Preparation method of zif-8 / aug peroxidase and application thereof in glutathione detection

Abstract

The application discloses a preparation method of ZIF-8 / Au peroxidase and application thereof in glutathione detection and belongs to the technical field of composite nanomaterials. 3.88g of 2-methyl imidazole is dissolved in 16mL of ultrapure water at room temperature, 200mg of 4mL of Zn(NO3)2 aqueous solution is quickly added under stirring, after the reaction is completed, nanocrystals are collected by centrifugation and washed, then the nanocrystals are dispersed in 20mL of ultrapure water, 48mM, 2.4mL of HAuCl4 is added under continuous stirring until the mixture is uniform, then 1mL of 0.1M of freshly prepared NaBH4 solution is added drop by drop under continuous stirring, after the solution color changes from bright yellow to wine red, the solution is left to stand, centrifuged and washed, and ZIF-8 / Au is obtained. ZIF-8 / Au has peroxidase-like activity, can catalyze the oxidation of a 3,3',5,5'-tetramethyl benzidine (TMB) and H2O2 reaction system to develop color (blue), after glutathione is added, the activity of the ZIF-8 / Au peroxidase-like enzyme can be inhibited, and the glutathione can be visually detected according to the color change of the reaction system.

Description

Technical fields:

[0001] This invention belongs to the field of composite nanomaterials technology, specifically relating to a method for preparing ZIF-8 / Au peroxidase and the application of the ZIF-8 / Au peroxidase in the detection of glutathione. Background technology:

[0002] Glutathione (GSH) is an antioxidant that maintains cellular redox homeostasis. It is a tripeptide thiol formed by the combination of glutamate, cysteine, and glycine. In particular, changes in glutathione levels are closely associated with several serious diseases, including Alzheimer's disease, diabetes, HIV / AIDS, and cancer.

[0003] To date, numerous analytical techniques have been established for the determination of glutathione, such as electrochemistry, surface-enhanced Raman scattering, high-performance liquid chromatography, optical sensing, and colorimetry. Compared to colorimetry, other detection methods suffer from drawbacks such as cumbersome sample pretreatment, complex operation, and lack of portability. Colorimetry offers advantages such as simplicity, cost-effectiveness, and ease of use. Therefore, this test has significant operational potential in remote areas and can be used without complex equipment. Enzyme catalysis is one of the most common methods in colorimetric determination, exhibiting good specificity and catalytic efficiency for substrates under mild conditions. However, it also has some significant drawbacks, such as complex purification processes, poor physicochemical stability, and high cost, thus limiting its further application. Biomimetic nanozymes (nanozymes) are nanomaterials with the functions of natural enzymes, possessing high stability, low cost, and tunable catalytic activity. Patents with publication numbers CN116786115A, CN116586055A, CN116559102A, and CN115753649A all disclose nanozymes for the detection of glutathione. AuNPs (gold nanoparticles) possess excellent catalytic properties. Existing technologies modify them to create composite nanomaterials for the detection of certain substances. For example, patent CN117517289A discloses a method for rapidly detecting nicotine isomers in e-cigarette liquids. This method uses reducing D-cysteine ​​carbon dots (DC-CDs) to prepare gold nanozymes (DC-CDs / AuNPs), which can catalyze the conversion of 3,3',5,5'-tetramethylbenzidine (TMB) to its oxidized form (oxTMB), and enhance... A patent with publication number CN108746660A discloses a method for preparing copper-gold-platinum composite nanomaterials and their application in the detection of cysteine, which strongly enhances the Raman signal of oxTMB. First, Cu@AuNPs are prepared, and then Cu@Au / Pt is prepared. The prepared Cu@Au / Pt composite nanomaterials have peroxidase-like activity, which can make the TMB and H2O2 system turn blue. The addition of cysteine ​​can inhibit the peroxidase-like activity. Based on the color change of the system, the concentration of cysteine ​​can be visually detected. Summary of the Invention:

[0004] The purpose of this invention is to provide a method for preparing ZIF-8 / Au peroxidase and its application in glutathione detection. By modifying it with ZIF-8 to generate composite nanomaterials, the problem of AuNPs being small in size and easily migrating and aggregating, thus affecting the activity of nanozymes, is solved.

[0005] To achieve the above objectives, the method for preparing ZIF-8 / Au peroxidase according to the present invention specifically includes the following steps:

[0006] 3.88 g of 2-methylimidazole was dissolved in 16 mL of ultrapure water at room temperature. 200 mg of 4 mL of Zn(NO3)2 aqueous solution was rapidly added with stirring. After stirring for 10 minutes, the nanocrystals were collected by centrifugation and washed three times. Then, the nanocrystals were dispersed in 20 mL of ultrapure water. 48 mM HAuCl4 was added with continuous stirring until the mixture was homogeneous. Then, 1 mL of freshly prepared 0.1 M NaBH4 solution was added dropwise with continuous stirring. After the solution color changed from bright yellow to wine red, it was allowed to stand. The solution was centrifuged and washed three times to obtain ZIF-8 / Au.

[0007] The ZIF-8 / Au peroxidase is spherical.

[0008] The ZIF-8 / Au peroxidase is used for the detection of glutathione. The specific method is as follows: ZIF-8 / Au, 3,3',5,5'-tetramethylbenzidine (TMB), H2O2 and NaAC-HAC buffer solution are added to the test solution containing glutathione. The reaction is carried out at room temperature. After the reaction is completed, the absorbance at 652 nm is detected. The concentration of GSH is calculated based on the absorbance and the constructed linear equation.

[0009] Specifically, the reaction was carried out at room temperature for 20 minutes.

[0010] The ZIF-8 / Au peroxidase was used for the detection of glutathione, with a detection limit of 0.28 μM.

[0011] ZIF-8 / Au exhibits peroxidase-like activity, catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 to produce a color change (blue). The addition of glutathione inhibits the activity of ZIF-8 / Au peroxidase, allowing for visual detection of glutathione based on the color change of the reaction system.

[0012] Compared with existing technologies, this invention has the following advantages: the metal-organic framework ZIF-8 has high thermal stability; using ZIF-8 as a carrier for AuNPs improves the utilization efficiency and storage stability of nanozymes; ZIF-8 / Au peroxidase can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) for colorimetric detection; glutathione can be detected based on colorimetry; and the concentration of GSH can be calculated based on absorbance and a constructed linear equation. This invention features fast detection speed, high accuracy of detection results, and simple operation. Attached image description:

[0013] Figure 1 These are photographs of ZIF-8 and ZIF-8 / Au from Example 1.

[0014] Figure 2 The images show the XRD patterns of ZIF-8 and ZIF-8 / Au prepared in Examples 1 and 2, where A represents Example 2 and B represents Example 1.

[0015] Figure 3 The images shown are scanning electron microscope (SEM) images (A) and transmission electron microscope (TEM) images (B) of ZIF-8 / Au in Example 1.

[0016] Figure 4 This is a diagram illustrating the peroxidase-like activity of ZIF-8 / Au in Example 1.

[0017] Figure 5 This study explores the feasibility of detecting glutathione in Example 1.

[0018] Figure 6 The graphs show the selectivity and anti-interference capabilities of ZIF-8 / Au for GSH detection in Example 1.

[0019] Figure 7 These are photographs of ZIF-8 / Au against different substances in Example 1.

[0020] Figure 8 This is a linear range diagram from Example 1.

[0021] Figure 9 The graph shows the detection limit determination in Example 1. Detailed implementation method:

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] I. Preparation of ZIF-8 / Au peroxidase

[0025] 2-Methylimidazole (3.88 g) was dissolved in ultrapure water (16 mL) at room temperature. A Zn(NO3)2 aqueous solution (200 mg, 4 mL) was rapidly added with stirring. After stirring for 10 minutes, the nanocrystals were collected by centrifugation and washed three times, then dispersed in ultrapure water (20 mL). HAuCl4 (48 mM, 2.4 mL) was added with continuous stirring until homogeneous, followed by dropwise addition of freshly prepared NaBH4 solution (1 mL, 0.1 M) with continuous stirring. The solution color changed from bright yellow to wine red, and then was allowed to stand. The solution was centrifuged and washed three times to obtain ZIF-8 / Au.

[0026] The ZIF-8 / Au scanning electron microscope (SEM) and transmission electron microscope (TEM) are as follows: Figure 3 As shown in the figure, the prepared ZIF-8 surface uniformly loads AuNPs to form a nanosphere structure. The XRD pattern (...) Figure 2 In B), sharp peaks appeared at 16.39°, 18.05°, 22.14°, 24.42°, and 26.70°, corresponding to the (013), (222), (114), (233), and (134) crystal planes of ZIF-8, respectively. This is consistent with the standard XRD diffraction peak simulation of ZIF-8, thus confirming the crystallinity of ZIF-8. After loading AuNPs, the intensity of the characteristic diffraction peaks of ZIF-8 decreased, and the diffraction peaks appeared at 38.3°, 44.6°, 64.7°, and 77.5°, corresponding to the (111), (200), (220), and (311) crystal planes of gold, respectively. This proves the successful loading of AuNPs.

[0027] II. Application of ZIF-8 / Au peroxidase in the detection of glutathione (GSH)

[0028] 1. Peroxidase performance of ZIF-8 / Au

[0029] A 3 mL system was prepared by sequentially adding 100 μL of 2 mg / mL ZIF-8 / Au complex, 100 μL of 100 mmol / L H₂O₂ solution, and 100 μL of 20 mmol / L TMB solution to acetate buffer (pH 4.5, 0.2 mol / L). The reaction was carried out at room temperature for 20 min. The TMB group and the H₂O₂ + TMB group served as controls. The peroxidase activity results of the ZIF-8 / Au complex are shown below. Figure 4As shown, the TMB system alone did not exhibit a characteristic absorption peak at 652 nm, indicating that TMB did not undergo oxidation. In the absence of ZIF-8 / Au, the TMB+H2O2 system showed a weak absorption peak at 652 nm, indicating that TMB could be weakly oxidized by H2O2. The absorbance at 652 nm significantly increased in the presence of ZIF-8 / Au, indicating that more oxidation products (oxTMB) were generated in the reaction system. In summary, these results demonstrate that the ZIF-8 / Au complex can catalyze the oxidation of TMB by H2O2, exhibiting activity similar to that of natural peroxidases.

[0030] 2. Exploring the feasibility of detecting glutathione

[0031] A 3 mL system was prepared by sequentially adding 100 μL of 2 mg / mL ZIF-8 / Au complex, 100 μL of 100 mmol / L H2O2 solution, 100 μL of 20 mmol / L TMB solution, and 100 μL of 1800 μmol / L glutathione solution to acetate buffer (pH 4.5, 0.2 mol / L). The reaction was carried out at room temperature for 20 min. The H2O2+TMB and H2O2+TMB+ZIF-8 / Au groups served as controls. Figure 5 As shown, the absorption of ultraviolet light at 652 nm wavelength decreased after the addition of GSH, thus proving that GSH detection is feasible. This is because GSH reacts with oxTMB to generate TMB.

[0032] 3. Investigation into the specificity and anti-interference ability of ZIF-8 / Au against GSH

[0033] 100 μL of 2 mg / mL ZIF-8 / Au, 100 μL of 20 mmol / L LTMB, and 100 μL of 100 mmol / L H2O2 were mixed in 0.2 M NaAC-HAC buffer at pH 4.5. Different solutions were prepared by dissolving 100 μL of other interfering substances (including MgCl2, CaCl2, NaCl, threonine (Thr), proline (Pro), ascorbic acid (AA), cysteine ​​(Cys), tryptophan (Trp), pyroglutamic acid (Cbz), and phenylalanine (Phe)) in ultrapure water, forming 3 mL systems (interference resistance: 100 μL of 1800 μmol / L GSH was added to each reaction). The reaction was carried out at room temperature for 20 min, and the UV spectrum at 652 nm was measured. The results are as follows: Figure 6 As shown, only GSH significantly decreased the UV absorbance of the ZIF-8 / Au mixture at 652 nm. The addition of other substances had almost no effect on the UV absorbance of the ZIF-8 / Au mixture, indicating that ZIF-8 / Au has good selectivity for GSH and can achieve specific recognition and detection of GSH. Figure 7 It can be understood that the color of the reaction system remains unchanged when other interfering substances are added, but when GSH is added, the color of the system changes from dark blue to light blue, becoming lighter.

[0034] Furthermore, since this method detects GSH based on the principle of catalytic oxidation and reduction of the substrate, high levels of strong oxidants or strong reducing agents (such as hydrogen peroxide, ascorbic acid, etc.) in the test sample should be avoided to prevent interference with the detection signal.

[0035] 4. Investigation of linear range and detection limit

[0036] Different concentrations of GSH standard solutions (0–60 μM) were added to acetate buffer (pH 4.5, 0.2 mol / L), 100 μL of 2 mg / mL ZIF-8 / Au complex, 100 μL of 100 mmol / L H₂O₂ solution, and 100 μL of 20 mmol / L TMB solution, respectively, to form mL systems. After thorough mixing, the solutions were reacted at room temperature for 20 min. The absorption spectra of the reaction solutions were measured using a UV-Vis absorption spectrometer, and the absorbance value at a wavelength of 652 nm was recorded. Figure 8 As shown, with increasing GSH concentration, the absorbance of the solution at 652 nm gradually decreased. The absorbance change (ΔA = A0 - A) showed a good linear relationship with the GSH concentration in the range of 0.5–60 μM, and the linear equation was ΔA = 0.16712 + 0.00951c(R). 2 =0.9764), the detection limit is 0.28 μM ( ). Figure 9 ).

[0037] Example 2

[0038] Preparation of ZIF-8 / Au peroxidase

[0039] 2-Methylimidazole (3.88 g) was dissolved in ultrapure water (16 mL) at room temperature. A Zn(NO3)2 aqueous solution (200 mg, 4 mL) was rapidly added with stirring. After stirring for 10 minutes, the nanocrystals were collected by centrifugation and washed three times, then dispersed in ultrapure water (5 mL). HAuCl4 (48 mM, 2.4 mL) was added with continuous stirring until homogeneous, followed by dropwise addition of freshly prepared NaBH4 solution (160 μL, 0.1 M) with continuous stirring. The solution color changed from bright yellow to wine red, and then was allowed to stand. The solution was centrifuged and washed three times to obtain ZIF-8 / Au.

[0040] Compared to Figure 3 Peak characteristics in A Figure 3 The sharper B-shape and clearer point of the tip indicate that the ZIF-8 / Au prepared in Example 1 has better crystallinity.

Claims

1. A method for detecting glutathione using ZIF-8 / Au peroxidase, characterized in that, The test is for glutathione levels that are not intended for therapeutic purposes. The ZIF-8 / Au peroxidase was prepared as follows: 3.88 g of 2-methylimidazole was dissolved in 16 mL of ultrapure water at room temperature. 200 mg of Zn(NO3)2 aqueous solution was rapidly added with stirring. After the reaction was complete, the nanocrystals were collected by centrifugation and washed. Then, they were dispersed in 20 mL of ultrapure water. 2.4 mL of 48 mM HAuCl4 was added with continuous stirring until the mixture was homogeneous. Then, 1 mL of freshly prepared 0.1 M NaBH4 solution was added dropwise with continuous stirring. After the solution color changed from bright yellow to wine red, it was allowed to stand, centrifuged, and washed to obtain ZIF-8 / Au.

2. The method for detecting glutathione using ZIF-8 / Au peroxidase according to claim 1, characterized in that, ZIF-8 / Au, 3,3',5,5'-tetramethylbenzidine, H2O2, and NaA were added to the test solution containing glutathione. C -HA C The reaction was carried out in a buffer solution at room temperature. After the reaction was completed, the absorbance at 652 nm was measured, and the concentration of GSH was calculated based on the absorbance and the constructed linear equation.

3. The method for detecting glutathione using ZIF-8 / Au peroxidase according to claim 2, characterized in that, React at room temperature for 20 minutes.

4. The method for detecting glutathione using ZIF-8 / Au peroxidase according to claim 3, characterized in that, The ZIF-8 / Au peroxidase was used for the detection of glutathione, with a detection limit of 0.28 μM.

Images