CN111024636A

CN111024636A - Colorimetric method for detecting glutathione based on CoOOH-TMB oxidation system

Info

Publication number: CN111024636A
Application number: CN202010030810.7A
Authority: CN
Inventors: 袁玲玲; 郭文娟
Original assignee: University of Jinan
Current assignee: Fosun diagnostic technology (Taizhou) Co.,Ltd.
Priority date: 2019-10-16
Filing date: 2020-01-13
Publication date: 2020-04-17
Anticipated expiration: 2040-01-13
Also published as: CN111024636B

Abstract

The disclosure relates to a method for detecting glutathione by a colorimetric method based on a CoOOH-TMB oxidation system and a kit thereof, wherein the method for detecting GSH comprises the following steps: mixing a sample to be detected with a cobalt oxyhydroxide nanosheet solution, and then incubating to obtain a mixed solution I; adding an acetic acid-acetate buffer solution and a tetramethylbenzidine solution into the mixed solution I, and uniformly mixing to obtain a mixed solution II; and (5) measuring the absorbance of the mixed solution II, namely measuring the content of the GSH. The method and the kit have the advantages of high sensitivity, good specificity, simple and convenient operation and low cost.

Description

Colorimetric method for detecting glutathione based on CoOOH-TMB oxidation system

Technical Field

The disclosure relates to a colorimetric method based on a CoOOH-TMB oxidation system for detecting glutathione, belonging to the field of biology and analytical chemistry.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Glutathione is a tripeptide containing gamma-amido bond and sulfhydryl group, consists of glutamic acid, cysteine and glycine, exists in almost every cell of the body, has two forms of reduction type (GSH) and oxidation type (GSSG), and is the most of reduced glutathione under physiological conditions. Glutathione can help to maintain normal immune system function, and has effects of resisting oxidation and removing toxic substance. The glutathione can be used in medicine, can be used as base material of functional food, and can be widely used in the functional food for delaying senility, enhancing immunity, resisting tumor, etc. Therefore, the method has various important meanings for detecting the content of the glutathione.

For the detection method of glutathione, the current commonly used methods include: fluorescence method, High Performance Liquid Chromatography (HPLC), Surface Enhanced Raman Spectroscopy (SERS), High Performance Capillary Electrophoresis (HPCE), enzyme-linked immunosorbent assay (ELISA), etc. Although these established methods have certain advantages in quantitative detection of glutathione, these detection methods require expensive instruments, complicated and time-consuming operation steps, high cost, and require professional personnel to operate.

Therefore, at present, there is still a need to develop a glutathione detection kit and a detection method which are simpler and lower in cost, etc., so as to be more suitable for scientific research and daily detection application.

Disclosure of Invention

Against the background art, the method is applied to various innovative products such as chemical drugs, biological products, traditional Chinese medicines, medical instruments and the like so as to obtain preclinical research and entrusted Contract Research (CRO) of clinical trials which are targeted for marketing approval. The method is mainly used for in-vitro diagnosis and detection in medical examination and inspection instruments and services, and the method for detecting the glutathione by the colorimetric method based on the CoOOH-TMB oxidation system is high in sensitivity, good in specificity, simple and convenient to operate and low in cost.

Specifically, the following technical scheme is adopted in the disclosure:

a first object of the present disclosure provides a kit for detecting GSH, the kit comprising:

cobalt oxyhydroxide (CoOOH) nanosheet solution or a substance in which a cobalt oxyhydroxide (CoOOH) nanosheet solution is disposed;

acetic acid-acetate buffer solution or buffer substance for preparing acetic acid-acetate buffer solution; and the combination of (a) and (b),

tetramethyl benzidine.

A second object of the present disclosure is to provide a kit for detecting GSSG, which comprises:

a GSH masking agent;

a GSSG alkaline reducing agent;

a neutralization reagent of the GSSG alkaline reducing agent;

tetramethylbenzidine (TMB).

In a third object of the present disclosure, there is provided a method of detecting GSH, the method comprising the steps of:

mixing a sample to be detected with a cobalt oxyhydroxide nanosheet solution, and then incubating to obtain a mixed solution I;

adding an acetic acid-acetate buffer solution and a tetramethylbenzidine solution into the mixed solution I, and uniformly mixing to obtain a mixed solution II;

and (5) measuring the absorbance of the mixed solution II, namely measuring the content of the GSH.

In a fourth object of the present disclosure, there is provided a method of detecting a GSSG, the method comprising the steps of:

adding a GSH masking agent into a sample to be detected, and uniformly mixing to obtain a mixed solution III;

adding a GSSG alkaline reducing agent into the mixed solution III, and uniformly mixing to obtain a mixed solution IV;

adding a neutralization reagent of the GSSG alkaline reducing agent into the mixed solution IV, and uniformly mixing to obtain a mixed solution V;

mixing the mixed solution five with a cobalt oxyhydroxide solution, and then incubating to obtain a mixed solution six;

adding an acetic acid-acetate buffer solution and a tetramethylbenzidine solution into the mixed solution VI, and uniformly mixing to obtain a mixed solution VII;

and (4) measuring the absorbance of the mixed solution seven, namely measuring the content of the GSSG.

A fifth object of the present disclosure provides a method of determining a ratio of GSH to GSSG, the method comprising the steps of:

determining the content of the GSH in the sample to be detected according to the method for detecting the GSH;

determining the content of GSSG in a sample to be detected according to the method for detecting GSSG;

the ratio of GSH to GSSH is calculated.

Compared with the related technology known by the inventor, one technical scheme of the present disclosure has the following beneficial effects:

the GSH and GSSH detection method and the kit disclosed by the invention have the advantages of high sensitivity, good specificity, simplicity and convenience in operation, low cost, no need of expensive instruments and biological reagents, and wide application prospect for detection of biological samples.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.

FIG. 1: synthesis of CoOOH and schematic diagrams for detecting GSH using CoOOH.

FIG. 2: transmission electron micrograph of CoOOH.

FIG. 3: a. AFM images of CoOOH; b. cross-sectional view of the corresponding thickness of CoOOH.

FIG. 4: a. infrared spectrum of CoOOH, and X-ray diffraction spectrum of CoOOH.

FIG. 5: after GSH solutions with different concentrations and a CoOOH nanosheet solution are mixed and reacted for 4h at 40 ℃, TMB and CoOOH remaining in the reaction are added to form an oxTMB absorption curve in the range of 0.1-6 mu M (a) and 8-300 mu M (c) and the corresponding change of the absorbance at 652nm along with the concentration of the GSH in the range of 0.1-6 mu M (b) and 8-300 mu M (d).

FIG. 6: reaction of remaining CoOOH with TMB in the range of 0.1-6 μm (a) and 8-300 μm (b) with different concentrations of GSH produced a calibration curve of the absorbance of oxTMB at 652nm as a function of GSH concentration.

FIG. 7: UV-vis absorption spectra of CoOOH nanosheets (a) and TMB (b).

FIG. 8: effect profile of Ascorbic Acid (AA) on GSH detection.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background art, some current commonly used glutathione detection methods have the problems of complicated and time-consuming operation steps, high cost, professional personnel operation and the like.

In order to solve the above technical problems, in a first exemplary embodiment of the present disclosure, there is provided a kit for detecting GSH, the kit including:

tetramethyl benzidine.

In one or more embodiments of the present disclosure, the substance configuring the cobalt oxyhydroxide (CoOOH) nanosheet solution is cobalt oxyhydroxide (CoOOH) nanosheet and water or polyethylene glycol.

In one or more embodiments of the present disclosure, the acetate-acetate may be sodium acetate-acetate (HAc-NaAc) having a pH of 3 to 4, and further, a pH of 4. The pH value of the buffer solution is set to be 3-4, and experiments prove that the system reacts for a long time under the acid environment condition, so that the system is beneficial to decomposing reducing substances such as ascorbic acid or destroying the molecular structures of the reducing substances, and the interference of the reducing substances such as the ascorbic acid is eliminated.

In one or more embodiments of the present disclosure, the kit for detecting GSH further comprises instructions providing specific instructions and precautions for the detection.

In a second exemplary embodiment of the present disclosure, there is provided a kit for detecting GSSG, comprising:

a GSH masking agent;

a GSSG alkaline reducing agent;

a neutralization reagent of the GSSG alkaline reducing agent;

tetramethylbenzidine (TMB).

In one or more embodiments of the present disclosure, the GSH masking agent may be a conventional agent in the art, including, but not limited to, N-ethylmaleimide (NEM), and the like.

In one or more embodiments of the present disclosure, the GSSG alkaline reducing agent may be a conventional agent in the art, including but not limited to sodium borohydride and the like.

In one or more embodiments of the present disclosure, the neutralizing agent of the GSSG alkaline reducing agent may be a conventional agent in the art, including but not limited to organic acids (e.g., acetic acid, etc.), and the like.

In one or more embodiments of the present disclosure, the acetate-acetate may be sodium acetate-acetate (HAc-NaAc) having a pH of 3 to 4, and further, a pH of 4.

In one or more embodiments of the present disclosure, the kit for detecting GSSG further comprises instructions for providing specific instructions and cautions for the detection.

In a third exemplary embodiment of the present disclosure, there is provided a method of detecting GSH, the method including the steps of:

(1) mixing a sample to be detected with a cobalt oxyhydroxide nanosheet solution, and then incubating to obtain a mixed solution I;

(2) adding an acetic acid-acetate buffer solution and a tetramethylbenzidine solution into the mixed solution I, and uniformly mixing to obtain a mixed solution II;

(3) and (5) measuring the absorbance of the mixed solution II, namely measuring the content of the GSH.

The CoOOH nanosheets can directly oxidize TMB to oxTMB (blue) with an absorption peak of 652nm and cause the color to change from colorless to blue, while GSH can decompose the CoOOH nanosheets to Co when GSH is present²⁺，Co²⁺Does not oxidize TMB, thereby reducing the production of oxTMB, resulting in a decrease in absorbance and bleaching of the blue color. The concentration of GSH is therefore readily determined by absorbance spectroscopy and observing the color change with the naked eye.

In one or more embodiments of the present disclosure, in step (1), the sample to be tested may be a biological sample or a non-biological sample.

The biological sample may be from a mammalian subject or a non-mammalian subject. The mammalian subject can be, for example, a human or other animal species. Biological samples include biological fluids such as whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, fecal matter, urine, spinal fluid, saliva, stool, cerebrospinal fluid, tears, mucus, and the like; biological tissue, such as hair, skin, cells from organs or other body parts, sections, or excised tissue; and so on. In many cases, the test sample is a cell, whole blood, plasma, or serum.

Non-biological samples include, but are not limited to, for example, foods, nutraceuticals, and the like, which can also be detected using GSH according to the principles described in this disclosure.

In one or more embodiments of the present disclosure, in step (1), the cobalt oxyhydroxide nanosheet solution is a mixed solution of cobalt oxyhydroxide nanosheets and water or polyethylene glycol;

furthermore, the cobalt oxyhydroxide nanosheet solution is a mixed solution composed of the cobalt oxyhydroxide nanosheets and polyethylene glycol, and in experimental research, the cobalt oxyhydroxide nanosheets are dispersed in the polyethylene glycol, so that the dispersing effect is more uniform, and the stabilization time of a subsequent detection system is longer, so that the detection result is more accurate and is obviously superior to that of a water system.

In one or more embodiments of the present disclosure, in step (1), the cobalt oxyhydroxide nanoplates can be conventionally prepared by prior art methods.

Preferably, the shape of the cobalt oxyhydroxide nanosheet is a regular or irregular hexagon, the side length of the hexagon is 50-150 nm, and the cobalt oxyhydroxide nanosheet with the shape can enable the detection effect to be more accurate and the sensitivity to be higher through experimental verification.

Correspondingly, a more preferable preparation method of the cobalt oxyhydroxide nanosheet comprises the following steps:

to Co (NO)₃)₂·6H₂And adding a NaOH solution into the O solution, ultrasonically dispersing the mixture, then adding a NaClO solution into the ultrasonically treated mixture, and then ultrasonically treating to obtain the cobalt oxyhydroxide nanosheet.

Wherein said Co (NO)₃)₂·6H₂The concentration of the O solution is 5-15 mmoL/L, and the concentration of the NaOH solution is 0.5-1.5moL/L, the concentration of NaClO solution is 0.5-1.5 moL/L, and Co (NO) is added₃)₂·6H₂The volume ratio of the O solution to the NaOH solution to the NaClO solution is 400-800: 100-150: 10-50.

In order to improve the dispersibility of the prepared CoOOH and the stability of a subsequent detection system, the obtained cobalt oxyhydroxide nanosheet is added with polyethylene glycol and subjected to ultrasonic treatment for 2-3 h.

In one or more embodiments of the present disclosure, in the step (1), the incubation temperature is 25 to 42 ℃, and the incubation time is 3 to 5 hours; in order to better exclude interference of other reducing substances in the system and improve detection specificity, the incubation temperature is preferably 40 ℃ and the incubation time is preferably 4 h.

In one or more embodiments of the present disclosure, the concentration of the cobalt oxyhydroxide nanosheet solution is 50 to 150 μ g/mL, and the concentration of the TMB solution is 2 to 8 mmoL/L. The volume ratio of the sample to be detected, the cobalt oxyhydroxide nanosheet solution and the TMB solution is (300-500): (300-500): (50-150).

In one or more embodiments of the present disclosure, the method for detecting GSH further includes a step of preparing a standard curve and a linear equation.

Specifically, a series of GSH standard solutions with different concentrations are prepared, the absorbance of the GSH standard solution with different concentrations is obtained according to the method for detecting the GSH, and a standard curve is drawn according to the absorbance to obtain a linear equation.

In a fourth exemplary embodiment of the present disclosure, there is provided a method of detecting a GSSG, the method including the steps of:

and operating according to the method for detecting the GSH, so that the content of the GSSG can be detected.

In one or more embodiments of the present disclosure, the method for detecting GSSA further includes a step of preparing a standard curve and a linear equation.

Specifically, a series of GSSA standard solutions with different concentrations are prepared, the absorbance of the GSSA standard solutions with different concentrations is obtained according to the method for detecting the GSSA, and a standard curve is drawn according to the absorbance to obtain a linear equation.

In a fifth exemplary embodiment of the present disclosure, there is provided a method of determining a ratio of GSH to GSSG, the method comprising the steps of:

the ratio of GSH to GSSH is calculated.

It should be noted that glutathione has important roles or meanings in both living or non-living organisms, such as reducing the level of free radicals in cells, maintaining the metabolic process, removing toxic substances generated in the metabolic process, balancing the redox pressure in cells, and the like, and the methods for detecting GSH and GSSG in the present disclosure are all non-diagnostic detection methods.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.

Example 1

Preparation of cobalt oxyhydroxide (CoOOH) nanosheet solution:

to 500. mu.L of Co (NO)₃)₂·6H₂To the O (10mM) solution was added 125. mu.L of NaOH (1.0M) solution, the mixture was sonicated for 1min, then 25. mu.L of NaClO (0.9M) sample was added to the sonicated mixture, and sonicated for 10min to yield a dark brown floc. In order to improve the dispersibility of the prepared CoOOH, polyethylene glycol is added into the mixture, and ultrasonic treatment is carried out for 2-3 hours to prepare a cobalt oxyhydroxide (CoOOH) nanosheet solution for later use.

And (3) obtaining the black brown CoOOH nanosheet through centrifugal washing, wherein the black brown CoOOH nanosheet is a projection electron microscope photo of the CoOOH nanosheet as shown in figure 2, and the obtained CoOOH nanosheet is hexagonal and has the side length of about 50-100 nm as can be seen from the figure. As shown in fig. 3, an AFM image and corresponding thickness profile of CoOOH nanoplates. FIG. 4 shows the infrared spectrum and X-ray diffraction spectrum of the CoOOH nanosheets. As shown in fig. 7, a, is the UV-vis absorption spectrum of CoOOH nanosheets.

Example 2

A kit for detecting GSH, the kit comprising:

cobalt oxyhydroxide (CoOOH) nanosheet solution;

acetic acid-sodium acetate buffer solution;

tetramethylbenzidine, shown as b in FIG. 7, is a UV-vis absorption spectrum.

A method for detecting GSH by using the kit comprises the following steps:

(1) preparing GSH standard solutions with different concentrations by adopting ultrapure water, and detecting the absorbance of the GSH standard solutions with different concentrations;

the specific detection method comprises the following steps:

A. 400 μ L of water and 400 μ L of CoOOH (100 μ g/mL) nanosheet solution were added to a centrifuge tube, the solution appeared pale brown yellow to CoOOH, 500 μ L of HAc-NaAc (pH 4) buffer solution (pH of the solution was controlled to 4), and 100 μ L of TMB (5mM) (for color development) were added to the centrifuge tube, and the mixture was mixed uniformly, and the absorbance thereof was measured and recorded as the absorbance of the original solution.

B. Respectively adding 400 mu L of GSH standard solution with different concentrations (0.1-6 mu moL/L, 8-300 mu moL/L) and 400 mu L of LCoOOH (100 mu g/mL) nanosheet solution into a centrifuge tube, wherein the solution is light brown yellow of CoOOH;

C. placing the centrifuge tube in an oven at 40 deg.C for 4h to allow GSH to reduce CoOOH to Co²⁺(ii) a After incubation for 4h, taking out the centrifuge tube, and lightening the color of the light brown yellow solution;

D. to a centrifuge tube, 500 μ L of HAc-NaAc (pH 4) buffer solution (pH of the solution was controlled to 4) and 100 μ LTMB (5mM) (for color development) were added and mixed well; turning blue after adding TMB;

E. transferring 200 mu L of the mixed solution, measuring the absorbance in a 96 micro-porous plate, and testing for three times in parallel;

the absorbance of the solution at a wavelength of 652nm was taken as the ordinate, and the concentration of the GSH standard solution as the abscissa, to establish a standard curve, as shown in fig. 5.

The difference between the original absorbance and the ordinate of the standard curve is taken as the new ordinate, and the concentration of the GSH standard solution is taken as the abscissa, so as to obtain the calibration curve shown in fig. 6.

(2) And (3) detecting GSH in the sample to be detected:

and (3) operating the specific detection method according to the method in the step (1) to obtain absorbance data, and substituting the absorbance data into a linear equation to calculate the concentration of the GSH in the sample to be detected.

And (3) test results:

1. as the concentration of added GSH increases, the production of oxTMB decreases, resulting in a decrease in absorbance at 652 nm; and the solution color can be observed by naked eyes to be sequentially lightened from blue;

2. the linear detection range of the method disclosed by the invention is 0.1-300 mu M, and the detection limit is 0.1 mu M, so that the practical detection application is met.

Example 3

The content detection of the GSH in the actual sample comprises the following steps:

(1) preparing SH-SY5Y cell extract: SH-SY5Y cells were washed once with PBS, harvested by centrifugation, and the supernatant was aspirated. A protein-removing reagent S solution was added in an amount 3 times the cell precipitation volume, and Vortex was performed sufficiently. The samples were then subjected to two rapid freeze-thaw cycles using liquid nitrogen and a 37 ℃ water bath. Placed at 4 ℃ or in an ice bath for 5 minutes. Centrifuge at 10,000g for 10min at 4 ℃. Taking supernatant for measuring total glutathione. The treated cell sample was diluted 100-fold with the protein-removing reagent S solution and then assayed.

Preparation of protein removal reagent S solution:

8 ml of ultrapure water was added to 0.4 g of the protein-removing reagent S provided in the kit, to prepare 8 ml of a 5% aqueous solution. Storing at 4 ℃.

(2) The content of GSH detected by the method in example 2 is 12.6878 + -1.81131 μmoL/L.

GSH in the SH-SY5Y cell extracting solution is detected by adopting a relatively mature commercial kit, the kit is a Biyuntian (Beyotime) total glutathione detection kit (product number S0052), the content of the GSH is detected to be 12.07053 +/-0.8551 mu moL/L, the relative difference between the GSH and the GSH is 5.11%, and no obvious difference exists. Therefore, the kit and the detection method provided by the disclosure have the technical effect equivalent to that of a mature commercial kit, the commercial kit is very expensive, and the kit provided by the disclosure is low in price and the detection method is simple and convenient.

Example 4

A kit for detecting GSSG, comprising:

n-ethylmaleimide;

sodium borohydride;

acetic acid;

cobalt oxyhydroxide (CoOOH) nanosheet solution;

acetic acid-sodium acetate buffer solution;

tetramethyl benzidine.

A method for detecting GSSG by using the kit comprises the following steps:

(1) preparing GSSG standard solutions with different concentrations by adopting ultrapure water, and detecting the absorbance of the GSSG standard solutions with different concentrations;

the specific detection method comprises the following steps:

A. respectively adding 40 mu g of N-ethylmaleimide into 400 mu L of GSSG standard solutions with different concentrations, uniformly mixing, and masking GSH;

B. adding 40 mu g of sodium borohydride into the mixed system obtained in the step A, uniformly mixing, and reducing GSSG into GSH;

C. adding 15 mu L of acetic acid into the mixed system in the step B, and neutralizing redundant sodium borohydride;

D. adding the mixed system in the step C and 400 mu L of CoOOH (100 mu g/mL) nanosheet solution into a centrifuge tube, wherein the solution is light brown yellow of CoOOH;

E. placing the centrifuge tube inOven incubation at 40 ℃ for 4h so that GSH can reduce CoOOH to Co²⁺(ii) a After incubation for 4h, taking out the centrifuge tube, and lightening the color of the light brown yellow solution;

F. to the tube was added 500. mu.L of HAc-NaAc (pH 4) buffer solution (pH 4 for control solution) and 100. mu.L of LTMB (5mM) for color development, and the mixture was mixed; turning blue after adding TMB;

G. transferring 200 mu L of the mixed solution, measuring the absorbance in a 96 micro-porous plate, and testing for three times in parallel;

and establishing a standard curve by taking the absorbance of the solution with the wavelength of 652nm as a vertical coordinate and the concentration of the GSSG standard solution as a horizontal coordinate to obtain a linear equation.

(2) Detecting GSSG in a sample to be detected:

the specific detection method is operated according to the method in the step (1) to obtain absorbance data, and the absorbance data is substituted into a linear equation to calculate the concentration of GSSG in the sample to be detected.

Example 5

Interference experiments:

to examine the selectivity of the disclosed methods for GSH, a series of interference tests were designed. The specific scheme is as follows:

the method of example 2 is adopted to detect the mixed solution of the interferent and the GSH, wherein the concentration of the interferent in the mixed solution is 0.01-2 mu moL/L, and the mixed solution is cysteine (Cys), N-acetylcysteine (NAC) or Ascorbic Acid (AA).

Specifically, as shown in FIG. 8, the absorbance at 652nm for the blank was 1.3866, the absorbance for the AA (concentration 2. mu. moL/L) added comparison was 1.3759, and the two lines are shown as overlapping in the figure. It can be seen that the addition of Ascorbic Acid (AA) does not interfere with the system. In practical application, the concentration of Ascorbic Acid (AA) in an organism is only one thousandth of GSH generally, and interference on a system is avoided, so that the accuracy of a result is influenced.

Experimental example 1

And (3) detecting GSH in the sample to be detected:

the specific detection method is as follows:

A. SH-SY5Y cell extracting solution in example 3 and 400 mu L of CoOOH (100 mu g/mL) nanosheet solution are added into a centrifuge tube, and the solution is light brown yellow of the CoOOH;

B. placing the centrifuge tube in an oven at 20 ℃ for incubation for 2 h; after the incubation is finished for 2h, taking out the centrifuge tube;

C. adding HAc-NaAc (pH 4) buffer solution (pH 5 of control solution, 100 μ L TMB (5mM) (for color development) into the above solution, mixing, adding TMB, and turning blue;

D. transferring 200 mu L of the mixed solution, measuring absorbance in a 96 micro-porous plate, testing in parallel for three times to obtain absorbance data, substituting the absorbance data into a linear equation obtained by the same method of the experimental example to calculate the concentration of the GSH in the SH-SY5Y cell extracting solution, and detecting to obtain the content of the GSH of 13.9543 +/-1.25127 mu moL/L.

The above embodiments are preferred embodiments of the present disclosure, but the embodiments of the present disclosure are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present disclosure should be regarded as equivalent replacements within the scope of the present disclosure.

Claims

1. A kit for detecting GSH, the kit comprising:

a cobalt oxyhydroxide nanosheet solution or a substance for preparing a cobalt oxyhydroxide nanosheet solution;

tetramethyl benzidine.

2. The kit according to claim 1, wherein the acetate-acetate salt is acetic acid-sodium acetate, and has a pH of 3 to 4, and further has a pH of 4;

further, the kit for detecting GSH also comprises instructions.

3. A kit for detecting GSSG is characterized by comprising:

a GSH masking agent;

a GSSG alkaline reducing agent;

a neutralization reagent of the GSSG alkaline reducing agent;

tetramethyl benzidine.

4. The kit of claim 3, wherein the GSH-masking agent is N-ethylmaleimide;

further, the GSSG alkaline reducing agent is sodium borohydride;

further, a neutralizing agent of the GSSG alkaline reducing agent is organic acid;

further, the acetic acid-acetate can be acetic acid-sodium acetate (HAc-NaAc) with the pH value of 3-4;

further, the kit for detecting GSSG also comprises an instruction manual.

5. A method of detecting GSH, the method comprising the steps of:

6. The method of claim 5, wherein the sample to be tested can be a biological sample or a non-biological sample;

further, the sample is a cell, whole blood, plasma, or serum;

further, the cobalt oxyhydroxide nanosheet solution is a mixed solution composed of cobalt oxyhydroxide nanosheets and water or polyethylene glycol;

further, the cobalt oxyhydroxide nanosheet solution is a mixed solution composed of cobalt oxyhydroxide nanosheets and polyethylene glycol;

furthermore, the shape of the cobalt oxyhydroxide nanosheet is regular or irregular hexagon, and the side length of the hexagon is 50-150 nm;

further, a method for preparing cobalt oxyhydroxide nanosheets, the method comprising the steps of:

to Co (NO)₃)₂·6H₂Adding a NaOH solution into the O solution, ultrasonically dispersing the mixture, then adding a NaClO solution into the ultrasonically treated mixture, and carrying out ultrasonic treatment to obtain a cobalt oxyhydroxide nano-sheet;

wherein said Co (NO)₃)₂·6H₂The concentration of the O solution is 5-15 mmoL/L, the concentration of the NaOH solution is 0.5-1.5 moL/L, the concentration of the NaClO solution is 0.5-1.5 moL/L, and Co (NO) is added₃)₂·6H₂The volume ratio of the O solution to the NaOH solution to the NaClO solution is 400-800: 100-150: 10-50.

7. The method according to claim 5, wherein the incubation temperature is 25 to 42 ℃ and the incubation time is 3 to 5 hours;

further, the incubation temperature is 40 ℃, and the time is 4 hours;

further, the concentration of the cobalt oxyhydroxide nanosheet solution is 50-150 mug/mL, and the concentration of the TMB solution is 2-8 mmoL/L;

further, the volume ratio of the sample to be detected, the cobalt oxyhydroxide nanosheet solution and the TMB solution is (300-500): (300-500): (50-150);

further, the method for detecting GSH further comprises the step of preparing a standard curve and a linear equation.

8. A method for detecting GSSG, which is characterized by comprising the following steps:

the method of detecting GSH according to any one of claims 5 to 7, wherein the content of GSSG can be detected.

9. The method of claim 8, further comprising: the method for detecting the GSSA further comprises the step of preparing a standard curve and a linear equation.

10. A method for determining the ratio of GSH to GSSG, comprising the steps of:

determining the content of GSH in a sample to be detected according to the method for detecting GSH in any one of claims 5-7;

determining the content of GSSG in a sample to be detected according to the method for detecting GSSG of claim 8 or 9;

the ratio of GSH to GSSH is calculated.