Glutathione detection reagent and application thereof

Abstract

The invention relates to the technical field of biological detection, and provides a glutathione detection reagent and application thereof. The glutathione reagent comprises an Au (I)-SG oligomer and a first cerium salt containing Ce < 4 + >, and the Au (I)-SG oligomer comprises a glutathione ligand and an Au element. After the glutathione reagent is mixed with GSH, Ce < 4 + > in the first cerium salt is reduced to release Ce < 3 + > ions, and the Ce < 3 + > ions induce in-situ synthesis of GSH (at) AuNCs, generate AIE characteristics and emit fluorescence, so that the content of GSH reacted with the glutathione detection reagent can be effectively detected through fluorescence detection, and the detection sensitivity is high, the specificity is good and the accuracy is high.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a glutathione detection reagent and its application. Background Art

[0002] Glutathione (GSH) is a substance with strong antioxidant, detoxification and cell protection effects. Its content in human cells is comparable to that of other biothiols. In addition, glutathione also has the magical effect of removing a variety of free radicals produced in human cells, including free radicals produced by metabolic disorders, tumors, cardiovascular diseases and neurodegenerative dysfunction (such as Alzheimer's disease, Parkinson's disease) and other diseases. Therefore, monitoring the level of glutathione in cells and body fluids can provide information on such oxidative stress-related diseases, and glutathione is of great significance in clinical biochemical analysis.

[0003] Currently available glutathione detection methods include high performance liquid chromatography, surface enhanced Raman scattering, electrochemical and electrochemiluminescence analysis methods, and fluorescence spectroscopy. Among them, the operation process of high performance liquid chromatography and surface enhanced Raman scattering is relatively cumbersome and requires experienced and skilled technicians to operate. The glutathione analysis methods based on electrochemistry and electrochemiluminescence have the disadvantages of poor accuracy, multi-step electrode functionalization, and time-consuming electrode cleaning process.

[0004] Although fluorescence spectroscopy has high accuracy, several organic fluorophores currently used as luminescent probes for monitoring glutathione include lucigenin, rhodamine-based fluorescent probes, and lanthanide-based organic / inorganic hybrid luminescent materials. These luminescent probes have problems such as photobleaching, toxicity, and complex synthesis processes. Among them, photobleaching can easily lead to weakening or disappearance of the fluorescence signal, and the detection results are prone to deviations. These characteristics hinder the practical application of luminescent probes such as lucigenin in real-time detection of glutathione. Therefore, it is urgent to provide a glutathione detection reagent with high detection accuracy. Summary of the invention

[0005] The purpose of the present application is to provide a glutathione detection reagent and its application, so as to solve the technical problem that the detection results of the fluorescent probe used in the glutathione detection in the prior art are prone to deviation.

[0006] At the same time, the embodiments of the present application also provide a glutathione detection kit and a glutathione content detection method.

[0007] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0008] In the first aspect, the present invention provides a glutathione detection reagent. The glutathione detection reagent of the present invention comprises an Au(I)-SG oligomer and a Ce 4+ The first cerium salt, wherein the Au(I)-SG oligomer includes a glutathione ligand and an Au element.

[0009] After the glutathione detection reagent is mixed with glutathione, Ce in the first cerium salt 4+ Reduction releases Ce 3+ ions, these Ce 3+ GSH@AuNCs are synthesized in situ by ion induction. The GSH@AuNCs have AIE characteristics and emit fluorescence, so that the content of glutathione reacting with the glutathione detection reagent can be detected by fluorescence detection, and the detection sensitivity is high, the specificity is good, and the accuracy is high.

[0010] In a second aspect, the present invention provides a glutathione detection kit. The glutathione detection kit of the present invention comprises the glutathione detection reagent described above.

[0011] Since the glutathione detection kit of the present application includes the glutathione detection reagent described above, based on the characteristics of high sensitivity, high accuracy and good specificity when the glutathione detection reagent described above is used to detect glutathione, the glutathione detection kit of the embodiment of the present application also has the characteristics of high sensitivity, high accuracy and good specificity for glutathione detection.

[0012] In a third aspect, the embodiments of the present application provide the use of the above glutathione detection reagent or glutathione detection kit in glutathione detection.

[0013] Based on the fact that the glutathione detection reagent and glutathione detection kit of the present application have good specificity for the detection of glutathione, and have high detection sensitivity and high result accuracy, the glutathione detection reagent and glutathione detection kit mentioned above can be used to effectively detect glutathione.

[0014] In a fourth aspect, the present application provides a method for detecting glutathione content. The method for detecting glutathione content in the present application comprises the following steps:

[0015] Mixing a glutathione detection reagent with a sample to be tested to obtain a premixed solution to be tested, performing a first redox treatment on the premixed solution to be tested to obtain a sample solution to be tested, and measuring the fluorescence intensity of the sample solution to be tested;

[0016] Mixing a glutathione detection reagent with a glutathione standard to obtain a standard premix solution, performing a second redox treatment on the standard premix solution to obtain a standard solution, and measuring the fluorescence intensity of the standard solution;

[0017] The glutathione content in the sample to be tested is calculated according to the fluorescence intensity of the sample solution to be tested, the glutathione concentration in the standard solution and the fluorescence intensity of the standard solution.

[0018] Wherein, the glutathione detection reagent is the glutathione detection reagent mentioned above.

[0019] The glutathione content detection method of the embodiment of the present application utilizes the aggregation-induced luminescence effect of GSH@AuNCs caused by glutathione to generate strong fluorescence, and within a certain range, the glutathione content is proportional to the fluorescence intensity, so the glutathione content in the sample to be tested can be detected by detecting the fluorescence intensity of the sample solution to be tested and the standard solution. The glutathione content detection method of the embodiment of the present application has high sensitivity, good specificity, and high accuracy of the detection result. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained like these drawings without paying any creative work.

[0021] Figure 1 The Ce(CO) provided in Example A1 3 ) 2 HRTEM image of nanosheets;

[0022] Figure 2 The Ce(CO) provided in Example A1 3 ) 2 AFM image of nanosheets;

[0023] Figure 3 For Example B1, Ce(CO 3 ) 2 Schematic diagram of in situ synthesis of GSH@AuNCs from nanosheets and GSH;

[0024] Figure 4 This is a schematic diagram of the fluorescence spectrum detection results of the standard solution of Example B1;

[0025] Figure 5 This is a schematic diagram of the standard curve of Example B1;

[0026] Figure 6 In Example B1, glutathione, Ce(CO3 ) 2 HRTEM image of in situ synthesized GSH@AuNCs after mixing nanosheets and Au(I)-SG oligomers;

[0027] Figure 7 In Example B1, glutathione and Ce(CO 3 ) 2 HRTEM image of nanosheets after mixing;

[0028] Figure 8 Selectivity diagram of the AIE-based gold nanocluster probe for GSH sensing. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0030] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0031] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, a~b (i.e. a and b), a~c, b~c, or a~b~c, where a, b, c can be single or multiple, respectively.

[0032] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0033] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0034] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0035] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0036] In order to solve the technical problem that the detection results of fluorescent probes used in glutathione detection in the prior art are prone to deviation, the present application proposes the following technical solution.

[0037] In a first aspect, the present invention provides a glutathione detection reagent. The glutathione detection reagent of the present invention comprises an Au(I)-SG oligomer and a Ce 4+ The first cerium salt, wherein the Au(I)-SG oligomer includes a glutathione ligand and an Au element.

[0038] The glutathione detection reagent of the embodiment of the present application can detect glutathione by utilizing the synergistic effect of redox reaction and aggregation-induced emission (AIE) effect. The glutathione detection reagent of the embodiment of the present application has low toxicity, high sensitivity for glutathione detection, good specificity, and high accuracy of the detection result.

[0039] Specifically, in the process of detecting glutathione using the glutathione detection reagent of the embodiment of the present application, the Ce 4+ The first cerium salt was used as a mediator and Ce was converted into 4+ Reduction to Ce 3+ , Ce 3+Induce Au(I)-SG oligomers to synthesize gold nanoclusters (GSH@AuNCs) in situ. GSH@AuNCs have AIE characteristics and can generate fluorescence under the action of excitation light. Therefore, the glutathione detection reagent of the embodiment of the present application can use glutathione to cause the aggregation-induced luminescence effect of GSH@AuNCs to generate strong fluorescence to achieve the detection of glutathione, and the detection sensitivity is high, the specificity is good, the accuracy and precision of the detection results are high, and the detection operation steps are few. In addition, the glutathione detection reagent of the embodiment of the present application has low toxicity and simple synthesis operation.

[0040] In some embodiments, the molar ratio of the glutathione ligand to the Au element in the Au(I)-SG oligomer can be (4:1) to (2:1). In an exemplary embodiment, the molar ratio of the glutathione ligand to the Au element in the Au(I)-SG oligomer can be 2:1, 3:1, 4:1, etc., which are typical but not limiting values, or any value between any two numerical ranges. In an exemplary embodiment, the Au(I)-SG oligomer can be prepared by mixing 0.8 mL of glutathione solution (50.0 mM) with 2.0 mL of HAuCl 4 The aqueous solution (10.0 mM) was reacted to obtain the product, at which the molar ratio of glutathione to Au element was 2:1.

[0041] In some embodiments, the Au(I)-SG oligomer in the glutathione detection reagent of the embodiment of the present application can be dissolved in a solvent such as an aqueous solvent to form an Au(I)-SG oligomer solution. In a further embodiment, the concentration of the Au(I)-SG oligomer solution can be 2mM. Controlling the concentration of the Au(I)-SG oligomer within this range further improves the detection sensitivity and accuracy. It should be noted that the Au(I)-SG oligomer solution can also be diluted to the target concentration before use.

[0042] In some embodiments, the Au(I)-SG oligomer can be prepared by a method comprising the following steps:

[0043] Au 3+ The solution is mixed with the first glutathione solution to obtain a first mixed solution, which is stirred under alkaline conditions to obtain Au(I)-SG oligomer.

[0044] The glutathione in the first glutathione solution contains sulfhydryl groups, which can bind Au 3+ Reduction to Au in solution + Then, the thiol groups in glutathione form coordination bonds with the Au element, thereby forming a complex to obtain Au(I)-SG oligomer. The preparation process of Au(I)-SG oligomer is simple and easy to operate, and the process conditions are easy to control.

[0045] In some embodiments, Au3+ Au in solution 3+ The molar ratio of Au to the glutathione contained in the first glutathione solution can be (1:2) to (1:4), and can be (1:2) to (1:3), (1:3) to (1:4). 3+ Au in solution 3+ The molar ratio of the glutathione contained in the first glutathione solution can be 1:2, 1:3, 1:4, etc., which are typical but not limiting values, or any value between any two numerical ranges. 3+ Au in solution 3+ The molar ratio of glutathione contained in the first glutathione solution is within this range, and the molar ratio of glutathione ligand to Au element in the prepared Au(I)-SG oligomer is further adjusted to further promote aggregation-induced emission and improve the sensitivity and accuracy of the glutathione detection result of the glutathione detection reagent in the embodiment of the present application.

[0046] In some embodiments, Au in the first mixed solution 3+ The concentration of Au in the first mixed solution can be 2mM; the concentration of glutathione in the first mixed solution can be 4mM. 3+ and glutathione concentration within this range, further controlling the reaction rate in the mixture and the molecular size of the Au(I)-SG oligomer, further promoting aggregation-induced emission, and enhancing the luminescence of gold nanoclusters, thereby further improving the sensitivity, accuracy and precision of the detection reagent of the embodiment of the present application for glutathione detection.

[0047] In some embodiments, Au 3+ The solution may include a chloroauric acid solution. The chloroauric acid solution has good solubility in aqueous solutions and can effectively provide Au for the reaction. 3+ .

[0048] In some embodiments, Au(I)-SG oligomers can be prepared by the following method: quickly mixing a first glutathione solution with a chloroauric acid solution to obtain a first mixed solution, and diluting the first mixed solution to 10 mL with water, and making the concentration of glutathione in the first mixed solution to be 4 mM, and Au 3+ The concentration of Au(I)-SG was 2 mM. The first mixed solution was stirred for five minutes to obtain a white precipitate, and then 0.2 mL of 0.5 M NaOH was quickly added while continuing to stir until the precipitate dissolved. The solution after the precipitate dissolved was kept at a constant stirring speed for 1 hour to obtain a transparent, colorless and non-luminescent Au(I)-SG oligomer.

[0049] In some embodiments, the first cerium salt contained in the glutathione detection reagent of the present application embodiment can be excessive, that is, the content of the first cerium salt contained is sufficient to make Ce 4+React with all the glutathione to be tested, where 1 mol of Ce 4+ 2 mol of glutathione to be tested is consumed. In the exemplary embodiment, when the first cerium salt is Ce(CO 3 ) 2 When the nanosheet is prepared, the reaction between the first cerium salt and the glutathione to be detected is shown in reaction equation (3). A sufficient amount of the first cerium salt allows the glutathione to be detected to react fully, further improving the accuracy of the detection result of the glutathione detection reagent of the present application embodiment.

[0050] In some embodiments, the first cerium salt may include Ce(CO 3 ) 2 In a further embodiment, Ce(CO 3 ) 2 The thickness of the nanosheet is 10 to 50 nm. 3 ) 2 The thickness of the nanosheet can be 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, etc., which are typical but not limiting values, or any value between any two numerical ranges. 3 ) 2 The thickness of the nanosheets is within this range, which promotes the 3 ) 2 Nanosheets dissolve to release Ce 4+ , in order to facilitate the reduction to Ce after the addition of GSH 3+ , promote the synthesis of gold nanoclusters, and enhance the fluorescence of gold nanoclusters to improve the sensitivity, accuracy and precision of glutathione detection.

[0051] In some embodiments, Ce(CO 3 ) 2 The nanosheets can be dispersed in solvents such as water to form Ce(CO 3 ) 2 Nanosheet dispersion. In a further embodiment, Ce(CO 3 ) 2 Ce(CO 3 ) 2 The concentration of the nanosheets may be 20 mg / mL.

[0052] In some embodiments, Ce(CO 3 ) 2 The nanosheets can be prepared by a preparation method comprising the following steps:

[0053] Step A10: Add CO 3 2- and / or HCO 3 - The carbonate solution and Ce3+ The cerium source solution is mixed and a precipitation reaction is performed to obtain Ce 2 (CO 3 ) 3 Nanosheets;

[0054] Step A20: Place Ce 2 (CO 3 ) 3 The nanosheets were oxidized under alkaline conditions to obtain Ce(CO 3 ) 2 Nanosheets.

[0055] In some embodiments, the carbonate solution may contain at least one of ammonium bicarbonate, ammonium carbonate, potassium bicarbonate, potassium carbonate, sodium bicarbonate, and sodium carbonate. These carbonates and bicarbonates have good solubility, and their solutions can provide carbonate and / or bicarbonate for the precipitation reaction.

[0056] In some embodiments, when the anion of the carbonate solution is HCO 3 - When the precipitation reaction of step A10 is shown in reaction equation (1):

[0057] 2Ce 3+ + 3HCO 3 - +8H 2 O → 3H + + Ce 2 (CO 3 ) 3 .8H 2 O (1).

[0058] In some embodiments, the CO in the carbonate solution 3 2- and / or HCO 3 - The concentration can be 96mM. Controlling CO in carbonate solution 3 2- and / or HCO 3 - concentration, combined with Ce in the cerium source solution 3+ concentration, effectively controlling the Ce obtained by precipitation reaction 2 (CO 3 ) 3 Nanosheet particle size, thereby controlling Ce 2 (CO 3 ) 3 The particle size of the nanosheets further promotes the formation of gold nanoclusters, enhances the fluorescence of the gold nanoclusters, and improves the sensitivity of the glutathione detection reagent of the embodiment of the present application to glutathione detection.

[0059] In some embodiments, the cerium source contained in the cerium source solution may include Ce(NO 3 ) 3 、CeCl 3 、Ce 2 (SO 4 ) 3 At least one of these cerium sources has good solubility and can efficiently provide Ce for the precipitation reaction. 3+ .

[0060] In some embodiments, Ce in the cerium source solution 3+ The concentration can be 32mM. Control the Ce in the cerium source solution 3+ concentration, combined with CO in carbonate solution 3 2- and / or HCO 3 - concentration, effectively controlling the Ce obtained by precipitation reaction 2 (CO 3 ) 3 Nanosheet particle size, thereby controlling Ce 2 (CO 3 ) 3 The particle size of the nanosheets promotes the formation of gold nanoclusters, enhances the fluorescence of the gold nanoclusters, and improves the sensitivity of the glutathione detection reagent of the embodiment of the present application to glutathione detection.

[0061] In some embodiments, the oxidation reaction in step A20 is as shown in reaction equation (2):

[0062] H 2 O+O 2 + CO 2 + Ce 2 (CO 3 ) 3 . → 2Ce(CO 3 ) 2 +2OH - (2).

[0063] In some embodiments, the glutathione detection reagent of the present application embodiment further includes a buffer, and the pH value of the buffer may be 6.5 to 7.5. In an exemplary embodiment, the buffer may include a HEPES buffer of pH 6.5. By adding a buffer to the glutathione detection reagent of the present application embodiment, the pH of the reaction system can be effectively controlled to be neutral or slightly acidic, which is conducive to enhancing the fluorescence intensity, thereby further improving the sensitivity of the detection and the accuracy of the detection results.

[0064] It should be noted that when the glutathione detection reagent in the embodiment of the present application includes a buffer solution, the buffer solution and the aforementioned Au(I)-SG oligomer and the first cerium salt can be separated from each other; or they can be mixed in pairs, for example, the Au(I)-SG oligomer can be dissolved in the buffer solution and the first cerium salt can be set separately; or the three can be separated from each other, and there is no specific limitation, and technicians in this field can make a choice according to actual needs.

[0065] In a second aspect, the present application provides a glutathione detection kit. The glutathione detection kit of the present application includes the glutathione detection reagent described above.

[0066] Based on the fact that the glutathione detection reagent described above has the characteristics of high sensitivity, high accuracy and good specificity for glutathione detection, the glutathione detection kit of the embodiment of the present application also has the characteristics of high sensitivity, good specificity and high accuracy of the detection result for glutathione detection.

[0067] In a third aspect, the embodiments of the present application provide the use of the above glutathione detection reagent or the above glutathione detection kit in glutathione detection.

[0068] Based on the glutathione detection reagent and glutathione detection kit of the embodiment of the present application, the glutathione detection reagent and glutathione detection kit have the characteristics of high sensitivity, high accuracy of detection results and good specificity for glutathione detection, so that the glutathione detection reagent and glutathione detection kit mentioned above have good application prospects in the detection of glutathione.

[0069] In some embodiments, the above glutathione detection reagent and / or glutathione detection kit can be used for quantitative detection of glutathione, and can also be used for qualitative detection of glutathione, which is not specifically limited, and those skilled in the art can make a selection according to actual needs.

[0070] In a fourth aspect, the present application embodiment provides a method for detecting glutathione content. The glutathione detection method of the present application embodiment comprises the following steps:

[0071] Step S10: mixing the glutathione detection reagent with the sample to be tested to obtain a premixed solution to be tested, performing a first oxidation-reduction treatment on the premixed solution to be tested to obtain a sample solution to be tested, and measuring the fluorescence intensity of the sample solution to be tested;

[0072] Step S20: mixing the glutathione detection reagent with the glutathione standard to obtain a standard premix solution, performing a second redox treatment on the standard premix solution to obtain a standard solution, and measuring the fluorescence intensity of the standard solution;

[0073] Step S30: Calculate the glutathione content in the sample to be tested according to the fluorescence intensity of the sample solution to be tested, the glutathione concentration in the standard solution and the fluorescence intensity of the standard solution.

[0074] Wherein, the glutathione detection reagent is the glutathione detection reagent mentioned above.

[0075] The glutathione content detection method of the present application embodiment utilizes glutathione to reduce Ce 4+ , release Ce 3+ , Ce 3+ Induced in-situ synthesis of GSH@AuNCs, GSH@AuNCs have aggregation-induced emission effect, can produce strong fluorescence, and within a specific range, the content of glutathione is proportional to the fluorescence intensity, so the concentration of glutathione in the sample solution to be tested can be detected by detecting the concentration and fluorescence intensity of the sample solution to be tested and the standard solution, and then the content of glutathione in the sample to be tested can be calculated. The glutathione content detection method of the embodiment of the present application has high sensitivity, good specificity, and high accuracy of the detection result.

[0076] Step S10:

[0077] In step S10, the glutathione detection reagent is mixed with the sample to be tested to obtain a premixed solution to be tested, and then a first redox treatment is performed to obtain a sample solution to be tested. When the sample to be tested contains glutathione, a redox reaction occurs between the glutathione to be tested and the first cerium salt to convert Ce 4+ Reduction to Ce 3+ , Ce 3+ Ions induce GSH@AuNCs to produce AIE characteristics and emit strong fluorescence. The fluorescence intensity of the sample solution to be tested is measured so that the glutathione concentration in the sample solution to be tested can be calculated based on the fluorescence intensity of the sample solution to be tested, thereby calculating the glutathione content in the sample to be tested.

[0078] In some embodiments, the equation of the first redox reaction in step A10 is shown in reaction equation (3):

[0079] 2GSH + Ce(CO 3 ) 2 + 2H + → GS-SG + Ce 3+ + 2H 2 O + 2CO 2 (3).

[0080] In some embodiments, the premixed solution to be tested may further include the buffer mentioned above. In an exemplary embodiment, a HEPES buffer having a pH of 6.5 may be added to the premixed solution.

[0081] In some embodiments, the temperature of the first redox treatment can be 30-40°C, optionally 30-35°C, 30-37°C, 35-40°C. In the exemplary embodiment, the temperature of the first redox treatment can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc., which are typical but non-limiting temperatures, or any temperature between any two numerical ranges. Controlling the temperature of the first redox treatment within this range further promotes the in situ synthesis of GSH@AuNCs, reduces the effect of protein denaturation on the fluorescence results, and improves the accuracy of the detection results.

[0082] In some embodiments, the time of the first redox treatment can be 1 to 2 hours. In the exemplary embodiment, the time of the first redox treatment can be a typical but non-limiting time such as 1 hour, 1.5 hours, 2 hours, or any time within any two numerical ranges. Controlling the time of the first redox treatment within this range further promotes the full reaction between the glutathione contained in the sample to be tested and the first cerium salt, further reduces the test error, and improves the accuracy of the test result.

[0083] In some embodiments, the excitation wavelength for measuring the fluorescence intensity of the sample solution to be tested can be 365 nm. By controlling the excitation wavelength within this range, the sensitivity of glutathione content detection can be further improved, and the accuracy of the test results can be improved. It should be noted that when the excitation wavelength is 365 nm, it means that the instrument setting parameter is 365 nm, and the actual excitation wavelength can be within the acceptable error range of the instrument.

[0084] In some embodiments, the emission wavelength for measuring the fluorescence intensity of the sample solution to be tested may include 500-600 nm, optionally 500-580 nm, 520-560 nm. In the exemplary embodiment, the emission wavelength may be 500 nm, 510 nm, 520 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 565 nm, 565 nm, 570 nm, 575 nm, 580 nm, 590 nm, 600 nm, etc., which are typical but non-limiting sizes, or any size within the two numerical ranges. By controlling the emission wavelength within this range, the fluorescence intensity at the same GSH@AuNCs concentration is further improved, and the sensitivity of glutathione content detection and the accuracy of the detection results are improved.

[0085] In some embodiments, in the glutathione content detection method of the embodiment of the present application, the applicable range of glutathione concentration can be 1-1000 μmol / L, which can be 50-1000 μmol / L, 50-500 μmol / L, 100-800 μmol / L, 400-1000 μmol / L. In a further embodiment, the concentration of glutathione contained in the sample solution to be tested can be 1-1000 μmol / L, which can be 50-1000 μmol / L, 50-500 μmol / L, 100-800 μmol / L, 400-1000 μmol / L. In the exemplary embodiment, the concentration of glutathione in the sample solution to be tested can be 1 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L, 500 μmol / L, 600 μmol / L, 700 μmol / L, 800 μmol / L, 900 μmol / L, 1000 μmol / L, etc., which are typical but non-limiting concentrations, or any concentration within any two numerical ranges. The concentration of glutathione in the sample solution to be tested is controlled within this range so that the concentration of glutathione is proportional to the fluorescence intensity, further improving the accuracy of the glutathione content detection results. It should be noted that the glutathione contained in the sample solution to be tested refers to the glutathione contained in the sample to be tested, and does not include the glutathione ligand in the Au(I)-SG oligomer.

[0086] Step S20:

[0087] The glutathione detection reagent is mixed with the glutathione standard product through step S20 to obtain a standard product premix solution, and the standard product solution is obtained after the second redox treatment, and the reaction equation of the second redox treatment is the same as the reaction equation of the first redox treatment. The fluorescence intensity of the standard product solution is measured so that the glutathione concentration in the sample solution to be tested can be calculated according to the fluorescence intensity of the sample solution to be tested, the fluorescence intensity of the standard product solution and the glutathione concentration in the standard product solution, so as to obtain the glutathione content contained in the sample to be tested. Among them, the glutathione standard product is a standard product with a known glutathione content, and the glutathione concentration in the standard product solution can be calculated according to the glutathione contained therein.

[0088] In some embodiments, when a buffer solution is added to the premixed solution to be tested, the same buffer solution may also be added to the premixed solution of the standard substance to reduce the error of the test result.

[0089] In some embodiments, the temperature of the second redox treatment can be the same as the temperature of the first redox treatment, for example, the temperature of the second redox treatment can be 30-40°C, optionally 30-35°C, 30-37°C, 35-40°C. In the exemplary embodiment, the temperature of the second redox treatment can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc., which are typical but non-limiting temperatures, or any temperature between any two numerical ranges. Controlling the temperature of the second redox treatment within this range further promotes the in situ synthesis of GSH@AuNCs in the standard solution, reduces the effect of protein denaturation caused by excessive temperature on the fluorescence results, and improves the accuracy of the detection results.

[0090] In some embodiments, the time of the second redox treatment can be the same as the time of the first redox treatment, for example, the time of the second redox treatment can be 1 to 2 hours. In the exemplary embodiment, the time of the second redox treatment can be a typical but non-limiting time such as 1 hour, 1.5 hours, 2 hours, or any time within any two numerical ranges. Controlling the time of the second redox treatment within this range further fully promotes the reaction of glutathione contained in the sample to be tested with the first cerium salt, further reduces the test error, and improves the accuracy of the test result.

[0091] In some embodiments, the excitation wavelength when measuring the fluorescence intensity of the standard solution can be the same as the excitation wavelength when measuring the fluorescence intensity of the sample solution to be tested. In an exemplary embodiment, the excitation wavelength for measuring the fluorescence intensity of the standard solution can also be 365 nm. By controlling the excitation wavelength within this range, the sensitivity of glutathione content detection is further improved, and the accuracy of the detection result is improved.

[0092] In some embodiments, the emission wavelength when determining the fluorescence intensity of the standard solution can be the same as the emission wavelength when determining the fluorescence intensity of the sample solution to be tested. The emission wavelength for determining the fluorescence intensity of the standard solution can include 500-600nm, optionally 500-580nm, 520-560nm. In the exemplary embodiment, the emission wavelength for determining the fluorescence intensity of the standard solution can be 500nm, 510nm, 520nm, 530nm, 535nm, 540nm, 545nm, 550nm, 555nm, 565nm, 565nm, 570nm, 575nm, 580nm, 590nm, 600nm and other typical but non-limiting sizes, or any size within the two numerical ranges. Controlling the emission wavelength within this range further improves the fluorescence intensity at the same concentration and improves the sensitivity and accuracy of glutathione content detection.

[0093] In some embodiments, the concentration of glutathione in the standard solution can be 1-1000 μmol / L, optionally 50-1000 μmol / L, 50-500 μmol / L, 100-800 μmol / L, 400-1000 μmol / L. In an exemplary embodiment, the concentration of glutathione in the sample solution to be tested can be 1 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L, 500 μmol / L, 600 μmol / L, 700 μmol / L, 800 μmol / L, 900 μmol / L, 1000 μmol / L, etc., which are typical but non-limiting concentrations, or any concentration within any two numerical ranges. The concentration of glutathione in the standard solution is controlled within this range so that the concentration of glutathione is proportional to the fluorescence intensity. When a standard curve is drawn with the concentration of the standard solution as the horizontal axis and the fluorescence intensity of the standard solution as the vertical axis, the concentration of glutathione in the standard solution and the fluorescence intensity have a good linear relationship within this range, further improving the accuracy of the glutathione content detection result. It should be noted that the glutathione contained in the standard solution refers to the glutathione contained in the glutathione standard added to the standard solution, and does not include the glutathione ligand in the Au(I)-SG oligomer.

[0094] In some embodiments, there may be only one standard solution, or there may be two or more standard solutions, without limitation.

[0095] It should be understood that there is no specific requirement for the execution order of step S10 and step S20. In some embodiments, step S10 may be executed first and then step S20; in other embodiments, step S20 may be executed first and then step S10; in other embodiments, step S10 and step S20 may also be executed at the same time. There is no specific limitation, and those skilled in the art may make a choice according to actual needs.

[0096] Step S30:

[0097] In step S30, the glutathione concentration of the sample solution to be tested is calculated according to the fluorescence intensity of the sample solution to be tested, and the glutathione concentration and fluorescence intensity in the standard solution, so as to further calculate the glutathione content in the sample to be tested.

[0098] It should be noted that the glutathione in the sample solution to be tested may include D-GSH, may include L-GSH, or may include both L-GSH and D-GSH, without specific limitation.

[0099] In some embodiments, when there is only one portion of the standard solution, the glutathione concentration of the sample solution to be tested can be calculated based on the principle that "the ratio of the fluorescence intensity of the standard solution to the glutathione concentration in the standard solution is equal to the ratio of the fluorescence intensity of the sample solution to the glutathione concentration in the sample solution to be tested". The glutathione concentration of the sample solution to be tested can be calculated based on the fluorescence intensity of the sample solution to be tested, and the glutathione concentration and fluorescence intensity in the standard solution, so that the glutathione content in the sample to be tested can be further calculated.

[0100] In some embodiments, when the standard solution is in multiple portions, for example, any number of portions such as 3, 4, 5, 6, 7, 8, 9, 10, 11, etc., a standard curve can be drawn with the concentration of glutathione in the standard solution as the horizontal coordinate and the fluorescence intensity of the standard solution as the vertical coordinate, so as to calculate the glutathione concentration in the sample solution to be tested according to the fluorescence intensity of the sample solution to be tested and the standard curve, and then calculate the glutathione content in the sample to be tested.

[0101] It should be noted that the fluorescence intensity of the sample solution to be tested in step S30 may be the fluorescence intensity after deducting the blank, and the fluorescence intensity of the standard solution may also be the fluorescence intensity after deducting the blank. The preparation method of the blank solution is the same as the preparation method of the sample solution to be tested or the standard solution, except that no sample to be tested or glutathione standard is added.

[0102] In order to enable the above implementation details and operations of the present application to be clearly understood by those skilled in the art, and to demonstrate the significant improvement in the performance of the glutathione detection reagent and its application in the embodiments of the present application, the above technical solution is illustrated by multiple embodiments below.

[0103] 1. Glutathione detection reagent

[0104] Example A1

[0105] This embodiment provides a glutathione detection reagent. The glutathione detection reagent of this embodiment includes Au(I)-SG oligomer and Ce 4+ The first cerium salt is Ce(CO 3 ) 2 Nanosheets.

[0106] The Au(I)-SG oligomer in this embodiment is composed of Au 3+ The solution is reacted with the first glutathione solution to obtain a product, wherein Au 3+ The solution is a 10.0 mM chloroauric acid aqueous solution, and the concentration of glutathione in the first glutathione solution is 50.0 mM. The preparation method of Au(I)-SG oligomer in this embodiment includes the following steps:

[0107] Step A1: Quickly inject 0.8 mL of the freshly prepared 50.0 mM first glutathione solution into a 2.0 mL 10.0 mM HAuCl 4 The first mixed solution was obtained by adding water immediately to make the total volume of the first mixed solution 10.0 mL. The first mixed solution was stirred for 5 minutes to obtain a white Au(I)-SG complex precipitate.

[0108] Step A2: Add 0.2 mL of 0.5 M sodium hydroxide solution to the first mixed solution containing the Au(I)-SG white complex in the above step A1 until the precipitate in the first mixed solution is dissolved, and stir the first mixed solution at a constant speed for 1 hour to obtain an Au(I)-SG oligomer solution containing Au(I)-SG oligomers, which are colorless, transparent, and non-luminescent.

[0109] In this embodiment, the first cerium salt is Ce(CO 3 ) 2 Nanosheets, the Ce(CO 3 ) 2 The preparation method of the nanosheets comprises the following steps:

[0110] Step B1: Take 1.4 mg of Ce(NO 3 ) 3 6H 2 O and 0.75 mg NH 4 HCO 3 , respectively dissolved in 50mL of water to obtain Ce 3+ of cerium source solution and carbonate solution.

[0111] Step B2: The cerium source solution and carbonate solution in step B1 were heated at 30°C for 2 minutes respectively, and then rapidly mixed under strong stirring. 3+ and HCO 3 - The final concentrations were 32 mM and 96 mM, respectively. The reaction mixture was stirred for 30 minutes, then stopped stirring, aged at 30°C for 24 hours, filtered, and the remaining solid was washed with water three times and dried at 80°C for 12 hours to obtain Ce(CO 3 ) 2 The Ce(CO 3 ) 2The images of nanosheets under high resolution transmission electron microscope (HRTEM) and atomic force microscope (AFM) are shown in Figure 2. Figure 1 and Figure 2 As shown. Figure 1 and Figure 2 As shown, Ce(CO 3 ) 2 The nanosheet has a nanosheet-like structure.

[0112] Step B3: Take Ce(CO 3 ) 2 20.0 mg of nanosheets were ultrasonically dispersed in 2.0 mL of water to prepare Ce(CO) with a concentration of 10 mg / mL. 3 ) 2 Nanosheet dispersion.

[0113] 2. Glutathione detection method

[0114] Example B1

[0115] This embodiment provides a glutathione detection method. The glutathione detection method of this example is the application of the glutathione detection reagent in Example A1 in glutathione detection.

[0116] The glutathione detection method of this embodiment is a glutathione content detection method, and the glutathione detection reagent used in this embodiment is the glutathione detection reagent in Example A1. Figure 3 As shown, the glutathione content detection method of this embodiment includes the following steps:

[0117] Step S1: Prepare multiple standard solutions. Take the Au(I)-SG oligomer solution and Ce(CO 3 ) 2 Nanosheet dispersion: 200 μL of Au(I)-SG oligomer solution and 30 μL of Ce(CO 3 ) 2 The nanosheet dispersion was fully mixed to obtain a nanosheet containing Au(I)-SG and Ce(CO 3 ) 2A second mixed solution of nanosheets. Add glutathione without content to the second mixed solution to obtain a standard premix. Add 200 μL of HEPES buffer with a concentration of 10 mM and a pH value of 6.5 to the standard premix, ultrasonically treat for 10 minutes, and then water bath at 37°C for 1 hour to obtain a standard solution. Among them, the amount of glutathione added to the standard premix is ​​such that the final concentration of glutathione in the standard solution ranges from 50 to 1000 μM. The specific concentrations of different standard solutions are as follows: Figure 4 shown.

[0118] Step S2: Prepare a blank solution. The preparation method of the blank solution is basically the same as the preparation method of the standard solution in step S1, except that the blank solution does not contain Au(I)-SG and Ce(CO 3 ) 2 Glutathione is added to the second mixture of nanosheets.

[0119] Step S3: After the standard solution of step S1 and the blank solution of step S2 are prepared, the fluorescence spectrum of the standard solution is immediately measured under the condition of an excitation wavelength of 365 nm. The fluorescence spectrum of the standard solution is as follows: Figure 4 The standard curve is drawn with the concentration of glutathione in the standard solution as the horizontal axis and the fluorescence intensity of the standard solution at a wavelength of 550nm as the vertical axis. The results are shown in Figure 5 shown.

[0120] Step S5: Determination of sample solution content and recovery rate. The human serum sample taken from the hospital was diluted 100 times, and the sample solution to be tested was prepared in the same way as the standard solution preparation method mentioned above, and the fluorescence was detected. Figure 5 The concentration of GSH in the diluted serum was calculated using the standard curve in to measure its recovery. Then, known concentrations of GSH (50, 200, and 400.0 μM) were added to the 100-fold diluted serum samples, and the concentration of GSH in the spiked serum samples was determined in the same manner. The recovery of the GSH biosensor in the spiked serum samples (i.e., the recovery of the content detection results) was then calculated. The results are shown in Table 1, and the recovery rates obtained ranged from 96.48 to 102.94%.

[0121] Table 1 Detection of GSH in diluted serum based on AuNCs probe

[0122]

[0123] The results shown in Table 1 indicate that the glutathione content determination method of this embodiment can effectively determine the GSH content in human serum.

[0124] like Figure 4 As shown ( Figure 4The schematic diagram of the fluorescence spectrum detection results of the standard solution in this example is that in the HEPES buffer at pH = 6.5, Au(I)-SG oligomer and Ce(CO 3 ) 2 After adding increasing concentrations of GSH to the mixture of nanosheets, the fluorescence spectrum of the synthesized GSH@AuNCs is shown in Figure 2. The fluorescence intensity of the standard solution is positively correlated with the glutathione concentration, and the maximum absorption wavelength is about 550nm. Figure 5 As shown ( Figure 5 is the standard curve of fluorescence intensity and concentration of the standard solution of this example, that is, in the HEPES buffer at pH = 6.5, in the presence of Au(I)-SG oligomer and Ce(CO 3 ) 2 After adding increasing concentrations of GSH to the mixture of nanosheets, the calibration curve of the fluorescence intensity of the synthesized GSH@AuNCs and the GSH concentration) was plotted. When the glutathione concentration of the standard solution was in the range of 50–1000 μM, Figure 5 As shown, the standard curve drawn with the concentration of the standard solution as the horizontal axis and the fluorescence intensity as the vertical axis is linear, and the standard curve shows a good linear relationship, which indicates that the applicable concentration range of the glutathione content determination method of the embodiment of the present application includes 50 to 1000 μM.

[0125] According to the above standard solution preparation method, glutathione and Ce(CO 3 ) 2 The nanosheets were sequentially added with Au(I)-SG oligomers to form in situ synthesized GSH@AuNCs, and the glutathione concentration was 2 mM. The glutathione and Ce(CO 3 ) 2 The particle structure of the nanosheets mixed with Au(I)-SG oligomers, that is, the glutathione and Ce(CO 3 ) 2 The structure of GSH@AuNCs synthesized in situ by mixing nanosheets with Au(I)-SG oligomers. Figure 6 As shown in the figure, the structure of GSH@AuNCs is similar to that of glutathione and Ce(CO 3 ) 2 The structure of the mixed nanosheets is compared with that of glutathione and Ce(CO 3 ) 2 HRTEM image of nanosheets after mixing Figure 7 shown.

[0126] like Figure 6 As shown ( Figure 6 for GSH and Ce(CO 3 ) 2HRTEM image of GSH@AuNCs synthesized in situ by sequentially adding Au(I)-SG oligomers to nanosheets. 3 ) 2 After mixing the nanosheets and Au(I)-SG oligomers, GSH@AuNCs with AIE properties were synthesized in situ. Figure 7 As shown, only glutathione and Ce(CO 3 ) 2 After the nanosheets were mixed, no structure with AIE properties was formed.

[0127] Example B2

[0128] Referring to the above Example B1, a linear calibration curve of a glutathione standard solution with a glutathione concentration range of 50 to 1000 μM was determined, and sample solutions with glutathione concentrations of 200.0 μM and 500.0 μM were taken for three consecutive measurements. The relative standard deviations of the measurement results were 2.3% and 2.8%, respectively, which indicates that the glutathione content detection method of the application example has good repeatability.

[0129] Example B3

[0130] This example investigates the specificity of the glutathione content detection in the present application example. The operation process is as follows:

[0131] Step G1: Take the Au(I)-SG oligomer solution and Ce(CO 3 ) 2 The nanosheet dispersion was prepared by referring to step S1 of Example B1 to prepare a standard solution with a glutathione concentration of 500 μM and a blank solution.

[0132] Step G2: Referring to the preparation method of step G1, K + 、Ag + Different metal ions or amino acids, as well as sugar with a concentration of 10.0 mM, or a bioreductant with a concentration of 500.0 μM were used to replace the GSH in the above step G1 to prepare different control solutions of substances. The types and concentrations of substances in each control solution are shown in Table 2.

[0133] Step G3: Detect the fluorescence spectra of the standard solution in step G1 and the different control solutions in step G2 respectively. The fluorescence intensity results of the standard solution and the control solution at 550nm are as follows: Figure 8 As shown, Figure 8 The PL in the equation represents the fluorescence intensity of the standard solution or other control solution. 0 represents the fluorescence intensity of the blank solution.

[0134] Table 2

[0135] Type of substance concentration Type of substance concentration Type of substance concentration GSH 500μM <![CDATA[Ba 2+ ]]> 1.0mM Proline (Pro.) 1.0mM <![CDATA[K + ]]> 1.0mM Glutamic acid (Glu.) 1.0mM Phenylalanine (Phe.) 1.0mM <![CDATA[Ag + ]]> 1.0mM Lactose (Lac.) 10.0mM Amino Acids (AA) 1.2mM <![CDATA[Zn 2+ ]]> 1.0mM Galactose (Gal.) 10.0mM Lipase (LiP.) 500μM <![CDATA[Co 2+ ]]> 1.0mM Fructose (Fru.) 10.0mM Cysteine ​​(Cys.) 1.0mM <![CDATA[Mg 2+ ]]> 1.0mM Glycine (Gly.) 1.0mM <![CDATA[H 2 THE 2 ]]> 800μM <![CDATA[Ni 2+ ]]> 1.0mM Alanine (Ala.) 1.0mM Reduced coenzyme I (NADH) 500μM <![CDATA[Cu 2+ ]]> 1.0mM Tyrosine (Tyr.) 1.0mM Reduced coenzyme II (NADPH) 500μM

[0136] like Figure 8 As shown, the metal ions and amino acids in Table 2 have no obvious fluorescence after being mixed with the glutathione detection reagent of Example A1, which means that these substances have no obvious interference with the detection of glutathione. The glutathione detection reagent of the embodiment of the present application has good anti-interference properties for glutathione detection, which means that the glutathione detection reagent of the embodiment of the present application has good specificity for glutathione detection.

[0137] Example B4

[0138] This example compares the sensitivity of different glutathione content detection methods, and the results are shown in Table 3.

[0139] Table 3

[0140]

[0141]

[0142] Note: GO, QD, GQDs, UCNPs, TAT-probe, DTFN, TP-N and CDs in Table 2 refer to graphene oxide, quantum dots, graphene quantum dots, upconversion nanoparticles, two-photon biothiol probes, dual-targeted fluorescent nanoprobes, phthalazinetrione derivatives, 2-(benzo[d]thiazol-2-yl)-4-hydroxyphthalazine-1(2H)-dimer monohydrate and carbon dots, respectively.

[0143] As shown in Table 2, the glutathione content detection method of the embodiment of the present application has high sensitivity, a detection limit of only 1.02 μM, a wide linear range, and good linearity when the glutathione concentration is above the detection limit and below 1000 μM. Therefore, the glutathione content detection method of the embodiment of the present application has a wider range of application and can be applied to the detection of samples with different glutathione contents.

[0144] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0145] References:

[0146] [1] Wang Y, Lu J, Tang L H, et al. Graphene oxide amplified electrogenerated chemiluminescence of quantum dots and its selective sensing for glutathione from thiol-containing compounds[J]. Analytical Chemistry, 2009, 81(23): 9710-9715.

[0147] [2] Dennany L, Gerlach M, O'Carroll S, et al. Electrochemiluminescence(ECL) sensing properties of water-soluble core-shell CdSe / ZnS quantum dots / Nafion composite films[J]. Journal of Materials Chemistry, 2011, 21(36): 13984-13990. Preliminary study on self-assembly, chiral optical properties and sensing of chiral gold nanoclusters.

[0148] [3] Kamidate T, Watanabe H. Peroxidase-catalysed luminol chemiluminescence method for the determination of glutathione[J]. Talanta, 1996, 43(10): 1733-1738.

[0149] [4] Tang S P, Yu C L, Qian L L, et al. Nitrogen-doped graphene quantum dots-MoS2 nanoflowers as a fluorescence sensor with an off / on switch for intracellular glutathione detection and fabrication of molecular logic gates[J]. Microchemical Journal, 2021, 171: 106786.

[0150] [5]Deng R R,Xie X J,Vendrell M,et al.Intracellular glutathionedetection using MnO2-nanosheetmodified upconversion Nanoparticles[J].Journalof the American Chemical Society,2011,133(50):20168-20171.

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[0155]

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Claims

1. A glutathione detection reagent, characterized in that Including Au(I)-SG oligomers and Ce 4+ The first cerium salt is prepared from Au(I)-SG oligomer, wherein the Au(I)-SG oligomer comprises a glutathione ligand and an Au element.

2. The glutathione detection reagent according to claim 1, characterized in that The glutathione detection reagent also includes a buffer solution, and the pH value of the buffer solution is 6.5-7.

5.

3. A glutathione detection kit, characterized in that, The glutathione detection kit comprises the glutathione detection reagent according to any one of claims 1-2.

4. Use of the glutathione detection reagent according to any one of claims 1 to 2 or the glutathione detection kit according to claim 3 in glutathione detection.

5. A method for detecting glutathione content, characterized in that: The steps include: Mixing a glutathione detection reagent with a sample to be tested to obtain a premixed solution to be tested, performing a first redox treatment on the premixed solution to be tested to obtain a sample solution to be tested, and measuring the fluorescence intensity of the sample solution to be tested; Mixing the glutathione detection reagent with a glutathione standard to obtain a standard premix solution, performing a second redox treatment on the standard premix solution to obtain a standard solution, and measuring the fluorescence intensity of the standard solution; Calculating the glutathione content in the sample to be tested according to the fluorescence intensity of the sample solution to be tested, the glutathione concentration in the standard solution and the fluorescence intensity of the standard solution; Wherein, the glutathione detection reagent is any one of the glutathione detection reagents in claims 1-2.

6. The method for detecting glutathione content as claimed in claim 5, characterized in that: The glutathione concentration contained in the standard solution is 1-1000 μmol / L.

7. The method for detecting glutathione content as claimed in claim 5, characterized in that: The pH value of the premixed solution to be tested is 6.5 to 7.5; and / or The pH value of the standard premix solution is 6.5-7.

5.

8. The method for detecting glutathione content according to any one of claims 5 to 7, characterized in that: The excitation wavelength for measuring the fluorescence intensity of the sample solution to be tested is 365 nm.

9. The method for detecting glutathione content according to any one of claims 5 to 7, characterized in that: The emission wavelength for measuring the fluorescence intensity of the sample solution to be tested includes 500-600 nm.

10. The method for detecting glutathione content according to any one of claims 5 to 7, characterized in that: The temperature of the first oxidation-reduction treatment is 30-40° C.; and / or The temperature of the second redox treatment is 30-40°C.

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