Three-mode biosensor for detecting glutathione and copper ions and construction method of three-mode biosensor

Abstract

The invention discloses a three-mode biosensor for detecting glutathione and copper ions and a construction method of the three-mode biosensor, and belongs to the field of biosensors. According to the sensor, ultraviolet spectrophotometry, Raman and a smart phone are combined, TMB is taken as a chromogenic substrate, Ag / MgMn2O4 and TMB are added into a NaAc buffer solution by utilizing the enzyme activity of Ag / MgMn2O4 oxide, GSH and Cu < 2 + > are detected, and when GSH exists, TMB oxide is inhibited to be TMbox < x >; and after Cu < 2 + > is added, the colorless TMB is re-oxidized into blue TMbox < ox >. The ultraviolet-Raman-smart phone three-mode biosensor for detecting GSH and Cu < 2 + > based on Ag / MgMn2O4 nano-enzyme is constructed, an ultra-sensitive new technology is provided for rapid and accurate detection of GSH and Cu < 2 + >, and the biosensor is especially suitable for concentration detection of GSH in urine and concentration detection of Cu < 2 + > in environmental lake water.

Description

Technical Field

[0001] The present invention belongs to the field of biosensors, and particularly relates to a triple-mode biosensor for detecting glutathione and copper ions and a construction method thereof. Background Art

[0002] Glutathione (GSH) is a tripeptide containing γ-amide bond and sulfhydryl group, which is an important regulatory metabolite in cells and has antioxidant effects in the liver, protecting liver cells from damage. Trace levels of aminothiol small biomolecules such as GSH are widely distributed in the tissues and body fluids of humans and animals, participating in various physiological activities such as cell composition and metabolism. Abnormal levels can lead to immune dysfunction and are related to diseases such as Alzheimer's disease, arteriosclerosis, and cardiovascular diseases. Therefore, it is particularly important to detect the content of GSH in urine. In addition, copper is a trace element transition metal, and with the rapid development of industry, it has a significant impact on both the environment and organisms. In water bodies, excessive Cu 2+ will pollute the water bodies and cause stress hazards to aquatic plants and animals. In the soil, using Cu 2+ to grow crops on severely polluted land will affect the growth of plants. Cu 2+ not only affects the growth of animals and plants in water, but also accumulates in water and sediments. Detecting the content of Cu 2+ in water bodies is a powerful measure to protect the environment. Currently, several methods for detecting Cu 2+ and GSH have been developed, such as fluorescence sensors, electrochemiluminescence methods, and fluorescence-colorimetric dual-mode methods, but these methods require a lot of time and cost. Therefore, using the ultraviolet-Raman-smartphone triple-mode detection method to detect Cu 2+ and GSH will be a powerful tool.

[0003] In the prior art, traditional catalysts such as natural enzymes have many limitations, such as poor stability, difficult preparation, high cost, and low specificity, which easily hinder their enzyme activity and cause their denaturation. As artificial enzymes, nanozymes have been widely studied and applied in practice due to their advantages such as simple preparation, high stability, and large-scale production. A large number of nanozymes have been proven to simulate different enzyme activities: peroxidase activity, catalase activity, superoxide dismutase activity. Many nano materials with enzyme activity have been reported, such as metal oxide nano materials, noble metal nano materials, carbon-based nano materials, and metal-organic framework nano materials. And AB2O4-type nano materials are mostly used in the battery field. Some AB2O4-type nano materials have been reported to be used in the field of biological detection based on their nanozyme activity, but there is no research report on Ag / MgMn2O4 in the field of nanozyme detection. Summary of the Invention

[0004] Aiming at the problems of complex operation, low sensitivity, small detection range and poor stability existing in the existing GSH and Cu 2+ detection technologies, the main object of the present invention is to provide a three-mode biosensor for detecting GSH and Cu 2+ to achieve rapid and accurate detection of GSH and Cu 2+ . Among them, GSH easily binds to metal sites to inhibit the activity of nanozymes, while Cu 2+ easily combines with GSH to form a complex, providing a super-sensitive new technology for detecting GSH and Cu 2+ and greatly improving the detection accuracy.

[0005] Another object of the present invention is to provide a construction method of the three-mode biosensor for detecting GSH and Cu 2+ . It is constructed based on the peroxidase activity of Ag / MgMn2O4 nanozymes, combining a smartphone, Raman and ultraviolet spectrophotometric colorimetric platforms, having both the advantages of a smartphone imaging system and image algorithm optimization, and the characteristics of Raman sensitivity and ultraviolet spectral colorimetric effect.

[0006] Another object of the present invention is to provide the application of the three-mode biosensor for detecting GSH and Cu 2+ in the rapid quantitative detection of GSH and Cu 2+ to achieve the instant detection and real-time monitoring of the concentrations of GSH and Cu 2+ .

[0007] To achieve the above objects, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided a three-mode biosensor for detecting GSH and Cu 2+ . Using TMB as a chromogenic substrate and utilizing the peroxidase activity of Ag / MgMn2O4, Ag / MgMn2O4 and TMB are added in a NaAc buffer solution, and GSH and Cu 2+ are detected by three modes of an ultraviolet spectrophotometer, a Raman spectrometer and a smartphone respectively;

[0009] When GSH is present, the oxidation of TMB to TMB ox is inhibited;

[0010] When Cu 2+ is added, the colorless TMB is re-oxidized to blue TMB ox , and an ultraviolet-Raman-smartphone three-mode biosensor for detecting GSH and Cu 2+ is constructed.

[0011] Preferably, the synthesis method of Ag / MgMn2O4 includes: first, synthesizing MgMn2O4 microflowers by an ethylene glycol-mediated solvothermal method, and then chemically reducing to synthesize Ag / MgMn2O4 with NaBH4, including: mixing an AgNO3 solution with an MgMn2O4 solution, reducing AgNO3 with NaBH4, and stirring to load Ag NPs onto the MgMn2O4 microflowers, thus obtaining the product.

[0012] Preferably, the linear ranges of GSH and Cu 2+ are 0.5 - 400 μM and 0.1 - 400 μM respectively, and the detection limits are 0.0875 μM and 0.06205 μM respectively.

[0013] In the second aspect of the present invention, a construction method of the three-mode biosensor for detecting GSH and Cu 2+ is provided, including the following steps:

[0014] (a) Synthesizing MgMn2O4 microflowers by an ethylene glycol-mediated solvothermal method;

[0015] (b) Chemically reducing to synthesize Ag / MgMn2O4 with NaBH4, including: mixing an AgNO3 solution with a solution of MgMn2O4 microflowers, reducing AgNO3 with a NaBH4 solution, and stirring to load Ag NPs onto the MgMn2O4 microflowers to obtain Ag / MgMn2O4;

[0016] (c) Taking Ag / MgMn2O4 and adding it to TMB and GSH for reaction, and taking GSH and Cu 2+ and adding them to the system of Ag / MgMn2O4 and TMB after the reaction to continue the reaction;

[0017] (d) Sampling the solution after the reaction in step (c), and detecting GSH and Cu 2+ with three modes of an ultraviolet spectrophotometer, Raman spectroscopy, and a smartphone respectively, to obtain an ultraviolet-Raman-smartphone three-mode biosensor for detecting GSH and Cu 2+ based on Ag / MgMn2O4 nanozyme.

[0018] Preferably, at room temperature, adding 50 μL of the system of Ag / MgMn2O4, TMB, and GSH to a NaAc buffer solution (pH = 4.0) to achieve the detection of GSH.

[0019] Preferably, at room temperature, adding 50 μL of a mixed solution of Ag / MgMn2O4, TMB, GSH, and Cu 2+ to a NaAc buffer solution (pH = 4.0) to achieve the detection of Cu 2+ ​

[0020] Preferably, the method for constructing the three-mode biosensor for detecting GSH and Cu 2+ comprises the following steps:

[0021] (1) Dissolve 2.5 g of PEG200 in ethylene glycol, and successively add Mg(CH3COO)2·4H2O and Mn(CH3COO)2·4H2O under stirring in a 40°C water bath for 2 h to obtain a precursor solution; the precursor solution is heat-treated in an autoclave at 200°C for 12 h to obtain a pale yellow intermediate, which is washed 5-6 times with ethanol and dried at 80°C for 12 h, and then calcined at 600°C for 5 h to obtain MgMn2O4 microflowers;

[0022] (2) At room temperature, dissolve 50 mg of MgMn2O4 microflowers in 40 mL of deionized water, mix the AgNO3 solution and the MgMn2O4 solution and stir for 1 h to form a mixed solution, slowly drop 5 mL of the NaBH4 solution into the mixed solution and stir for 1 h, and after centrifuging to collect the precipitate, obtain Ag / MgMn2O4;

[0023] (3) First, take 60 μL of the Ag / MgMn2O4 supernatant and add 50 μL of TMB and 50 μL of GSH and react for 30 min, then take GSH and Cu 2+ react for 40 min, and then add Ag / MgMn2O4 and TMB and continue to react for 30 min to obtain.

[0024] More preferably, in step (1), the ethylene glycol is 75 mL, Mg(CH3COO)2·4H2O is 0.27 g, Mn(CH3COO)2·4H2O is 0.612 g, and the heating rate is 5°C / min.

[0025] Preferably, in step (2), the concentration of the AgNO3 solution is 0.0118 M, and the concentration of the NaBH4 solution is 0.0235 M.

[0026] Preferably, in step (3), the GSH solution is first prepared into a solution with an initial concentration of 0.1 mM and diluted to a concentration range of 0.5-400 μM; Cu 2+ The solution is first prepared into a solution with an initial concentration of 0.1 mM and diluted to a concentration range of 0.1-400 μM.

[0027] Preferably, in step (3), the concentration of the Ag / MgMn2O4 solution is 0.08 mg / mL, and the concentration of the TMB solution is 0.8 mM.

[0028] In the third aspect of the present invention, there is provided the application of the three-mode biosensor in the rapid quantitative detection of GSH and Cu 2+ ​

[0029] Preferably, the three - mode biosensor for the rapid quantitative detection of GSH and Cu 2+ includes the following methods:

[0030] Colorimetric detection of GSH by smartphone includes: placing the reaction solution in a dark box (with stable light and isolated from external interference conditions), placing the smartphone above the dark box for taking pictures, using the smartphone APP to read the RGB values of the taken pictures, and performing linear fitting on the RGB values;

[0031] Colorimetric detection of Cu by smartphone 2+ includes: placing the reaction solution in a dark box (with stable light and isolated from external interference conditions), placing the smartphone above the dark box for taking pictures, using the smartphone APP to read the RGB values of the taken pictures, and converting the RGB values into grayscale values for linear fitting;

[0032] The ultraviolet absorption scanning range of the ultraviolet spectrophotometer is 500 - 800 nm, and the Raman spectrum scanning range of the Raman spectrometer is 200 - 2000 cm -1 .

[0033] Preferably, the three - mode biosensor is used for the detection of GSH concentration in urine and Cu 2+ concentration in environmental lake water.

[0034] The present invention constructs an ultraviolet - Raman - smartphone three - mode biosensor based on Ag / MgMn2O4 nanozyme for the detection of GSH and Cu 2+ , and its working principle is: the peroxidase - like activity of Ag / MgMn2O4 mainly comes from O2 ·- . When GSH is present, it binds to the metal sites of Ag / MgMn2O4, inhibiting the peroxidase - like activity of Ag / MgMn2O4, thereby inhibiting the oxidation of colorless TMB to blue TMB ox , and the characteristic peak at 652 nm of TMB in the ultraviolet spectrophotometer decreases, and the characteristic peak at 1612 cm ox also decreases in Raman intensity; when Cu -1 is added, Cu 2+ combines with GSH to form a complex, inhibiting the coordination of GSH and metal sites, releasing the active sites, and enabling the oxidation of colorless TMB to blue TMB 2+ , the ultraviolet absorption peak of TMB at 652 nm gradually increases, and at 1612 cm ox , the Raman peak also increases; ox at 1612 cm -1The Raman peaks at [specific location] gradually increase, with dual-mode signal output of colorimetry and Raman method. At the same time, combined with the smartphone colorimetric sensing platform, the RGB values of the experimental images are read by the smartphone, and data analysis is carried out through PS software to achieve the instant detection and real-time monitoring of GSH and Cu 2+ concentrations.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] 1. The present invention constructs a UV-Raman-smartphone triple-mode biosensor for detecting GSH and Cu 2+ by using the peroxidase activity of Ag / MgMn2O4 oxide. The rapid and accurate detection of GSH and Cu 2+ is realized by using three modes of ultraviolet, Raman and smartphone. It is a super-sensitive new technology that can be used to detect the GSH concentration in human urine and the Cu 2+ concentration in environmental lake water, greatly improving the sensitivity and intelligence of detection.

[0037] 2. The real-time detection of GSH and Cu 2+ is realized by using the visual detection system that reads the RGB values by the smartphone and converts them into grayscale values. It has good stability and accuracy and can be used for the intelligent detection of GSH and Cu 2+ in daily life.

[0038] 3. Compared with the traditional biosensors for detecting GSH and Cu 2+ , the triple-mode sensor constructed by the present invention can resist other interfering substances (such as Gly, Glu, Ser, Arg, Trp, Asp, Glucose, Cholesterol) for the detection of GSH, and can resist other interfering ions (such as K 2+ , Na + , Mn + , Ca 2+ , Fe 2+ , Mg 2+ , Zn 2+ , Zn 2+ ) for the detection of Cu

[0039] It has good anti-interference and selectivity, greatly improving the detection accuracy.

[0040] 4. The present invention can adopt multiple responses to improve the detection accuracy, and has the advantages of high portability, low cost, intelligence, etc., solving the problem that the information may be unstable and incorrect due to completely relying on one mode for quantitative measurement. 2+The detection limits are 0.08913 μM, 0.06205 μM, and 0.09783 μM respectively. By combining the detection of precision instruments and smartphones, the real-time monitoring in daily life is realized, and it has a wide application prospect in the fields of biomedicine and environmental detection. 2+ Brief Description of the Drawings

[0041] Figure 1 Schematic diagram of the ultraviolet-Raman-smartphone triple-mode biosensor for detecting GSH and Cu prepared in Example 1 2+

[0042] Figure 2 Response curve of the ultraviolet colorimetric sensor system prepared in Example 1 to GSH

[0043] Figure 3 Response curve of the Raman sensing system prepared in Example 1 to GSH

[0044] Figure 4 Response curve of the ultraviolet colorimetric sensor system prepared in Example 1 to Cu 2+

[0045] Figure 5 Response curve of the Raman sensing system prepared in Example 1 to Cu 2+

[0046] Figure 6 Response curve of the smartphone sensor system prepared in Example 1 to GSH

[0047] Figure 7 Response curve of the smartphone sensor system prepared in Example 1 to Cu 2+

[0048] Figure 8 Test results of the selectivity and anti-interference of the triple-mode biosensor prepared in Example 1

[0049] Figure 9 Test results of the repeatability of the triple-mode biosensor prepared in Example 1 Detailed Description of the Invention

[0050] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in combination with specific embodiments and drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0051] Example 1

[0052] This example presents a UV-Raman-smartphone triple-mode biosensor for detecting GSH and Cu 2+ The preparation process is as follows Figure 1 shown: Ag / MgMn2O4 is prepared by an ethylene glycol-mediated solvothermal method and a chemical reduction method using NaBH4 as a reducing agent. At room temperature, 50 μL of Ag / MgMn2O4, TMB, and GSH are added to a NaAc buffer solution (pH = 4.0) to achieve the detection of GSH; at room temperature, 50 μL of Ag / MgMn2O4, TMB, and a mixed solution of GSH and Cu 2+ are added to a NaAc buffer solution (pH = 4.0) to achieve the detection of Cu 2+ and thus obtain a UV-Raman-smartphone triple-mode biosensor. The steps are as follows

[0053] The UV-Raman-smartphone triple-mode sensor is prepared based on the peroxidase-like activity of Ag / MgMn2O4. The preparation method is as follows

[0054] Dilute a 0.1 mM GSH solution with deionized water. The diluted GSH solution concentration is 0.5 - 400 μM. Add the GSH solution to the Ag / MgMn2O4 / TMB system and react for 30 min. The UV absorption scanning range is 500 - 800 nm to detect the GSH UV absorption peak( Figure 2 ), and the Raman spectrum range is 200 - 2000 cm -1 to detect the GSH Raman peak( Figure 3 ).

[0055] Dilute a 0.1 mM copper sulfate solution with deionized water. The diluted copper sulfate solution concentration is 0.1 - 400 μM. After the copper sulfate solution reacts with the GSH solution for 40 min, add it to the Ag / MgMn2O4 / TMB system and react for 30 min. The UV absorption scanning range is 500 - 800 nm to detect the Cu 2+ UV absorption peak( Figure 4 ), and in the Raman spectrum range of 200 - 2000 cm -1 to detect the Cu 2+ Raman peak( Figure 5 ).

[0056] The construction of the smartphone colorimetric sensing method is as follows

[0057] Dilute the prepared GSH solution to a concentration of 0.5 - 300 μM. Add the copper sulfate solution and the GSH solution to the Ag / MgMn2O4 / TMB system. After 30 min, place it in a dark box. Place the smartphone above the dark box to take a photo, and then use the smartphone APP to read the RGB values of the experimental photo taken. Perform linear fitting on the corresponding RGB values ( Figure 6 ).

[0058] Dilute the prepared copper sulfate solution to a concentration of 0.1 - 300 μM. Add the copper sulfate solution to the Ag / MgMn2O4 / TMB system. After 30 min, place it in a dark box. Place the smartphone above the dark box to take a photo, and then use the smartphone APP to read the RGB values of the experimental photo taken. Convert the corresponding RGB values to grayscale values and perform linear fitting ( Figure 7 ).

[0059] As can be seen from Figure 2 and 3 , the peak value of the ultraviolet spectrophotometer at 625 nm gradually decreases as the GSH concentration increases. The concentration of GSH is 0.5 - 400 μM. As the GSH concentration increases, the Raman peak value becomes weaker and weaker, and the corresponding color gradually changes from blue to colorless ( Figure 5 ), indicating that the sensor can sensitively detect GSH.

[0060] As can be seen from Figure 4 and 5 , the peak value of the ultraviolet spectrophotometer at 625 nm gradually increases as the concentration of Cu 2+ increases. The concentration of Cu 2+ is 0.1 - 400 μM. As the GSH concentration increases, the Raman peak value becomes stronger and stronger, and the corresponding color gradually changes from colorless to blue ( Figure 7 ), indicating that the sensor can sensitively detect Cu 2+ .

[0061] As can be seen from Figure 8 , when GSH and interfering substances coexist, it can be observed that the absorbance value of the reaction system is relatively low, indicating that other substances do not interfere with the GSH detection sensor. When there is no GSH and only interfering substances exist in the reaction system, the sensor hardly responds to other substances, indicating that the sensor has excellent selectivity for GSH. Similarly, the sensor also has good anti-interference and selectivity for the determination of Cu 2+ .

[0062] As Figure 9 shown, select 10 groups of samples for reproducibility testing, all adding the same concentration of GSH or Cu 2+ , and it can be observed that the absorbance values basically remain the same, indicating that the sensor has good reproducibility.

[0063] Effect Example 1

[0064] Application of the ultraviolet-Raman-smartphone triple-mode biosensor prepared in Example 1 in detecting GSH in human urine and Cu in environmental lake water 2+ The application steps are as follows:

[0065] To exclude potential interfering factors in urine, the urine was first centrifuged, pre-filtered through a 0.22 μm membrane, diluted 100-fold with phosphate buffer, and the treated urine was used for subsequent analysis. In addition, lake water in the school was collected, and the concentration of Cu in the lake water was quantitatively analyzed, and the sediment in the lake water was filtered using a 0.22 μm filter membrane. 2+ The GSH solution was diluted to different concentrations with deionized water. The GSH solution was added to the Ag / MgMn2O4 / TMB reaction system and reacted for 30 min. Similarly, the copper sulfate solution was diluted with deionized water. After the copper sulfate solution reacted with the GSH solution for 40 min, it was added to the Ag / MgMn2O4 / TMB system and reacted for 30 min. After the reaction, it was recorded with an ultraviolet spectrophotometer and a Raman spectrometer. At the same time, for the GSH detection sensor, the reacted solution was placed in a dark box (stable light source), the smartphone was placed above the dark box for taking pictures, and the RGB values of the taken experimental pictures were read with the smartphone APP for linear fitting; for the Cu

[0066] detection sensor, the corresponding RGB values were converted into grayscale values for linear fitting. 2+ As shown in Tables 1 and 2, the ultraviolet-Raman-smartphone triple-mode was used to detect GSH in real human urine and Cu in environmental lake water. The standard addition method was used to detect the content of GSH in human urine and Cu in environmental lake water, and it had a good recovery rate, proving the feasibility of the sensor in the detection of actual samples and its potential application value in the fields of biosensing and environmental detection.

[0067] Table 1: Analysis of urine samples by the GSH triple-mode sensor 2+ Table 2: Analysis of lake water samples by the Cu 2+ triple-mode sensor

[0068] Table 1: Urine sample analysis of the GSH triple-mode sensor

[0069]

[0070]

[0071] Table 2: Lake water sample analysis of the Cu 2+ triple-mode sensor

[0072]

[0073]

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A triple-mode biosensor for detecting glutathione and copper ions, characterized in that, Using TMB as the chromogenic substrate and taking advantage of the peroxidase activity of Ag / MgMn2O4 oxide, Ag / MgMn2O4 and TMB are added to the NaAc buffer, and GSH and Cu are detected respectively in three modes: using an ultraviolet spectrophotometer, a Raman spectrometer, and a smartphone. 2+ Detection is carried out. When GSH is present, the oxidation of TMB to TMB is inhibited ox ; When Cu is added 2+ the colorless TMB is re-oxidized to blue TMB ox and a UV-Raman-smartphone triple-mode biosensor for detecting GSH and Cu 2+ is constructed.

2. The triple-mode biosensor for detecting glutathione and copper ions according to claim 1, wherein The synthesis method of Ag / MgMn2O4 includes: first, synthesizing MgMn2O4 microflowers by an ethylene glycol-mediated solvothermal method, and then chemically reducing to synthesize Ag / MgMn2O4 with NaBH4, including: mixing an AgNO3 solution and an MgMn2O4 solution, reducing AgNO3 with NaBH4, stirring, and loading Ag NPs onto the MgMn2O4 microflowers to obtain the product.

3. The three-mode biosensor for detecting glutathione and copper ions according to claim 1, characterized in that, GSH and Cu 2+ The linear ranges are 0.5 - 400 μM and 0.1 - 400 μM respectively, and the detection limits are 0.0875 μM and 0.06205 μM respectively.

4. The construction method of the three-mode biosensor for detecting glutathione and copper ions according to any one of claims 1 to 3, characterized in that, It includes the following steps: (a) Synthesizing MgMn2O4 microflowers by an ethylene glycol-mediated solvothermal method; (b) Chemically reducing to synthesize Ag / MgMn2O4 with NaBH4, including: mixing an AgNO3 solution and an MgMn2O4 solution, reducing AgNO3 with NaBH4, stirring, and loading Ag NPs onto the MgMn2O4 microflowers to obtain Ag / MgMn2O4; (c) Add Ag / MgMn2O4 to carry out a reaction with TMB and GSH. Take GSH and Cu 2+ After the reaction, continue the reaction in the system of Ag / MgMn2O4 and TMB; (d) Take samples of the solution after the reaction in step (c), and detect GSH and Cu using three modes: ultraviolet spectrophotometer, Raman spectroscopy, and smartphone respectively. 2+ Perform detection.

5. The construction method of the triple-mode biosensor for detecting glutathione and copper ions according to claim 4, wherein, At room temperature, detecting GSH in a system of 50 μL of Ag / MgMn2O4, TMB, and GSH in a NaAc buffer solution with pH = 4.0; At room temperature, 50 μL of the mixed solution of Ag / MgMn2O4, TMB, and GSH and Cu was added to the NaAc buffer solution with pH = 4.0 for the detection of Cu 2+ 2+ detection.​ 6. The construction method of the triple-mode biosensor for detecting glutathione and copper ions according to claim 4 or 5, characterized in that, The method for constructing a three-mode biosensor for detecting GSH and Cu 2+ comprises the following steps: (1) Dissolve 2.5 g of PEG200 in ethylene glycol, and then successively add Mg(CH3COO)2·4H2O and Mn(CH3COO)2·4H2O under stirring in a 40 °C water bath for 2 h to obtain a precursor solution; the precursor solution is heat-treated in an autoclave at 200 °C for 12 h to obtain a pale yellow intermediate, which is washed 5 - 6 times with ethanol and dried at 80 °C for 12 h, and then calcined at 600 °C for 5 h to obtain MgMn2O4 microflowers; (2) At room temperature, dissolve 50 mg of MgMn2O4 microflowers in 40 mL of deionized water, mix and stir the AgNO3 solution and the MgMn2O4 solution for 1 h to form a mixed solution, slowly drop 5 mL of the NaBH4 solution into the mixed solution and stir for 1 h, and after centrifuging to collect the precipitate, obtain Ag / MgMn2O4; (3) First, take 60 μL of the supernatant of Ag / MgMn2O4, add 50 μL of TMB and 50 μL of GSH, and react for 30 min. Then, take GSH and Cu 2+ react for 40 min, and then add Ag / MgMn2O4 and TMB and continue to react for 30 min.

7. The construction method of the triple-mode biosensor for detecting glutathione and copper ions according to claim 6, characterized in that, In step (1), the amount of ethylene glycol is 75 mL, the amount of Mg(CH3COO)2·4H2O is 0.27 g, the amount of Mn(CH3COO)2·4H2O is 0.612 g, and the heating rate is 5 °C / min; and / or in step (2), the concentration of the AgNO3 solution is 0.0118 M, and the concentration of the NaBH4 solution is 0.0235 M; In step (3), the GSH solution is first prepared into a solution with an initial concentration of 0.1 mM and diluted to a concentration range of 0.5 - 400 μM; the Cu 2+ solution is first prepared into a solution with an initial concentration of 0.1 mM and diluted to a concentration range of 0.1 - 400 μM. Preferably, in step (3), the concentration of the Ag / MgMn2O4 solution is 0.08 mg / mL, and the concentration of the TMB solution is 0.8 mM.

8. Use of the three-mode biosensor for detecting glutathione and copper ions according to any one of claims 1 to 3 in the rapid quantitative detection of GSH and Cu 2+ ions.

9. The application according to claim 8, wherein The method for rapidly and quantitatively detecting GSH and Cu by the three-mode biosensor includes: 2+ ​ Colorimetric detection of GSH with a smartphone includes: placing the reaction solution in a dark box, placing the smartphone above the dark box to take a photo, using a smartphone APP to read the RGB values of the taken photo, and performing linear fitting on the RGB values; Colorimetric detection of Cu by smartphone 2+ , including: placing the reaction solution in a dark box, placing the smartphone above the dark box to take a photo, using the smartphone APP to read the RGB values of the taken photo, and converting the RGB values into grayscale values for linear fitting; The ultraviolet absorption scanning range of the ultraviolet spectrophotometer is 500 - 800 nm, and the Raman spectrum scanning range of the Raman spectrometer is 200 - 2000 cm -1 .

10. The three-mode biosensor for detecting glutathione and copper ions is used for detecting the GSH concentration in urine and the Cu 2+ concentration in environmental lake water.

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