CuMn bimetallic nano-enzyme, preparation method of portable hydrogel of CuMn bimetallic nano-enzyme and application of portable hydrogel in detection of ascorbic acid

Abstract

The invention belongs to the technical field of materials, and particularly relates to a CuMn bimetallic nano-enzyme, a preparation method of portable hydrogel of the CuMn bimetallic nano-enzyme and application of the portable hydrogel in ascorbic acid detection. A preparation method of the portable hydrogel ball comprises the following steps: preparing a CuMn bimetallic nano-enzyme into a turbid liquid, ultrasonically dispersing sodium alginate into the CuMn bimetallic nano-enzyme turbid liquid, sucking sol by using a dropper, putting the sol into a tube cover of a plastic centrifugal tube, dropwise adding a calcium chloride solution, and continuously shaking to form the hydrogel ball. The CuMn bimetallic nano-enzyme material disclosed by the invention has rich active sites and a relatively large specific surface area, and the material has a relatively low detection limit on ascorbic acid detection. The hydrogel can immobilize the enzyme and can improve the catalytic activity and stability of the enzyme. The hydrogel has porosity and can promote molecular diffusion, and the hydrogel is very perfect as a substrate due to optical clarity. And the color change of the device can be combined with a smart phone to carry out on-site portable visual detection.

Description

Technical Field

[0001] The invention relates to the field of material technology, and in particular to a preparation method of a CuMn bimetallic nanozyme and a portable hydrogel thereof, and application of the nanozyme in detecting ascorbic acid. Background Art

[0002] At present, the methods for detecting ascorbic acid (AA) include electrochemical method, liquid chromatography, fluorescence analysis and spectrophotometry. Compared with the above methods, colorimetry has attracted more and more attention due to its high sensitivity, low cost and simplicity. Usually, it converts the detection substance into a color change visible to the naked eye to detect the content of a certain substance, thus avoiding the use of expensive instruments and complicated operations. In the presence of H2O2 solution, CuMn bimetallic nanozyme can effectively oxidize the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the corresponding blue product oxidized tetramethylbenzidine (oxTMB), which can undergo redox reaction with AA, thereby causing obvious color change. Based on the above research principle, a new method for rapid and sensitive detection of AA content was 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-like activity and provides potential application prospects in colorimetric sensing applications.

[0004] Hydrogels can immobilize enzymes, which can improve the catalytic activity and stability of enzymes. Hydrogels are porous and can promote molecular diffusion, and hydrogels are perfect as substrates due to their optical clarity. And their color changes can be combined with smartphones. Therefore, a new method for rapid, portable and visual detection of AA content has been established. Summary of the invention

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

[0006] A CuMn bimetallic nanozyme, the preparation method of which comprises the following steps:

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

[0008] 2) Preparation of hollow structured Mn nanozyme: dispersing the Mn nanozyme in deionized water and stirring, adding tannic acid aqueous solution, heating, centrifuging and collecting the product, washing, soaking the precipitate in a hydrochloric acid solution, washing, and obtaining a hollow structured Mn nanozyme;

[0009] 3) The hollow structured Mn nanozyme was ultrasonically dispersed in deionized water, and a Cu(NO3)2·3H2O aqueous solution was added under stirring. After continued stirring, the precipitate was collected by centrifugation, washed, and dried to obtain the 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; and the aging time is 24h.

[0011] Furthermore, in the above-mentioned CuMn bimetallic nanozyme, 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 performed in an 85°C oil bath for 6 hours; and the concentration of the hydrochloric acid solution is 0.24 mol / L.

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

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

[0014] Furthermore, in the above-mentioned portable hydrogel ball, the concentration of the CuMn bimetallic nanozyme suspension is 0.1 mg / mL.

[0015] Furthermore, in the above-mentioned portable hydrogel ball, the mass ratio of sodium alginate to CuMn bimetallic nanozyme is 30:1.

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

[0017] The above-mentioned CuMn bimetallic nanozyme is used in the detection of ascorbic acid, and the method is as follows: using a CuMn bimetallic nanozyme with a concentration of 1 mg / mL, ascorbic acid is detected under the conditions of pH 4.5 and temperature 20°C: CuMn bimetallic nanozyme, TMB, and H2O2 solution are added, and after reacting for 15 minutes, the ascorbic acid solution to be detected is added, and the concentration of ascorbic acid is detected by colorimetry.

[0018] The portable hydrogel ball is used in the detection of ascorbic acid as follows: TMB and H2O2 solution are dripped into the portable hydrogel ball, and after 15 minutes of reaction, the ascorbic acid solution to be detected is dripped into the portable hydrogel ball, and the concentration of ascorbic acid is detected by colorimetry.

[0019] Beneficial effects of the present invention:

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

[0021] 2. In order to detect on-site in time, a portable and visualized hydrogel ball was prepared based on the CuMn bimetallic nanozyme material. The hydrogel can immobilize the enzyme and improve the catalytic activity and stability of the enzyme. The hydrogel has porosity that can promote molecular diffusion, and the hydrogel is perfect as a substrate due to its optical clarity. And its color change can be used in conjunction with a smartphone, thus establishing a direct and compatible detection colorimetric method for on-site portable detection.

[0022] 3. The preparation method of the present invention has low production cost, is easy to carry, and can be used for portable visual detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0027] Figure 5This is the UV image of CuMn bimetallic nanozyme detecting AA.

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

[0029] Figure 7 This is the result diagram of AA detection of different actual samples by CuMn bimetallic nanozyme.

[0030] Figure 8 This is the image of CuMn bimetallic nanozyme portable hydrogel and its corresponding RGB image.

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

[0032] Fig.10 This is a diagram of the experimental operation process of CuMn bimetallic nanozyme portable hydrogel detection of AA. DETAILED DESCRIPTION

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

[0034] 1) 147 mg Mn(Ac)2·4H2O and 220 mg trisodium citrate were dissolved in 40 mL deionized water to form solution A, and 132 mg K3[Fe(CN)6] was dissolved in 60 mL deionized water to form solution B. Solution B was then poured into solution A with magnetic stirring. After stirring for 1 min, the resulting solution was aged at room temperature for 24 h, the precipitate was collected by centrifugation, washed several times with water and ethanol, and dried at 70 °C to obtain Mn nanozyme.

[0035] 2) First, 30 mg of the prepared Mn nanozyme was dispersed in 100 mL of deionized water, and then, after stirring, 50 mL of tannic acid aqueous solution (1.5 g) was added to the suspension, and the obtained uniform dispersion was heated in an oil bath at 85°C for 6 hours, the product was collected by centrifugation, washed several times with water and ethanol, and then, the precipitate was soaked in 0.24 mol / L hydrochloric acid solution for 10 min, washed several times with deionized water and ethanol to obtain Mn (hollow structure) nanozyme.

[0036] 3) Synthesis of CuMn bimetallic nanozyme from the obtained Mn (hollow structure) nanozyme: 30 mg of the prepared Mn (hollow structure) nanozyme was ultrasonically dispersed in 10 mL of deionized water, and 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 the CuMn bimetallic nanozyme.

[0037] (II) Testing

[0038] 1. Use SEM to study and analyze the morphology of CuMn bimetallic nanozyme.

[0039] like Figure 1 As shown, the CuMn bimetallic nanozyme has a hollow cubic structure and presents a good morphology.

[0040] Figure 2 These are the XRD diagrams of CuMn bimetallic nanozyme, Mn nanozyme, 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, the CuMn bimetallic nanozyme catalyzes the oxidation of colorless TMB to blue oxTMB, indicating that the CuMn bimetallic nanozyme exhibits amazing peroxidase-like catalytic activity. At 652nm, the absorbance of the H2O2-TMB system is low. In contrast, after the addition of the CuMn bimetallic nanozyme, a significant change with UV-visible absorption peak characteristics appeared in the solution, indicating that the CuMn bimetallic nanozyme has peroxidase-like activity and can catalyze the oxidation of TMB initiated by H2O2.

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

[0044] like Figure 4 , is to optimize the temperature, pH of the buffer solution and time. The optimum temperature is 20℃, the optimum pH is 4.5 and the optimum time is 15min.

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

[0046] 1. Use concentration is 1mg mL -1 The CuMn bimetallic nanozyme was used to detect ascorbic acid AA at pH 4.5 and 20°C. 30 μL CuMn bimetallic nanozyme, 50 μL TMB (10 mM), 50 μL 15% H2O2 solution and 3770 μL buffer solution were added. After 15 minutes of reaction, 100 μL AA solution of different concentrations was added and the absorbance was detected at a wavelength of 652 nm.

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

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

[0049] The concentration used is 1 mg mL -1 The CuMn bimetallic nanozyme was tested on different beverages at pH 4.5 and 20°C. 30 μL CuMn bimetallic nanozyme, 50 μL TMB (10 mM), 50 μL 15% H2O2 solution and 3770 μL buffer solution were added. After 15 minutes of reaction, 100 μL of different beverages were added respectively, and the absorbance was detected at a wavelength of 652 nm.

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

[0051] Example 4 Detection of AA concentration based on a portable hydrogel ball platform.

[0052] The CuMn bimetallic nanozyme was prepared into a suspension with a concentration of 0.1 mg / mL, 300 mg of sodium alginate was dispersed in 100 mL of the CuMn bimetallic nanozyme suspension, and the obtained viscous liquid was immersed in a calcium chloride solution with a concentration of 1 wt% to obtain hydrogel balls.

[0053] 50 μL of 10 mM TMB and 50 μL of 15% hydrogen peroxide were dropped on the hydrogel, and after 15 min of reaction, 100 μL of AA solutions with different concentrations were dropped on the hydrogel, and the color was captured by a smartphone for further analysis using a color-assisted application.

[0054] In order to achieve the actual quantitative detection of AA by hydrogel spheres, we used a smartphone-driven signal readout system that can easily read the detected AA content with the naked eye. The smartphone-driven signal readout system can automatically generate a linear fit of R, G, and B to AA concentration based on the inherent different calculation modes. Fig. 9 , the 0.3R+0.59G+0.11B mode has the best linear correlation, and the corresponding equation is 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 balls, the detection method of AA is more effective and convenient in on-site detection.

Claims

1. A CuMn bimetallic nanozyme, characterized in that: The preparation method thereof comprises the following steps: 1) Preparation of Mn nanozyme: Mn(Ac)2·4H2O and trisodium citrate were dissolved in deionized water to form solution A, K3[Fe(CN)6] was dissolved in deionized water to form solution B, solution B was poured into solution A with magnetic stirring, and after stirring, aged at room temperature, the precipitate was collected by centrifugation, washed, and dried to obtain Mn nanozyme; 2) Preparation of hollow structured Mn nanozyme: dispersing the Mn nanozyme in deionized water and stirring, adding tannic acid aqueous solution, heating, centrifuging and collecting the product, washing, soaking the precipitate in a hydrochloric acid solution, washing, and obtaining a hollow structured Mn nanozyme; 3) The hollow structured Mn nanozyme was ultrasonically dispersed in deionized water, and a Cu(NO3)2·3H2O aqueous solution was added under stirring. After continued stirring, the precipitate was collected by centrifugation, washed, and dried to obtain the 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; and the aging time is 24 h.

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 performed in an 85° C. oil bath for 6 h; 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 structured 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 to 4 is prepared into a suspension, sodium alginate is ultrasonically dispersed in the CuMn bimetallic nanozyme suspension to obtain a viscous liquid, the viscous liquid is drawn with a dropper and placed in the cap of a 10 mL plastic centrifuge tube, a calcium chloride solution is added and continuously shaken to form hydrogel balls.

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

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

1.

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

9. The use of the CuMn bimetallic nanozyme according to any one of claims 1 to 4 in detecting ascorbic acid, characterized in that: The method is as follows: Use CuMn bimetallic nanozyme with a concentration of 1 mg / mL to detect ascorbic acid at pH 4.5 and temperature 20°C: Add CuMn bimetallic nanozyme, TMB, and H2O2 solution, wait for 15 minutes, then add the ascorbic acid solution to be detected, and detect the concentration of ascorbic acid by colorimetry.

10. Use of the portable hydrogel ball according to any one of claims 5 to 8 in detecting 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, an ascorbic acid solution to be detected is dropped into the portable hydrogel ball, and the concentration of ascorbic acid is detected by colorimetry.

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