A method for detecting glutathione based on Mn-CDs nanozyme colorimetric sensing technology

Abstract

The present invention discloses a method for detecting glutathione based on the Mn-CDs nanozyme colorimetric sensing technology, belonging to the field of molecular detection technology. Based on the peroxidase-like activity of manganese-doped carbon quantum dots (Mn-CDs), under acidic conditions, it can catalyze TMB to generate ox-TMB, turning the solution from colorless to blue. Ox-TMB has an absorption peak at 652 nm. As an antioxidant, glutathione can inhibit the redox reaction catalyzed by Mn-CDs, resulting in the solution turning from blue to colorless and causing a decrease in absorbance. Based on this, a standard curve is established to achieve the quantitative detection of glutathione. The system constructed by this method is stable, has low environmental sensitivity, rapid reaction, wide detection range, low detection limit, and strong specificity, providing new method experience for the clinical detection of glutathione.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly to a method for detecting glutathione based on Mn-CDs nanozyme colorimetric sensing technology. Background Art

[0002] Glutathione is an endogenous active tripeptide containing γ-amide bond and sulfhydryl group, composed of glutamic acid, cysteine and glycine, and has two forms: reduced form (GSH) and oxidized form (GSSH). Under physiological conditions, 98% exists in the reduced form. It exists in almost every cell of the body. Its active group sulfhydryl group is easy to combine with certain drugs, toxins, etc., making it have an integrative detoxification effect. Especially, glutathione in hepatocytes can participate in biotransformation. The sulfhydryl group also combines with free radicals in the body, which can directly reduce free radicals into acidic substances, thereby accelerating the excretion of free radicals and counteracting the damage of free radicals to important organs. In addition, it also plays an important role in maintaining the in vivo redox level, protecting the activity of sulfhydryl enzymes, maintaining the stability of the erythrocyte membrane structure, regulating cell cycle, cell proliferation, apoptosis and other cell processes, and can regulate lymphocyte function and immune response. The level of glutathione is related to many diseases such as cancer, heart disease, AIDS and neurodegenerative diseases. Therefore, it is of great significance to determine the content of glutathione in body fluids.

[0003] At present, the GSH detection methods include high performance liquid chromatography, mass spectrometry, electrochemical analysis, fluorescence sensing method and calorimetry, etc. However, most of these methods need to be equipped with special instruments for detection, and have the disadvantages of complex operation, high analysis cost and narrow detection range. In contrast, the colorimetric analysis method has the advantages of low cost, simplicity and easy operation. In addition to not requiring specific precision instruments, its color change can be directly observed with the naked eye, which is an ideal method. As an artificial enzyme, nanozyme has the advantages of low preparation cost, strong operation stability, high catalytic activity and low sensitivity to environmental conditions. It reflects the concentration with the solution color sensing signal. Compared with ELISA, it does not rely on antigen-antibody reaction and does not rely on the participation of natural enzymes to directly develop color.

[0004] Patent publication number CN 116559102A (publication date: August 8, 2023) discloses a rapid detection method for glutathione and cysteine. Based on the activity of Fe-N-C@Hemin nanozyme and using the inhibition principle of sulfhydryl small molecules on the peroxidase-like activity of Fe-N-C@Hemin, the detection ability of Fe-N-C@Hemin nanozyme for glutathione and cysteine is verified, but it is used in the technical field of food safety detection; CN 117624519A (publication date: March 1, 2024) belongs to the field of photoelectrochemical analysis, with a relatively high cost, and is used for the detection of glutathione in bread and milk. Its application in actual serum samples is still unknown.

[0005] CN 117430770 A (Publication Date: January 23, 2024) discloses a method for detecting glutathione, uric acid, and L-cysteine based on the photo-responsive peroxidase-like activity of TPy COF. However, the detection range of GSH is 0 - 60 μM, the detection range is narrow, the material preparation is complex, and the operating conditions require high light catalysis. CN 116586055 A (Publication Date: August 15, 2023) discloses a method for preparing a manganese-based nanozyme using yam polysaccharide as a template and for detecting glutathione and Cr 6+ (or Hg 2+ ) ions. The detection range of GSH is 5 - 40 μM. Similarly, the detection range is narrow, and it has not been used for the detection of actual samples, and the anti-interference ability in actual samples is unknown.

[0006] In view of the current situation of the above-mentioned existing detection methods, that is, quantitative methods such as high-performance liquid chromatography, mass spectrometry, and electrochemical analysis have defects such as the need to purchase specific instruments, high costs, long time consumption, and unsuitability for large-scale applications. Electrochemical analysis methods and fluorescence sensing methods require the preparation of specific and expensive electrochemical cells and complex fluorescence probe preparations. In addition, they are also relatively cumbersome in dealing with background interference and sample pretreatment, with limited applications. Moreover, existing colorimetric methods based on artificial enzymes have low detection ranges, complex preparation of some materials, and high requirements for operating conditions. The present invention provides a method for detecting glutathione with low cost, wide detection range, fast analysis speed, applicable to existing instruments in clinical laboratories, without the need for additional instrument purchase costs, and convenient operation. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting glutathione based on the Mn-CDs nanozyme colorimetric sensing technology to solve the problems existing in the above-mentioned prior art. Based on the peroxidase-like activity of manganese-doped carbon quantum dots (Mn-CDs), under acidic conditions, it can catalyze 3,3',5,5'-tetramethylbenzidine (TMB) to generate ox-TMB, turning the solution from colorless to blue. Ox-TMB has an absorption peak at 652 nm. As an antioxidant, glutathione can inhibit the redox reaction catalyzed by Mn-CDs, resulting in the solution turning from blue to colorless and causing a decrease in absorbance. Based on this, a standard curve is established to achieve the quantitative detection of glutathione. The system constructed by this method is stable, less sensitive to the environment, has a rapid reaction, a wide detection range, a low detection limit, and strong specificity, providing new method experience for the clinical detection of glutathione.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for detecting glutathione based on a manganese-doped carbon quantum dot nanozyme colorimetric sensing technology. Under acidic conditions, the manganese-doped carbon quantum dot nanozyme catalyzes the redox reaction of 3,3',5,5'-tetramethylbenzidine to generate a blue solution; the addition of glutathione inhibits the redox reaction and the blue solution fades; the content of glutathione is determined by using the color change of the solution with or without glutathione as the signal output.

[0010] Preferably, it includes the following steps:

[0011] (1) Mix the sodium acetate-acetic acid buffer solution, 3,3',5,5'-tetramethylbenzidine solution and manganese-doped carbon quantum dot solution, add the sample to be tested, react, and measure the absorbance A at 652 nm; use water as the blank control and measure the absorbance A0 at 652 nm.

[0012] (2) Calculate ΔA according to ΔA = A0 - A; substitute ΔA into the standard curve to calculate the content of glutathione in the sample to be tested.

[0013] Preferably, the pH of the sodium acetate-acetic acid buffer solution is 3.5 ± 0.2; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 3 - 5 mM; the concentration of the manganese-doped carbon quantum dot solution is 1 mg / mL.

[0014] Preferably, the volume ratio of the sodium acetate-acetic acid buffer solution, the 3,3',5,5'-tetramethylbenzidine solution, the manganese-doped carbon quantum dot solution and the sample solution to be tested is 26:1:2:1.

[0015] Preferably, the temperature of the reaction is 20°C - 35°C and the time is 20 - 40 min.

[0016] Preferably, within the range of 0.0005 - 0.8 mM, the standard curve is ΔA = 0.9129c + 0.0412.

[0017] The present invention also provides a kit for detecting glutathione based on a manganese-doped carbon quantum dot nanozyme colorimetric sensing technology, and the kit contains a sodium acetate-acetic acid buffer solution, a 3,3',5,5'-tetramethylbenzidine solution and a manganese-doped carbon quantum dot solution.

[0018] Preferably, the pH of the sodium acetate-acetic acid buffer solution is 3.5 ± 0.2; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 3 - 5 mM; the concentration of the manganese-doped carbon quantum dot solution is 1 mg / mL.

[0019] The present invention discloses the following technical effects:

[0020] (1) The reagents used in the detection method of the present invention are simple and inexpensive; the experimental operation is convenient. Only Mn-CDs nanozyme and TMB are needed for the reaction. By utilizing the oxidase activity, it can avoid the defect of insufficient stability caused by the easy decomposition of H2O2 required for peroxidase-like activity in previous studies. It can be operated at room temperature, has low environmental sensitivity, can be detected using conventional instruments in existing clinical laboratories, without the need to purchase additional large experimental equipment, and can be combined with the current clinical laboratory pipeline equipment to achieve large-scale detection in a short time.

[0021] (2) The detection method of the present invention is based on the excellent oxidase-like activity of Mn-CDs nanozyme, with rapid reaction. The entire reaction can be completed within 20 minutes to obtain the detection result. Compared with traditional detection methods, the detection time is faster, meeting the requirements of rapid detection. Preliminary qualitative analysis can be carried out by visual observation, and the detection of absorbance at 652 nm can accurately and quickly reflect the quantitative result.

[0022] (3) The detection method of the present invention has a wide detection range, reaching 3 orders of magnitude, low detection limit, high sensitivity, and strong specificity, which can meet the detection of actual serum samples, provide some method experience for the construction of subsequent colorimetric sensors, and has potential for clinical application. Brief Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 It is a comparison chart of the oxidase-like catalytic activities of Mn-CDs and CDs in Example 1. The inset is the appearance diagram of the system solution containing Mn-CDs (blue) and CDs (colorless);

[0025] Figure 2 It is the ultraviolet-visible absorption spectrum diagram of different system solutions in Example 2. The inset is the appearance diagram of different system solutions;

[0026] Figure 3 It is the optimization result diagram of the detection conditions in Example 3; A is the optimization result of the optimal pH of NaAc-HAc buffer solution; B is the optimization result of the optimal color development concentration of TMB solution; C is the optimization result of the optimal reaction time; D is the optimization result of the optimal reaction temperature;

[0027] Figure 4 It is the standard curve diagram for the detection of glutathione by the colorimetric sensing method based on Mn-CDs nanozyme in Example 4;

[0028] Figure 5 This is the specific verification result of detecting glutathione by the Mn-CDs nanozyme colorimetric sensing method in Example 5. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] Verification of the peroxidase-like activity of Mn-CDs

[0036] In order to study the oxidase-like catalytic activity of Mn-CDs (prepared according to the method in the reference "Bifunctional Mn-Doped N-Rich Carbon Dots with Tunable Photoluminescence and Oxidase-Mimetic Activity Enabling Bimodal Ratiometric Colorimetric / Fluorometric Detection of Nitrite"), under the same conditions, CDs without Mn doping were used for comparison and TMB was used as the substrate to explore its absorbance at 652nm.

[0037] The specific operations are:

[0038] In 2600 μL of NaAc-HAc solution at pH 3.5, 100 μL of 3 mM TMB solution was added, and then 200 μL of 1 mg / mL Mn-CDs dispersion and CDs dispersion were added thereto, mixed thoroughly and incubated for 20 min, and then the UV-visible absorption spectrum was obtained using a spectrophotometer. Figure 1 As shown in the figure, under the same conditions, the mixed solution with Mn-CDs changed from colorless to blue and had a higher absorption at 652nm, while the mixed solution with CDs had almost no color change and almost no absorption at 652nm. Therefore, at the same concentration, Mn-CDs showed better oxidase-like activity than CDs.

[0039] Example 2

[0040] Feasibility of colorimetric sensing for glutathione (GSH) detection based on Mn-CDs nanozymes

[0041] In order to verify the feasibility of the colorimetric sensing method of Mn-CDs nanozymes for detecting GSH, the UV-visible absorption spectra of different system solutions were used for detection. The specific operation is as follows:

[0042] Set up the following solution:

[0043] NaAc-HAc+TMB: add 100 μL of 3 mM TMB solution to 2900 μL of NaAc-HAc solution at pH 3.5;

[0044] NaAc-HAc+TMB+GSH: add 100 μL of 3 mM TMB solution to 2800 μL of NaAc-HAc solution at pH 3.5, and then add 100 μL of 1 mM GSH solution;

[0045] Mn-CDs + NaAc-HAc + TMB: Add 100 μL of 3 mM TMB solution to 2700 μL of NaAc-HAc solution with pH = 3.5, and then add 200 μL of 1 mg / mL Mn-CDs dispersion;

[0046] Mn-CDs + NaAc-HAc + GSH: Add 200 μL of 1 mg / mL Mn-CDs dispersion to 2700 μL of NaAc-HAc solution with pH = 3.5, and then add 100 μL of 1 mM GSH solution;

[0047] Mn-CDs + NaAc-HAc + TMB + GSH: Add 100 μL of 3 mM TMB solution and 200 μL of 1 mg / mL Mn-CDs dispersion to 2600 μL of NaAc-HAc solution with pH = 3.5. After mixing, add 100 μL of 1 mM GSH solution.

[0048] After incubating the solutions of the above systems at room temperature for 20 min, detect them with a spectrophotometer at 652 nm. The results are as Figure 2 shown. NaAc-HAc + TMB shows that in the NaAc-HAc buffer, TMB itself has no absorption peak at 652 nm (NaAc-HAc + TMB); NaAc-HAc + TMB + GSH shows that after adding GSH, it does not affect the color and absorbance of the solution; Mn-CDs + NaAc-HAc + TMB shows that using TMB as the substrate, the color of the solution turns blue after adding Mn-CDs, showing a strong absorption peak at 652 nm, and the absorbance reaches 0.913. In contrast, the color of the solution of the system Mn-CDs + NaAc-HAc + GSH does not change, indicating that the presence of GSH cannot directly react with Mn-CDs, nor can it reflect the peroxidase-like catalytic activity of Mn-CDs nanozyme; Mn-CDs + NaAc-HAc + TMB + GSH shows that when using TMB as the substrate in the NaAc-HAc buffer system, adding Mn-CDs nanozyme and then adding GSH, the color of the system solution is lighter blue than that of Mn-CDs + NaAc-HAc + TMB, showing a relatively strong absorption peak at 652 nm, and the absorbance reaches 0.352, indicating that GSH can reduce the value of the absorbance of the solution, and the difference in absorbance reflects the concentration of GSH solution. Based on this, a standard curve is established for detecting GSH.

[0049] Example 3

[0050] Optimization of detection conditions for colorimetric sensing method based on Mn-CDs nanozyme for detecting glutathione (GSH)

[0051] Detection system: In 1300 μL of NaAc-HAc buffer solution, add 50 μL of TMB solution. Then, add 100 μL of Mn-CDs dispersion solution with a concentration of 1 mg / mL, mix well. Finally, add 50 μL of GSH solution with a concentration of 0.1 mM, and measure the absorbance of this system at 652 nm. The pH, TMB concentration, reaction time, and temperature during the reaction were optimized respectively. The specific operations are as follows:

[0052] (1) Prepare the above detection system with NaAc-HAc buffer solution within the pH range of 3.0±0.2 - 6.0±0.2. The absorbance detection results of different pH systems are as Figure 3 shown in A of. It can be seen that as the acidity of pH slowly weakens, the absorbance of the system first increases and then decreases. When pH = 3.5±0.2, the absorbance reaches the maximum value of 0.353. Therefore, the optimal pH of this detection system is determined to be 3.5±0.2.

[0053] (2) Explore the influence of TMB concentration on the detection system under the pH in step (1). Prepare the above detection system with TMB solutions with concentrations of 1, 3, 5, 8, 10, 13, and 15 mM. The absorbance detection results of different concentration TMB systems are as Figure 3 shown in B of. It can be seen that as the TMB concentration increases, the absorbance of the system first increases and then decreases. When the TMB concentration is between 3 - 5 mM, the color development is significant and the absorbance reaches the maximum. When the TMB concentration is 3 mM, the absorbance value is 0.215. Therefore, the optimal color development concentration is 3 mM for TMB;

[0054] (3) Under the optimal pH and TMB concentration, that is, pH = 3.5±0.2, TMB = 3 mM, explore the influence of the color development time on the detection system. Measure the absorbance of the system every 5 minutes. The results are as Figure 3 shown in C of. It can be seen that within 5 - 40 minutes, as the reaction time extends, GSH, as an antioxidant, inhibits the redox reaction catalyzed by the peroxidase-like activity of Mn-CDs with TMB as the substrate, resulting in a decrease in the absorbance of the solution at 652 nm. From 20 minutes to 40 minutes, the reaction is stable and the absorbance remains unchanged. Therefore, 20 minutes is the optimal reaction time.

[0055] (4) Under the optimal pH, TMB concentration, and reaction time conditions, explore the influence of the reaction temperature on the detection system. Incubate the detection system at 20, 25, 30, 35, 40, and 45 °C respectively. The absorbance detection results of different reaction temperature systems are as Figure 3 shown in D of. It can be seen that when the reaction temperature is between 20 °C and 35 °C, the absorbance of the system basically remains unchanged. Therefore, the detection is carried out at room temperature.

[0056] Example 4

[0057] Standard curve for the detection of glutathione (GSH) based on the colorimetric sensing method of Mn-CDs nanozyme

[0058] Construct a standard curve for the detection of GSH based on the colorimetric sensing method of Mn-CDs nanozyme. The specific operation is as follows:

[0059] Add 100 μL of 3 mM TMB solution to 2600 μL of NaAc-HAc buffer solution with pH = 3.5. Then, add 200 μL of 1 mg / mL Mn-CDs dispersion. After thorough mixing, add 100 μL of GSH solutions with concentrations of 0, 0.5, 0.2, 0.4, 0.6, and 0.8 mM respectively. Mix well and react at room temperature for 20 min. Measure the absorbance A of the above solutions at 652 nm using a microplate reader. Calculate the difference in absorbance ΔA and establish a standard curve, where ΔA = A0 - A, and A0 is the measurement result when the concentration of the GSH solution is 0.

[0060] The results are as Figure 4 shown. It can be seen that GSH can effectively reduce TMB to oxTMB, resulting in the fading of blue color and the increase in the difference in absorbance. In the range of 0.0005 - 0.8 mM, there is a good linear relationship between GSH and ΔA. The linear equation is ΔA = 0.9129c + 0.0412, and the correlation coefficient R2 = 0.9922. The detection limit LOD is calculated to be 0.167 μM based on 3 times the signal-to-noise ratio.

[0061] Example 5

[0062] Specificity verification for the detection of glutathione (GSH) based on the colorimetric sensing method of Mn-CDs nanozyme

[0063] In a mixed solution containing Mn-CDs (200 μL, 1 mg / mL), TMB (100 μL, 3 mM), and NaAc-HAc buffer solution (2600 μL, pH = 3.5 ± 0.2), common interferents and GSH (100 μL, 500 μM) are added respectively. The interferents include KCl, NaCl, CaCl2, MgCl2, ZnCl2, glycine, L-aspartic acid, urea, glucose, BSA, and GSH. Measure the absorbance value A at 652 nm. Use the addition of 100 μL of deionized water in the system as a control, and measure the absorbance value A0 at 652 nm. Calculate the difference in absorbance ΔA according to ΔA = A0 - A. The detection results are as Figure 5As shown, it can be seen that the detection system of the present invention has a high response to glutathione, with ΔA reaching 0.577, while the ΔA values of other interfering substances do not exceed 0.1, and in the actual samples, the concentrations of the interfering substances are lower than those used in the tests of this example. This indicates that the detection method constructed by the present invention has high selectivity and strong anti-interference ability, that is, it has high specificity for the detection of glutathione.

[0064] Example 6

[0065] Precision evaluation of the detection of glutathione (GSH) based on the Mn-CDs nanozyme colorimetric sensing method

[0066] To evaluate the precision of the constructed Mn-CDs nanozyme colorimetric sensing method, glutathione at three concentrations of low, medium, and high were selected. According to the method described in Example 4, 10 samples of each concentration were detected every day for 3 consecutive days, and the average value and standard deviation were calculated. The precision was evaluated by the relative standard deviation. The results are shown in Table 1. The within-batch precision was 0.92% - 5.82%, and the between-batch precision was 5.63% - 17.87%. The detection method constructed by the present invention has good precision.

[0067] Table 1 Results of precision analysis

[0068]

[0069] Example 7

[0070] Accuracy evaluation of the detection of glutathione (GSH) based on the Mn-CDs nanozyme colorimetric sensing method

[0071] To evaluate the accuracy of the constructed Mn-CDs nanozyme colorimetric sensing method, the spiked recovery rate of GSH was determined by the standard addition recovery test. GSH standard samples of 0.2, 0.4, and 0.6 mM were selected as the test samples, and spiked test samples were prepared by adding 0.4 mM GSH in a volume ratio of 1:1. The actual added concentration was 0.2 mM. The test samples and spiked test samples were prepared into a detection system according to the method described in Example 4 (the test samples were mixed with water in a volume ratio of 1:1 and then detected). Each sample was tested 3 times independently, and the results were averaged. The results are shown in Table 2. The spiked recovery rate of the samples was in the range of 97.5% - 108.5%, and the relative standard deviation RSD was in the range of 3.5% - 6.5%. The recovery rate of the standard samples was good. Therefore, the accuracy of the detection of glutathione based on the Mn-CDs nanozyme colorimetric sensing method is high and has a certain feasibility.

[0072] Table 2 Results of accuracy analysis

[0073]

[0074] Example 8

[0075] Verification of the Mn-CDs Nanozyme-based Colorimetric Sensing Method for Detecting Actual Serum Samples

[0076] To evaluate the application of the constructed Mn-CDs nanozyme-based colorimetric sensing method in clinical practice, glutathione in actual serum samples was detected and the spiked recovery rate was determined. Three actual serum samples were selected as test samples, and spiked test samples were prepared by adding 0.4 mM GSH at a volume ratio of 1:1. The actual added concentration was 0.2 mM. The detection system was prepared according to the method described in Example 4 (the test sample was mixed with water at a volume ratio of 1:1 and then detected). Each sample was tested independently 3 times, and the results were averaged. As shown in Table 3, the spiked recovery rate of the samples was in the range of 96.5%-108.5%, and the RSD was in the range of 0.7%-3.0%. The Mn-CDs nanozyme-based colorimetric sensing method for detecting glutathione obtained a good spiked recovery rate in actual serum samples, with RSD ≤ 3% and high accuracy, indicating certain potential in actual clinical applications.

[0077] Table 3 Analysis Results of Spiked Recovery Tests for Actual Serum Samples

[0078]

[0079] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting glutathione based on a colorimetric sensing technique using manganese-doped carbon quantum dot nanozymes, characterized in that, Under acidic conditions, manganese-doped carbon quantum dot nanozyme catalyzes the redox reaction of 3,3',5,5'-tetramethylbenzidine to generate a blue solution; the redox reaction is inhibited by adding glutathione, and the blue solution fades; the content of glutathione is determined by using the color change of the solution with or without glutathione as the signal output; The method includes the following steps: (1) Mix sodium acetate - acetic acid buffer solution, 3,3',5,5'-tetramethylbenzidine solution and manganese-doped carbon quantum dot solution, add the sample to be tested, react, and measure the absorbance A at 652 nm; use water as the blank control and measure the absorbance A0 at 652 nm; (2) Calculate ΔA according to ΔA = A0 - A; substitute ΔA into the standard curve to calculate the content of glutathione in the sample to be tested; The pH of the sodium acetate - acetic acid buffer solution is 3.5 ± 0.2; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 3 - 5 mM; the concentration of the manganese-doped carbon quantum dot solution is 1 mg / mL; In the range of 0.0005 - 0.8 mM, the standard curve is ΔA = 0.9129c + 0.0412.

2. The method according to claim 1, wherein The volume ratio of the sodium acetate - acetic acid buffer solution, the 3,3',5,5'-tetramethylbenzidine solution, the manganese-doped carbon quantum dot solution and the sample solution to be tested is 26:1:2:

1.

3. The method according to claim 1, characterized in that The temperature of the reaction is 20°C - 35°C, and the time is 20 - 40 min.

4. A kit for detecting glutathione based on a manganese-doped carbon quantum dot nanozyme colorimetric sensing technique, characterized in that, The kit contains sodium acetate - acetic acid buffer solution, 3,3',5,5'-tetramethylbenzidine solution and manganese-doped carbon quantum dot solution.

5. The kit according to claim 4, wherein, The pH of the sodium acetate - acetic acid buffer solution is 3.5 ± 0.2; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 3 - 5 mM; the concentration of the manganese-doped carbon quantum dot solution is 1 mg / mL.

Images