Preparation method of silver-modified hollow vanadium pentoxide micro-sphere oxide enzyme and application of silver-modified hollow vanadium pentoxide micro-sphere oxide enzyme in detection of ascorbic acid in human serum

Abstract

The invention relates to a preparation method of a silver-modified hollow vanadium pentoxide micro-sphere oxide enzyme, which is characterized by comprising the following steps: dissolving V2O5 and H2C2O4. 2H2O in water, and stirring at 80 DEG C until a clear blue VOC2O4 solution is formed; dropwise adding the VOC2O4 solution into isopropanol, transferring the solution into a stainless steel high-pressure reaction kettle, and keeping at 200 DEG C for 2.5 hours to obtain a VOC2O4 and isopropanol mixed solution; centrifuging the obtained VOC2O4 and isopropanol mixed solution to perform solid-liquid separation, and repeatedly washing with ethanol to obtain a precipitate; calcining the precipitate obtained in the step (3) to obtain V2O5; dispersing the V2O5 obtained in the step (4) in deionized water to obtain a V2O5 suspension; compared with the prior art, the preparation method of the silver-modified hollow vanadium pentoxide micro-sphere oxide-like enzyme and the application of the silver-modified hollow vanadium pentoxide micro-sphere oxide-like enzyme in detection of ascorbic acid in human serum have the advantages that a novel Ag-modified V2O5 nano-enzyme with a hollow micro-sphere structure is creatively designed and synthesized, the nano-enzyme shows excellent oxidase-like activity, and the nano-enzyme can be applied to detection of ascorbic acid in human serum. And the colorimetric detection capability on ascorbic acid is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and particularly to a method for preparing a silver-modified hollow vanadium pentoxide microsphere oxidase and an application thereof in detecting ascorbic acid in human serum. Background Art

[0002] Ascorbic acid (AA), also known as vitamin C, is a key natural compound in multiple redox processes in biological cells and organisms. Its antioxidant properties are essential for improving immunity, preventing cancer, and protecting LDL cholesterol from oxidation. Moreover, ascorbic acid deficiency may increase susceptibility to various diseases, including the common cold, cardiovascular problems, and mental health disorders. Therefore, accurate and efficient detection of ascorbic acid levels is essential for health monitoring and disease prevention.

[0003] A variety of methods have been developed for the detection of ascorbic acid, including high-performance liquid chromatography, fluorescence, gas chromatography, and electrochemical techniques. Although these methods have certain advantages, such as high sensitivity and specificity, they also have disadvantages such as expensive equipment, complex procedures, and time-consuming processes. These limitations highlight the need to develop cost-effective and environmentally friendly alternatives. Among the many emerging alternatives, colorimetric analysis has received significant attention due to its simplicity, rapid results, high cost-effectiveness, and ease of visual detection. In recent years, enzyme-like nanomaterials, or nanozymes, have emerged as effective colorimetric tools.

[0004] Advances in nanochemistry and nanocatalysis have driven the rapid development of nanomaterials with enzyme-mimicking functions. These nanozymes often surpass natural enzymes in terms of stability, catalytic activity, versatility, and cost-effectiveness. In particular, nanozymes with oxidase-like activity have shown great potential in the development of biosensors for biomolecule detection. Despite significant progress in nanozyme research, challenges remain in fully understanding the enzymatic activity, sensing mechanism, and detection methods of nanozyme-based biosensors. The main problems currently facing nanozyme research include: 1) the enzyme-like catalytic activity of nanozymes is relatively low compared to natural enzymes; 2) the types of nanozymes are limited.

[0005] In recent years, nanozymes with enzyme-like properties have become strong candidates for the development of new detection platforms due to their unique catalytic activity, high stability and low cost. Among these nanozymes, vanadium pentoxide (chemical formula: V 2 O 5 Nanozymes of V have attracted much attention due to their inherent oxidase-like activity, which can promote the colorimetric detection of various biomolecules including AA. Moreover, the V can be further improved by structural modification and the introduction of other functional materials. 2 O 5 Catalytic efficiency of nanozymes. Summary of the invention

[0006] Based on the problems or shortcomings existing in the prior art, the purpose of the present invention is to provide a method for preparing a silver-modified hollow vanadium pentoxide microsphere oxidase and its application in detecting ascorbic acid in human serum, so as to achieve the purpose of improving the activity of nanozyme oxidase and enhancing the colorimetric detection ability of ascorbic acid.

[0007] The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase provided by the present invention is characterized by comprising the following steps: Step (1), V 2 O 5 and H 2 C 2 O 4 ·2H 2 O is dissolved in water and stirred at 80℃ until a clear blue VOC is formed. 2 O 4 Solution; Step (2): VOC 2 O 4 The solution was added dropwise into isopropanol and stirred for 40 minutes. The solution was transferred to a stainless steel autoclave and maintained at 200°C for 2.5 hours to obtain VOC 2 O 4 Mixed with isopropyl alcohol; Step (3), take the VOC obtained in step (2) 2 O 4 Mix the mixture with isopropanol, centrifuge to separate the solid and liquid, and wash repeatedly with ethanol to obtain a precipitate; Step (4): calcine the precipitate obtained in step (3) to obtain V 2 O 5 ; Step (5), take V obtained in step (4) 2 O 5 Dispersed in deionized water, V 2 O 5 Suspension; Step (6), to V 2 O 5 Silver nitrate solution was added to the suspension and ultrasonic treatment was performed for 60 minutes to obtain V 2 O 5 Mixture solution; Step (7), adding sodium borohydride solution to the V obtained in step (6) 2 O 5 The mixture solution was placed in an ice bath for 60 minutes; Step (8), centrifugation to separate the solid and liquid, and collecting the Ag-modified V 2 O 5The precipitate was repeatedly washed with deionized water; Step (9), vacuum drying, to obtain Ag-modified V 2 O 5 .

[0008] Furthermore, in step (1), the commodity category V 2 O 5 0.60g, H 2 C 2 O 4 ·2H 2 O is 1.25 g and water is 40 mL.

[0009] Furthermore, the VOC 2 O 4 The solution is 9 mL and the isopropanol is 60 mL.

[0010] Furthermore, the calcination condition in step (4) is calcination at 350° C. for 120 minutes.

[0011] Furthermore, in step (5), V 2 O 5 The volume of the solution is 300 mg and the volume of deionized water is 300 mL.

[0012] Furthermore, in step (6), 0.8 mL of 0.1 mol·L -1 Silver nitrate solution.

[0013] Furthermore, in step (7), 60 mL of 1 mmol·L -1 Sodium borohydride solution.

[0014] Furthermore, the vacuum drying condition in step (9) is drying at 40°C for 12 hours.

[0015] The present invention also provides an application method of silver-modified hollow vanadium pentoxide microsphere oxidase for detecting ascorbic acid in human serum, which is characterized by comprising the following steps: (a) Ag-modified V 2 O 5 (30 μg·mL -1 , 100 μL), TMB (0.15 mM, 100 μL) and HAc-NaAc buffer (50 mM, pH=2.5, 1600 μL), 200 μL of ascorbic acid of different concentrations were added, the reaction solution was incubated in a 35°C water bath for 40 min, the UV-visible absorption spectrum of the detection solution between 300 nm and 800 nm was recorded, and the absorbance value at 652 nm was recorded; (b) Drawing of standard curve: within a certain range, draw a standard curve with AA concentration as the horizontal axis and absorbance difference (ΔA) as the vertical axis; (c) Determine ΔA of the ascorbic acid solution to be tested by UV-visible spectroscopy, and compare it with the standard curve in step (b) to obtain the ascorbic acid concentration in the solution to be tested.

[0016] Furthermore, the range of step (b) is 1–20 μM.

[0017] The invention provides a method for preparing a silver-modified hollow vanadium pentoxide microsphere-like oxidase and its application in detecting ascorbic acid in human serum. Compared with the prior art, a novel hollow microsphere-structured Ag-modified V 2 O 5 The nanozyme exhibited excellent oxidase-like activity and enhanced the colorimetric detection ability of ascorbic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The Ag-modified V prepared by the present invention 2 O 5 (a) X-ray diffraction pattern (XRD), (b) X-ray photoelectron spectroscopy (XPS), and (f) elemental distribution of oxidase-like enzymes; Figure 2 (a) Ag-modified V prepared in the present invention 2 O 5 Scanning electron microscope image of (a) Ag-modified V 2 O 5 Transmission electron microscopy images of (c) Ag-modified V 2 O 5 High-resolution transmission electron microscopy images of (d) Ag-modified V 2 O 5 Energy spectrum mapping image of Figure 3 Figure 2 shows the light absorption characteristics of different reaction systems at 652 nm and the changes in the reaction solution: (a) Ag-modified V 2 O 5 + TMB + buffer; (b) TMB + buffer; (c) Ag-modified V 2 O 5 + buffer, the inset shows the color changes of each system; Figure 4 (a) UV-visible spectrometer spectra of the system after adding different concentrations of AA, and the inset is a photo of the system after adding different concentrations of AA (0–120 μM); (b) the linear relationship between the absorbance intensity at 652 nm and the AA concentration. DETAILED DESCRIPTION Example

[0019] The Ag-modified V used in the method of the present invention 2 O 5 It was prepared as follows: (1) 0.60 g of commercial V 2 O 5 and 1.25 g of H 2 C 2 O 4 ·2H 2 O was dissolved in 40 mL of water and stirred at 80 °C until a clear blue VOC was formed. 2 O 4 Solution. (2) Add 9 mL of VOC 2 O 4 The solution was added dropwise into 60 mL of isopropanol and stirred for 40 minutes. The solution was then transferred to a stainless steel autoclave and kept at 200 °C for 2.5 hours to obtain VOC 2 O 4 (3) The solid and liquid were separated by centrifugation and washed with ethanol to obtain a precipitate. (4) After calcination at 350°C for 120 minutes, V 2 O 5 (5) 300 mg of the V 2 O 5 Disperse in 300 mL of deionized water. (6) Add 0.8 mL of 0.1 mol·L -1 (7) Add 60 mL of 1 mmol·L silver nitrate solution and ultrasonicate for 60 minutes to ensure homogeneity. -1 Sodium borohydride solution was added to the mixture, and the reaction mixture was placed in an ice bath for 60 minutes to promote the reaction. (8) After the reaction was completed, the solid and liquid were separated by centrifugation to collect the Ag-modified V 2 O 5 The product was washed repeatedly with deionized water. (9) The product was dried under vacuum at 40°C for 12 hours to obtain the final product Ag-modified V 2 O 5 .

[0020] Ag modified V 2 O 5 Oxidase characterization data Figure 1 As shown: Figure 1 (a) is the XRD spectrum of the synthesized sample. It can be seen from the figure that all the diffraction peaks of the synthesized sample can be compared with the orthorhombic phase V 2 O 5The test results are consistent with the standard cards of Ag (JCPDS, No. 41-1426) and Ag (JCPDS, No. 41-1402).

[0021] The types and valence states of the elements in the synthesized samples were further studied by XPS. Figure 1 (be) shown. Full spectrum ( Figure 1 b) shows that only Ag, V, O and C exist in the synthesized sample, among which the C signal originates from the amorphous carbon in the calibration process. This indicates that only Ag, V and O exist in the synthesized sample. The 2p spectrum peaks of V are located at 517.28 eV and 524.92 eV, which are consistent with V 5+ The presence of additional peaks at 515.88 eV and 523.6 eV may be related to the residual VO 2 The resulting V 4+ related( Figure 1 c). Figure 1 d shows the characteristic binding energies of Ag at 374.1 eV (Ag 3d 3 / 2) and 368.1 eV (Ag 3d 5 / 2), respectively. The O 1s peak at 530.12 eV is attributed to the Ag-modified V 2 O 5 The peak at 530.7 eV is attributed to the adsorption of V on Ag-modified V 2 O 5 The experimental results further prove that the sample we synthesized is Ag-modified V 2 O 5 ( Figure 1 e). Figure 1 f shows the presence of Ag, V, and O elements in the sample.

[0022] Figure 2 The Ag-modified V 2 O 5 The morphology, structure and element distribution characteristics of nanomaterials. Scanning electron microscope image ( Figure 2 a) shows the Ag-modified V 2 O 5 The particles are spherical in shape, with a size of approximately 1 to 3 micrometers and a tendency to aggregate. Transmission electron microscopy image ( Figure 2 b) The hollow structure of these particles was further confirmed by high-resolution transmission electron microscopy images ( Figure 2 c) reveals that Ag-modified V 2 O 5 The crystal lattice structure of Ag nanoparticles was successfully modified on V nanoparticles.2 O 5 Surface. Spectral element mapping image ( Figure 2 d) shows the distribution of V, O, and Ag within the particle, proving that Ag is 2 O 5 These results provide a basis for the successful preparation of Ag-modified V 2 O 5 Provides strong structural and compositional evidence.

[0023] The prepared Ag-modified V was detected by catalyzing the typical oxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB, colorless) and dissolved oxygen to generate oxidized TMB (blue). 2 O 5 Whether it has peroxidase-like catalytic activity. Figure 3 As shown, the synthesized Ag-modified V 2 O 5 When introduced into TMB solution, the originally colorless solution turns into bright blue. Figure 3 As shown in the inset; and a clear absorption peak was observed at 652 nm, which is the characteristic of ox-TMB. In contrast, there was no significant color change in the other groups, and the reaction system had no obvious absorption peak at 652 nm. These findings confirm that Ag-modified V 2 O 5 The effective catalytic effect of Ag-modified V in TMB oxidation, leading to the formation of ox-TMB, was demonstrated. 2 O 5 Intrinsic oxidase-like activity.

[0024] Nanozymes catalyze the oxidation of TMB to produce bright blue ox-TMB, but this catalytic oxidation process can be gradually lightened or even faded by ascorbic acid, which has a strong reducing property. Based on the above process, a simple and efficient colorimetric method for detecting ascorbic acid was constructed. The typical detection process is as follows: (a) In the Ag-modified V 2 O 5 (30 μg·mL -1, 100 μL), TMB (0.15mM, 100 μL) and HAc-NaAc buffer (50 mM, pH=2.5, 1600 μL), add 200 μL of ascorbic acid of different concentrations, incubate the reaction solution in a 35°C water bath for 40 min, record the UV-visible absorption spectrum of the detection solution between 300 nm-800 nm, and record the absorbance value at 652 nm. (b) Drawing of the standard curve: In the range of 1–20 μM, draw a standard curve with AA concentration as the horizontal axis and absorbance difference (ΔA) as the vertical axis; (c) Use UV-visible spectroscopy to measure ΔA of the ascorbic acid solution to be tested, and compare it with the standard curve described in step (b) to obtain the ascorbic acid concentration in the solution to be tested.

[0025] like Figure 4 As shown in Figure A, as the amount of AA added increases, the absorbance of the system at 652 nm gradually decreases, and the absorbance difference (ΔA) at 652 nm is positively correlated with the AA concentration. Figure 4 As shown in B, in the range of 1–20 μM, ΔA showed a good linear relationship with AA concentration, ΔA = 0.01588*X + 0.01290, the R² value was 0.9961, and the detection limit was 0.103 μM (S / N=3).

[0026] The spike recovery method was used to investigate the accuracy of the established AA colorimetric detection method in actual samples. The results are shown in the table. The recovery rate in the spiked samples was 97.78%~99.90%, and the RSD was 1.68%~2.29%. The above results show that based on V 2 O 5 The detection method for AA in serum established by nanozymes has good accuracy and stability.

[0027] Detection of AA in human serum The above is only an embodiment of the present invention, and is not any formal or substantial limitation of the present invention. For those skilled in the art, without departing from the method of the present invention, modifications, supplements and substitutions can be made, and these modifications, supplements and substitutions should also be within the scope of protection of the present invention. Any equivalent changes made by those skilled in the art using the technical content disclosed above without departing from the spirit and scope of the present invention are all equivalent embodiments of the present invention; any equivalent changes made to the above embodiments based on the essential technology of the present invention, modifications, supplements and substitutions, still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing silver-modified hollow vanadium pentoxide microsphere oxidase, It is characterized in that The following steps are involved: Step (1), V 2 O 5 and H 2 C 2 O 4 ·2H 2 O is dissolved in water and stirred at 80℃ until a clear blue VOC is formed. 2 O 4 Solution; Step (2): VOC 2 O 4 The solution was added dropwise into isopropanol and stirred for 40 minutes. The solution was transferred to a stainless steel autoclave and maintained at 200°C for 2.5 hours to obtain VOC 2 O 4 Mixed with isopropyl alcohol; Step (3), take the VOC obtained in step (2) 2 O 4 Mix the mixture with isopropanol, centrifuge to separate the solid and liquid, and wash repeatedly with ethanol to obtain a precipitate; Step (4): calcine the precipitate obtained in step (3) to obtain V 2 O 5 ; Step (5), take V obtained in step (4) 2 O 5 Dispersed in deionized water, V 2 O 5 Suspension; Step (6), to V 2 O 5 Silver nitrate solution was added to the suspension and ultrasonic treatment was performed for 60 minutes to obtain V 2 O 5 Mixture solution; Step (7), adding sodium borohydride solution to the V obtained in step (6) 2 O 5 The mixture solution was placed in an ice bath for 60 minutes; Step (8), centrifugation to separate the solid and liquid, and collecting the Ag-modified V 2 O 5 The precipitate was repeatedly washed with deionized water; Step (9), vacuum drying, to obtain Ag-modified V 2 O 5 .

2. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that Step (1) V 2 O 5 0.60g, H 2 C 2 O 4 ·2H 2 O is 1.25 g and water is 40 mL.

3. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that VOC in step (2) 2 O 4 The solution is 9 mL and the isopropanol is 60 mL.

4. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that The calcination conditions in step (4) are calcination at 350° C. for 120 minutes.

5. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that Step (5) Weigh V 2 O 5 The volume of the solution is 300 mg and the volume of deionized water is 300 mL.

6. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that Step (6) Add 0.8 mL of 0.1 mol·L -1 Silver nitrate solution.

7. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that Step (7) Add 60 mL of 1 mmol·L -1 Sodium borohydride solution.

8. The method for preparing the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1, It is characterized in that The vacuum drying condition in step (9) is drying at 40°C for 12 hours.

9. An application method of the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 1 for detecting ascorbic acid in human serum, It is characterized in that The following steps are involved: (a) Ag-modified V 2 O 5 (30 μg·mL -1 , 100 μL), TMB (0.15 mM, 100 μL) and HAc-NaAc buffer (50 mM, pH=2.5, 1600 μL), 200 μL of ascorbic acid of different concentrations were added, the reaction solution was incubated in a 35°C water bath for 40 min, the UV-visible absorption spectrum of the detection solution between 300 nm and 800 nm was recorded, and the absorbance value at 652 nm was recorded; (b) Drawing of standard curve: within a certain range, draw a standard curve with AA concentration as the horizontal axis and absorbance difference (ΔA) as the vertical axis; (c) Determine ΔA of the ascorbic acid solution to be tested by UV-visible spectroscopy, and compare it with the standard curve in step (b) to obtain the ascorbic acid concentration in the solution to be tested.

10. The application method of the silver-modified hollow vanadium pentoxide microsphere oxidase according to claim 9 for detecting ascorbic acid in human serum, It is characterized in that The range described in step (b) is 1–20 μM.

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