A method for analyzing and detecting GSH based on vanadyl pentoxide oxidase

Abstract

The application relates to the field of biological detection technology, in particular to a preparation method of a vanadyl pentoxide oxidase and application of the vanadyl pentoxide oxidase in detection of glutathione in human serum. The preparation method of the vanadyl pentoxide oxidase comprises the following steps: step one, dissolving NH4VO3, polyether P123 and HCl in anhydrous ethanol; step two, solvent thermal treatment; step three, collecting black precipitates; step four, washing the black precipitates with distilled water for several times; step five, drying; and step six, heating the collected precipitates. The application provides the preparation method of the vanadyl pentoxide oxidase, and the prepared vanadyl pentoxide is applied to the detection of glutathione in human serum, so that the cost is low, and the prepared vanadyl pentoxide has good accuracy and stability.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a GSH analysis and detection method based on vanadium pentoxide-based oxidases. Background Technology

[0002] Glutathione (GSH) is the most important small-molecule bioactive oligopeptide in the body's antioxidant defense system and the most abundant low-molecular-weight thiol in cells, possessing significant antioxidant and detoxification functions. Abnormal GSH levels in humans are associated with cancer, Parkinson's disease, AIDS, Alzheimer's disease, and cardiovascular diseases. Therefore, accurate detection of GSH in the human body is of great significance in disease diagnosis. Currently, commonly used methods for GSH detection include high-performance liquid chromatography (HPLC), fluorescence spectroscopy, electrochemical analysis, and colorimetric detection. However, most of these methods have certain drawbacks, such as being time-consuming, requiring large sample sizes, expensive equipment, and skilled personnel. Colorimetric methods, on the other hand, have attracted widespread attention from researchers due to their low cost, rapid reaction, and visual observation capabilities. The colorimetric detection of glutathione is mainly based on its inhibitory effect on the catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB).

[0003] However, natural enzymes catalyzing the above reactions suffer from drawbacks such as poor stability, high cost, and sensitivity to environmental conditions, limiting their widespread application. Nanomaterials with enzyme-like activity (nanozymes), due to their high stability, low cost, and ease of preparation, have become a research hotspot in recent years. Currently, the main challenges facing nanozyme research include: 1) the enzyme-like catalytic activity of nanozymes is relatively low compared to natural enzymes; and 2) the variety of nanozymes is limited. Therefore, improving the activity of existing nanozymes, developing novel nanozymes, and exploring their applications remain key focuses in the field of nanozymes.

[0004] Vanadium pentoxide (V₂O₅), as a transition metal oxide, is widely studied in fields such as electrode materials for energy storage devices due to its abundant natural content, low production cost, and rich redox chemical, optical, and electrical properties. Recently, V₂O₅ has been reported to possess enzyme-mimicking activity. However, most detection methods based on the enzyme-mimicking properties of V₂O₅ typically involve H₂O₂, a destructive oxidant. Research on biomolecular detection based on the oxidase-like properties of V₂O₅, which do not require H₂O₂, is currently limited. Therefore, it is essential to develop a simple and efficient colorimetric method for the detection of GSH based on the oxidase-like activity of V₂O₅. Summary of the Invention

[0005] This invention provides a method for GSH analysis and detection based on vanadium pentoxide-based oxidases, aiming to prepare vanadium pentoxide-based oxidases and develop a simple and efficient colorimetric detection method for GSH.

[0006] This invention provides a method for the analysis and detection of GSH based on vanadium pentoxide-based oxidase. The vanadium pentoxide-based oxidase is obtained through the following steps: Step 1, dissolving NH4VO3, polyether P123, and HCl in anhydrous ethanol; Step 2, solvothermal treatment; Step 3, collecting the black precipitate; Step 4, washing several times with distilled water; Step 5, drying; and Step 6, heating the collected precipitate. The method is characterized by adding 200 μL of GSH-containing solution to a detection system consisting of 100 μL of V2O5 (30 μg / mL), 100 μL of 0.4 mM TMB, and 1600 μL of 50 mM HAc-NaAc buffer (pH 2.5). The reaction solution is incubated in a 35°C water bath for 40 min. The UV-Vis absorption spectrum of the detection solution between 300 nm and 800 nm is recorded and displayed on a 652 nm display. The absorbance value at nm was used to plot the curves of absorbance versus GSH concentration based on the experimental data. The curves showed a linear correlation in the range of 1–30 μM, with a detection limit of 0.28 μM.

[0007] Furthermore, the NH4VO3 is 0.9 g, the polyether P123 with a molecular weight of 5800 is 1.5 g, the HCl with a concentration of 2 mol / L has a volume of 6 mL, and the anhydrous ethanol has a volume of 90 mL.

[0008] Furthermore, the solvothermal treatment is performed at 200°C for 24 hours.

[0009] Furthermore, the heating is performed by placing the furnace in a muffle furnace at 300°C for 2 hours.

[0010] This invention provides a method for the analysis and detection of glutathione based on vanadium pentoxide-based oxidases. The prepared vanadium pentoxide is applied to the detection of glutathione in human serum, which is low in cost and has good accuracy and stability. Attached Figure Description

[0011] Figure 1 The images show (A) X-ray diffraction pattern (XRD), (B) X-ray photoelectron spectroscopy (XPS), and (C) scanning electron microscope (SEM) images (elemental distribution) of the prepared V2O5 mimic enzyme.

[0012] Figure 2The light absorption characteristics of different reaction systems at 652 nm and the changes in the reaction solutions are shown (a, V2O5+TMB+buffer; b, V2O5+buffer; c, TMB+buffer).

[0013] Figure 3 The images show the UV-Vis absorption spectra of the detection system with different concentrations of GSH and the corresponding photographs (A), and the curves showing the difference between the GSH concentration and the absorbance of the detection system (the interlude is the linear interval) (B).

[0014] Figure 4 This is a diagram illustrating the synthesis and detection mechanism of V2O5-type oxidases and GSH. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] The present invention provides a method for preparing vanadium pentoxide-based oxidases for the analysis and detection of GSH. The method involves dissolving 0.9 g NH₄VO₃, 1.5 g polyether P123 (Mw=5800), and 6 mL HCl (2 mol·L⁻¹) in 90 mL anhydrous ethanol. The solution is then subjected to solvothermal treatment at 200°C for 24 h. The resulting black precipitate is collected, washed several times with distilled water, and dried at 60°C for 4 h. The collected precipitate is then heated in a muffle furnace at 300°C for 2 h to obtain the final product.

[0017] Physical characterization data of V2O5 nanozymes, such as Figure 1 As shown: Figure 1 (A) shows the XRD pattern of the synthesized sample. As can be seen from the figure, all diffraction peaks of the synthesized sample match the standard card of orthorhombic V₂O₅ (JCPDS, No. 41-1426), and the intensity of the diffraction peaks is relatively high, indicating that the synthesized sample has good crystallinity. This demonstrates that we have successfully synthesized orthorhombic V₂O₅ with good crystallinity.

[0018] We further investigated the types and valence states of elements contained in the synthesized samples using XPS, and the results are as follows: Figure 1As shown in (B), the full spectrum reveals that the synthesized sample contains only three elements: V, O, and C. The C signal originates from amorphous carbon during the calibration process. This indicates that the synthesized sample contains only V and O. The inset shows that the peaks with binding energies of 517 and 525 eV correspond to V, O, and C, respectively. 5+ 2p 3 / 2 and 2p 1 / 2 This indicates that the V element in the synthesized sample has a +2 valence. The experimental results further confirm that the sample we synthesized is V₂O₅.

[0019] Figure 1 (C) shows a typical SEM image of the synthesized sample. As can be seen from the image, the synthesized V₂O₅ has a cuboid structure with a width of tens to hundreds of nanometers and a length of hundreds of nanometers. Elemental distribution shows that V and O elements (Figures C1 and C2) are present and uniformly distributed in the synthesized sample.

[0020] like Figure 2 The image shows the light absorption characteristics of different reaction systems at 652 nm and the changes in the reaction solutions (a, V₂O₅ + TMB + buffer; b, V₂O₅ + buffer; c, TMB + buffer). The catalytic activity of the prepared V₂O₅ was assessed by reacting the typical substrate of catalytic oxidase, 3,3',5,5'-tetramethylbenzidine (TMB, colorless), with dissolved oxygen to form oxidized TMB (blue). From... Figure 2 The illustration shows the color changes of the solutions in different systems: the V2O5+TMB+buffer system shows a more obvious blue color; the V2O5+buffer and TMB+buffer systems do not show any obvious color changes. Figure 2 These are the UV-Vis absorption spectra of the three reaction systems. The V2O5+buffer and TMB+buffer systems show almost no absorption peaks at 372 nm and 652 nm, while the V2O5+TMB+buffer system shows relatively obvious absorption peaks at 372 nm and 652 nm with good peak shape. This indicates that the synthesized V2O5 can catalyze the oxidation of TMB to oxidized TMB and has oxidase-like activity.

[0021] A GSH analysis and detection method based on the oxidase properties of V₂O₅ was established. Nanozymes catalyze the oxidation of TMB to produce a bright blue ox-TMB. However, this catalytic oxidation process can be inhibited by the highly reducing GSH, causing the blue color of the solution to gradually fade or even disappear. Based on this process, a simple and efficient colorimetric method for GSH detection was constructed. A typical detection procedure is as follows: 200 μL of GSH at different concentrations is added to a detection system consisting of V₂O₅ (30 μg·mL⁻¹, 100 μL), TMB (0.4 mM, 100 μL), and HAc-NaAc buffer (50 mM, pH=2.5, 1600 μL). The reaction solution is incubated in a 35℃ water bath for 40 min. The UV-Vis absorption spectra of the detection solution between 300 nm and 800 nm are recorded, and the absorbance value at 652 nm is also recorded.

[0022] like Figure 3 As shown in Figure A, with increasing GSH concentration, the absorbance of the system at 652 nm gradually decreases, and the absorbance difference (ΔA) at 652 nm is positively correlated with the GSH concentration. Figure 3 As shown in Figure B, within the range of 1–30 μM, ΔA showed a good linear relationship with GSH concentration, with the linear equation being ΔA = 0.01390*X + 0.07276, the regression coefficient R² = 0.998, and the detection limit being 0.28 μM (S / N = 3).

[0023] The accuracy of the established colorimetric method for the detection of glutathione in real samples was investigated using the spiked recovery method. The results are shown in the table below. The recovery rate in spiked samples was 97.1%–101.7%, and the RSD was 0.9%–2.4%. These results indicate that the method for the detection of GSH in serum based on V₂O₅ nanozymes is effective.

[0024] The method has good accuracy and stability.

[0025] Detection of GSH in serum

[0026]

[0027] The above embodiments are provided for those skilled in the art to implement or use the present invention. Those skilled in the art can make various modifications or changes to the above embodiments without departing from the inventive concept of the present invention. Therefore, the protection scope of the present invention is not limited to the above embodiments, but should be the maximum scope that conforms to the innovative features mentioned in the claims.

Claims

1. A method for the analysis and detection of GSH based on vanadium pentoxide-based oxidase, wherein the vanadium pentoxide-based oxidase is obtained through the following steps: Step 1, dissolving NH4VO3, polyether P123, and HCl in anhydrous ethanol; Step 2, solvothermal treatment; Step 3, collecting the black precipitate; Step 4, washing several times with distilled water; Step 5, drying; and Step 6, heating the collected precipitate, characterized in that: In a detection system consisting of 100 μL of V₂O₅ (30 μg / mL), 100 μL of TMB (0.4 mM), and 1600 μL of HAc-NaAc buffer (50 mM, pH 2.5), 200 μL of GSH-containing solution was added. The reaction solution was incubated in a 35°C water bath for 40 min. The UV-Vis absorption spectra of the detection solution in the range of 300 nm to 800 nm were recorded, and the absorbance value at 652 nm was also recorded. Based on the experimental data, a curve was plotted to show a linear correlation between absorbance and GSH concentration in the range of 1–30 μM, with a detection limit of 0.28 μM.

2. The method for GSH analysis and detection based on vanadium pentoxide-based oxidases according to claim 1, characterized in that: The composition includes 0.9 g of NH4VO3, 1.5 g of polyether P123 with a molecular weight of 5800, 6 mL of HCl with a concentration of 2 mol / L, and 90 mL of anhydrous ethanol.

3. The method for GSH analysis and detection based on vanadium pentoxide-based oxidases according to claim 1, characterized in that, The solvothermal treatment was performed at 200°C for 24 hours.

4. The method for GSH analysis and detection based on vanadium pentoxide-based oxidases according to claim 1, characterized in that, The heating process involves placing the furnace at 300°C in a muffle furnace for 2 hours.

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