and particularly relates to a B, N-MoSe2-based bimetallic nanometer enzyme as well as a preparation method and application thereof, the preparation method comprising the following steps: uniformly mixing B, N-MoSe2 dispersion liquid and metal salt mixed solution, performing heating reaction to obtain a mixed reaction liquid; adding a reducing agent into the mixed reaction liquid to perform in-situ reduction reaction, and obtaining B, N-MoSe2@PdCu-based bimetallic nanometer enzyme. The preparation method has the advantages of simple preparation process, mild reaction condition and green environmental protection; and the prepared B, N-MoSe2@PdCu-based bimetallic nanometer enzyme has excellent peroxidase-like activity and can efficiently catalyze hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to develop color.

Description

Technical Field

[0001] This invention relates to the field of nanozyme catalysis and colorimetric sensing technology, and particularly to a B,N-MoSe2-based bimetallic nanozyme, its preparation method, and its applications. Background Technology

[0002] Hydrogen peroxide (H2O2) is an important reactive oxygen species in living organisms, participating in various physiological processes such as cell signaling and oxidative stress. Abnormal H2O2 concentrations are closely related to various diseases (such as cancer and neurodegenerative diseases). Simultaneously, H2O2 is also a key detection indicator in environmental and food fields. Therefore, achieving rapid and accurate detection of H2O2 has significant scientific and practical value.

[0003] Colorimetric sensing has become one of the mainstream technologies for H2O2 detection due to its advantages such as simple operation, low cost, and visualized results. Its core relies on catalysts with peroxidase-like activity. Traditional natural enzymes (such as horseradish peroxidase) have drawbacks such as easy inactivation, high preparation cost, and difficulty in large-scale production; single metal nanozymes (such as Pd and Cu-based nanozymes) have limited catalytic activity and poor stability.

[0004] Therefore, developing a simple bimetallic nanozyme and its preparation method, and using it to construct a highly sensitive H2O2 colorimetric sensing platform, has become an urgent technical problem to be solved. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems, this invention provides a B,N-MoSe2-based bimetallic nanozyme, its preparation method, and its applications. The B,N-MoSe2-based bimetallic nanozyme exhibits excellent peroxidase-like activity, its preparation method is simple, and it can be used for efficient catalysis of TMB colorimetric development. Simultaneously, it can also be used to construct a highly sensitive and stable H2O2 colorimetric detection method.

[0006] In one aspect, the present invention provides a method for preparing a B,N-MoSe2-based bimetallic nanozyme, comprising: preparing a B,N-MoSe2 support; dissolving the B,N-MoSe2 support in water to obtain a B,N-MoSe2 dispersion; preparing a metal salt mixture of soluble Pd salt and soluble Cu salt; uniformly mixing the B,N-MoSe2 dispersion and the metal salt mixture, and heating the mixture to obtain a mixed reaction solution; and then using a nanozyme capable of reducing Pd... 2+ Cu 2+ A reducing agent with metallic ions is added to the mixed reaction solution to carry out an in-situ reduction reaction, yielding B,N-MoSe2@PdCu-based bimetallic nanozymes.

[0007] In some embodiments of the present invention, the step of preparing the B,N-MoSe2 support includes: calcining a mixture of melamine, boric acid and molybdenum selenide at 300-400°C for 4-6 hours, for example, 4.2 hours, 4.6 hours, 5.2 hours, 5.6 hours, 5.8 hours, etc., to obtain the B,N-MoSe2 support; wherein the mass ratio of melamine, boric acid and molybdenum selenide is (4-6):(4-6):1, for example, 4:4:1, 4.5:4.5:1, 5.2:5.2:1, 5.6:5.6:1, 5.8:5.8:1, etc.

[0008] In some embodiments of the present invention, the soluble Pd salt includes at least one of K2PdCl4, PdCl2, and Pd(NO3)2; and / or, the soluble Cu salt includes at least one of copper chloride, copper sulfate, and copper nitrate.

[0009] In some embodiments of the present invention, the molar ratio of Pd to Cu in the metal salt mixture is 1:(1~9), for example, it can be 1:1.5, 1:3.6, 1:4.8, 1:5.8, 1:8.6, etc.

[0010] In some embodiments of the present invention, the molar ratio of B,N-MoSe2 support, Pd element and Cu element in the heating reaction is (6~25):1:(1~9), for example, it can be 6.8:1:1.8, 10.8:1:2.9, 15.8:1:4.1, 19.6:1:6.8, 23.6:1:8.9, etc.; and / or, the heating reaction temperature is 60~80℃, for example, it can be 62℃, 68℃, 71℃, 74℃, 78℃, etc.; and / or, the heating reaction time is 4~6h, for example, it can be 4.3h, 4.8h, 5.3h, 5.6h, 5.9h, etc.

[0011] In some embodiments of the present invention, the reducing agent in the in-situ reduction reaction is sodium borohydride, and / or the temperature of the in-situ reduction reaction is 20~25°C, for example, 21°C, 22°C, 23°C, 23.5°C, 24°C, etc.; and / or the time of the in-situ reduction reaction is 1~10h, for example, 1.3h, 4.8h, 6.3h, 7.6h, 9.8h, etc.

[0012] In another aspect, the present invention provides a B,N-MoSe2-based bimetallic nanozyme obtained using the above-described preparation method.

[0013] In some embodiments of the present invention, the B,N-MoSe2-based bimetallic nanozyme is magnetic.

[0014] In another aspect, the present invention provides an application of the above-mentioned B,N-MoSe2-based bimetallic nanozyme in the quantitative detection of hydrogen peroxide.

[0015] In some embodiments of the present invention, the application is for the quantitative detection of hydrogen peroxide in the colorimetric reaction of hydrogen peroxide oxidizing 3,3',5,5'-tetramethylbenzidine. The method for detecting hydrogen peroxide content includes: adding B,N-MoSe2@PdCu-based bimetallic nanozyme and 3,3',5,5'-tetramethylbenzidine sequentially to Tris-HCl buffer solution with pH 4-6 (e.g., 4.1, 4.6, 5.3, 5.6, 5.9, etc.) to obtain a basic detection solution; adding H2O2 standard solutions of different concentrations to the basic detection solution and incubating for 8-15 min (e.g., 9 min, 10 min, 11 min, 12 min, 14 min, etc.); adding sulfuric acid stop solution and measuring the absorbance value at 452 nm; fitting the linear relationship between H2O2 concentration and absorbance to obtain a standard curve; adding the H2O2 solution to be tested to the basic detection solution and incubating for 8-15 min; adding sulfuric acid stop solution and measuring the absorbance value at 452 nm. The absorbance value at nm is used to calculate the concentration of H2O2 in the test solution.

[0016] The technical solution provided by this invention has the following advantages: The B,N-MoSe2-based bimetallic nanozymes of this invention exhibit excellent catalytic activity. The B,N-MoSe2-based bimetallic nanozymes use B,N-MoSe2 as a carrier and load Pd and Cu bimetallic active sites. B and N co-doping regulates the electronic structure of B,N-MoSe2, and the Pd and Cu bimetals produce a synergistic catalytic effect, significantly enhancing the peroxidase-like activity of the nanozymes. The catalytic efficiency is far higher than that of B,N-MoSe2, B,N-MoSe2@Cu, and B,N-MoSe2@Pd.

[0017] The B,N-MoSe2-based bimetallic nanozymes of this invention are magnetic, allowing for rapid separation and recovery using magnetism. The B,N-MoSe2-based bimetallic nanozymes exhibit high stability, with their enzyme activity remaining almost unchanged even after multiple uses.

[0018] The preparation method of B,N-MoSe2-based bimetallic nanozymes in this invention has the following characteristics: (1) The preparation process is simple and controllable: a stepwise reaction process is adopted, and the product can be obtained by mixing and standing, heating and stirring and low-temperature reduction. The reaction conditions are mild and the steps are easy to operate, which is suitable for large-scale production; (2) The raw material ratio is clear: the amount of each raw material, the solvent volume and the reaction parameters are all optimal ratios to ensure Pd 2+ Cu 2+The ions are fully adsorbed and reduced on the B,N-MoSe2 substrate, resulting in products with uniform morphology and sufficient exposure of active sites.

[0019] The B,N-MoSe2-based bimetallic nanozymes of this invention exhibit excellent catalytic activity. B and N co-doping modulates the electronic structure of MoSe2, resulting in a synergistic catalytic effect between Pd and Cu bimetals, significantly enhancing the peroxidase-like activity of the nanozymes. The catalytic efficiency is far higher than that of B,N-MoSe2 or B,N-MoSe2@Cu and B,N-MoSe2@Pd. The superior catalytic activity of the B,N-MoSe2-based bimetallic nanozymes is due to the large specific surface area of ​​the two-dimensional layered structure of B,N-MoSe2, which facilitates the dispersion of Pd and Cu metals. Furthermore, doping MoSe2 with B and N elements allows for flexible control of the electronic properties of B,N-MoSe2, optimizing the synergistic effect between Pd and Cu.

[0020] The application of B,N-MoSe2-based bimetallic nanozymes in this invention has the following characteristics: (1) Excellent detection performance: The constructed H2O2 colorimetric sensing platform has a wide linear range (0~6 Mm), a low detection limit (0.21 mM), good stability, and strong anti-interference ability; (2) Broad application prospects: It can be used for rapid detection of H2O2 in biological samples and environmental water samples. It is easy to operate and the results are visualized. No large instruments are required, making it suitable for on-site detection and clinical diagnosis; (3) Rapid recycling: Since B,N-MoSe2-based bimetallic nanozymes are magnetic, they can be rapidly separated from the reaction solution by utilizing their magnetic properties after the reaction. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0022] 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, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0023] Figure 1 This is a TEM image of the B,N-MoSe2-based bimetallic nanozyme of Example 1 of the present invention. Figure 2 This is a comparison of the UV-Vis absorption spectra of different systems used to verify the peroxidase-like activity of B,N-MoSe2@PdCu in Example 2 of the present invention. Figure 3Comparison of UV-Vis absorption spectra of B,N-MoSe2 loaded on different carriers (single / bimetallic) in Example 2 of the present invention; Figure 4 The Michaelis constant K for B,N-MoSe2@Cu with respect to the H2O2 substrate in Example 2 of this invention. m and the maximum reaction rate V max Relevant data; Figure 5 The Michaelis constant K for B,N-MoSe2@Cu with respect to the TMB substrate in Example 2 of this invention is... m and the maximum reaction rate V max Relevant data; Figure 6 The Michaelis constant K for B,N-MoSe2@Pd with respect to the H2O2 substrate in Example 2 of this invention. m and the maximum reaction rate V max Relevant data; Figure 7 The Michaelis constant K of B,N-MoSe2@Pd with respect to the TMB substrate in Example 2 of this invention. m and the maximum reaction rate V max Relevant data; Figure 8 The Michaelis constant K for B,N-MoSe2@PdCu with respect to the H2O2 substrate in Example 2 of this invention. m and the maximum reaction rate V max Relevant data; Figure 9 The Michaelis constant K for B,N-MoSe2@PdCu with respect to the TMB substrate in Example 2 of this invention. m and the maximum reaction rate V max Relevant data; Figure 10 The Michaelis constant K for B,N-MoSe2@Cu, B,N-MoSe2@Pd, and B,N-MoSe2@PdCu with respect to the H2O2 substrate in Example 2 of this invention is given. m and the maximum reaction rate V max A dual Y-axis plot; Figure 11 The Michaelis constant K for B,N-MoSe2@Cu, B,N-MoSe2@Pd, and B,N-MoSe2@PdCu with respect to the TMB substrate in Example 2 of this invention is... m And a dual Y-axis plot of the maximum reaction rate Vmax; Figure 12 Macroscopic comparison of the magnetic properties of B,N-MoSe2, B,N-MoSe2@Cu, B,N-MoSe2@Pd and B,N-MoSe2@PdCu prepared in Example 2 of the present invention. Figure 13 To verify the stability of B,N-MoSe2@PdCu prepared in Example 2 of this invention; Figure 14 The following are the UV-Vis absorption spectra of a series of H2O2 standard solutions of the present invention, in Example 3 of this invention. Figure 15 Standard curves were plotted for a series of H2O2 standard solutions of different concentrations in Example 3 of this invention. Detailed Implementation

[0024] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0025] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0026] According to an embodiment of the present invention, a method for preparing B,N-MoSe2-based bimetallic nanozymes is provided, comprising the following steps: (1) Preparation of B,N-MoSe2 support: Melamine, boric acid and molybdenum selenide were mixed evenly and then calcined at high temperature in a tube furnace to obtain B,N-MoSe2 support; (2) Preparation of B,N-MoSe2 dispersion: The B,N-MoSe2 support obtained in step (1) is dispersed in water to obtain B,N-MoSe2 dispersion; (3) Preparation of metal salt mixture: K2PdCl4 and CuCl2·2H2O are dissolved in water to obtain metal salt mixture; (4) Heating reaction: The B,N-MoSe2 dispersion obtained in step (2) is mixed evenly with the metal salt mixture obtained in step (3), and after standing, it is stirred and heated to obtain a mixed reaction solution; (5) Low-temperature in-situ reduction: Add the reducing agent to the mixed reaction solution obtained in step (4) to carry out in-situ reduction reaction and obtain crude product; (6) Product collection: After filtering, washing and drying the crude product obtained in step (5), B,N-MoSe2@PdCu-based bimetallic nanozyme is obtained.

[0027] In some embodiments of the present invention, the preparation method of the B,N-MoSe2 support in step (1) includes the following steps: ① First, accurately weigh 4 g of melamine, 4 g of boric acid and 1 g of large piece of molybdenum selenide powder, and mix them in a beaker containing 100 mL of anhydrous ethanol.

[0028] ② Next, place the beaker in an ultrasonic cleaner and sonicate for 1 hour to ensure the three substances are evenly mixed. Change the water every half hour to prevent the water temperature from becoming too high.

[0029] ③Then, the obtained black mixed solution was placed in a forced-air drying oven and dried at 80 °C for 3 h to allow the anhydrous ethanol to evaporate completely.

[0030] ④ Finally, the obtained mixed powder was spread evenly in a ceramic boat and placed in a quartz tube furnace under nitrogen protection for calcination. The temperature was raised to 400℃ and calcined for 4 hours. After the furnace temperature cooled naturally to room temperature, the gray-black product B,N-MoSe2 was removed.

[0031] In some embodiments of the present invention, in step (2), the B,N-MoSe2 dispersion is prepared by weighing 0.15 g of B,N-MoSe2 powder and dispersing it in 15 mL of water, and then sonicating or stirring until the dispersion is uniform to obtain the B,N-MoSe2 dispersion.

[0032] In some embodiments of the present invention, in step (3), the metal salt mixture is prepared by weighing 0.1 g of K2PdCl4 powder and 0.1092 g of CuCl2·2H2O powder and dissolving them in 3 mL of water, stirring until completely dissolved to obtain the metal salt mixture.

[0033] In some embodiments of the present invention, the heating reaction step (4) includes: uniformly mixing the B,N-MoSe2 dispersion obtained in step (2) with the metal salt mixture obtained in step (3), and letting it stand at room temperature (20°C~25°C) for 30 min to allow Pd to react. 2+ Cu 2+ The ions were fully adsorbed by the B,N-MoSe2 support, and then the mixture was stirred at 60°C for 6 hours to obtain a mixed reaction solution.

[0034] In some embodiments of the present invention, in step (5), the low-temperature in-situ reduction reaction includes: cooling the mixed reaction solution obtained in step (4) to room temperature, slowly adding 10 mL of 0.5 M reducing agent NaBH4 solution, and continuously stirring at room temperature for 1 hour, thereby removing the adsorbed Pd through NaBH4. 2+ Cu 2+ The ions are reduced to the metallic element to obtain the crude product.

[0035] In some embodiments of the present invention, the step of collecting the product in step (6) includes: centrifuging and filtering the crude product obtained in step (5), washing it three times each with deionized water and anhydrous ethanol to remove unreacted raw materials and impurities, and then vacuum drying it at 80°C for 6 hours to obtain B,N-MoSe2@PdCu-based bimetallic nanozyme powder.

[0036] In some embodiments of the present invention, in step (6), the obtained B,N-MoSe2@PdCu-based bimetallic nanozymes exhibit peroxidase catalytic activity. This is mainly due to the following two reasons: Firstly, in the B,N-MoSe2 support, the B and N doping regulates the band structure of MoSe2, resulting in a large number of oxygen vacancies and unsaturated active sites, which enhances the electron transport rate and optimizes the adsorption capacity for H2O2 and TMB. At the same time, the two-dimensional layered structure of B,N-MoSe2 provides a large specific surface area, exposing sufficient catalytic sites. Secondly, in the PdCu bimetallic alloy modification, the synergistic electronic effect generated by the PdCu metal alloy regulates the Fermi level of the B,N-MoSe2@PdCu-based bimetallic nanozymes, reducing the reaction energy barrier for H2O2 decomposition and TMB oxidation. The Pd and Cu bimetallic sites can efficiently capture H2O2 and cleave the OO bond to generate highly active hydroxyl radicals (・OH) or high-valence metal active intermediates, significantly enhancing the peroxidase-like catalytic activity, which is far superior to single-metal modification or pure MoSe2 substrate.

[0037] According to an embodiment of the present invention, an application of B,N-MoSe2-based bimetallic nanozyme in the colorimetric detection of H2O2 is provided, specifically including the following steps: (1) Preparation of basic detection solution: Mix B,N-MoSe2@PdCu-based bimetallic nanozyme and 3,3´,5,5´-tetramethylbenzidine (TMB) to obtain basic detection solution; (2) Plotting the standard curve: Add H2O2 standard solutions of different concentrations (0~6 Mm) to the basic test solution obtained in step (1), incubate at 30℃ for 10 min, then add 100 μL of 2 M sulfuric acid stop solution, and measure the absorbance value at 452 nm; plot the H2O2 concentration as the abscissa and the absorbance value as the ordinate to fit a linear standard curve, R0 2 >0.99; (3) Detect the sample to be tested: Add the H2O2 solution to be tested to the basic test solution obtained in step (1), incubate at 30°C for 10 min, measure the absorbance value, and substitute it into the standard curve obtained in step (2) to calculate the concentration of H2O2 in the test solution.

[0038] In some embodiments of the present invention, the final concentration of B,N-MoSe2@PdCu-based bimetallic nanozyme in the basic detection solution in step (1) is 0.03 g / mL.

[0039] In some embodiments of the present invention, in the linear standard curve obtained in step (3), the linear range of H2O2 detection is 0~6 mM, and the detection limit is 0.21 mM.

[0040] The following detailed examples will provide a further explanation.

[0041] The TMB colorimetric reagent kit uses the TMB two-component kit from Zhuangmeng Biotechnology.

[0042] Example 1B: Preparation of N-MoSe2@PdCu-based bimetallic nanozymes (1) Weigh 4 g of melamine, 4 g of boric acid, and 1 g of large pieces of molybdenum selenide powder, and mix them in a beaker containing 100 mL of anhydrous ethanol. Next, place the beaker in an ultrasonic cleaner and sonicate for 1 h to ensure uniform mixing of the three substances. Change the water every half hour to prevent excessive water temperature. Then, place the resulting black mixed solution in a forced-air drying oven at 80℃ for 3 h to allow the anhydrous ethanol to completely evaporate. Next, spread the resulting mixed powder evenly in a ceramic boat and place it in a quartz tube furnace under nitrogen protection for calcination. The temperature is programmed to rise to 400℃ and calcined for 4 h. After the furnace temperature cools naturally to room temperature, remove the gray-black product (B,N-MoSe2) for later use.

[0043] (2) Weigh 0.15 g of B,N-MoSe2 powder, add 15 mL of deionized water, and stir magnetically for 30 min until it is evenly dispersed to obtain B,N-MoSe2 dispersion; (3) Weigh 0.1 g K2PdCl4 powder and 0.1092 g CuCl2·2H2O powder, add 3 mL deionized water, and stir magnetically until completely dissolved to obtain a metal salt mixture. (4) Pour the B,N-MoSe2 dispersion and the metal salt mixture into the same reaction vessel, stir magnetically for 10 min until uniformly mixed, let stand at room temperature for 30 min, and then stir at 60℃ for 6 hours. (5) After the reaction is complete, the mixed reaction solution is cooled to room temperature naturally. 10 mL of 0.5 M NaBH4 aqueous solution is slowly added dropwise under stirring. After the addition is complete, the mixture is stirred at room temperature for 1 hour to obtain the crude product. (6) The crude product is centrifuged and filtered. The filter residue is washed three times with deionized water and then washed twice with anhydrous ethanol to remove unreacted impurities such as K2PdCl4, CuCl2·2H2O and NaBH4. (7) Place the washed filter residue in a vacuum drying oven at 60℃ and dry for 6~10 hours to obtain B,N-MoSe2@PdCu-based bimetallic nanozymes.

[0044] Figure 1 The image shows a TEM image of the B,N-MoSe2-based bimetallic nanozyme prepared in step (7).

[0045] Example 2 Peroxidase-like activity verification Prepare the following systems (using Zhuangmeng Biotechnology's ELISA substrate B kit, solution A is TMB, and solution B is H2O2. Maintain a total volume of 3 mL for each system): System 1: TMB System 2: TMB + H2O2 System 3: B,N-MoSe2 (0.03 g / mL) + TMB + H2O2 System 4: B,N-MoSe2@Cu (0.03 g / mL) + TMB + H2O2 The preparation method of B,N-MoSe2@Cu is the same as that of B,N-MoSe2@PdCu in Example 1, except that 0.1092 g of CuCl2·2H2O powder is added and K2PdCl4 powder is not added.

[0046] System 5: B,N-MoSe2@Pd (0.03 g / mL) + TMB + H2O2 The preparation method of B,N-MoSe2@Pd is the same as that of B,N-MoSe2@PdCu in Example 1, except that 0.1g of K2PdCl4 powder is added and CuCl2·2H2O powder is not added.

[0047] System 6: B,N-MoSe2@PdCu (0.03 g / mL) + TMB + H2O2 The preparation method of B,N-MoSe2@PdCu is exactly the same as that in Example 1.

[0048] After incubating each system in a 30 ℃ constant temperature water bath for 50 min, the UV-Vis absorption spectra of each system in the 500~800 nm range were measured, and the results are as follows. Figure 2 and Figure 3 As shown. Among them, Figure 2 The UV-Vis absorption spectra of different systems were used to verify the peroxidase-like activity of B,N-MoSe2@PdCu; Figure 3Comparison of UV-Vis absorption spectra of B,N-MoSe2 supported on different supports (single / bimetallic) demonstrates the activity enhancement effect of Pd and Cu bimetallic synergistic catalysis. Figure 2 As shown, system 6 exhibits a strong characteristic absorption peak at 652 nm, while the other systems show low absorbance or no obvious characteristic peaks. This demonstrates that the B,N-MoSe2@PdCu-based bimetallic nanozyme prepared in this invention has optimal peroxidase-like activity and is the core component for catalyzing the oxidation of TMB by H2O2.

[0049] Therefore, by verifying the peroxidase-like activity of the nanozyme using UV-Vis absorption spectroscopy, the following conclusions can be drawn: ① Control group: TMB, TMB+H2O2, B,N-MoSe2+TMB+H2O2, B,N-MoSe2@Cu+TMB+H2O2, and B,N-MoSe2@Pd+TMB+H2O2 all showed no obvious characteristic absorption peaks or had low absorbance; ② Experimental group: The B,N-MoSe2@PdCu+TMB+H2O2 system showed a strong characteristic absorption peak at 652 nm, indicating that the bimetallic nanozyme can efficiently catalyze the oxidation of TMB by H2O2 to generate blue oxTMB, and has excellent peroxidase-like activity.

[0050] Experiments on enzyme activity The Michaelis constant K of B,N-MoSe2@Cu with respect to H2O2 substrate m and the maximum reaction rate V max Relevant data such as Figure 4 As shown; the Michaelis constant K of B,N-MoSe2@Cu with respect to the TMB substrate. m and the maximum reaction rate V max Relevant data such as Figure 5 As shown.

[0051] The Michaelis constant Km and maximum reaction rate V of B,N-MoSe2@Pd with respect to the H2O2 substrate are also relevant. max Relevant data such as Figure 6 As shown. The Michaelis constant Km and maximum reaction rate V of B,N-MoSe2@Pd with respect to the TMB substrate are also shown. max Relevant data such as Figure 7 As shown.

[0052] The Michaelis constant K of B,N-MoSe2@PdCu with respect to H2O2 substrate m and the maximum reaction rate V max Relevant data such as Figure 8 As shown; the Michaelis constant K of B,N-MoSe2@PdCu for the TMB substrate. m and the maximum reaction rate V max Relevant data such as Figure 9 As shown.

[0053] According to the above Figure 4 , Figure 6 and Figure 8 The data yielded the Michaelis constant K for B,N-MoSe2@Cu, B,N-MoSe2@Pd, and B,N-MoSe2@PdCu with respect to the H2O2 substrate. m and the maximum reaction rate V max The dual Y-axis plot is Figure 10 .like Figure 10 As shown, B,N-MoSe2@PdCu exhibits a larger Vt for H2O2 substrate compared to the other two materials. max B,N-MoSe2@Cu exhibits better substrate compatibility, while B,N-MoSe2@Pd shows poorer substrate compatibility. The addition of Cu and Pd doping improves Vo. max .

[0054] According to the above Figure 5 , Figure 7 and Figure 9 The data obtained yielded the Michaelis constant K for B,N-MoSe2@Cu, B,N-MoSe2@Pd, and B,N-MoSe2@PdCu with respect to the TMB substrate. m and the maximum reaction rate V max The dual Y-axis plot is Figure 11 .like Figure 11 As shown, B,N-MoSe2@PdCu exhibits a larger Vt for TMB substrates compared to the other two materials. max B,N-MoSe2@Pd exhibits better substrate compatibility, while B,N-MoSe2@Cu shows poorer substrate compatibility. Pd doping improves substrate compatibility, while Cu doping increases Vo. max .

[0055] Magnetic verification of B,N-MoSe2@PdCu Take 20 mg of B,N-MoSe2 from system 3, B,N-MoSe2@Cu from system 4, B,N-MoSe2@Pd from system 5, and B,N-MoSe2@PdCu from system 6 into centrifuge tubes and add distilled water to a final volume of 9 mL. Mix thoroughly. Place a magnet near one side of each centrifuge tube in turn. After 5 minutes, observe the phenomena. The specific results are as follows: Figure 12 As shown.

[0056] according to Figure 12The results show that B,N-MoSe2@PdCu can be manipulated and enriched on one side of the centrifuge tube with the aid of an external magnetic field, while the other three materials do not exhibit this phenomenon. These results indicate that B,N-MoSe2@PdCu possesses excellent magnetic separation performance.

[0057] Stability of B,N-MoSe2@PdCu-based bimetallic nanozymes Prepare a Tris-HCl buffer solution with a pH of 5. Take 1 mL of the buffer solution in a test tube, add 500 μL each of components A and B of the TMB kit, mix well, and then add 5 mg of B,N-MoSe2@PdCu. After reacting for 10 min, separate the material using a magnet. Measure the absorbance of the solution at a wavelength of 652 nm. Then, wash the separated material three times with distilled water and ethanol, and dry it. Repeat the above method seven times. The results are as follows: Figure 13 As shown.

[0058] according to Figure 13 The analysis of the results leads to the following conclusion: the absorbance of the solution remained essentially unchanged during the seven repeated uses of B,N-MoSe2@PdCu, demonstrating the good catalytic performance of B,N-MoSe2@PdCu.

[0059] Example 3 Quantitative detection of H2O2 (1) Preparation of basic detection solution: Take several centrifuge tubes containing 1 mL Tris-HCl buffer, and add 500 μL of 0.03 g / mL B,N-MoSe2@PdCu and 500 μL of diluted TMB (V(TMB):V(H2O)=1:3) solution respectively. This mixture is the basic detection solution. (2) Preparation of H2O2 standard solution: Dilute 30% H2O2 stock solution with deionized water to prepare a series of H2O2 solutions (0~5.5 mM), and add 500 μL of the standard solution to the prepared basic test solution. (3) After incubating the series of H2O2 standard solutions obtained in step (2) in a 30℃ constant temperature water bath for 10 min, add 100 μL of 2 M sulfuric acid stop solution. Measure the absorbance of each system at 452 nm using a UV-Vis spectrophotometer. The specific test results are as follows: Figure 14 As shown; (4) Plotting the standard curve: Plot the absorbance values ​​obtained in step (3) with H2O2 concentration (mM) on the x-axis and absorbance at 452 nm on the y-axis, and perform linear fitting to obtain the standard curve: y = 0.07802x + 0.03437(R) 2=0.99528), where y is absorbance and x is H2O2 concentration. The standard curve is shown below. Figure 15 As shown.

[0060] (5) Sample testing: After incubating the sample in a 30℃ constant temperature water bath for 10 min, add 100 μL of 2 M sulfuric acid stop solution and measure the absorbance at 452 nm using a UV-Vis spectrophotometer. Substitute the obtained absorbance value into the standard curve obtained in step (4) to calculate the concentration of H2O2.

[0061] In summary, the B,N-MoSe2-based bimetallic nanozymes, their preparation methods, and applications in the embodiments of the present invention utilize B,N-MoSe2 as a carrier to load Pd and Cu bimetallic active sites. The nanozymes are prepared through a stepwise process of mixing and settling, heating and stirring, and reduction with a reducing agent. The preparation process is simple, the reaction conditions are mild, and it is environmentally friendly. The prepared B,N-MoSe2@PdCu-based bimetallic nanozyme exhibits excellent peroxidase-like activity, efficiently catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide, showing a characteristic absorption peak at 652 nm. Furthermore, the B,N-MoSe2-based bimetallic nanozyme demonstrates high stability. A colorimetric sensing platform constructed based on this nanozyme enables highly sensitive quantitative detection of hydrogen peroxide (H2O2), with a linear range of 0–6 mM and a detection limit as low as 0.21 mM. This overcomes the problems of insufficient catalytic activity and poor detection stability associated with traditional nanozymes, showing promising application prospects in fields such as biological detection and environmental monitoring.

[0062] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0063] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a B,N-MoSe2-based bimetallic nanozyme, characterized in that, include: Preparation of B,N-MoSe2 support; The B,N-MoSe2 support was dissolved in water to obtain a B,N-MoSe2 dispersion; Preparation of a metal salt mixture containing soluble Pd and soluble Cu salts; The B,N-MoSe2 dispersion and the metal salt mixture were uniformly mixed and heated to obtain a mixed reaction solution. Will be able to restore Pd 2+ Cu 2+ A reducing agent with metallic ions is added to the mixed reaction solution to carry out an in-situ reduction reaction, yielding a B,N-MoSe2@PdCu-based bimetallic nanozyme.

2. The preparation method according to claim 1, characterized in that, The steps for preparing the B,N-MoSe2 support include: A mixture of melamine, boric acid and molybdenum selenide was calcined at 300-400℃ for 4-6 hours to obtain B,N-MoSe2 support; The mass ratio of melamine, boric acid and molybdenum selenide is (4~6):(4~6):

1.

3. The preparation method according to claim 1 or 2, characterized in that, The soluble Pd salt includes at least one of K2PdCl4, PdCl2, and Pd(NO3)2; And / or, the soluble Cu salt includes at least one of copper chloride, copper sulfate, and copper nitrate.

4. The preparation method according to any one of claims 1 to 3, characterized in that, In the metal salt mixture, the molar ratio of Pd to Cu is 1:(1~9).

5. The preparation method according to any one of claims 1 to 4, characterized in that, During the heating reaction, The mass ratio of B,N-MoSe2 support, Pd element and Cu element is (6~25):1:(1~9); And / or, the temperature for heating the reaction is 60~80℃; And / or, the heating reaction time is 4~6 hours.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In in-situ reduction reactions, The reducing agent is sodium borohydride; And / or, the temperature for the in-situ reduction reaction is 20~25℃; And / or, the in-situ reduction reaction takes 1 to 10 hours.

7. A B,N-MoSe2-based bimetallic nanozyme obtained by the preparation method according to any one of claims 1 to 6.

8. The B,N-MoSe2-based bimetallic nanozyme according to claim 7, characterized in that, The B,N-MoSe2-based bimetallic nanozyme is magnetic.

9. The application of the B,N-MoSe2-based bimetallic nanozyme as described in claim 7 or 8 in the quantitative detection of hydrogen peroxide.

10. The application according to claim 9, characterized in that, The application is for the quantitative detection of hydrogen peroxide in the colorimetric reaction of hydrogen peroxide with 3,3',5,5'-tetramethylbenzidine. The method for detecting hydrogen peroxide content includes: B,N-MoSe2@PdCu-based bimetallic nanozyme and 3,3´,5,5´-tetramethylbenzidine were added sequentially to Tris-HCl buffer solution with pH 4-6 to obtain the basic detection solution. H2O2 standard solutions of different concentrations were added to the basic detection solution and incubated for 8-15 min. After adding sulfuric acid stop solution, the absorbance value was measured at 452 nm. The linear relationship between H2O2 concentration and absorbance was fitted to obtain the standard curve. Add the H2O2 solution to be tested to the basic detection solution, incubate for 8-15 min, add sulfuric acid stop solution, and calculate the concentration of H2O2 in the test solution by measuring the absorbance at 452 nm.

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