A method for detecting glutathione by colorimetric electrochemical dual-mode based on polyethylene glycol and polyethyleneimine modified manganese dioxide as signal tag

Abstract

The application discloses a method for detecting glutathione by using polyethylene glycol and polyethylene imine modified manganese dioxide as a signal tag, and the method comprises the following steps: step 1, preparing PEI-PEG@MnO2; step 2, preparing an electrode; step 3, colorimetric detection; and step 4, electrochemical detection. The PEI and PEG of the application are used in cooperation to increase the specific surface area and improve the catalytic oxidation performance of the manganese dioxide nanoflower.

Description

Technical Field

[0001] This invention relates to a colorimetric electrochemical dual-mode detection method for glutathione based on polyethylene glycol and polyethyleneimine-modified manganese dioxide as a signal tag. Background Technology

[0002] To date, various methods exist for the detection of glutathione (GSH) in food, including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). However, these methods suffer from drawbacks such as expensive equipment, complex operation, and cumbersome pretreatment, limiting their application in glutathione determination. Biosensors, including capillary biosensors, colorimetric biosensors, fluorescence biosensors, and electrochemical biosensors, have emerged as the most promising and attractive alternative platforms for glutathione detection in food. Colorimetric sensors, as a mature detection technology, allow direct visual observation of signal changes without requiring expensive and complex signal transduction equipment. Electrochemical sensors achieve lower limits of detection (LOD) and wider linear ranges through electrochemical methods. Single-mode systems are susceptible to interference from complex environments (e.g., colorimetric analysis is affected by background color, and electrodes are affected by surface contamination). Dual-mode systems, through a "double chain of evidence," significantly reduce the risk of false positives or false negatives due to instrument errors or human negligence. In food (such as milk and juice) or clinical samples (such as whole blood and serum) testing, matrix effects are significant. Dual-mode detection can utilize the high selectivity of electrochemistry and the intuitiveness of colorimetry to avoid interference from matrix color. It also avoids the problems of traditional precision testing instruments being bulky and unusable on-site. With its ease of operation, good selectivity, and portability, it provides a promising method for glutathione detection. Summary of the Invention

[0003] The purpose of this invention is to provide a colorimetric electrochemical dual-mode detection method for glutathione based on polyethylene glycol and polyethyleneimine-modified manganese dioxide as a signal tag.

[0004] To achieve the above and other related objectives, the technical solution provided by this invention is: a colorimetric electrochemical dual-mode detection method for glutathione based on polyethylene glycol and polyethyleneimine-modified manganese dioxide as a signal tag, comprising:

[0005] Step 1: Preparation of PEI-PEG@MnO2

[0006] Polyethyleneimine and polyethylene glycol were added to deionized water, mixed evenly, and then manganese acetate was added and stirred. Sodium hydroxide solution was then added and stirred continuously at room temperature. The mixture was washed with deionized water and freeze-dried under vacuum to obtain PEI-PEG@MnO2.

[0007] Step 2: Electrode preparation

[0008] PEI-PEG@MnO2 powder was dispersed in a chitosan solution, and the resulting PEI-PEG@MnO2x suspension was dropped onto the surface of a pretreated glassy carbon electrode and then dried at room temperature.

[0009] Step 3: Colorimetric Detection

[0010] Mix PBS buffer solution, 3,3',5,5'-tetramethylbenzidine, PEI-PEG@MnO2 and the sample to be tested, react at room temperature, and then use a UV spectrophotometer to measure the colorimetric signal value at 652 nm.

[0011] Step 4: Electrochemical detection

[0012] The electrode prepared in step 2 was used as the working electrode, the AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. The electrolyte included 3,3',5,5'-tetramethylbenzidine and PBS buffer. The PBS buffer solution, 3,3',5,5'-tetramethylbenzidine and the sample to be tested were mixed and reacted at room temperature. Then, the electrode prepared in step 2 was used as the anode and the platinum electrode was used as the cathode for measurement.

[0013] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0014] This invention relates to a colorimetric and electrochemical dual-mode biosensor for detecting glutathione (GSH) in food. Using manganese acetate oxidized with NaOH as the core, and modifying manganese dioxide with polyethyleneimine (PEI) and polyethylene glycol (PEG), a novel PEI-PEG@MnO2 nanomaterial is formed as a signal tag, exhibiting superior catalytic performance. Glutathione (GSH) is a reducing agent. The addition of GSH inhibits the catalytic activity of PEI-PEG@MnO2 for TMB. Attached Figure Description

[0015] Figure 1 Material characterization: (AB) TEM images of PEI-PEG@MnO2; (C) TEM image of MnO2; (D) EDS image of PEI-PEG@MnO2.

[0016] Figure 2Feasibility analysis of colorimetric and electrochemical methods: (A) UV-Vis absorption spectra of TMB / MnO2-TMB / PEI@MnO2-TMB / PEI-PEG@MnO2-TMB / PEI-PEG@MnO2-TMB-GSH; (B) CV spectra of PEI-PEG@MnO2 in 0.1M PBS buffer, 5µM TMB, and 20µM GSH solution.

[0017] Figure 3 To optimize experimental conditions for the colorimetric sensor: (A) PEI addition amount (B) PEI-PEG@MnO2 concentration (C) pH of PBS buffer (D) Reaction time of PEI-PEG@MnO2 with TMB

[0018] Figure 4 Effect of scan rate on the electrocatalytic oxidation of GSH: (A) Different scan rates (10~200 mVs) on PEI-PEG@MnO2 / GCE -1 (A) CV curve at time (B) Relationship between peak current and scan rate on PEI-PEG@MnO2 / GCE

[0019] Figure 5 The effect of pH on the electrocatalytic oxidation of GSH: (A) CV curves of PEI-PEG@MnO2 / GCE in 0.1M PBS buffer containing 1mM GSH, with pH varying from 7 to 9; (B) E pa Linear relationship between pH value

[0020] Figure 6 Colorimetric detection of GSH: (A) UV-Vis absorption spectrum of GSH detected by PEI-PEG@MnO2; (B) Linearity graph of GSH determination.

[0021] Figure 7 Electrochemical detection of GSH: (A) CV response of the sensor to different concentrations of GSH; (B) E pa Linear relationship between GSH concentration and concentration

[0022] Figure 8 To assess the selectivity and stability of the colorimetric sensor, we consider (A) the sensor's specificity to different interfering substances; and (B) the sensor's stability at different storage times.

[0023] Figure 9 For the selectivity and repeatability of electrochemical sensors: (A) Specificity of the sensor to different interfering substances; (B) Reproducibility of the sensor to different electrodes.

[0024] Figure 10 This is a process flow diagram of the present invention. Detailed Implementation

[0025] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0026] Please see Figure 1-10 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0027] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0028] Reagents and materials:

[0029] Manganese acetate tetrahydrate (MnC4H6O4.4H2O), sodium hydroxide (NaOH), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), potassium chloride (KCl), sodium chloride (NaCl), manganese acetate, zinc acetate, ferric chloride, copper acetate, glacial acetic acid, phenylalanine, cysteine, lysine, glutamic acid, glycine, leucine, lactose, glucose, and fructose were purchased from Sinopharm Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Chitosan, polyethylene glycol (PEG), polyethyleneimine (PEI), and glutathione (GSH) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).

[0030] The technical solution of the present invention will be further described in detail below with reference to examples.

[0031] Example 1: A colorimetric electrochemical dual-mode detection method for glutathione based on polyethylene glycol and polyethyleneimine-modified manganese dioxide as signal tags.

[0032] (1)Synthetic materials:

[0033] Preparation of PEI-PEG@MnO2

[0034] Dissolve 1g of polyethyleneimine and 1g of polyethylene glycol in 10mL of deionized water, sonicate for 10min, mix well, add manganese acetate (100mM, 2mL) and stir for 10min, then add sodium hydroxide solution (70mM, 10mL), and stir vigorously at room temperature for 12h. Wash three times with deionized water, freeze-dry under vacuum to obtain a dark brown precipitate.

[0035] (2) Fabrication of biosensors

[0036] 1. Pretreatment

[0037] Before testing, prepare glutathione solutions of different concentrations from 0.01mM to 10mM, and dissolve the reagent in 1mM deionized water.

[0038] Before electrode modification, the GCE was polished with 0.3 and 0.05 μm alumina powder, respectively, then sonicated in ultrapure water and ethanol, and finally dried with nitrogen to obtain a smooth, mirror-like electrode surface. PEI-PEG@MnO2 powder was weighed and dispersed in a 5% chitosan solution. 5 µL of the well-dispersed PEI-PEG@MnO2 (2 mg / mL) solution was added dropwise to the electrode surface and dried at room temperature for 2 h.

[0039] 2. Colorimetric detection

[0040] The reaction system was supplemented with 370 µL of 0.1M PBS buffer solution (pH=5), 10 µL of TMB (2.5mM), 10 µL of PEI-PEG@MnO2 (1mg / mL), and 10 µL of GSH solution. After reacting at room temperature for 8 min, the colorimetric signal value was measured at 652 nm using a UV spectrophotometer.

[0041] 3. Electrochemical detection

[0042] 5 mL of 0.1 M PBS buffer solution (pH=8), 10 µL of TMB (2.5 mM), and 10 µL of GSH at different concentrations were added to the reaction system. The reaction was carried out at room temperature for 3 min before measurement.

[0043] (3) Conclusion

[0044] 1. Morphological characterization of materials

[0045] like Figure 1 As shown in Figures AB, the morphology and size of the prepared nanocomposite material were characterized by transmission electron microscopy (TEM). PEI-PEG@MnO2 consists of spherical nanoparticles with an average particle size of 198 nm and distinct boundaries, exhibiting good dispersibility. Figure 1The morphology and size of unmodified MnO2 were characterized by C. MnO2 consisted of near-spherical nanoparticles with an average particle size of 98.5 nm and indistinct boundaries. (Elemental distribution map (EDS) was used.) Figure 1 The D-values ​​show that Mn, O, and N elements are uniformly distributed in PEI-PEG@MnO2.

[0046] 2. Feasibility Analysis

[0047] To verify the enhancement effect of PEI-PEG@MnO2 on colorimetric signals, the colorimetric signals of MnO2, PEI@MnO2, and PEI-PEG@MnO2 at 652 nm in 0.1 M PBS buffer solution were compared. Figure 2 As shown in Figure A, PEI-PEG@MnO2 exhibits a higher absorption peak at 652 nm compared to both MnO2 and PEI@MnO2. Using the colorimetric signal from PEI-PEG@MnO2 can improve the analytical performance of the colorimetric sensor.

[0048] To verify the enhancement effect of PEI-PEG@MnO2 / GCE on electrochemical signals, the CV signal of the oxidation peak in 0.1 M PBS buffer solution after the addition of GSH was compared. Figure 2 Measurements at a 50 mV scan rate showed that the addition of GSH resulted in a higher peak current in the oxidation peak compared to the addition of TMB. Experimental results indicate that the electrochemical signal amplification strategy using PEI-PEG@MnO2 / GCE can improve the analytical performance of the sensor.

[0049] 3. Condition Optimization

[0050] To achieve the most sensitive analytical performance of the sensor, several key variables were optimized, including the relative amount of PEI added, material concentration, pH of the PBS buffer solution, reaction time, the effect of scan rate on electrocatalytic oxidation, and the effect of pH on electrocatalytic oxidation. For example... Figure 3 As shown, the final selected relative addition amount of PEI was 1g, and the optimal material concentration was 1mg / mL. -1 The optimal pH of the PBS buffer solution is 5, and the optimal reaction time with TMB is 8 min.

[0051] like Figure 4 As shown, the scan rates (50~250 mV s) were investigated. -1 The effect of PEI-PEG@MnO2 / GCE on the catalytic oxidation behavior. Figure 4 It can be seen that the scan rate affects the peak potential difference (ΔE). p ) and peak current (I pa The effect of ΔE pThe peak current increases with increasing scan rate, and is linearly related to the square root of the scan rate. Figure 4 B), the linear regression equation is I pa =-3.6894E-9v 1 / 2 -2.2145E-7 proves that the reaction process is a diffusion-controlled catalytic reaction.

[0052] Furthermore, according to Faraday's law, it is expressed as I pa =(nFQv) / 4RT (where n is the number of electrons involved in the electrochemical reaction, Q is the total charge in the reaction, v is the scan rate, and F is the Faraday constant [96485 Cmo -l R is the gas constant, with units of [8.314 J / K]. -1 mol -l [T is the temperature [298K]), when the scan rate is 50 mVs -1 At that time, the number of electrons transferred (n) was calculated to be 1.96. This indicates that in the electrochemical reaction, two electrons (n≈2) are transferred.

[0053] like Figure 5 As shown, PEI-PEG@MnO2 / GCE exhibits pH dependence in its catalytic oxidation. Figure 5 As shown in A, the redox peak potential (E) pa The value shifts negatively with increasing pH, and there is a linear correlation between it and pH. Figure 5 B), the linear regression equation is E pa =0.463-0.0398pH (R 2 =0.987). The slope is -0.0395 V pH. -1 It is close to the theoretical Nernst value of 0.059VpH. -1 This indicates that the electrode process involves two electrons (with...) Figure 4 (The results were consistent) and the transfer of two protons. Furthermore, by Figure 5 As can be seen from A, the absolute value of the oxidation peak current reaches its maximum at pH 8, and then the catalytic oxidation reaction weakens as the pH value increases.

[0054] 4. Analytical performance

[0055] like Figure 6 As shown in Figure A, the absorbance signal at 652 nm gradually decreases with increasing glutathione concentration, reaching a plateau at 50 µM glutathione concentration. Figure 6 Figure B shows a good linear relationship between the absorbance at 652 nm and the glutathione concentration in the range of 0.025 µM-30 µM, and the calculated detection limit is 0.213 µM (S / N = 3).

[0056] like Figure 7 As shown, Figure 7 As shown in Figure A, the CV signal of PEI-PEG@MnO2 / GCE increases with the increase of GSH concentration, and eventually reaches a plateau. Figure 7 Figure B shows a good linear relationship between the current value and the GSH concentration in the range of 0.02 µM to 120 µM, and the calculated detection limit is 0.742 × 10⁻⁶. -11 M (S / N = 3).

[0057] 5. Selectivity, stability, and repeatability

[0058] The colorimetric signals of phenylalanine, cysteine, lysine, glutamic acid, glycine, leucine, lactose, glucose, fructose, KCl, NaCl, manganese acetate, ferric chloride, zinc acetate, copper acetate, and the target glutathione (GSH) were measured. Figure 8 A) The selectivity of the developed colorimetric biosensor was investigated. It was found that only the target glutathione exhibited a high colorimetric response, indicating that the sensor has good selectivity. Furthermore, after the successfully prepared colorimetric sensor was placed for 1, 3, 5, 7, 9, 11, 13, and 15 days, colorimetric detection of glutathione was performed, and the absorbance at a wavelength of 652 nm was recorded. Figure 8 As shown in Figure B, the RSD values ​​of the colorimetric sensor after different storage times were all less than 5%, which is within an acceptable range. This indicates that the prepared colorimetric sensor has good storage stability.

[0059] The CV signals of phenylalanine, cysteine, glycine, leucine, lactose, glucose, fructose, KCl, NaCl, manganese acetate, zinc acetate, and the target glutathione (GSH) were measured. Figure 9 (A) The selectivity of the developed electrochemical biosensor was investigated. It was found that only the target GSH exhibited a high current response, indicating that the sensor has good selectivity. Furthermore, as... Figure 9 As shown in Figure B, the reproducibility of this biosensor was investigated. Five different batches of the biosensor were constructed and measured three times under the same experimental conditions. The RSD of the peak current values ​​among the five biosensors was approximately 2.99%, indicating good reproducibility.

[0060] 6. Testing of actual samples

[0061] Purchased Dangshan pears and milk were used as real samples to verify the sensor's performance in actual samples. The samples were then processed. The fat and protein in the milk formed an emulsion, obstructing the light path in the cuvette, and this had to be completely removed. The milk was refrigerated at 4°C for 15 minutes, followed by freeze centrifugation, discarding the top layer of solid fat.

[0062] A small amount of acetic acid was added to lower the pH to the isoelectric point of the protein, causing it to coagulate and clump. An equal volume of acetonitrile was added, and the mixture was vortexed and centrifuged. The sample was centrifuged at 10,000 rpm for 10 min, filtered through a 0.22 μm filter, and then diluted with PBS buffer. The supernatant was then used for spiked recovery experiments. 0, 12.5, and 25 μM glutathione were added to the treated samples for spiked recovery experiments. The results are shown in Table 1. The colorimetric method showed recoveries of 97.47%–105.43% for spiked Dangshan pear juice and milk samples, with RSDs of 0.65%–4.73%. The electrochemical method showed recoveries of 97.79%–101.26% for spiked Dangshan pear juice and milk samples, with RSDs of 0.56%–2.87%. The experimental results indicate that the sensor developed in this study can be used for actual sample detection and has a certain degree of reliability and accuracy.

[0063] Table 1. Recovery analysis of glutathione in actual samples

[0064]

[0065] Manganese dioxide nanospheres are a type of manganese dioxide material with a unique spherical nanostructure, combining the advantages of small size effect, high specific surface area, and spherical structure of nanomaterials. The surface exposes a large amount of Mn. 4 ⁺ / Mn³⁺ active sites enhance adsorption, catalysis, and electrochemical activity. Multiple valence states of manganese (Mn²⁺ / Mn³⁺ / Mn) 4 (⁺) This endows it with reversible redox capabilities, enabling it to participate in various electron transfer reactions. The hydrophilic long chains of PEG form steric hindrance, preventing nanosphere aggregation; the cationic amine groups (-NH2) of PEI are anchored to the surface of manganese dioxide nanospheres (MnO2 is negatively charged) through electrostatic interactions, increasing the specific surface area and forming a dual stable structure of "electrostatic anchoring + steric hindrance" with PEG. This ensures uniform dispersion in the aqueous phase, and the steric hindrance of the polymer reduces non-specific adsorption at active sites and enhances the activity of its oxidase (OXD) nanozymes. Multiple performance improvements have been achieved in colorimetric and electrochemical detection, addressing the shortcomings of dispersibility and stability of pure manganese dioxide nanospheres, and introducing new properties such as targeted binding and signal amplification through the introduction of functional groups in the polymer.

[0066] This invention provides a mild, simple, highly catalytically active, and stable PEI-PEG@MnO2 synthesis method.

[0067] PEI and PEG work synergistically to increase the specific surface area and improve the catalytic oxidation performance of manganese dioxide nanoflowers.

[0068] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A colorimetric electrochemical dual-mode detection method for glutathione based on polyethylene glycol and polyethyleneimine-modified manganese dioxide as a signal tag, characterized in that, include: Step 1: Preparation of PEI-PEG@MnO2 Polyethyleneimine and polyethylene glycol were added to deionized water, mixed evenly, and then manganese acetate was added and stirred. Sodium hydroxide solution was then added and stirred continuously at room temperature. The mixture was washed with deionized water and freeze-dried under vacuum to obtain PEI-PEG@MnO2. Step 2: Electrode preparation PEI-PEG@MnO2 powder was dispersed in a chitosan solution, and the resulting PEI-PEG@MnO2x suspension was dropped onto the surface of a pretreated glassy carbon electrode and then dried at room temperature. Step 3: Colorimetric Detection Mix PBS buffer solution, 3,3',5,5'-tetramethylbenzidine, PEI-PEG@MnO2 and the sample to be tested, react at room temperature, and then use a UV spectrophotometer to measure the colorimetric signal value at 652 nm. Step 4: Electrochemical detection The electrode prepared in step 2 was used as the working electrode, the AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. The electrolyte included 3,3',5,5'-tetramethylbenzidine and PBS buffer. The PBS buffer solution, 3,3',5,5'-tetramethylbenzidine and the sample to be tested were mixed and reacted at room temperature. The measurement was then performed using the electrode prepared in step 2 as the anode and a platinum electrode as the cathode.

Images