A CuMn bimetallic nanozyme and its portable hydrogel preparation method and its application in the detection of ascorbic acid.

Abstract

This invention belongs to the field of materials technology, specifically relating to a CuMn bimetallic nanozyme and its preparation method with a portable hydrogel, and its application in the detection of ascorbic acid. A portable hydrogel sphere is prepared as follows: a CuMn bimetallic nanozyme is prepared into a suspension; sodium alginate is ultrasonically dispersed in the CuMn bimetallic nanozyme suspension; the sol is drawn up with a dropper and placed in the cap of a plastic centrifuge tube; calcium chloride solution is added dropwise while continuously shaking to form a hydrogel sphere. The CuMn bimetallic nanozyme material of this invention has abundant active sites and a large specific surface area, and this material has a low detection limit for ascorbic acid. The hydrogel can immobilize the enzyme, improving its catalytic activity and stability. The hydrogel's porosity promotes molecular diffusion, and its optical clarity makes it an ideal substrate. Furthermore, its color changes can be combined with a smartphone for on-site, portable, and visual detection.

Description

Technical Field

[0001] This invention relates to the field of materials technology, specifically to a CuMn bimetallic nanozyme and its preparation method with a portable hydrogel, and its application in the detection of ascorbic acid. Background Technology

[0002] Currently, methods for detecting ascorbic acid (AA) include electrochemical methods, liquid chromatography, fluorescence analysis, and spectrophotometry. Compared with these methods, colorimetric methods have attracted increasing attention due to their high sensitivity, low cost, and simplicity. Typically, they convert the analyte into a visible color change to detect its content, thus avoiding the use of expensive instruments and complex operations. In the presence of H₂O₂ solution, CuMn bimetallic nanozymes can effectively oxidize the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the corresponding blue product, oxidized tetramethylbenzidine (oxTMB), which can undergo a redox reaction with AA, causing a significant color change. Based on this research principle, a rapid and sensitive new method for detecting AA content is established.

[0003] Nanozymes are unique metal-organic frameworks with abundant redox active sites and open coordination structures. The emergence of bimetallic nanozymes reveals their unique enzyme-mimicking activity, offering potential applications in colorimetric sensing.

[0004] Hydrogels can immobilize enzymes, enhancing their catalytic activity and stability. Their porosity promotes molecular diffusion, and their optical clarity makes them ideal substrates. Furthermore, their color-changing properties can be integrated with smartphones. Therefore, a rapid, portable, and visualized new method for detecting AA content can be established. Summary of the Invention

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A CuMn bimetallic nanozyme is prepared by the following steps:

[0007] 1) Preparation of Mn nanozymes: Mn(Ac)2·4H2O and trisodium citrate were dissolved in deionized water to form solution A, and K3[Fe(CN)6] was dissolved in deionized water to form solution B. Solution B was magnetically stirred and poured into solution A. After stirring, the mixture was aged at room temperature, the precipitate was collected by centrifugation, washed, and dried to obtain Mn nanozymes.

[0008] 2) Preparation of hollow Mn nanozymes: Mn nanozymes were dispersed in deionized water and stirred. Tannic acid aqueous solution was added, heated, and the product was collected by centrifugation. The product was washed, and the precipitate was soaked in hydrochloric acid solution and washed to obtain hollow Mn nanozymes.

[0009] 3) The hollow Mn nanozyme was ultrasonically dispersed in deionized water, and Cu(NO3)2·3H2O aqueous solution was added while stirring. After stirring, the precipitate was collected by centrifugation, washed and dried to obtain CuMn bimetallic nanozyme.

[0010] Furthermore, in the above-mentioned CuMn bimetallic nanozyme, in step 1), the mass ratio of Mn(Ac)2·4H2O, trisodium citrate, and K3[Fe(CN)6] is 147:220:132; the aging time is 24h.

[0011] Furthermore, in step 2) of the aforementioned CuMn bimetallic nanozyme, the concentration of the tannic acid aqueous solution is 0.03 g / mL; the mass ratio of Mn nanozyme to tannic acid is 1:50; the heating is carried out in an oil bath at 85°C for 6 hours; and the concentration of the hydrochloric acid solution is 0.24 mol / L.

[0012] Furthermore, in step 3) of the aforementioned CuMn bimetallic nanozyme, the concentration of the Cu(NO3)2·3H2O aqueous solution is 24.2 mg / mL; the mass ratio of the hollow Mn nanozyme to Cu(NO3)2·3H2O is 15:121.

[0013] A portable hydrogel ball is prepared as follows: CuMn bimetallic nanozyme of any one of the above is prepared into a suspension, sodium alginate is ultrasonically dispersed into CuMn bimetallic nanozyme suspension to obtain a viscous liquid, the viscous liquid is drawn into the cap of a 10mL plastic centrifuge tube with a dropper, calcium chloride solution is added dropwise and shaken continuously to form a hydrogel ball.

[0014] Furthermore, the concentration of the CuMn bimetallic nanozyme suspension in the aforementioned portable hydrogel sphere is 0.1 mg / mL.

[0015] Furthermore, in the aforementioned portable hydrogel sphere, the mass ratio of sodium alginate to CuMn bimetallic nanozyme is 30:1.

[0016] Furthermore, in the aforementioned portable hydrogel ball, the concentration of the calcium chloride solution is 1 wt%.

[0017] The application of the aforementioned CuMn bimetallic nanozyme in the detection of ascorbic acid is as follows: A CuMn bimetallic nanozyme with a concentration of 1 mg / mL is used to detect ascorbic acid under the conditions of pH 4.5 and a temperature of 20℃. CuMn bimetallic nanozyme, TMB, and H2O2 solution are added, and after a reaction time of 15 min, the ascorbic acid solution to be detected is added. The concentration of ascorbic acid is then determined by colorimetry.

[0018] The application of the aforementioned portable hydrogel spheres in the detection of ascorbic acid is as follows: TMB and H2O2 solutions are dropped into the portable hydrogel spheres. After the reaction is completed within 15 minutes, the ascorbic acid solution to be detected is dropped into the portable hydrogel spheres, and the concentration of ascorbic acid is detected by colorimetric method.

[0019] The beneficial effects of this invention are:

[0020] 1. The bimetallic nanozyme material of this invention possesses abundant active sites and a large specific surface area, which greatly enhances the peroxidase-like activity of the nanozyme and exhibits superior catalytic performance for hydrogen peroxide. Due to its coordination effect with ascorbic acid, this material has a low detection limit for ascorbic acid.

[0021] 2. To enable timely on-site detection, portable and visualized hydrogel spheres were fabricated based on CuMn bimetallic nanozyme materials. The hydrogel immobilizes the enzyme, enhancing its catalytic activity and stability. Its porosity promotes molecular diffusion, and its optical clarity makes it an ideal substrate. Furthermore, its color changes can be integrated with smartphones, establishing a direct and compatible colorimetric detection method for portable on-site detection.

[0022] 3. The preparation method described in this invention has low production cost, is easy to carry, and allows for portable visual detection. Attached Figure Description

[0023] Figure 1 This is a scanning electron microscope image of a CuMn bimetallic nanozyme.

[0024] Figure 2 These are the XRD patterns of CuMn bimetallic nanozymes, Mn (hollow structure) nanozymes, and Mn nanozymes.

[0025] Figure 3 This is a comparison chart of the enzyme activities of CuMn bimetallic nanozymes, Mn (hollow structure) nanozymes, and Mn nanozymes.

[0026] Figure 4 This is a graph showing the optimization of temperature, pH, and time (from left to right) for CuMn bimetallic nanozymes.

[0027] Figure 5This is the UV spectrum of CuMn bimetallic nanozyme detection of AA.

[0028] Figure 6 This is a graph showing the linear relationship between the absorbance and concentration of AA detected by CuMn bimetallic nanozymes.

[0029] Figure 7 This is a graph showing the AA test results of CuMn bimetallic nanozymes on different actual samples.

[0030] Figure 8 These are images of CuMn bimetallic nanozyme portable hydrogels and their corresponding RGB images.

[0031] Figure 9 This is a linear relationship graph between the R, G, B values ​​and AA integrated in the smartphone in Example 4.

[0032] Figure 10 This is a diagram illustrating the experimental procedure for detecting AA using a portable hydrogel containing CuMn bimetallic nanozymes. Detailed Implementation

[0033] Example 1: Preparation of a bimetallic nanozyme material (I) Preparation of CuMn bimetallic nanozymes

[0034] 1) Dissolve 147 mg Mn(Ac)2·4H2O and 220 mg trisodium citrate in 40 mL of deionized water to form solution A, and dissolve 132 mg K3[Fe(CN)6] in 60 mL of deionized water to form solution B. Then, pour solution B into solution A with a magnetic stirrer, stir for 1 min, age the resulting solution at room temperature for 24 h, collect the precipitate by centrifugation, wash several times with water and ethanol, and dry at 70 °C to obtain Mn nanozyme.

[0035] 2) First, disperse 30 mg of the prepared Mn nanozyme in 100 mL of deionized water. Then, after stirring, add 50 mL of tannic acid aqueous solution (1.5 g) to the suspension. Heat the resulting uniform dispersion in an oil bath at 85 °C for 6 hours. Collect the product by centrifugation, wash it several times with water and ethanol, and then soak the precipitate in 0.24 mol / L hydrochloric acid solution for 10 min. Wash it several times with deionized water and ethanol to obtain Mn (hollow structure) nanozyme.

[0036] 3) Synthesis of CuMn bimetallic nanozymes from the obtained Mn (hollow structure) nanozymes: 30 mg of the prepared Mn (hollow structure) nanozymes were ultrasonically dispersed in 10 mL of deionized water. Then, 10 mL of Cu(NO3)2·3H2O aqueous solution (242 mg) was added under stirring. After stirring at room temperature for 0.5 h, the precipitate was collected by centrifugation, washed several times with water and ethanol, and dried at 70 °C to obtain CuMn bimetallic nanozymes.

[0037] (II) Testing

[0038] 1. The morphology of CuMn bimetallic nanozymes was studied and analyzed using SEM.

[0039] like Figure 1 As shown, CuMn bimetallic nanozymes have a hollow cubic structure and exhibit a good morphology.

[0040] Figure 2 These are XRD patterns of CuMn bimetallic nanozymes, Mn nanozymes, and Mn (hollow structure) nanozyme materials.

[0041] 2. Evaluation of the peroxidase activity of CuMn bimetallic nanozymes.

[0042] analyze Figure 3 Only when TMB and hydrogen peroxide coexist do the CuMn bimetallic nanozymes catalyze the oxidation of colorless TMB to blue oxTMB, indicating that the CuMn bimetallic nanozymes exhibit remarkable peroxidase-like catalytic activity. At 652 nm, the absorbance of the H2O2-TMB system is relatively low. In contrast, the addition of CuMn bimetallic nanozymes resulted in a significant change in the solution's UV-Vis absorption peak characteristics, indicating that the CuMn bimetallic nanozymes possess peroxidase-like activity and can catalyze the H2O2-induced oxidation of TMB.

[0043] 3. Optimization of peroxidase activity of CuMn bimetallic nanozymes.

[0044] like Figure 4 The optimal temperature, pH of the buffer solution, and time were optimized, with the optimal temperature being 20℃, the optimal pH being 4.5, and the optimal time being 15min.

[0045] Example 2 uses CuMn bimetallic nanozymes to detect ascorbic acid.

[0046] 1. Use a concentration of 1 mg / mL -1 CuMn bimetallic nanozymes were used to detect ascorbic acid (AA) under conditions of pH 4.5 and temperature 20℃. 30 μL of CuMn bimetallic nanozyme, 50 μL of TMB (10 mM), 50 μL of 15% H2O2 solution, and 3770 μL of buffer solution were added. After reacting for 15 min, 100 μL of AA solutions of different concentrations were added, and the absorbance was measured at 652 nm.

[0047] 2. The detection mechanism mainly involves the coordination of Mn and Cu with ascorbic acid, such as... Figure 5 As the concentration of AA increases, the absorbance signal weakens. For example... Figure 6 There is a linear relationship between absorbance and concentration.

[0048] Example 3 uses CuMn bimetallic nanozymes to detect actual samples.

[0049] Use a concentration of 1 mg / mL -1 CuMn bimetallic nanozymes were used to detect different beverages under pH 4.5 and 20℃ conditions. 30 μL of CuMn bimetallic nanozyme, 50 μL of TMB (10 mM), 50 μL of 15% H2O2 solution, and 3770 μL of buffer solution were added. After reacting for 15 min, 100 μL of different beverages were added, and the absorbance was measured at 652 nm.

[0050] We used Wahaha mineral water as a blank solution and measured the absorbance of Nongfu Spring mineral water, Jasmine Honey Tea, Scream, Iced Black Tea, Jasmine First Snow, and Pulse. We then converted the absorbance to concentration using a linear equation, and the results are as follows: Figure 7 As shown.

[0051] Example 4: Detection of AA concentration using a portable hydrogel ball platform.

[0052] CuMn bimetallic nanozymes were prepared into a suspension with a concentration of 0.1 mg / mL. 300 mg of sodium alginate was dispersed in 100 mL of CuMn bimetallic nanozyme suspension. The resulting viscous liquid was then immersed in a 1 wt% calcium chloride solution to obtain hydrogel spheres.

[0053] 50 μL of 10 mM TMB and 50 μL of 15% hydrogen peroxide were dropped onto the hydrogel. After reacting for 15 min, 100 μL of AA solutions of different concentrations were dropped onto the hydrogel. The colors were captured by a smartphone and further analyzed using a color-assisted application.

[0054] To achieve the actual quantitative detection of AA on hydrogel spheres, we used a smartphone-driven signal readout system, which allows for easy visual reading of the AA content. This smartphone-driven signal readout system, based on inherent different calculation modes, can automatically generate linear fits between R, G, and B values ​​and the AA concentration. For example... Figure 9 The 0.3R+0.59G+0.11B model exhibits the best linear correlation, with the corresponding equation being y = 23.21071 + 0.33373x(R) 2 =0.99). Due to the unique catalytic activity of CuMn bimetallic nanozymes and the smartphone-driven signal readout system based on CuMn bimetallic nanozyme hydrogel spheres, this detection AA method is more effective and convenient for on-site detection.

Claims

1. A CuMn bimetallic nanozyme, characterized in that, Its preparation method includes the following steps: 1) Preparation of Mn nanozymes: Mn(Ac)2·4H2O and trisodium citrate were dissolved in deionized water to form solution A, and K3[Fe(CN)6] was dissolved in deionized water to form solution B. Solution B was magnetically stirred and poured into solution A. After stirring, the mixture was aged at room temperature, the precipitate was collected by centrifugation, washed, and dried to obtain Mn nanozymes. 2) Preparation of hollow Mn nanozymes: Mn nanozymes were dispersed in deionized water and stirred. Tannic acid aqueous solution was added, heated, and the product was collected by centrifugation. The product was washed, and the precipitate was soaked in hydrochloric acid solution and washed to obtain hollow Mn nanozymes. 3) The hollow Mn nanozyme was ultrasonically dispersed in deionized water, and Cu(NO3)2·3H2O aqueous solution was added while stirring. After stirring, the precipitate was collected by centrifugation, washed and dried to obtain CuMn bimetallic nanozyme.

2. The CuMn bimetallic nanozyme according to claim 1, characterized in that, In step 1), the mass ratio of Mn(Ac)2·4H2O, trisodium citrate, and K3[Fe(CN)6] is 147:220:132; the aging time is 24h.

3. The CuMn bimetallic nanozyme according to claim 1, characterized in that, In step 2), the concentration of the tannic acid aqueous solution is 0.03 g / mL; the mass ratio of Mn nanozyme to tannic acid is 1:50; the heating is carried out in an oil bath at 85°C for 6 hours; and the concentration of the hydrochloric acid solution is 0.24 mol / L.

4. The CuMn bimetallic nanozyme according to claim 1, characterized in that, In step 3), the concentration of the Cu(NO3)2·3H2O aqueous solution is 24.2 mg / mL; the mass ratio of the hollow Mn nanozyme to Cu(NO3)2·3H2O is 15:

121.

5. A portable hydrogel ball, characterized in that, The preparation method is as follows: the CuMn bimetallic nanozyme described in any one of claims 1-4 is prepared into a suspension, sodium alginate is ultrasonically dispersed into the CuMn bimetallic nanozyme suspension to obtain a viscous liquid, the viscous liquid is drawn up with a dropper and placed in the cap of a 10mL plastic centrifuge tube, calcium chloride solution is added dropwise and shaken continuously to form hydrogel spheres.

6. A portable hydrogel ball according to claim 5, characterized in that, The concentration of the CuMn bimetallic nanozyme suspension was 0.1 mg / mL.

7. A portable hydrogel ball according to claim 5, characterized in that, The mass ratio of sodium alginate to CuMn bimetallic nanozyme is 30:

1.

8. A portable hydrogel ball according to claim 5, characterized in that, The concentration of the calcium chloride solution is 1 wt%.

9. The application of the CuMn bimetallic nanozyme according to any one of claims 1-4 in the detection of ascorbic acid, characterized in that, The method is as follows: CuMn bimetallic nanozyme with a concentration of 1 mg / mL was used to detect ascorbic acid under the conditions of pH 4.5 and temperature 20℃: CuMn bimetallic nanozyme, TMB and H2O2 solution were added, and after the reaction was completed for 15 min, the ascorbic acid solution to be tested was added, and the concentration of ascorbic acid was detected by colorimetric method.

10. The application of the portable hydrogel spheres according to any one of claims 5-8 in the detection of ascorbic acid, characterized in that, The method is as follows: TMB and H2O2 solutions are dropped into a portable hydrogel ball. After reacting for 15 minutes, the ascorbic acid solution to be tested is dropped into the portable hydrogel ball, and the concentration of ascorbic acid is detected by colorimetry.

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