Enhanced colorimetric method for rapidly and selectively detecting glutathione and kit thereof

Abstract

The invention discloses an enhanced colorimetric method for rapidly and selectively detecting glutathione and a kit thereof, and belongs to the technical field of biochemical detection. The method comprises the following steps: preparing a prussian blue solution, a Turnish blue solution, a ferrous chloride solution or a ferric chloride solution as a nano-enzyme catalysis solution; mixing the catalytic solution with an ABTS solution and a hydrogen peroxide solution according to a specific proportion to form a blank reaction solution; adding a glutathione-containing to-be-detected sample into the solution to carry out a chromogenic reaction; and determining the content of glutathione by determining the absorbance difference between the blank and the reaction solution to be detected in a specific wavelength range. The method is mainly used for rapid, selective and quantitative detection of glutathione in the fields of food, medicine and biological analysis.

Description

Technical Field

[0001] This invention belongs to the field of biochemical detection technology. More specifically, this invention relates to an enhanced colorimetric method and kit for rapid and selective detection of glutathione. Background Technology

[0002] Glutathione (GSH) is an important biological antioxidant, and the accurate detection of its content is crucial in the food, pharmaceutical, and bioanalytical fields. Currently, nanozyme-based colorimetric methods are receiving widespread attention due to their ease of operation and low cost.

[0003] In existing technologies, the detection of GSH using the peroxidase activity of Prussian blue nanozymes has been reported. For example, prior art 1 (Xiaocheng Lvetal., New Journal of Chemistry, 2024) discloses the catalytic oxidation of TMB by H2O2 based on mSiO2@PB nanocomposite materials, while GSH inhibits this colorimetric reaction, thereby achieving the detection of GSH. Prior art 2 (Wang Jiawei et al., Journal of Liaocheng University, 2019) also uses a Prussian blue nanocube catalytic TMB-H2O2 system, where GSH inhibits the colorimetric reaction through the dual effects of Fe³⁺ on the nanozyme surface and reduced oxidized TMB.

[0004] However, these existing technologies all belong to the "suppression-based" detection mode. These methods generally suffer from narrow linear ranges and poor selectivity due to interference from other antioxidant molecules in the sample. Furthermore, TMB itself is carcinogenic, and its oxidation products have poor solubility, which may affect the stability and safety of the detection. Therefore, there is an urgent need in the field to develop a rapid detection method for GSH that is highly selective and more safe and reliable. Summary of the Invention

[0005] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0006] The present invention provides an enhanced colorimetric method and kit for rapid and selective detection of glutathione. One objective of this invention is to provide a non-inhibitory method for quantitative detection of GSH based on absorbance or RGB colorimetric enhancement signals, enabling rapid and wide linear range detection of GSH, and allowing analysis to be performed via portable devices such as smartphones.

[0007] This invention provides an enhanced colorimetric method for rapid and selective detection of glutathione, comprising the following steps: Using water as a solvent, prepare solutions of Prussian blue, Tönnies blue, ferrous chloride, or ferric chloride as nanozyme catalytic solutions; The nanozyme catalytic solution was mixed with ABTS solution and hydrogen peroxide solution in a certain proportion to form a blank reaction solution; The test sample containing glutathione was added to the blank reaction solution, mixed thoroughly, and then subjected to a colorimetric reaction to obtain the test reaction solution. The absorbance of the blank reaction solution and the test reaction solution was measured in the wavelength range of 400 nm to 550 nm. The content of glutathione in the sample to be tested is determined based on the absorbance difference between the blank reaction solution and the test reaction solution. The ABTS solution concentration is 5-15 mmol / L, and the amount added in each 400 μL total reaction volume is 30-50 μL. The concentration of the hydrogen peroxide solution is 40-60 mmol / L, and the amount added is 15-25 μL per 400 μL of total reaction volume; The amount of the nanozyme catalytic solution added is 15-50 μL per 400 μL total reaction volume.

[0008] According to another aspect of the present invention, the content of glutathione in the sample to be tested is determined by obtaining the RGB colorimetric value of the reaction solution to be tested and calculating the colorimetric I value based on the RGB colorimetric value of the reaction solution to be tested. The chromaticity I value is calculated using the formula I = 0.3R + 0.59G + 0.11B, where R, G, and B are the values ​​of the red, green, and blue channels of the photograph of the reaction solution to be tested, respectively.

[0009] Preferably, the nanozyme catalytic solution is a Prussian blue solution, which is prepared by mixing a ferric chloride solution with a concentration of 1-500 μmol / L with a potassium ferrocyanide solution of the same concentration range at a volume ratio of 1:1 to 4:3; the amount of the nanozyme catalytic solution added to every 400 μL of total reaction volume is 15-25 μL.

[0010] Preferably, the nanozyme catalytic solution is a Tunchausen blue solution, which is prepared by mixing a ferrous chloride solution with a concentration of 1-500 μmol / L with a potassium ferricyanide solution of the same concentration range at a volume ratio of 1:2 to 3:2; the amount of the nanozyme catalytic solution added to every 400 μL of total reaction volume is 15-25 μL.

[0011] Preferably, the nanozyme catalytic solution is a ferrous chloride solution or a ferric chloride solution, with a concentration of 1-50 μmol / L, and the amount added in every 400 μL of total reaction volume is 30-50 μL.

[0012] Preferably, the reaction temperature for the colorimetric reaction is 20-50℃, and the reaction time is 1-30 min.

[0013] Preferably, the absorbance is the value measured at a wavelength of 418 nm for the reaction solution.

[0014] Preferably, the step of determining the glutathione content in the sample to be tested includes: Plot a first standard curve between the concentration of the glutathione standard solution and the difference in absorbance, or plot a second standard curve between the concentration of the glutathione standard solution and the colorimetric I value; The absorbance difference or colorimetric I value of the sample to be tested is substituted into the first standard curve or the second standard curve to calculate the glutathione content.

[0015] The present invention also provides a kit for the above-described enhanced colorimetric method, comprising a nanozyme catalytic solution component, an ABTS solution component, and a hydrogen peroxide solution component; The nanozyme catalytic solution is composed of one of Prussian blue solution, Tönnies blue solution, ferrous chloride solution, or ferric chloride solution. The concentration of the ABTS solution component is 5-15 mmol / L, and the preparation volume is 30-50 μL added to every 400 μL of total reaction volume; The concentration of the hydrogen peroxide solution component is 40-60 mmol / L, and the amount used is 15-25 μL added to every 400 μL of total reaction volume.

[0016] Preferably, the nanozyme catalytic solution is a Prussian blue solution, and the Prussian blue solution contains a stabilizer; the stabilizer is a mixture of trehalose and polyvinylpyrrolidone; wherein the concentration of trehalose in the Prussian blue solution is 5-20 g / L, and the concentration of polyvinylpyrrolidone in the Prussian blue solution is 1-5 g / L; the amount of Prussian blue solution containing the stabilizer added is 15-25 μL per 400 μL of total reaction volume.

[0017] The present invention has at least the following beneficial effects: This invention achieves "enhanced" colorimetric detection of glutathione by constructing a reaction system with a nanozyme catalytic solution composed of Prussian blue, Thunberg's blue, ferrous chloride, or ferric chloride solution, and an ABTS solution and a hydrogen peroxide solution. By utilizing the reducing properties of glutathione, in the colorimetric reaction of ABTS oxidized by nanozyme-catalyzed hydrogen peroxide oxidation, glutathione does not inhibit but significantly enhances the colorimetric signal. The mechanism of this enhancement lies in the fact that glutathione can act as an electron donor, effectively regulating or regenerating the valence state of iron in the active center of the nanozyme, accelerating the catalytic cycle of iron ions between Fe²⁺ and Fe³⁺, thereby increasing the peroxidase-like activity of the nanozyme. This leads to more ABTS being oxidized into ABTS cation radicals with strong absorption near 418 nm, resulting in a deeper solution color and increased absorbance. This mechanism, based on the enhancement of catalytic activity by reducing substances, differs from the behavior of common substances such as ascorbic acid and cysteine, thus endowing this method with high selectivity for glutathione detection.

[0018] This invention enhances the catalytic reaction of nanozymes with GSH to form a deeper-colored ABTSox by ABTS and H2O2. Leveraging the positive correlation between ABTSox and GSH content, a convenient and effective enhanced colorimetric method for GSH detection is developed. The reagents are simple and readily available, the operation is straightforward, requiring no complex procedures such as heating, the experiment is easily reproducible, the reaction time is short, and it can be performed under simple conditions, significantly reducing reagent, time, and equipment costs. The enhanced colorimetric method has a wide linear range. Most common antioxidants such as ascorbic acid and cysteine, as well as thiol molecules and amino acids, do not enhance the colorimetric reaction of ABTS by nanozymes and do not interfere with GSH detection.

[0019] In this kit, the addition of a Prussian blue solution stabilized by a mixture of trehalose and polyvinylpyrrolidone (PVP) effectively prevents the aggregation and sedimentation of Prussian blue nanoparticles. This significantly improves the stability of the nanozyme catalytic solution during storage, ensuring the consistency of the kit's shelf life and detection performance. In summary, this technical solution, by constructing a specific enhanced catalytic colorimetric system and optimizing its components and conditions, achieves highly selective, wide linear range, safe, rapid, stable, and cost-effective quantitative detection of glutathione.

[0020] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the principle of the enhanced colorimetric method for rapid and selective detection of glutathione according to the present invention. Figure 2 This is an illustration of the selection of metal types for Prussian blue, Tönnies blue and other types of Prussian blue in this invention. Prussian blue is abbreviated as PB. Figure 3 The image shows the ultraviolet absorption spectrum of Embodiment 3 of the present invention. In the figure, a is a mixed solution of [ABTS+PB], b is a mixed solution of [ABTS+H2O2+GSH], c is a mixed solution of [ABTS+H2O2], d is a mixed solution of [ABTS+H2O2+PB], and e is a mixed solution of [ABTS+H2O2+PB+GSH].

[0022] Figure 4 This is a schematic diagram of the UV-Vis absorption spectrum and corresponding solution photographs for the quantitative detection of GSH by spectroscopic method in Example 4 of the present invention. In the figure, A and Q are mixed solutions of [ABTS+H2O2+PB] with added GSH at concentrations of 0, 6.25 nM, 12.5 nM, 31.25 nM, 62.5 nM, 0.625 μM, 1.25 μM, 3.125 μM, 6.25 μM, 12.5 μM, 18.75 μM, 25 μM, 31.25 μM, 43.75 μM, 62.5 μM, 125 μM, and 625 μM, respectively. Figure 5 This is a schematic diagram of the relationship between absorbance and concentration in Example 5 of the present invention; Figure 6 This is a schematic diagram of the relationship between RGB values ​​and concentration in Embodiment 6 of the present invention; Figure 7 This is an interference test of the reaction system in Example 7 of the present invention; Figure 8 The graphs show the color development and absorbance bars of Prussian blue nanozyme solutions with and without stabilizers in the reaction system, indicating the presence or absence of GSH. Figure 9 Bar charts showing the absorbance of different solvents with and without GSH; Figure 10 A standard curve plotting RGB values ​​versus GSH concentrations for samples of pig liver, pig blood, pork, chicken liver, and spinach; Figure 11 The Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) spectra of PB are shown. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0024] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0025] Based on extensive practical research, this invention provides an enhanced colorimetric method for the rapid and selective detection of glutathione. This detection method enhances the colorimetric development of ABTS by catalyzing the oxidation of Prussian blue, Thunberg blue, ferrous chloride, and ferric chloride solutions with H2O2 through GSH, establishing an enhanced colorimetric method to achieve quantitative analysis of GSH, thereby effectively improving the detection range, sensitivity, and selectivity.

[0026] The first aspect of this invention provides an enhanced colorimetric method for rapid and selective detection of glutathione, comprising the following steps: Prepare Prussian blue, Tönnies blue, ferrous chloride solution or ferric chloride solution as nanozyme catalytic solution; The nanozyme catalytic solution was mixed with ABTS solution and hydrogen peroxide solution in a certain proportion to form a blank reaction solution; The test sample containing glutathione was added to the blank reaction solution, mixed thoroughly, and then subjected to a colorimetric reaction to obtain the test reaction solution. The absorbance of the blank reaction solution and the test reaction solution were measured in the wavelength range of 400 nm to 550 nm, respectively, or the RGB colorimetric value of the test reaction solution was obtained. The content of glutathione in the sample to be tested is determined based on the absorbance difference between the blank reaction solution and the test reaction solution, or based on the chromaticity I value calculated from the RGB chromaticity value of the test reaction solution. The chromaticity I value is calculated using the formula I = 0.3R + 0.59G + 0.11B, where R, G, and B are the values ​​of the red, green, and blue channels of the photograph of the reaction solution to be tested, respectively. The ABTS solution concentration is 5-15 mmol / L, and the amount added in each 400 mL total reaction volume is 30-50 μL. The concentration of the hydrogen peroxide solution is 40-60 mmol / L, and the amount added is 15-25 μL per 400 μL of total reaction volume; The amount of the nanozyme catalytic solution added is 15-50 μL per 400 μL total reaction volume.

[0027] The enhanced colorimetric method for rapid and selective detection of glutathione provided by this invention may include: mixing Prussian blue with ABTS and H2O2 solutions to obtain a blank solution; adding GSH standard solutions of different concentrations to the blank solution; measuring the absorbance of the blank solution and the GSH standard solution using a UV-Vis spectrophotometer; and taking a photo of the GSH standard solution with a mobile phone to obtain the R, G, and B values ​​of the photo's colorimetry. Based on the absorbance difference and colorimetry R, ​​G, and B values ​​of the GSH standard solution and the blank solution provided in the above steps, a relationship curve is plotted between the absorbance difference and colorimetry R, ​​G, and B values ​​and the GSH concentration. The GSH content in the sample to be tested is then determined based on the absorbance difference and colorimetry R, ​​G, and B values ​​of the sample to be tested.

[0028] In the enhanced colorimetric method for rapid and selective detection of glutathione described above, the step of providing the content of GSH in the test solution can be a qualitative detection, i.e., providing whether the test sample contains GSH, or it can be a quantitative detection, i.e., providing the specific content of GSH in the test sample.

[0029] In the above-mentioned enhanced colorimetric method for rapid and selective detection of glutathione, the concentrations of FeCl3 solution and K4[Fe(CN)6] solution can be 1 μM to 5 μM, 5 μM to 10 μM, 10 μM to 50 μM, 50 μM to 100 μM, 500 μM to 500 μM, or higher.

[0030] In the enhanced colorimetric method for rapid and selective detection of glutathione described above, Prussian blue solution is prepared by uniformly mixing FeCl3 solution and K4[Fe(CN)6] solution at a volume ratio of 4:3. Alternatively, FeCl3 solution and K4[Fe(CN)6] solution can be mixed at a volume ratio of 1:1 or other molar ratios.

[0031] In the enhanced colorimetric method described above for rapid and selective detection of glutathione, the reagents and reaction system are all carried out in ultrapure water. Commonly used solvents in existing technologies are typically weakly acidic or neutral buffer solutions, such as acetate-sodium acetate buffer or phosphate buffer. Furthermore, the pH of the reaction system can be 4.4–4.6, 4.6–4.8, 4.8–5.0, 5.0–5.5, 5.5–6.0, 6.0–6.5, or 6.5–7.0.

[0032] In the enhanced colorimetric method described above for rapid and selective detection of glutathione, the GSH content in the sample to be tested is typically within the range of 6.25 nM to 625 μM, 6.25 nM to 10 nM, 10 nM to 50 nM, 50 nM to 100 nM, 100 nM to 500 nM, 500 nM to 1 μM, 1 μM to 10 μM, 10 μM to 50 μM, 50 μM to 100 μM, 100 μM to 500 μM, 100 μM to 625 μM, or a better range.

[0033] The nanozyme catalytic solution is a Tönnies blue solution (FeCl2 and K3[Fe(CN)6] mixed in a molar ratio of 1:2 or 3:2), or a mixture of MnCl2, CoCl2, NiCl2 and K3[Fe(CN)6] in a volume ratio of 3:2 to generate a Prussian blue-like solution, or a FeCl2 solution or a FeCl3 solution.

[0034] In some embodiments of the present invention, the colorimetric reagent is 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) (abbreviated as ABTS), and an ABTS solution of 10 mmol / L is prepared using ultrapure water as the solvent. 30% H2O2 is taken and diluted with ultrapure water to prepare a 50 mmol / L H2O2 solution.

[0035] In some embodiments of the present invention, an appropriate amount of GSH standard is taken and prepared into GSH solutions of different concentrations using ultrapure water as a solvent.

[0036] Suitable solvents are typically weakly acidic or neutral buffer solutions, such as sodium acetate buffer or phosphate buffer.

[0037] A second aspect of this invention provides a glutathione detection kit, applicable to the enhanced colorimetric method for rapid and selective detection of glutathione provided in the first aspect of this invention. The kit typically includes nanozyme catalytic solution components, ABTS solution components, and hydrogen peroxide solution components. The nanozyme catalytic solution is composed of one of Prussian blue solution, Tönnies blue solution, ferrous chloride solution, or ferric chloride solution. The concentration of the ABTS solution component is 5-15 mmol / L, and the preparation volume is 30-50 μL added to every 400 μL of total reaction volume; The concentration of the hydrogen peroxide solution component is 40-60 mmol / L, and the amount used is 15-25 μL added to every 400 μL of total reaction volume.

[0038] In some embodiments of the present invention, the nanozyme catalytic solution is a Prussian blue solution, and the Prussian blue solution contains a stabilizer; the stabilizer is a mixture of trehalose and polyvinylpyrrolidone; wherein the concentration of trehalose in the Prussian blue solution is 5-20 g / L, and the concentration of polyvinylpyrrolidone in the Prussian blue solution is 1-5 g / L; the amount of Prussian blue solution containing the stabilizer added is 15-25 μL per 400 μL of total reaction volume.

[0039] The enhanced colorimetric method and related detection kit for rapid and selective detection of glutathione provided by this invention involve the generation of a colorimetric signal by the oxidation of glutathione (ABTS) by H2O2 catalyzed by low concentration of Prussian blue promoted by GSH. The color gradually deepens with increasing concentration of added GSH. The G, R, and B values ​​of the colorimetry are obtained by measuring absorbance and taking photographs to achieve quantitative determination of GSH. The detection linear range of this method is 12.5 nM to 62.5 μM, and the detection limit can reach 6.5 nmol / L. It has the characteristics of low detection limit, wide linear range, and high selectivity, and has broad application prospects in food detection, medical diagnosis and testing, and has good industrialization prospects.

[0040] The present invention will be further illustrated by the following examples, but these examples do not limit the scope of protection of the present invention.

[0041] Example 1 Preparation of Prussian Blue (PB): A 10 mM ferric chloride stock solution and a 10 mM potassium ferrocyanide stock solution were prepared using ultrapure water as the solvent. These stock solutions were then serially diluted with ultrapure water to prepare 100 μM ferric chloride working solutions and 100 μM potassium ferrocyanide working solutions, respectively. The 100 μM ferric chloride working solution and the 100 μM potassium ferrocyanide working solution were mixed thoroughly at room temperature at a volume ratio of 4:3 to obtain the Prussian Blue (PB) solution.

[0042] Preparation of Thunbyl Blue (TB): 10 mM ferrous chloride and 10 mM potassium ferricyanide solutions were prepared separately using ultrapure water as the solvent. These solutions were then serially diluted to prepare 100 μM ferrous chloride and 100 μM potassium ferricyanide solutions. The 100 μM ferrous chloride and 100 μM potassium ferricyanide solutions were then mixed at a volume ratio of 3:2 to prepare the TB solution. This mixing process can be carried out at room temperature.

[0043] Preparation of PB(Ni): 10 mM nickel chloride and 10 mM potassium ferricyanide solutions were prepared using ultrapure water as the solvent. These solutions were then serially diluted to prepare 100 μM nickel chloride and 100 μM potassium ferricyanide solutions. The 100 μM nickel chloride and 100 μM potassium ferricyanide solutions were then mixed at a volume ratio of 3:2 to prepare the PB(Ni) solution. This mixing process can be carried out at room temperature.

[0044] Preparation of PB(Co): 10 mM cobalt chloride and 10 mM potassium ferricyanide solutions were prepared using ultrapure water as the solvent. These solutions were then serially diluted to prepare 100 μM cobalt chloride and 100 μM potassium ferricyanide solutions. The 100 μM cobalt chloride and 100 μM potassium ferricyanide solutions were then mixed at a volume ratio of 3:2 to prepare the PB(Co) solution. This mixing process can be carried out at room temperature.

[0045] Preparation of PB(Mn): 10 mM manganese chloride and 10 mM potassium ferricyanide solutions were prepared using ultrapure water as the solvent. These solutions were then serially diluted to prepare 100 μM manganese chloride and 100 μM potassium ferricyanide solutions. The 100 μM manganese chloride and 100 μM potassium ferricyanide solutions were then mixed at a volume ratio of 3:2 to prepare the PB(Mn) solution. This mixing process can be carried out at room temperature.

[0046] Example 2 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution were placed in centrifuge tubes, respectively. 320 μL of ultrapure water and 320 μL of 62.5 μmol / L GSH solution were added to each tube, followed by 20 μL of Prussian blue, Thunb. blue, PB(Ni), PB(Co), and PB(Mn) solutions (prepared in Example 1). The mixtures were thoroughly mixed and reacted in a 35°C water bath for 10 min. The UV absorption spectra of the mixed solutions in the 400-550 nm range were then measured using a UV-Vis spectrophotometer. The absorption spectra of the sample solutions with and without added GSH are shown below. Figure 2 As shown. By Figure 2 (PB), (TB), (PB(Ni)), (PB(Co)), (PB(Mn)) show that in the system containing PB, the absorbance measured at 418 nm is the largest difference between the absorbance measured with and without GSH in the sample solution with added GSH.

[0047] Take 40 μL of 10 mM ABTS solution and 32 μL of 50 mM H2O2 solution into centrifuge tubes, respectively. Add 40 μL of ultrapure water and 40 μL of 0.1 mM GSH solution. Then add 40 μL of solutions of 1 mM ferric chloride, 1 mM ferrous chloride, 1 mM copper chloride, 1 mM aluminum chloride, 1 mM chromium trichloride, 1 mM cadmium chloride, 10 mM cobalt chloride, 10 mM manganese chloride, 10 mM magnesium chloride, 10 mM nickel chloride, and 10 mM zinc chloride, respectively. Mix well. Make up the reaction volume to 400 μL with ultrapure water. After reacting at room temperature for 25 min, measure the UV absorption spectrum of the above mixed solution in the 400-550 nm range using a UV-Vis absorption spectrophotometer. The absorption spectra of the sample solutions with and without added GSH are shown below. Figure 2 As shown in (M). By Figure 2 (M) indicates that in the system containing ferrous chloride, the absorbance difference at 418 nm is greatest between the sample solution with added GSH and the sample solution without added GSH. Similarly, in the ferric chloride system, the absorbance difference at 418 nm between the sample solution with added GSH and the sample solution without added GSH is also significant.

[0048] Depend on Figure 2 It is evident that the presence of iron ions in the system and the valence state of iron ions are also key factors in this method.

[0049] Example 3 Take 40 μL of 10 mM ABTS solution and 20 μL of Prussian blue (prepared in Example 1) into a centrifuge tube, add 340 μL of ultrapure water, mix and shake for 10 s, react in a 35°C water bath for 10 min, and then measure the UV absorption spectrum of the above mixed solution in the 400-550 nm range using a UV-Vis absorption spectrophotometer. Figure 3 As shown in curve a.

[0050] Take 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution into centrifuge tubes, respectively, add 340 μL of 62.5 μmol / L GSH solution, mix and shake for 10 s, react in a 35℃ water bath for 10 min, and then measure the UV absorption spectrum of the above mixed solution in the 400-550 nm range using a UV-Vis absorption spectrophotometer. Figure 3 As shown in curve b.

[0051] <c>Take 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution into centrifuge tubes, add 340 μL of ultrapure water, mix and shake for 10 s, react in a 35℃ water bath for 10 min, and then measure the UV absorption spectrum of the above mixed solution in the 400-550 nm range using a UV-Vis absorption spectrophotometer. Figure 3 As shown in curve c.

[0052] <d>Take 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution into centrifuge tubes, add 20 μL of Prussian blue (prepared in Example 1) and 320 μL of ultrapure water, mix and shake for 10 s, react in a 35°C water bath for 10 min, and then measure the UV absorption spectrum of the above mixed solution in the 400-550 nm range using a UV-Vis absorption spectrophotometer. Figure 3 As shown by curve d.

[0053] <e>Take 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution into centrifuge tubes, respectively. Add 20 μL of Prussian blue (prepared in Example 1) and 320 μL of 62.5 μmol / L GSH solution, mix and shake for 10 s. After reacting in a water bath at 35℃ for 10 min, measure the UV absorption spectrum of the above mixed solution in the 400-550 nm range using a UV-Vis absorption spectrophotometer. Figure 3 As shown by curve e.

[0054] Depend on Figure 3 It can be seen that when only ABTS and Prussian blue (prepared in Example 1) are present in the system, the absorbance of the solution is weak. Only when Prussian blue (prepared in Example 1), ABTS, and H2O2 are added does the result show a more obvious absorption peak. However, when GSH solution is added, the system shows a strong absorption peak at 418 nm.

[0055] Example 4 PB solution was prepared by mixing 100 μM ferric chloride and 100 μM potassium ferrocyanide solutions at a volume ratio of 4:3 at room temperature.

[0056] 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution were respectively placed in centrifuge tubes. 320 μL of GSH standard solutions of different concentrations were added, followed by 20 μL of PB solution (obtained in the previous step). The mixtures were thoroughly mixed and reacted at room temperature for 5 min. The UV absorption spectra of the above mixed solutions in the 400-550 nm range were then measured using a UV-Vis absorption spectrophotometer. The UV absorption spectra and corresponding solution photographs obtained from the measurements of the standard samples with different GSH concentrations are shown below. Figure 4 As shown.

[0057] Example 5 From the UV absorption spectrum of Example 4 above, the difference between the absorbance of standard samples with different GSH concentrations (0~625 μM) measured at 418 nm and the absorbance of the solution without GSH (ΔA = A - A0, where A is the solution with added GSH and A0 is the solution without GSH) and the concentration curve results are as follows: Figure 5 As shown. The concentration was determined within a detection range of 0 μmol / L - 625 μmol / L. Figure 5 It can be seen that there is a good linear relationship between the GSH concentration in the standard (12.5 nM~62.5 μM) and the measured absorbance difference, and the detection limit can reach 6.5 nmol / L.

[0058] Example 6 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution were respectively added to 96-well plates. 320 μL of GSH standard solutions of different concentrations were added, followed by 20 μL of PB solution (Example 4). The mixture was thoroughly mixed and reacted at room temperature for 5 min. This was repeated three times in parallel. The plates were placed in a dark chamber, and an LED white light was turned on. The solutions in the 96-well plates were photographed using a mobile phone, and the chromaticity values ​​(R, G, B) of the photographs were obtained. The I value was calculated using the chromaticity value relationship I = 0.3R + 0.59G + 0.11B. Figure 6 It can be seen that there is a good linear relationship between the GSH concentration (12.5 nM~62.5 μM) in the standard sample and the chromaticity I value.

[0059] Example 7 Example 7 aims to verify the selectivity of the detection method for glutathione (GSH) detection, i.e., to assess whether other potentially coexisting substances would interfere with the determination of GSH. The specific experimental procedures are as follows: 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution were placed in a 96-well plate, followed by the addition of 20 μL of Prussian blue solution (as a nanozyme catalyst, prepared in Example 1). Next, 320 μL of solutions of different potential interfering substances were added to each well. These substances included: 5... Sodium sulfite (Na₂SO₃), sodium nitrite (NaNO₂), sodium chloride (NaCl), fructose (Fru), tannic acid (TA), sucrose (Suc), tea polyphenols (GTP), caffeic acid (CA), gallic acid (GA), resveratrol (Res), arginine (Arg), cysteine ​​(Cys), cystine (Cys₂), histidine (His), phenylalanine (Phe), leucine (Leu), glucose (Glu), propyl gallate (PG), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), ascorbic acid (Vc). Hydroquinone (HQ); 1 mM of glycine (Gly), tryptophan (Trp), threonine (Thr), valine (Val), isoleucine (Ile), alanine (Ala), serine (Ser), citrulline (Cit), glutamine (Gln), proline (Pro), hydroxyproline (Hyp), lysine (Lys), methionine (Met), tyrosine (Tyr), magnesium chloride (MgCl2), and catechol (CT); and 100 µM of aspartic acid (Asp), tartaric acid, and glutamate (Glu); and 10 µM of oxidized glutathione (GSSG). Two controls were also included: a blank control (320 μL of ultrapure water) and a positive control (320 μL of 35 µM GSH solution). All reaction systems were mixed thoroughly at room temperature and reacted for 5 minutes. The absorbance of each well was then measured at 418 nm using a microplate reader. The degree of interference of each substance was assessed by calculating the absorbance difference between the solution containing the potential interfering substance and the blank control solution, and comparing it with the absorbance difference caused by the positive control (GSH). Experimental results are as follows: Figure 7 As shown, among the 42 substances tested, most substances, including common antioxidants (such as ascorbic acid, vitamin C), amino acids (such as cysteine, arginine, etc.), and sugars, did not cause significant changes in absorbance. Although aspartic acid, tartaric acid, glutamic acid, and oxidized glutathione (GSSG) also showed enhanced color development, their color development effect was significantly weaker than that of GSH at the same concentration. By experimentally determining a reasonable absorbance difference threshold, GSH can be effectively distinguished from these interfering substances. The above results indicate that this detection method has good selectivity for GSH relative to many common components in biological samples. When the aforementioned few interfering substances are known and controlled, it can be effectively applied to the detection of GSH in complex samples such as food and medicine. The nanozyme catalyst used in this example is pre-synthesized Prussian blue, the preparation method of which is detailed in Example 1.

[0060] In summary, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0061] Example 8 A kit for detecting glutathione was prepared using Prussian blue (PB) solution as a nanozyme catalytic solution, prepared according to the method in Example 1. The test was conducted at room temperature (25°C), simulating a routine laboratory testing environment. The performance of the kit was tested immediately after preparation.

[0062] Test method: 1. Reagent preparation: Nanozyme catalytic solution: Prussian blue (PB) solution was freshly prepared according to the method described in Example 1.

[0063] ABTS solution: Prepare an ABTS solution with a concentration of 10 mmol / L using ultrapure water.

[0064] Hydrogen peroxide solution: Dilute 30% H2O2 with ultrapure water to a concentration of 50 mmol / L.

[0065] GSH standard solution: A GSH standard working solution with a concentration of 20.0 μmol / L was precisely prepared using ultrapure water and used as the sample to be tested.

[0066] 2. Testing steps: Add 40 μL of ABTS solution (10 mmol / L), 20 μL of H2O2 solution (50 mmol / L), and 20 μL of Prussian blue (PB) solution to a centrifuge tube. Add 320 μL of 20.0 μmol / L GSH standard working solution to the mixture, vortex until homogeneous, and react at room temperature for 5 minutes. Simultaneously, perform a blank experiment using ultrapure water instead of the GSH standard solution.

[0067] The absorbance values ​​(A and A0) of the test reaction solution and the blank reaction solution at 418 nm were measured using a UV-Vis spectrophotometer, and the absorbance difference ΔA = A - A0 was calculated.

[0068] 3. Results and Calculations: To assess the reliability of the method, we conducted three independent parallel experiments.

[0069] The absorbance differences (ΔA) measured in three parallel experiments were 0.268, 0.275, and 0.271, respectively.

[0070] The calculated average GSH concentration was 19.56 μmol / L, with a standard deviation (SD) of 0.32 μmol / L.

[0071] Compared with the true value (20.0 μmol / L), the relative error was -2.2%, and the recovery rate was 97.8%.

[0072] The specific calculation process is as follows: The linear regression equation of the standard curve used for quantitative calculation, established according to Example 5, is: y = 0.05301 +0.01116x (where y is ΔA and x is the GSH concentration in μmol / L). Substitute the three measured ΔA values ​​into the formula to calculate the concentration respectively: X1= (0.268 - 0.05301) / 0.01116 ≈ 19.26 μmol / L; X2= (0.275 - 0.05301) / 0.01116 ≈ 19.89 μmol / L; X3= (0.271 - 0.05301) / 0.01116 ≈ 19.53 μmol / L; Average concentration = (19.26 + 19.89 + 19.53) / 3 ≈ 19.56 μmol / L; Standard deviation (SD) ≈ 0.32 μmol / L; Recovery rate = (19.56 / 20.0) × 100% = 97.8%; 4. Results: The results of this experiment show that the kit of the present invention achieves a recovery rate of 97.8% for the detection of 20.0 μmol / L GSH standard solution, and the relative standard deviation (RSD) of three parallel determinations is 1.64% (RSD = SD / mean value × 100% = 0.32 / 19.56 × 100%), demonstrating good accuracy and precision. This demonstrates the reliability of the method and kit of the present invention in practical detection. It should be noted that freshly prepared PB solution was used in this experiment. For the long-term stability of commercial kits, please refer to the evaluation results of Examples 9 and 10.

[0073] Example 9 To verify the effect of stabilizer on the color development of Prussian blue (PB) nanozyme solution, the test method is as follows: 1. Reagent preparation: Preparation of PB working solution: Prussian blue (PB) solution was prepared according to the method in Example 1 (i.e., prepared by mixing 100 μM FeCl3 and 100 μM K4[Fe(CN)6] in a volume ratio of 4:3). This solution was directly used as the reference PB working solution for this stability test.

[0074] Preparation of stabilized PB solution: Take four 5 mL portions of PB and place them in 10 mL centrifuge tubes. Process as follows: Group A (the present invention group): Add 50mg trehalose and 10mg PVPK30, vortex until completely dissolved, and mix evenly.

[0075] Group B (Single Trehalose Group): Only 50 mg of trehalose was added and dissolved by vortexing.

[0076] Group C (single PVP group): Add only 10mg PVPK30 and dissolve by vortex.

[0077] Group D (blank control group): No stabilizers were added.

[0078] All groups were tested for colorimetric appearance and absorbance under the condition of "with or without 50 μM GSH". The reaction conditions were room temperature, 5 min, and a total reaction volume of 400 μL (40 μL ABTS + 20 μL H2O2 + 20 μL PB solution + 320 μL water / GSH). 2. Test and experimental conditions: ① Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, and 20 μL of PB(A). 320 μL of water.

[0079] ② Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB(A), and 320 μL of 50 μM GSH.

[0080] ③ Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB (B), and 320 μL of water.

[0081] ④ Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB (B), and 320 μL of 50 μM GSH.

[0082] ⑤ Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB(C), and 320 μL of water.

[0083] ⑥ Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB(C), and 320 μL of 50 μM GSH.

[0084] ⑦ Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB(D), and 320 μL of water.

[0085] ⑧ Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB(D), and 320 μL of 50 μM GSH.

[0086] 3. Test the color development results and absorbance on the day of preparation, as follows: Figure 8 As shown: 1) Color and Appearance Comparison: All groups showed blue under the "with GSH" condition, but there were some differences in appearance: Group A (mixed stabilizer): The solution is uniform and transparent, with a consistent blue color depth, no turbidity or layering, and good color stability; Group B (trehalose only): The solution is clear and transparent, with a blue depth similar to Group A, but its uniformity is slightly inferior to Group A (slight concentration fluctuations visible to the naked eye). Group C (PVP only): The solution is clear, but the blue color is significantly paler, and its color intensity is lower than that of Groups A and B, which can be directly distinguished by the naked eye. Group D (without stabilizer): The solution is clear, and the blue color depth is similar to that of Groups A and B, but the stability is poor without the addition of a stabilizer.

[0087] 2) Absorbance difference analysis uses "absorbance with GSH and absorbance without GSH" as the core indicator (reflecting the color enhancement effect of GSH): Group A: The absorbance difference is the largest (Figure 8 shows that the absorbance of "with added GSH" is significantly higher than that of Groups B and C, and also better than that of Group D), indicating that the mixed stabilizer does not interfere with the enhancing effect of GSH on the catalytic activity of PB, and because the PB nanoparticles are uniformly dispersed, the catalytic efficiency is stable and the color signal is the strongest. Group B: The absorbance difference is slightly lower than that of Group A, indicating that trehalose alone can maintain the catalytic activity of PB, but lacks the steric hindrance effect of PVP. PB particles have a slight tendency to aggregate, resulting in a slight decrease in catalytic efficiency. Group C: The absorbance difference is the smallest, which is consistent with the phenomenon of "pale blue". This is because PVP alone is not enough to protect the dispersion of PB and there is no solubilizing effect of trehalose. The catalytic activity of PB is weakened and the enhancement effect of GSH cannot be fully reflected. Group D: The absorbance difference is similar to that of Group B, but the absence of a stabilizer means that PB particles are prone to agglomeration in the future (see test results in Example 10), and the performance is acceptable only in the "instant color development" stage.

[0088] The test results show that the mixed stabilizer of trehalose and PVP can significantly improve the color uniformity and signal intensity of PB nanozyme solution without interfering with the GSH-enhanced color development mechanism. Its synergistic effect (trehalose solubilization + PVP dispersion) is better than that of a single stabilizer. A single PVP stabilizer will weaken the catalytic activity of PB, resulting in a lighter color development, which does not meet the requirements of the detection system for "color development intensity". The PB solution (Group A) containing mixed stabilizers fully meets the requirements of the detection system of this invention in the "instant color development" stage.

[0089] Example 10 Stability study of Prussian blue nanozyme solution containing stabilizer, test method: 1. Reagent preparation: Preparation of PB working solution: Prussian blue (PB) solution was prepared according to the method in Example 1 (i.e., prepared by mixing 100 μM FeCl3 and 100 μM K4[Fe(CN)6] in a volume ratio of 4:3). This solution was directly used as the reference PB working solution for this stability test.

[0090] Preparation of stabilized PB solution: Take four 10 mL portions of the above PB working solution and place them in 15 mL centrifuge tubes. Process them as follows: Sample A (the present invention group): Add 100 mg of trehalose (final concentration 10 g / L) and 20 mg of PVP K30 (final concentration 2 g / L), vortex until completely dissolved, and mix evenly.

[0091] Comparative Example B (Single Trehalose Group): Only 100 mg of trehalose (final concentration 10 g / L) was added and dissolved by vortexing.

[0092] Comparative Example C (single PVP group): only 20 mg PVP K30 (final concentration 2 g / L) was added and dissolved by vortexing.

[0093] Comparative Example D (blank control group): No stabilizers were added.

[0094] All sample solutions should be sealed and stored.

[0095] 2. Accelerated aging test and testing conditions: All the sample tubes were placed in a constant temperature incubator at 40℃ (±0.5℃) for accelerated aging tests to simulate the effect of long-term storage at room temperature.

[0096] Samples were taken on day 0 (initial), day 7, and day 14 for testing. Before each test, the sample tubes were removed from the oven, cooled to room temperature (25°C), and then gently vortexed for 30 seconds to ensure sample homogeneity.

[0097] 3. Stability assessment indicators and specific testing methods: Catalytic activity assessment: The detection method was strictly followed in Example 4. 20 μL of the PB solution to be tested at each time point was taken and added to a system containing 40 μL of 10 mM ABTS, 20 μL of 50 mM H2O2, and 320 μL of 20 μM GSH standard solution.

[0098] After reacting at 25°C for 5 minutes, the absorbance at 418 nm was immediately measured using a UV-Vis spectrophotometer (A). A blank value (A0) was determined using ultrapure water instead of the GSH standard solution.

[0099] Calculate the absorbance difference ΔA = A - A0. Using the ΔA value of each group of samples on day 0 as a baseline (100%), calculate the catalytic activity retention rate at subsequent time points. Each sample was measured in triplicate, and the average value was taken.

[0100] Physical stability assessment: Visual observation: Under a pure white background and the same lighting conditions, observe and record whether blue precipitate appears in each sample solution, and whether there are changes in solution color uniformity or transparency.

[0101] Particle size measurement: Dynamic light scattering (DLS) nanoparticle size analyzer was used to perform three parallel measurements on each sample. The average hydrated particle size (Z-Average, d.nm) and polydispersity index (PDI) were recorded to accurately quantify the degree of nanoparticle aggregation. The measurement results are shown in Table 1.

[0102] Table 1: The experimental results show that the PB solution in the blank control group (D) has extremely poor stability. After 14 days, it loses about 60% of its activity and undergoes severe aggregation and sedimentation. This indicates that the aggregation problem of Prussian blue nanoparticles affects its use.

[0103] The single stabilizer groups (B and C) improved stability to some extent, but the effect was limited. Trehalose (B) mainly provided long-term thermodynamic stability, but was insufficient in physically inhibiting initial aggregation; PVP (C) provided immediate steric hindrance, but its long-term protective effect was poor. After 14 days, the activity retention rate of both was significantly lower than 80%, and significant aggregation occurred.

[0104] The present invention group (A) exhibits a significant synergistic enhancement effect. The combination of trehalose and PVP simultaneously provides long-term stability protection (trehalose) and immediate steric stabilization (PVP). After 14 days of accelerated aging, the catalytic activity retention rate is still over 95%, and the solution remains uniform and clear throughout, with only a slight increase in particle size and PDI, demonstrating excellent stability.

[0105] Example 11 To verify whether the buffer solution is suitable for the GSH-enhanced detection system of this invention, the following test method was used: (Comparison of "water system" and "acetate buffer systems with different pH values"). 1. Reagent preparation: Preparation of PB working solution: Prussian blue (PB) solution was prepared according to the method in Example 1 (i.e., prepared by mixing 100 μM FeCl3 and 100 μM K4[Fe(CN)6] in a volume ratio of 4:3).

[0106] 2. Test and experimental conditions: Water system: 2 groups (without GSH: 40μL ABTS + 20μL H2O2 + 20μL PB + 320μL water; with GSH: 320μL replaced with 50μM GSH); Buffer systems: 5 groups (acetate buffers at pH 3.6, 4.0, 4.4, 4.8, and 5.2; the amounts of other reagents are the same as in the water system, except that "water / water + GSH" is replaced with "buffer solution / buffer solution + GSH"). The reaction conditions were room temperature and 5 min. The absorbance at 418 nm was measured, and the core indicator was "absorbance with GSH - absorbance without GSH" (ΔA).

[0107] Water system 1: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB, and 320 μL of water, and react at room temperature for 5 minutes.

[0108] Water system 2: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of PB, 320 μL of water, and 20 μL of 50 μM GSH, and react at room temperature for 5 minutes.

[0109] Buffer system 3: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, and 320 μL of pH 3.6 acetate buffer, and react at room temperature for 5 minutes.

[0110] Buffer system 4: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, 20 μL of 50 μM GSH, and 300 μL of pH 3.6 acetate buffer, and react at room temperature for 5 minutes.

[0111] Buffer system 5: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, and 320 μL of pH 4.0 acetate buffer, and react at room temperature for 5 minutes.

[0112] Buffer system 6: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, 20 μL of 50 μM GSH, and 300 μL of pH 4.0 acetate buffer, and react at room temperature for 5 minutes.

[0113] Buffer system 7: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, and 320 μL of pH 4.4 acetate buffer, and react at room temperature for 5 minutes.

[0114] Buffer system 8: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, 20 μL of 50 μM GSH, and 300 μL of pH 4.4 acetate buffer, and react at room temperature for 5 minutes.

[0115] Buffer system 9: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, and 320 μL of pH 4.8 acetate buffer, and react at room temperature for 5 minutes.

[0116] Buffer system 10: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, 20 μL of 50 μM GSH, and 300 μL of pH 4.8 acetate buffer, and react at room temperature for 5 minutes.

[0117] Buffer system 11: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, and 320 μL of pH 5.2 acetate buffer, and react at room temperature for 5 minutes.

[0118] Buffer system 12: Take 40 μL of 10 mmol / L ABTS solution, 20 μL of 50 mmol / L H2O2 solution, 20 μL of LPB, 20 μL of 50 μM GSH, and 300 μL of pH 5.2 acetate buffer, and react at room temperature for 5 minutes.

[0119] 3. Refer to the absorbance test results as follows: Figure 9 As shown, although ABTS is oxidized better under weakly acidic conditions and Prussian blue is stable under weakly acidic conditions, in the GSH-enhanced Prussian blue-catalyzed ABTS-H2O2 colorimetric reaction system of this invention, the difference in absorbance between the presence and absence of GSH in the acetate buffer environment at pH 4.0 is extremely small, which does not meet the requirements of the GSH-enhanced Prussian blue-catalyzed ABTS-H2O2 colorimetric reaction system of this invention.

[0120] The GSH-enhanced colorimetric detection system of this invention is not suitable for acetate buffer environments. Even in the weakly acidic pH range (3.6-5.2) where ABTS and PB are theoretically stable, the ΔA value is significantly reduced due to the interference of the buffer solution on the catalytic mechanism, making it difficult to meet quantitative requirements. The water system is the optimal solvent choice for this detection system: it has no additional ion interference and can ensure that the enhancing effect of GSH on the catalytic activity of PB is fully exerted.

[0121] Example 12 Verify the applicability of the enhanced colorimetric method of this invention for detecting glutathione (GSH) in real-world complex food samples: The pig liver, pig blood, pork, chicken liver, and spinach were homogenized separately using a grinder. The specific procedures were as follows: 3g of each sample was weighed and dispersed in 10mL of ultrapure water and sonicated for 30 minutes. Then, the resulting solutions were centrifuged at 7000 rpm for 10 minutes. 3mL of the supernatant was collected and added to 6mL of acetonitrile, and stirred vigorously for 30 minutes. The resulting solutions were then centrifuged again (7000 rpm for 10 minutes). The resulting supernatant was transferred to a rotary evaporator to remove the acetonitrile and filtered through a 0.22 μm microporous membrane. Finally, the sample solutions were appropriately diluted: pig blood 10-fold, pig liver 100-fold, pork 100-fold, and chicken liver 100-fold. Similarly, spinach was ground and juiced using a grinder. The resulting spinach juice was centrifuged at 7000 rpm for 10 minutes, the supernatant was collected, and filtered through a 0.22 μm microporous membrane. The resulting filtrate was diluted 50-fold.

[0122] Different concentrations of GSH (0-50 μM) were added to the diluted samples using the standard addition method. 40 μL of 10 mM ABTS solution and 20 μL of 50 mM H2O2 solution were added to each well of a 96-well plate. Then, 20 μL of GSH standard solution of different concentrations was added, followed by 20 μL of PB solution (Example 1) and 300 μL of ultrapure water. The mixture was thoroughly mixed and reacted at room temperature for 5 min. This process was repeated three times in parallel. The plates were placed in a dark chamber with an LED white light on, and the solutions in the 96-well plates were photographed using a mobile phone to obtain the chromaticity R, G, and B values. The I value was calculated using the chromaticity relationship I = 0.3R + 0.59G + 0.11B. As shown in the figure below, there is a good linear relationship between the GSH concentration (12.5 nM~62.5 M) in the diluted standard samples and the chromaticity I value. The reaction was carried out at room temperature for 5 min. R, G, and B values ​​at different concentrations were captured by photographing. I was calculated at each concentration, and a standard curve of I versus GSH concentration was plotted. The results are as follows: Figure 10 As shown in Table 2, the recovery rates were calculated using the aforementioned standard curve, where the GSH concentration varied from 5.0 to 25.0 μM. The actual glutathione detection results in the samples are shown in Table 2. Table 2: Results of Glutathione Detection in Actual Samples As can be seen from the results in Table 2, the recoveries of low-concentration spikes (5 μM) were all between 95.8% and 104.2% (e.g., 95.8% recovery for 5 μM in pig liver and 104.2% recovery for 5 μM in spinach). This shows that the reaction system of the present invention still has high accuracy in detecting GSH even when the content in the sample is low, and is applicable to actual samples.

[0123] This embodiment achieves quantitative analysis by taking photos with a mobile phone and calculating RGB colorimetry. This design demonstrates the "convenient detection" effect of the present invention. This embodiment does not require professional instruments (such as enzyme-linked immunosorbent assay readers or spectrophotometers), but only a mobile phone (a conventional device) and a dark box (easy to set up), making it suitable for food processing sites, grassroots testing institutions, and other scenarios.

[0124] Example 13 Characterization of PB: The PB solution obtained in Example 1 was divided into multiple 1.5 mL centrifuge tubes, each containing 500 μL of PB solution. The tubes were centrifuged at 10,000 rpm for 30 minutes, and the precipitates were collected. The precipitates from multiple tubes were combined and dried overnight in a vacuum drying oven at 35°C. The dried precipitate was then used for Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) measurements. Figure 11 As shown. The FT-IR image is located at approximately 3453 cm⁻¹. -1 There is a broad and strong absorption band at 2072 cm⁻¹, corresponding to the OH stretching vibration of water molecules (hydrogen bonding causes the broad peak). -1 A strong absorption peak appears at 1637 cm⁻¹, corresponding to the stretching vibration of C≡N; -1 The point is a bending vibration of HOH; ~596 cm -1 and ~500 cm -1 The nearby absorption peaks are attributed to the bending vibrations of the Fe-C≡N-Fe bridging bonds in the PB core framework. These results confirm that the PB precipitate possesses a complete three-dimensional coordination polymer network structure, and XPS full-spectrum analysis also confirms the presence of Fe, O, N, K, and C elements in PB.

[0125] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.< / e> < / d> < / c>

Claims

1. An enhanced colorimetric method for rapid and selective detection of glutathione, characterized in that, Includes the following steps: Using water as a solvent, prepare solutions of Prussian blue, Tönnies blue, ferrous chloride, or ferric chloride as nanozyme catalytic solutions; The nanozyme catalytic solution was mixed with ABTS solution and hydrogen peroxide solution in a certain proportion to form a blank reaction solution; The test sample containing glutathione was added to the blank reaction solution, mixed thoroughly, and then subjected to a colorimetric reaction to obtain the test reaction solution. The absorbance of the blank reaction solution and the test reaction solution was measured in the wavelength range of 400 nm to 550 nm. The content of glutathione in the sample to be tested is determined based on the absorbance difference between the blank reaction solution and the test reaction solution. The ABTS solution concentration is 5-15 mmol / L, and the amount added in each 400 μL total reaction volume is 30-50 μL. The concentration of the hydrogen peroxide solution is 40-60 mmol / L, and the amount added is 15-25 μL per 400 μL of total reaction volume; The amount of the nanozyme catalytic solution added is 15-50 μL per 400 μL total reaction volume.

2. The enhanced colorimetric method according to claim 1, characterized in that, The nanozyme catalytic solution is a Prussian blue solution, which is prepared by mixing a ferric chloride solution with a concentration of 1-500 μmol / L with a potassium ferrocyanide solution of the same concentration range at a volume ratio of 1:1 to 4:3; the amount of Prussian blue solution added is 15-25 μL per 400 μL of total reaction volume.

3. The enhanced colorimetric method according to claim 1, characterized in that, The nanozyme catalytic solution is a Tunchausen blue solution, which is prepared by mixing a ferrous chloride solution with a concentration of 1-500 μmol / L with a potassium ferricyanide solution of the same concentration range at a volume ratio of 1:2 to 3:2; the amount of Tunchausen blue solution added is 15-25 μL per 400 μL of total reaction volume.

4. The enhanced colorimetric method according to claim 1, characterized in that, The nanozyme catalytic solution is a ferrous chloride solution or a ferric chloride solution, with a concentration of 1-50 μmol / L. The amount of ferrous chloride solution or ferric chloride solution added is 30-50 μL per 400 μL of total reaction volume.

5. The enhanced colorimetric method according to claim 1, characterized in that, The reaction temperature for the colorimetric reaction is 20-50℃, and the reaction time is 1-30 min.

6. The enhanced colorimetric method according to claim 1, characterized in that, The absorbance is the value measured at a wavelength of 418 nm for the reaction solution.

7. The enhanced colorimetric method according to claim 1, characterized in that, The steps for determining the glutathione content in the sample to be tested include: Plot a first standard curve between the concentration of the glutathione standard solution and the difference in absorbance. The absorbance difference of the sample to be tested is substituted into the first standard curve to calculate the glutathione content.

8. The enhanced colorimetric method according to claim 1, characterized in that, The RGB colorimetric value of the test reaction solution is obtained, and the colorimetric I value is calculated based on the RGB colorimetric value of the test reaction solution to determine the glutathione content in the test sample. The chromaticity I value is calculated using the formula I = 0.3R + 0.59G + 0.11B, where R, G, and B are the values ​​of the red, green, and blue channels of the photograph of the reaction solution to be tested, respectively. The steps for determining the glutathione content in the sample to be tested include: Plot a second standard curve between the concentration of the glutathione standard solution and the colorimetric I value; The chromaticity I value of the sample to be tested is substituted into the second standard curve to calculate the glutathione content.

9. A kit for implementing the enhanced colorimetric method according to any one of claims 1 to 8, characterized in that, This includes nanozyme catalytic solution components, ABTS solution components, and hydrogen peroxide solution components; The nanozyme catalytic solution is composed of one of Prussian blue solution, Tönnies blue solution, ferrous chloride solution, or ferric chloride solution. The concentration of the ABTS solution component is 5-15 mmol / L, and the preparation volume is 30-50 μL added to every 400 μL of total reaction volume; The concentration of the hydrogen peroxide solution component is 40-60 mmol / L, and the amount used is 15-25 μL added to every 400 μL of total reaction volume.

10. The reagent kit according to claim 9, characterized in that, The nanozyme catalytic solution is composed of Prussian blue solution, and the Prussian blue solution contains a stabilizer; the stabilizer is a mixture of trehalose and polyvinylpyrrolidone; wherein the concentration of trehalose in the Prussian blue solution is 5-20 g / L, and the concentration of polyvinylpyrrolidone in the Prussian blue solution is 1-5 g / L; the amount of Prussian blue solution containing the stabilizer added is 15-25 μL per 400 μL of total reaction volume.

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