A method for detecting reduced glutathione using a colorimetric sensor based on Fe3O4@o-ppyNPs.

Abstract

The application discloses a method for detecting reduced glutathione based on a colorimetric sensor constructed by Fe3O4@o-ppy NPs, and comprises the following steps: constructing Fe3O4@o-ppy NPs; constructing a GSH colorimetric sensor: adding GSH, TMB and Fe3O4@o-ppy NPs with different concentrations into an acetic acid-sodium acetate buffer solution, incubating, obtaining a detection system, measuring the absorbance value of the detection system at 650 nm, taking the concentration of GSH as the abscissa and the absorbance value at 650 nm as the ordinate, and establishing a GSH standard curve; measuring the absorbance value of an unknown reduced glutathione sample at 650 nm, and substituting the absorbance value into the GSH standard curve to obtain the concentration of GSH in the sample. The application constructs a colorimetric sensor with high sensitivity and high selectivity, the linear range of GSH is 0-600 muM, and the detection limit is 1.4 muM.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection and relates to a method for detecting reduced glutathione based on a colorimetric sensor. More specifically, it relates to a method for detecting reduced glutathione based on a colorimetric sensor constructed from Fe3O4@o-ppy NPs. Background Technology

[0002] Nanozymes are nanomaterials with catalytic activity similar to natural enzymes. Nanozymes possess advantages such as high stability, simple preparation, cost-effectiveness, and ease of storage, and are gradually becoming alternatives to natural enzymes. Nanozymes exhibit high activity and ease of modification, leading to their wide application in fields such as biosensing, environmental protection, antibacterial, antitumor, and cell protection. Among various nanozymes, magnetic nanozymes stand out in sensor construction due to their unique analytical advantages. Magnetic nanozymes are catalytically active and magnetic nanomaterials. Using simple magnetic separation in analytical systems allows for real-time control of catalytic reactions and purification of the analytical system, reducing interference and improving detection accuracy. Therefore, magnetic nanozymes offer numerous advantages and have broad application prospects.

[0003] Polypyrrole is a star material for supercapacitors and a key representative of conductive polymers, possessing better flexibility and higher density than other polymers, which helps improve its electron transport performance. Nanomaterials modified with polypyrrole can exhibit enhanced nanozyme activity, further expanding their applications in biosensing and tumor therapy. Previous studies have used concentrated nitric acid to oxidize polypyrrole to obtain oxygen-functionalized polypyrrole nanozymes, but this method is relatively dangerous.

[0004] Nanozymes have been applied in biosensing, environmental protection, and anti-tumor therapy. Particularly in the field of biosensing, they can generate various response signals, including colorimetric, fluorescence, electrochemical, and chemiluminescent signals. Among these, colorimetric sensors are simple, fast, intuitive, versatile, and easy to widely apply. They achieve quantitative detection of targets by measuring ultraviolet-visible absorbance values, or can directly monitor color changes after a colorimetric solution reaction with the naked eye. However, gradients of a single color are difficult for the human eye to distinguish. By adding different color indicators during the reaction process, several color gradients can be formed, making them easier for the human eye to differentiate.

[0005] Reduced glutathione (GSH) is an important endogenous antioxidant widely found inside and outside human cells. It scavenge free radicals and is closely related to human health. The normal endogenous GSH level in adults is 0.8–1.1 mM. Imbalances in GSH levels are associated with various clinical diseases, such as cardiovascular disease, Parkinson's disease, diabetes, and many types of cancer. Therefore, GSH can be used as a key biomarker and prognostic indicator for these diseases, making its analysis necessary.

[0006] Currently, methods for detecting reduced glutathione (GSH) generally suffer from drawbacks such as low sensitivity, time-consuming detection, high cost, and cumbersome procedures. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing detection methods, such as low sensitivity, long detection time, high cost, and cumbersome procedures. A novel magnetic nanozyme Fe3O4@o-ppy NPs with high oxidase-like activity was synthesized, and a colorimetric sensing system for detecting GSH was established using Fe3O4@o-ppy NPs. This method can detect GSH simply, quickly, intuitively, and with high sensitivity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A magnetic nanoenzyme Fe3O4@o-ppy NPs is prepared by synthesizing Fe3O4 NPs from ferrous chloride tetrahydrate (FeCl2·4H2O) and ferric chloride hexahydrate (FeCl3·6H2O), then using Fe3O4 NPs as a precursor to synthesize Fe3O4@ppy NPs with pyrrole, and finally performing oxygen functionalization treatment on Fe3O4@ppy NPs using sodium hydroxide solution.

[0010] Specifically, the Fe3O4@o-ppy NPs are prepared by the following method:

[0011] Step (a): Ferrous chloride tetrahydrate and ferric chloride hexahydrate were dissolved in ultrapure water and heated to 70–90°C under N2 protection with stirring. Ammonia water was added dropwise to adjust the pH of the solution to 9–11, and the reaction was maintained at this temperature for 30–60 minutes. After the reaction was completed, nanoparticles were obtained by magnetic separation. The nanoparticles were washed successively with anhydrous ethanol and deionized water, and dried under vacuum at 60°C to obtain Fe3O4 NPs. The molar ratio of ferrous chloride tetrahydrate to ferric chloride hexahydrate was 0.5:1, and the volume ratio of ferrous chloride tetrahydrate to ultrapure water was 1:30–1:60 mmol / mL.

[0012] Step (b): Pyrrole and Fe3O4 NPs were dissolved in ultrapure water and stirred for 30 minutes. Ammonium persulfate was added, and the mixture was reacted in an ice-water bath for 3 hours. The particles were separated by magnetic separation, washed successively with anhydrous ethanol and deionized water, and dried under vacuum at 60°C to obtain Fe3O4@ppyNPs. At room temperature, Fe3O4@ppyNPs were subjected to oxygen functionalization treatment with sodium hydroxide solution for 30–60 minutes. The product was separated by magnetic separation, washed with deionized water, and dried under vacuum at 60°C to obtain Fe3O4@o-ppy NPs.

[0013] The ratio of pyrrole to Fe3O4 NPs was 1.5:1 μL / mg; the ratio of Fe3O4 NPs to ultrapure water was 1.6:1 mg / mL; the ratio of pyrrole to ammonium persulfate was 60:1 μL / mmol; the ratio of Fe3O4@ppyNPs to sodium hydroxide solution was 1:1 mg / mL; and the concentration of sodium hydroxide solution was 0.2–1.0 M.

[0014] In step (a), specifically, ferrous chloride tetrahydrate and ferric chloride hexahydrate are dissolved in ultrapure water, heated to 80°C under N2 protection while stirring, ammonia water is added dropwise, the pH of the solution is adjusted to 10, and the reaction continues for 45 minutes.

[0015] In step (b), preferably, the concentration of the sodium hydroxide solution is 0.5M sodium hydroxide.

[0016] Preferably, the oxygen functionalization treatment takes 40 to 60 minutes.

[0017] Most preferably, the oxygen functionalization treatment time is 50 minutes.

[0018] A method for detecting reduced glutathione using a colorimetric sensor constructed based on highly oxidase-active Fe3O4@o-ppy NPs, comprising the following steps:

[0019] Step (1): Construct Fe3O4@o-ppy NPs;

[0020] Step (2) Construction of reduced glutathione (GSH) colorimetric sensor: Add different concentrations of GSH, 3,3',5,5'-tetramethylbenzidine (TMB) and Fe3O4@o-ppy NPs to acetate-sodium acetate buffer solution, incubate at 40-50℃ for 1-10 minutes to obtain the detection system, measure the absorbance value of the detection system at 650nm by UV-Vis spectrophotometry, and establish a GSH standard curve with the concentration of GSH as the x-axis and the absorbance value at 650nm as the y-axis;

[0021] Step (3) Colorimetric sensor sample detection: Measure the absorbance value of the unknown reduced glutathione at 650 nm in the sample to be tested according to step (2), and substitute the absorbance value at 650 nm into the GSH standard curve in step (2) to obtain the concentration of reduced glutathione in the sample to be tested.

[0022] In step (2), the acetate-sodium acetate buffer solution is an acetate-sodium acetate buffer solution with a pH of 3.0 to 5.0, preferably an acetate-sodium acetate buffer solution with a pH of 4.0.

[0023] Preferably, the incubation temperature is 45°C and the incubation time is 5 minutes.

[0024] In the detection system, the final concentration of Fe3O4@o-ppy NPs is 0.05–0.25 μg / mL, preferably 0.2 μg / mL; the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5–1.0 mM, preferably 0.75 mM; and the concentration of GSH is 0–600 μM, specifically selected from 0, 100, 200, 300, 400, 500, and 600 μM.

[0025] Specifically, a reduced glutathione colorimetric sensor was constructed: 50 μL of TMB solution and 50 μL of Fe3O4@o-ppyNPs solution were added to 350 μL of acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs; 10 μL of GSH solution of different concentrations was added to 40 μL of acetate-sodium acetate buffer, followed by 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs, and incubated to obtain the detection system.

[0026] In step (3), sample detection: 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution were added to 350 μL of acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs; 10 μL of the test sample with unknown GSH concentration was added to 40 μL of acetate-sodium acetate buffer, and then 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs was added. After incubation, the detection system was obtained; the absorbance value at 650 nm was measured by UV-Vis spectrophotometry, and the absorbance value was substituted into the GSH standard curve in step (2) to obtain the GSH concentration in the test sample.

[0027] As a preferred technical solution for the method of detecting reduced glutathione using a colorimetric sensor based on Fe3O4@o-ppy NPs described in this invention, a reduced glutathione (GSH) colorimetric sensor is constructed as follows: Different concentrations of GSH, 3,3',5,5'-tetramethylbenzidine (TMB), and Fe3O4@o-ppy NPs are added to an acetate-sodium acetate buffer solution, and the solution is incubated at 40–50°C for 1–10 minutes. Amaranth is then added to obtain the detection system. The absorbance value of the detection system at 650 nm is measured by ultraviolet-visible spectrophotometry. A GSH standard curve is established with the concentration of GSH as the x-axis and the absorbance value at 650 nm as the y-axis.

[0028] The acetate-sodium acetate buffer solution is an acetate-sodium acetate buffer solution with a pH of 3.0 to 5.0, preferably an acetate-sodium acetate buffer solution with a pH of 4.0.

[0029] Preferably, the incubation temperature is 45°C and the incubation time is 5 minutes.

[0030] In the detection system, the final concentration of Fe3O4@o-ppy NPs is 0.05–0.25 μg / mL, preferably 0.2 μg / mL; the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5–1.0 mM, preferably 0.75 mM; the concentration of GSH is 0–600 μM, specifically selected from 0, 100, 200, 300, 400, 500, and 600 μM; and the concentration of amaranth is 25–50 μg / mL, preferably 40 μg / mL.

[0031] Specifically, a reduced glutathione colorimetric sensor was constructed as follows: 50 μL of TMB solution and 50 μL of Fe3O4@o-ppyNPs solution were added to 350 μL of acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs; 10 μL of GSH solution of different concentrations was added to 40 μL of acetate-sodium acetate buffer, followed by 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs, incubation, and then 16 μL of amaranth red solution was added to obtain the detection system.

[0032] As another technical solution of the method for detecting reduced glutathione according to the present invention, a method for detecting reduced glutathione based on a colorimetric sensor constructed from Fe3O4@o-ppyNPs includes the following steps:

[0033] Step (1): Construct Fe3O4@o-ppy NPs;

[0034] Step (2) Construction of a reduced glutathione (GSH) visual colorimetric sensor: Different concentrations of GSH, 3,3',5,5'-tetramethylbenzidine (TMB) and Fe3O4@o-ppy NPs were added to an acetate-sodium acetate buffer solution and incubated at 40-50℃ for 1-10 minutes. Amaranth red was added to obtain the detection system. As the GSH concentration changed from 0 to 600 μM, the color of the detection system showed a significant color gradient change from red to bright blue. Based on the different colors corresponding to different GSH concentrations, a GSH visual colorimetric sensor was constructed. By adding amaranth red, the visual color changed from a single blue change to a rich gradient change from red to bright blue.

[0035] Step (3), Sample detection: Obtain the color of the detection system of the test sample containing unknown reduced glutathione according to step (2), compare it with the GSH visual colorimetric sensor in step (2), and predict the concentration range of reduced glutathione in the test sample.

[0036] In step (2), the acetate-sodium acetate buffer solution is an acetate-sodium acetate buffer solution with a pH of 3.0 to 5.0, preferably an acetate-sodium acetate buffer solution with a pH of 4.0.

[0037] Preferably, the incubation temperature is 45°C and the incubation time is 5 minutes.

[0038] In the detection system, the final concentration of Fe3O4@o-ppy NPs is 0.05–0.25 μg / mL, preferably 0.2 μg / mL; the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5–1.0 mM, preferably 0.75 mM; the concentration of GSH is 0–600 μM, specifically selected from 0, 100, 200, 300, 400, 500, and 600 μM; and the concentration of amaranth is 25–50 μg / mL, preferably 40 μg / mL.

[0039] Specifically, a reduced glutathione visual colorimetric sensor was constructed: 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution were added to 350 μL of acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs; 10 μL of GSH solution of different concentrations was added to 40 μL of acetate-sodium acetate buffer, followed by 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs, incubation, and then 16 μL of amaranth red solution was added to obtain the detection system.

[0040] In step (3), specifically, sample detection: 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution are added to 350 μL of acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs; 10 μL of the test sample with unknown GSH concentration is added to 40 μL of acetate-sodium acetate buffer, and then 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs is added. After incubation, 16 μL of amaranth red solution is added to obtain the detection system; the color of the detection system is compared with the GSH visual colorimetric sensor in step (4) to predict the concentration of reduced glutathione in the test sample.

[0041] The test samples were selected from whole blood and cells.

[0042] When the sample to be tested is whole blood, mix the whole blood with 10% trichloroacetic acid at a volume ratio of 1:1, cool in an ice bath for 10 minutes, centrifuge, and collect the supernatant.

[0043] When the sample to be tested is selected from cells, the cell particles are mixed with 10% trichloroacetic acid, gently stirred, centrifuged, and the supernatant is collected.

[0044] This invention uses ultrapure water or acetate-sodium acetate buffer to prepare TMB solution, Fe3O4@o-ppy NPs solution, GSH solution, and amaranth red solution, respectively.

[0045] The detection mechanism of the method of the present invention ( Figure 1 Fe3O4@o-ppy NPs exhibit oxidase-like activity and can catalyze the formation of superoxide radicals from free O2. ·- Based on the oxidase-like activity of Fe3O4@o-ppy NPs, a colorimetric sensor for the quantitative determination of GSH was established. In the absence of GSH, the oxidase-like activity of Fe3O4@o-ppy NPs catalyzes the formation of superoxide radicals (O2- ... ·- The process oxidizes TMB to oxidized TMB (oxTMB), which has strong UV absorption at 650 nm. However, when GSH is present, the amount of oxTMB generated in the system decreases, and the absorbance at 650 nm is significantly reduced.

[0046] Compared with the prior art, the present invention has the following significant advantages:

[0047] 1. Polypyrrole has unique sp... 2 The hybrid carbon atom arrangement in this invention utilizes a sodium hydroxide degradation method to oxygen-functionalize polypyrrole nanoparticles Fe3O4@ppyNPs, which is safer and simpler. During the chemical corrosion of polypyrrole, the polypyrrole chains undergo nucleophilic attack by OH-, rearranging the pyrrole rings and generating carbonyl and hydroxyl groups, which serve as active sites for oxidative dehydrogenation reactions. This invention prepares a novel nanoenzyme Fe3O4@o-ppy NPs modified with oxygen-containing functional groups. Fe3O4@o-ppy NPs exhibit high oxidase-like activity, along with excellent stability and reusability.

[0048] 2. This invention constructs a colorimetric sensor for detecting reduced glutathione based on Fe3O4@o-ppy NPs. It has the advantages of high sensitivity and high selectivity. The linear range of reduced glutathione is 0-600 μM and the detection limit is 1.4 μM.

[0049] 3. This invention constructs a visual colorimetric sensor for detecting reduced glutathione based on Fe3O4@o-ppy NPs. Different concentration ranges of reduced glutathione correspond to different colors. Based on the visual colorimetric sensor constructed in this invention, the concentration range of reduced glutathione in a sample can be obtained visually.

[0050] 4. The colorimetric sensor constructed in this invention can be successfully applied to the determination of reduced glutathione in human whole blood samples and cells, and can also be used as a drug screening method. Attached Figure Description

[0051] Figure 1 This invention describes the mechanism of quantitative determination of reduced glutathione using a colorimetric sensor constructed based on Fe3O4@o-ppy NPs.

[0052] Figure 2 TEM images of Fe3O4 NPs (A) and Fe3O4@o-ppy NPs (C) synthesized in Example 1; normal particle size distribution diagrams of Fe3O4 NPs (B) and Fe3O4@o-ppy NPs (D).

[0053] Figure 3 The image shows the X-ray photoelectron spectrum of Fe3O4@o-ppy NPs synthesized in Example 1.

[0054] Figure 4 High-resolution N1s spectra of Fe3O4@o-ppy NPs obtained by NaOH treatment for different times are shown; where (A) 0h (Control, i.e., Fe3O4@ppy NPs); (B) NaOH treatment for 1h; (C) NaOH treatment for 4h.

[0055] Figure 5 The infrared spectra of Fe3O4@o-ppy NPs obtained by treating with NaOH for different times are shown.

[0056] Figure 6 The results show the effect of different NaOH treatment times on the activity of Fe3O4@o-ppy NPs pseudooxidase.

[0057] Figure 7 The standard curves of adding different concentrations of GSH to the detection system constructed in Example 3 are shown.

[0058] Figure 8 This is a comparison chart of the selectivity of the detection system constructed in Example 3 for GSH.

[0059] Figure 9 The results show the effect of pH on the catalytic activity of Fe3O4@o-ppy NPs pseudooxidase.

[0060] Figure 10 The Michaelis curves of Fe3O4@o-ppy NPs are shown.

[0061] Figure 11 The detection results of the detection system constructed in Example 3 in actual cell samples are shown in the figure. Detailed Implementation

[0062] The technical solution of the present invention will be described in more detail with reference to the accompanying drawings and specific embodiments. Although the following are preferred embodiments of the present invention, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0063] The inventors studied the properties of Fe3O4@o-ppy NPs using a colorimetric method, and found that Fe3O4@o-ppy NPs exhibited strong oxidase-like activity. Fe3O4@o-ppy NPs, TMB, and different concentrations of GSH were incubated together, and the absorbance values ​​of the samples were measured to establish a standard curve.

[0064] Example 1

[0065] Step (a): Dissolve 0.172 g FeCl2·4H2O and 0.472 g FeCl3·6H2O in 40 mL of ultrapure water, stir continuously, heat to 80 °C under N2 protection, maintain the temperature at 80 °C, add ammonia water (25%) dropwise until the pH of the above solution reaches 10, and continue the reaction for 45 minutes; after the reaction is completed, obtain nanoparticles by magnetic separation, wash the nanoparticles with anhydrous ethanol and deionized water in sequence, and dry them under vacuum at 60 °C to obtain Fe3O4 NPs;

[0066] Step (b): Dissolve 480 μL of pyrrole and 320 mg of Fe3O4 NPs sequentially in 200 mL of ultrapure water, stir at room temperature for 30 minutes, then add 8 mL of 1 M ammonium persulfate solution, and react in an ice-water bath for 3 hours; obtain black particles by magnetic separation, wash the black particles with anhydrous ethanol and deionized water, and dry under vacuum at 60 °C for 12 hours to obtain Fe3O4@ppyNPs; add 200 mg of Fe3O4@ppyNPs to 200 mL of 0.5 M sodium hydroxide solution, stir at room temperature for 50 minutes, separate the solid product by magnetic separation, wash with deionized water, and dry under vacuum at 60 °C to obtain magnetic nanozyme Fe3O4@o-ppyNPs.

[0067] The Fe3O4 NPs obtained in step (a) and the Fe3O4@o-ppy NPs obtained in step (b) were characterized.

[0068] TEM images show that the Fe3O4 NPs are spherical. Figure 2 A), while Fe3O4@o-ppy NPs look like pomegranates, with Fe3O4 NPs embedded like seeds in o-ppy (oxygen-functionalized polypyrrole). Figure 2 C). The average particle size of Fe3O4 NPs is 14.5 ± 1.98 nm. Figure 2B), the average particle size of Fe3O4@o-ppy NPs is 83.2±6.43 nm. Figure 2 D).

[0069] X-ray photoelectron spectroscopy indicates that Fe3O4@o-ppy NPs contain C, N, O, and Fe elements. Figure 3 According to high-resolution N1s spectral data, Fe3O4@ppy NPs do not contain C=N-( Figure 4 A). With the extension of NaOH chemical corrosion time (1h and 4h respectively), the C=N- content increased rapidly, reaching 11.8 at% at 4h, indicating that the original pyrrole rings had rearranged. Figure 4 B and 4C). The infrared spectrum of Fe3O4@o-ppy NPs indicates that it is located at 1696 -1 carbonyl group and 1096 cm -1 The content of hydroxyl groups gradually increased with the reaction time of NaOH. Figure 5 This further clarifies that the oxygen-containing groups generated by the ppy (polypyrrole) chain of Fe3O4@o-ppy NPs undergo rearrangement.

[0070] The above characterizations all demonstrate the successful synthesis of Fe3O4@o-ppy NPs.

[0071] Example 2

[0072] The effect of different oxygen functionalization times of Fe3O4@ppy NPs treated with sodium hydroxide solution on the activity of Fe3O4@o-ppy NPs pseudooxidase was investigated.

[0073] Fe3O4@ppyNPs were synthesized using the methods described in steps (a) and (b) of Example 1. 200 mg of Fe3O4@ppyNPs was added to 200 mL of 0.5 M sodium hydroxide solution, and the mixture was stirred at room temperature for 20 min, 30 min, 40 min, 50 min, 1 h, 2 h, 4 h, 6 h, and 8 h. The solid product was separated by magnetic separation, washed with deionized water, and dried under vacuum at 60 °C to obtain Fe3O4@o-ppy NPs.

[0074] The oxidase-like activity of the obtained Fe3O4@o-ppy NPs was investigated: Fe3O4@o-ppy NPs obtained from different oxygen functionalization times were prepared into aqueous solutions of the same concentration; 50 μL of Fe3O4@ppy NPs aqueous solution and 50 μL of TMB aqueous solution were added to 400 μL of acetate-sodium acetate buffer and incubated at 45℃ for 5 minutes to obtain different detection systems. In the detection systems, the final concentration of Fe3O4@o-ppy NPs was 0.2 μg / mL and the final concentration of TMB was 0.75 mM; the absorbance value at 650 nm of each detection system was measured, and the highest absorbance value among all detection systems was taken as 100% to calculate the relative enzyme activity.

[0075] Relative activity = (Absorbance value of the detection system / Maximum absorbance value) × 100%

[0076] like Figure 6 As shown, the pseudo-oxidase activity of Fe3O4@o-ppy NPs was highest when the sodium hydroxide solution was used to treat Fe3O4@o-ppy NPs with oxygen for 30-60 minutes. Considering both treatment time and pseudo-oxidase activity, the treatment time was further optimized to 40-60 minutes. The pseudo-oxidase activity of Fe3O4@o-ppy NPs was highest when the oxygen functionalization treatment was 50 minutes.

[0077] Example 3

[0078] A method for detecting reduced glutathione using a colorimetric sensor based on Fe3O4@o-ppy NPs includes the following steps:

[0079] Step (1): Dissolve 0.172g FeCl2·4H2O and 0.472g FeCl3·6H2O in 40mL of ultrapure water, stir continuously, heat to 80℃ under N2 protection, maintain the temperature at 80℃, add ammonia water (25%) dropwise until the pH of the solution reaches 10, and continue the reaction for 45 minutes; after the reaction is completed, obtain nanoparticles by magnetic separation, wash the nanoparticles with anhydrous ethanol and deionized water in sequence, and dry them under vacuum at 60℃ to obtain Fe3O4 NPs;

[0080] Step (2): Dissolve 480 μL of pyrrole and 320 mg of Fe3O4 NPs in 200 mL of ultrapure water, stir at room temperature for 30 minutes, then add 8 mL of 1 M ammonium persulfate solution and react in an ice-water bath for 3 hours; obtain black particles by magnetic separation, wash the black particles with anhydrous ethanol and deionized water in sequence, and dry under vacuum at 60 °C for 12 hours to obtain Fe3O4@ppy NPs; add 200 mg of Fe3O4@ppy NPs to 200 mL of 0.5 M sodium hydroxide solution, stir at room temperature for 50 minutes, separate the solid product by magnetic separation, wash with deionized water, and dry under vacuum at 60 °C to obtain magnetic nanozyme Fe3O4@o-ppyNPs;

[0081] Step (3) Construction of a reduced glutathione (GSH) colorimetric sensor: TMB solution, Fe3O4@o-ppy NPs solution, GSH solution, and amaranth red solution were prepared using ultrapure water. 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution were added to 350 μL of pH 4 acetate-sodium acetate buffer to obtain an acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs. 10 μL of GSH aqueous solution of different concentrations was added to 40 μL of pH 4 acetate-sodium acetate buffer, followed by 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs. The mixture was incubated at 45°C for 5 minutes, and then 16 μL of amaranth red solution was added to obtain the detection system. In the detection system, Fe3O4@o-ppy... The final concentration of NPs was 0.2 μg / mL, the concentration of TMB was 0.75 mM, the concentrations of GSH were 0, 100, 200, 300, 400, 500, and 600 μM, and the concentration of amaranth red was 40 μg / mL.

[0082] The ultraviolet absorption curve of the detection system in the 500-800 nm range was measured by ultraviolet-visible spectrophotometry. Figure 7 A) Obtain the absorbance value of the detection system at 650 nm; construct a GSH standard curve with GSH concentration as the x-axis and absorbance value at 650 nm as the y-axis. Figure 7 B): Y=-0.00241[GSH]+1.4929, R 2 =0.9966, detection limit is 1.4 μM;

[0083] Step (4), Sample Detection: Referring to step (3), add 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution to 350 μL of pH4 acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs; add 10 μL of the test sample with unknown GSH concentration to 40 μL of pH4 acetate-sodium acetate buffer, then add 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs, incubate at 45℃ for 5 minutes, add 16 μL of amaranth solution to obtain the detection system; in the detection system, Fe3O4@o-ppy The final concentration of NPs was 0.2 μg / mL, the concentration of TMB was 0.75 mM, and the concentration of amaranth red was 40 μg / mL. The absorbance of the detection system at 650 nm was measured by UV-Vis spectrophotometry. The absorbance value was substituted into the GSH standard curve established in step (3) to obtain the GSH concentration in the sample to be tested.

[0084] Step (5) Constructing the GSH visual colorimetric sensor: Following step (3), the detection system is obtained, such as... Figure 7 As shown in C, as the GSH concentration changes from 0 to 600 μM, the color of the detection system exhibits a significant red-bright blue gradient change. A visual colorimetric sensor for GSH is constructed based on the different colors corresponding to different GSH concentrations.

[0085] Step (6) Sample detection: The detection system is obtained according to step (4). The color of the detection system is compared with the GSH visual colorimetric sensor in step (5) to predict the concentration range of reduced glutathione in the sample to be tested.

[0086] Example 4

[0087] The selectivity of the colorimetric sensor for GSH was investigated.

[0088] The selectivity of the colorimetric sensor was examined by comparing it with common interfering substances such as oxidized glutathione (GSSG), cystine (CYSS), bovine serum albumin (BSA), glutamic acid (Glu), glycine (Gly), methionine (Met), tryptophan (Try), tyrosine (Tyr), lysine (Lys), homocysteine ​​(Hcy), and cysteine ​​(Cys).

[0089] TMB solution, Fe3O4@o-ppy NPs solution, GSH solution, interfering agent solution, and amaranth red solution were prepared using ultrapure water. 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution were added to 350 μL of pH 4 acetate-sodium acetate buffer to obtain acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs. 10 μL of GSH solution or interfering agent solution was added to 40 μL of pH 4 acetate-sodium acetate buffer, followed by 450 μL of acetate-sodium acetate buffer containing TMB and Fe3O4@o-ppy NPs. The mixture was incubated at 45°C for 5 minutes, and then 16 μL of amaranth red solution was added to obtain the detection system. In the detection system, the final concentration of Fe3O4@o-ppyNPs was 0.2 μg / mL, the final concentration of TMB was 0.75 mM, the final concentration of GSH or interfering substances was 600 μM, and the concentration of amaranth red was 40 μg / mL. The absorbance of the detection system at 650 nm was measured and recorded. An equal volume of ultrapure water was used as a blank control instead of GSH solution.

[0090] See results Figure 8 Compared to the blank control and other interfering substances, the colorimetric sensor exhibits high specificity for GSH, except for Cys. Although high concentrations of Cys also lead to a decrease in absorbance, its concentration (μM level) is significantly lower than the GSH concentration (mM level) in biological systems (such as blood sample systems and intracellular systems), therefore its interference with GSH detection is negligible. These results demonstrate that the colorimetric sensor based on Fe3O4@o-ppy NPs has high selectivity for GSH and can distinguish GSH from other non-target substances.

[0091] Example 5

[0092] The effect of different pH values ​​on the catalytic activity of Fe3O4@o-ppy NPs pseudooxidase was investigated.

[0093] TMB solution and Fe3O4@o-ppy NPs solution were prepared using ultrapure water. 50 μL of TMB solution and 50 μL of Fe3O4@o-ppy NPs solution were added to 400 μL of acetate-sodium acetate buffer solutions with pH values ​​of 2, 3, 4, 5, 6, 7, and 8, respectively. The mixture was thoroughly mixed, resulting in a final concentration of 0.2 μg / mL for Fe3O4@o-ppy NPs and 0.75 mM for TMB. The mixture was incubated at 45°C for 5 minutes, and the absorbance at 650 nm was measured.

[0094] See results Figure 9The results indicate that the catalytic activity of Fe3O4@o-ppy NPs oxidase is pH-dependent, exhibiting good catalytic activity at pH 3–5, with optimal activity at pH 4. Therefore, pH 4 was ultimately chosen as the optimal reaction pH.

[0095] Example 6

[0096] To quantify the catalytic efficiency and affinity of the material, the steady-state kinetic parameters, including the Michaelis constant (Km), were calculated using the double reciprocal method. m ) and maximum initial velocity value V max K m The lower the value, the higher the affinity between the mimic enzyme and the matrix. max The higher the value, the higher the saturation reaction rate.

[0097] 50 μL of TMB aqueous solutions of different concentrations and 50 μL of Fe3O4@o-ppy NPs aqueous solutions were added to 400 μL of acetate-sodium acetate buffer solution at pH 4. The mixture was thoroughly mixed to achieve final TMB concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mM, and a final Fe3O4@o-ppy NPs concentration of 0.2 μg / mL. The mixture was incubated at 45°C for 5 minutes, and the absorbance at 650 nm was measured. The reaction rate was calculated using these absorbance values ​​and substituted into 1 / V = (K... m / V max (1 / [S])+1 / V max In the process, a double reciprocal curve is obtained, and the Michaelis constant K is calculated using the intercept and slope. m and maximum initial velocity V max .

[0098] See results Figure 10 K of Fe3O4@o-ppy NPs m =0.052mM, V max =1.96×10 -6 Ms -1 .

[0099] Comparing the K of the Fe3O4@o-ppy NPs of this invention with other reported materials possessing oxidase-like activity m and V max The results are shown in Table 1. Fe3O4@o-ppy NPs have a small K0 m With a larger V max This indicates that the Fe3O4@o-ppy NPs of the present invention have excellent oxidase-like activity.

[0100] Table 1. K values ​​of different materials with mimetic oxidase activity m and V max

[0101]

[0102] References:

[0103] [1]Wan Y, Qi P, Zhang D, et al. Manganese oxide nanowire-mediated enzyme-linked immunosorbent assay[J]. Biosensors and Bioelectronics. 2012, 33(1): 69-74.

[0104] [2]Asati A, Santra S, Kaittanis C, et al. Oxidase-Like Activity of Polymer-Coated Cerium Oxide Nanoparticles[J]. Angewandte Chemie International Edition. 2009, 48(13): 2308-2312.

[0105] [3]Yu C, Chen T, Jiang J, et al. Lysozyme-directed synthesis of platinum nanoclusters as a mimic oxidase[J]. Nanoscale. 2014, 6(16): 9618-9624.

[0106] [4]Qiu N, Liu Y, Guo R. Electrodeposition-Assisted Rapid Preparation of Pt Nanocluster / 3D Graphene Hybrid Nanozymes with Outstanding Multiple Oxidase-Like Activity for Distinguishing Colorimetric Determination of Dihydroxybenzene Isomers[J]. ACS Applied Materials & Interfaces. 2020, 12(13): 15553-15561.

[0107] [5]He W, Liu Y, Yuan J, et al.Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays[J].Biomaterials.2011,32(4):1139-1147.

[0108] Application Example 1

[0109] Tests were performed on whole blood samples from healthy individuals.

[0110] Whole blood was mixed with 10% trichloroacetic acid at a volume ratio of 1:1, cooled in an ice bath for 10 minutes to obtain protein precipitation, centrifuged at 12000 rpm for 15 minutes, and the supernatant was used as the test sample. 0, 800, 1000, and 1200 μM GSH standards were added to the test sample, respectively, and the absorbance value at 650 nm was measured according to the method in Example 3. The absorbance was then substituted into the GSH standard curve established in Example 3 to obtain the GSH standard concentration in the sample. Each sample was measured three times, and the average value was taken. The RSD and recovery rate were calculated (see Table 2).

[0111] Table 2. GSH recovery rate in whole blood (n=3)

[0112]

[0113] Application Example 2

[0114] Measurement of GSH in cells

[0115] Endothelial cells were selected and included normal cells, cells stimulated under oxidative stress (i.e., by the inflammatory cytokine interleukin-2 (IL-2), and IL-2 cells treated with rosuvastatin. Cells were cultured in 60 mm culture dishes. Cells were trypsinized and washed with pH 7.4 phosphate buffer, and approximately 2 × 10⁶ cells were collected by centrifugation. 5 Cells were counted using a cell counter in the buffer solution after centrifugation. The cells were mixed with 10% trichloroacetic acid and gently stirred for 6 hours. Protein particles were removed by centrifugation, and the supernatant containing GSH was collected. The absorbance of the supernatant at 650 nm was measured according to the method in Example 3. The absorbance was then substituted into the GSH standard curve established in Example 3 to obtain the GSH concentration in the sample. Each sample was measured three times, and the average value was taken.

[0116] like Figure 11 As shown, the GSH content in cells under oxidative stress (il-2) was higher than that in normal cells (Control). The GSH level in inflammatory cells (il+station) treated with rosuvastatin returned to normal.

[0117] This indicates that the method of the present invention can not only be used for the detection of GSH in cells, but also as a means of drug screening.

[0118] The embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A magnetic nanoenzyme Fe3O4@o-ppy NPs, characterized in that: The magnetic nanozyme Fe3O4@o-ppy NPs were prepared by the following method: Step (a): Ferrous chloride tetrahydrate and ferric chloride hexahydrate were dissolved in ultrapure water and heated to 70-90°C under N2 protection with stirring. Ammonia water was added dropwise to adjust the pH of the solution to 9-11, and the reaction was maintained at this temperature for 30-60 minutes. After the reaction was completed, nanoparticles were obtained by magnetic separation. The nanoparticles were washed with ethanol and deionized water in sequence and dried under vacuum at 60°C to obtain Fe3O4NPs. The molar ratio of ferrous chloride tetrahydrate to ferric chloride hexahydrate was 0.5:

1. Step (b): Pyrrole and Fe3O4 NPs were dissolved in ultrapure water and stirred until homogeneous. Ammonium persulfate was added, and the mixture was reacted in an ice-water bath for 3 hours. The particles were separated magnetically, washed successively with anhydrous ethanol and deionized water, and dried under vacuum at 60°C to obtain Fe3O4@ppyNPs. Fe3O4@ppyNPs were then subjected to oxygen functionalization treatment with sodium hydroxide solution at room temperature for 30–60 minutes. The product was magnetically separated, washed with deionized water, and dried under vacuum at 60°C to obtain magnetic nanozyme Fe3O4@o-ppyNPs. The ratio of pyrrole to Fe3O4 NPs was 1.5:1 μL / mg; the ratio of pyrrole to ammonium persulfate was 60:1 μL / mmol; and the concentration of sodium hydroxide solution was 0.2–1.0 M.

2. The magnetic nanoenzyme Fe3O4@o-ppy NPs according to claim 1, characterized in that: In step (a), ferrous chloride tetrahydrate and ferric chloride hexahydrate are dissolved in ultrapure water, heated to 80°C while stirring under N2 protection, ammonia water is added dropwise to adjust the pH of the solution to 10, and the reaction is continued for 45 minutes. In step (b), the concentration of the sodium hydroxide solution is 0.5M; the oxygen functionalization treatment time is 40-60 minutes.

3. The magnetic nanoenzyme Fe3O4@o-ppy NPs according to claim 2, characterized in that: In step (b), the oxygen functionalization treatment takes 50 minutes.

4. A method for detecting reduced glutathione using a colorimetric sensor constructed based on the magnetic nanoenzyme Fe3O4@o-ppy NPs according to any one of claims 1-3, characterized in that: Includes the following steps: Step (1): Construct the magnetic nanoenzyme Fe3O4@o-ppy NPs according to any one of claims 1-3; Step (2) Construction of a reduced glutathione colorimetric sensor: Add different concentrations of reduced glutathione, 3,3',5,5'-tetramethylbenzidine and magnetic nanozyme Fe3O4@o-ppy NPs to an acetate-sodium acetate buffer solution and incubate at 40-50℃ for 1-10 minutes to obtain the detection system. Measure the absorbance of the detection system at 650 nm using UV-Vis spectrophotometry. Establish a reduced glutathione standard curve with the concentration of reduced glutathione as the x-axis and the absorbance at 650 nm as the y-axis. Step (3) Colorimetric sensor sample detection: Measure the absorbance value of the unknown reduced glutathione sample at 650 nm according to the detection method in step (2), substitute the absorbance value at 650 nm into the reduced glutathione standard curve in step (2), and obtain the concentration of reduced glutathione in the sample.

5. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 4, characterized in that: In step (2), the pH of the acetate-sodium acetate buffer solution is 3.0 to 5.

0.

6. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 5, characterized in that: In step (2), the pH of the acetate-sodium acetate buffer solution is 4.

0.

7. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 4, characterized in that: The incubation temperature is 45°C, and the incubation time is 5 minutes.

8. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 4, characterized in that: In the aforementioned detection system, the final concentration of the magnetic nanozyme Fe3O4@o-ppy NPs is 0.05–0.25 μg / mL; the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5–1.0 mM; and the concentration of reduced glutathione is 0–600 μM.

9. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 8, characterized in that: In the aforementioned detection system, the final concentration of the magnetic nanozyme Fe3O4@o-ppy NPs is 0.2 μg / mL.

10. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 8, characterized in that: In the detection system described above, the concentration of 3,3',5,5'-tetramethylbenzidine is 0.75 mM.

11. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 4, characterized in that: Construction of a reduced glutathione colorimetric sensor: Different concentrations of reduced glutathione, 3,3',5,5'-tetramethylbenzidine, and magnetic nanozyme Fe3O4@o-ppyNPs were added to an acetate-sodium acetate buffer solution and incubated at 40–50 °C for 1–10 minutes. Amaranth was then added to obtain the detection system. The absorbance of the detection system at 650 nm was measured by UV-Vis spectrophotometry. A standard curve of reduced glutathione was established with the concentration of reduced glutathione as the x-axis and the absorbance at 650 nm as the y-axis. In the detection system, the final concentration of the magnetic nanozyme Fe3O4@o-ppy NPs is 0.05–0.25 μg / mL; the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5–1.0 mM; the concentration of reduced glutathione is 0–600 μM; and the concentration of amaranth red is 25–50 μg / mL.

12. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 11, characterized in that: In the aforementioned detection system, the final concentration of the magnetic nanozyme Fe3O4@o-ppy NPs is 0.2 μg / mL.

13. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 11, characterized in that: In the detection system described above, the concentration of 3,3',5,5'-tetramethylbenzidine is 0.75 mM.

14. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 11, characterized in that: In the detection system, the concentration of reduced glutathione is 0, 100, 200, 300, 400, 500, and 600 μM.

15. The method for detecting reduced glutathione using a colorimetric sensor constructed based on magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 11, characterized in that: In the aforementioned detection system, the concentration of amaranth red is 40 μg / mL.

16. A method for detecting reduced glutathione using a visual colorimetric sensor constructed based on the magnetic nanoenzyme Fe3O4@o-ppy NPs according to any one of claims 1-3, characterized in that: Includes the following steps: Step (1): Construct the magnetic nanoenzyme Fe3O4@o-ppy NPs according to any one of claims 1-3; Step (2): Constructing a visual colorimetric sensor for reduced glutathione: Add different concentrations of reduced glutathione, 3,3',5,5'-tetramethylbenzidine, and magnetic nanozyme Fe3O4@o-ppy NPs to an acetate-sodium acetate buffer solution. Incubate at 40-50℃ for 1-10 minutes, then add amaranth red to obtain the detection system. In the detection system, the final concentration of magnetic nanozyme Fe3O4@o-ppy NPs is 0.05-0.25 μg / mL, the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5-1.0 mM, and the concentration of amaranth red is 25-50 μg / mL. Based on the different colors corresponding to different concentrations of reduced glutathione, a visual colorimetric sensor for reduced glutathione is constructed. Step (3) Sample detection: Obtain the color of the detection system of the test sample containing unknown reduced glutathione according to the detection method in step (2), and compare it with the reduced glutathione visual colorimetric sensor in step (2) to predict the concentration range of reduced glutathione in the test sample.

17. The method for detecting reduced glutathione using a visual colorimetric sensor constructed from magnetic nanoenzymes Fe3O4@o-ppy NPs according to claim 16, characterized in that: In step (2), the final concentration of the magnetic nanozyme Fe3O4@o-ppyNPs in the detection system is 0.2 μg / mL; the concentration of 3,3',5,5'-tetramethylbenzidine is 0.75 mM; the concentration of reduced glutathione is 0-600 μM; and the concentration of amaranth red is 40 μg / mL.

Images