Application method of 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster electrochemiluminescence probe

Abstract

The invention discloses a use method of a 2-sulfydryl-1, 3, 4-thiadiazole-gold nanocluster electrogenerated chemiluminescence probe, a 2-sulfydryl-1, 3, 4-thiadiazole ligand is used as a reducing agent or a protective agent to obtain a multifunctional gold nanocluster, and during detection, N, N-diisopropylethanolamine is used as a co-reaction agent. The invention has the advantages of strong light-emitting signal, high quantum yield, low light-emitting potential and the like, thereby having favorable application prospects in the fields of biosensing and the like.

Description

Technical Field

[0001] This invention belongs to the field of nanotechnology, and in particular relates to a method for using a 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe. Background Technology

[0002] Electrochemiluminescence (ECL) refers to the electrochemical reaction that occurs when a specific potential is applied to an electrode surface, causing chemical substances near the electrode to generate high-energy intermediates or free radicals. Due to its advantages such as no need for external light excitation, low background noise, wide dynamic range, and strong controllability, it has shown irreplaceable application value in fields such as biosensing, clinical diagnosis, environmental monitoring, and food safety. Electrochemiluminescent materials are the core functional components of electrochemiluminescence systems, and their role spans the entire process of electron transfer, energy transfer, and luminescence. In recent years, electrochemiluminescent materials with excellent biocompatibility and high sensitivity have sparked a research boom in the fields of in vitro diagnostics and biosensing.

[0003] Gold nanoclusters (AuNCs), as ultrasmall metallic nanomaterials (<3 nm) with sizes between atoms and nanoparticles, have become a cutting-edge research hotspot in electrochemiluminescence materials due to their discrete electronic energy levels, tunable luminescence properties, excellent biocompatibility, and high surface activity, showing great potential in catalysis, optics, and biomedicine. However, as electrochemiluminescence probes, gold nanoclusters are still limited by their high excitation potential, single function, and co-reactant dependence. Therefore, researching and preparing low-potential gold nanocluster probes without co-reactants can significantly expand the application of electrochemiluminescence sensors in materials science and biomedicine.

[0004] The applicant previously filed a Chinese patent application (application number 202510678448.7) for a gold nanocluster electrochemiluminescence probe and its preparation method. This probe utilizes 2-mercapto-1,3,4-thiadiazole ligands as reducing or protecting agents to obtain multifunctional gold nanoclusters. A glassy carbon electrode is used as the working electrode. The 2-mercapto-1,3,4-thiadiazole ligands are added to an electrolyte containing phosphate buffer; or the 2-mercapto-1,3,4-thiadiazole ligands are modified onto a glassy carbon electrode and used as the working electrode, with phosphate buffer as a co-reactant. This probe can generate electrochemiluminescence signals without other co-reactants, exhibiting low luminescence potential, high quantum yield, and good biocompatibility.

[0005] Through further research, the applicant discovered that using a co-reactant when employing this type of gold nanocluster electrochemiluminescence probe for detection can yield unexpected technical effects and greatly improve detection efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a method for using a 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe.

[0007] The technical solution adopted in this invention is as follows: a method for using a 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescence probe, wherein a multifunctional gold nanocluster is obtained by using 2-mercapto-1,3,4-thiadiazole ligand as a reducing agent or a protecting agent, characterized in that: a quaternary ammonium salt is used as a co-reactant during detection.

[0008] The quaternary ammonium salt is N,N-diisopropylethanolamine.

[0009] Furthermore, the method of using the 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe involves modifying a glassy carbon electrode with a 2-mercapto-1,3,4-thiadiazole ligand and using it as the working electrode, with a phosphate buffer containing N,N-diisopropylethanolamine as the co-reactant.

[0010] The 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe of this invention is prepared by the following steps:

[0011] 1) The synthesis steps of 2-mercapto-1,3,4-thiadiazole gold nanoclusters are as follows: Sodium hydroxide and chloroauric acid solution are added to 2-mercapto-1,3,4-thiadiazole solution, mixed well, and placed in a constant temperature water bath at 25~70℃ for constant temperature reaction; after the reaction is completed, the solution is purified by dialysis to obtain an aqueous solution of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters, which is stored in a refrigerator at 4℃ in the dark.

[0012] 2) Polish the glassy carbon electrode with Al2O3 powder until it is smooth and mirror-like, then put it into HNO3 solution (1:1), anhydrous ethanol, and deionized water in sequence for ultrasonic cleaning for 3 minutes, and blow it dry with nitrogen; take the aqueous solution of 2-mercapto-1,3,4-thiadiazole gold nanoclusters obtained in step 1) and drop it onto the surface of the treated glassy carbon electrode, and dry it at room temperature to obtain the gold nanocluster modified glassy carbon electrode.

[0013] Further, in step 1) above, the concentration of sodium hydroxide is 0.1~0.8 mol / L; the concentration of chloroauric acid solution is 0.005~0.07 mol / L; the concentration of 2-mercapto-1,3,4-thiadiazole solution is 0.001~0.70 mol / L; and the water bath constant temperature reaction is less than 4.0 hours.

[0014] The electrochemiluminescence probe of this invention is a solid-phase probe, in which 2-mercapto-1,3,4-thiadiazole gold nanoclusters (hereinafter referred to as gold clusters) are modified on an electrode. The method for generating and detecting the electrochemiluminescence signal is as follows:

[0015] A three-electrode system was used for testing. A glassy carbon electrode modified with a gold nanocluster probe was used as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. The buffer solution was phosphate buffer, and KCl was used as the electrolyte. The electrodes were inserted into a buffer solution containing the co-reactant N,N-diisopropylethanolamine. A specific scanning voltage was applied, and the photomultiplier tube voltage was set to 450 V–650 V. Electrochemiluminescence radiation was generated on the surface of the working electrode. The relative electrochemiluminescence efficiency was measured to be 259.6%.

[0016] Specifically, the present invention adopts the following technical solution:

[0017] (I) Preparation of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters

[0018] The synthesis steps of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters are as follows: Sodium hydroxide solution (0.1–0.8 mol / L) and chloroauric acid solution (0.005–0.07 mol / L) were added to a 2-mercapto-1,3,4-thiadiazole solution (0.001–0.70 mol / L). After mixing, the solution was placed in a constant temperature water bath at 25–70 °C for 0–4.0 hours. After the reaction was completed, the solution was purified by dialysis to obtain an aqueous solution of 2-mercapto-1,3,4-thiadiazole gold nanoclusters, which was then stored at 4 °C in the dark.

[0019] (II) Preparation of electrodes modified with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters

[0020] The glassy carbon electrode was polished to a smooth mirror surface using Al₂O₃ powders of 1.0 μm, 0.3 μm, and 0.05 μm thicknesses, respectively. It was then sequentially immersed in a 1:1 solution of HNO₃, anhydrous ethanol, and deionized water, followed by ultrasonic cleaning for 3 minutes, and dried under nitrogen. 4 μL of an aqueous solution of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters was added dropwise to the treated glassy carbon electrode surface and allowed to dry at room temperature to obtain the gold nanocluster-modified glassy carbon electrode.

[0021] (III) Generation and detection of electrochemiluminescence signals from 2-mercapto-1,3,4-thiadiazole-gold nanocluster probes

[0022] A three-electrode system was used for testing. A glassy carbon electrode modified with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters or 2-mercapto-1,3,4-thiadiazole-gold nanoclusters probes was used as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. These electrodes were immersed in a buffer solution containing a co-reactant. Cyclic voltammetry was used, with an initial potential of 0 V and a termination potential of 0.8 V. The photomultiplier tube voltage was set to 450 V–650 V, and the electrochemiluminescence signal generated on the surface of the working electrode was detected.

[0023] The advantages of this invention are:

[0024] (1) This invention uses 2-mercapto-1,3,4-thiadiazole-gold nanoclusters as luminescent materials and takes advantage of their good optical and electrical properties. Quaternary ammonium salts are used as co-reactants to prepare high-performance electrochemiluminescent probes. The preparation method is simple and easy to operate and has good reproducibility.

[0025] (2) The anodic electrochemiluminescence system based on 2-mercapto-1,3,4-thiadiazole-gold nanoclusters prepared in this invention has the advantages of strong luminescence signal, high quantum yield and low luminescence potential, and therefore has good application prospects in the fields of biosensing. Attached Figure Description

[0026] Figure 1 The UV-Vis absorption spectrum of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters is shown.

[0027] Figure 2 The fluorescence spectrum of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0028] Figure 3 Electrochemiluminescence-time curves of glassy carbon electrodes modified with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters.

[0029] Figure 4 Electrochemiluminescence-potential curves of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0031] Example 1

[0032] Add 0.6 mL of 0.3 mol / L sodium hydroxide and 2 mL of 0.05 mol / L chloroauric acid solution to 2 mL of 0.06 mol / L 2-mercapto-1,3,4-thiadiazole solution, mix well, and stir at 30 ℃ for 1.5 hours. After the reaction, the solution is purified by dialysis using a dialysis bag with a molecular weight cutoff of 3500 to obtain an aqueous solution of 2-mercapto-1,3,4-thiadiazole gold nanoclusters. Detect an appropriate amount of the 2-mercapto-1,3,4-thiadiazole-protected gold nanocluster solution using a UV-Vis spectrophotometer. The results show that the absorption peak of the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters is at 300 nm (see...). Figure 1 ).

[0033] Example 2

[0034] Add 0.6 mL of 0.3 mol / L sodium hydroxide and 2 mL of 0.05 mol / L chloroauric acid solution to 2 mL of 0.06 mol / L 2-mercapto-1,3,4-thiadiazole solution, mix well, and stir at 30℃ for 1.5 hours. After the reaction, the solution is purified by dialysis using a dialysis bag with a molecular weight cutoff of 3500 to obtain an aqueous solution of 2-mercapto-1,3,4-thiadiazole gold nanoclusters. Detect an appropriate amount of the 2-mercapto-1,3,4-thiadiazole-protected gold nanocluster solution using a fluorescence spectrometer. The results show that the excitation peak of the 2-mercapto-1,3,4-thiadiazole-gold nanoclusters is at 335 nm, and the fluorescence emission peak is at 550 nm (see [link to relevant documentation]). Figure 2 ).

[0035] Example 3

[0036] Add 0.6 mL of 0.3 mol / L sodium hydroxide and 2 mL of 0.05 mol / L chloroauric acid solution to 2 mL of 0.06 mol / L 2-mercapto-1,3,4-thiadiazole solution, mix well, and stir at 30℃ for 1.5 hours. After the reaction, the reaction solution is purified by dialysis using a dialysis bag with a molecular weight cutoff of 3500 to obtain an aqueous solution of 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. Polish and grind a 3 mm diameter glassy carbon electrode sequentially with 1.0 μm, 0.3 μm, and 0.05 μm Al2O3 powders until a smooth mirror surface is achieved. Then, ultrasonically clean the electrode for 3 minutes in HNO3 solution (concentrated nitric acid to water volume ratio of 1:1), anhydrous ethanol, and deionized water, and dry it with nitrogen gas. 4 μL of a 2-mercapto-1,3,4-thiadiazole-gold nanocluster solution was dropped onto the surface of a pre-treated glassy carbon electrode and dried at room temperature to obtain a 2-mercapto-1,3,4-thiadiazole-gold nanocluster modified glassy carbon electrode. This electrode was then inserted into a 0.1 mol / L pH 11.5 phosphate buffer solution containing 0.001 mol / L N,N-diisopropylethanolamine and 0.1 mol / L KCl. Cyclic voltammetry was used with an initial potential of 0 V, a termination potential of 0.8 V, and a scan rate of 0.1 V / s. The photomultiplier tube voltage was set to 600 V, and the electrochemiluminescence signal generated on the working electrode surface was detected, yielding a good electrochemiluminescence signal. The relationship curves between the light signal and time and between the light signal and potential are shown in [reference needed]. Figure 3 , Figure 4 .

[0037] Example 4

[0038] A glassy carbon electrode was modified with 2-mercapto-1,3,4-thiadiazole-gold nanoclusters. The prepared gold nanocluster probe-modified glassy carbon electrode was then inserted into a 0.1 mol / L phosphate buffer solution (pH 11.5) containing 0.001 mol / L N,N-diisopropylethanolamine and 0.1 mol / L KCl. Cyclic voltammetry was used, with a linear scan voltage ranging from 0 V to 1.0 V at a scan rate of 0.2 V / s. The photomultiplier tube voltage was set to 550 V. The electrochemiluminescence signal (I) generated on the working electrode surface was detected, and the corresponding charge was Q. f Additionally, a 3 mm diameter glassy carbon electrode was polished and ground sequentially with 1.0 μm, 0.3 μm, and 0.05 μm Al₂O₃ powders until a smooth mirror surface was achieved. It was then sequentially immersed in HNO₃ solution (1:1), anhydrous ethanol, and deionized water for ultrasonic cleaning for 3 minutes, and dried with N₂. A three-electrode system was used, with the bare glassy carbon electrode as the working electrode, a platinum wire electrode as the counter electrode, and Ag / AgCl as the reference electrode. The bare glassy carbon electrode was inserted into a solution containing 1.0 mmol / L ruthenium bipyridine [Ru(bpy)₃].2+ In an acetonitrile solution containing 0.1 mol / L tetrabutylperchlorate, a scanning voltage of -1.0 to -1.8 V was applied at a scanning rate of 0.2 V / s. The photomultiplier tube voltage was set to 550 V. The electrochemiluminescence signal (I°) generated on the surface of the working electrode was detected, and the corresponding charge was Q°. f From formula Φ ECL =Φ° ECL (IQ° f / I°Q f The electrochemiluminescence efficiency Φ of the obtained 2-mercapto-1,3,4-thiadiazole-gold nanocluster probe was calculated. ECL It is 259.6%.

[0039] Example 5

[0040] The enhancing effect of the co-reactant N,N-diisopropylethanolamine on different probes.

[0041] Ruthenium bipyridine Ru(bpy)3 2+ The solution was dissolved in phosphate-buffered saline (PBS) at pH 11.5, with a final homogeneous concentration of 1 μM. The ECL signal measured in pure PBS was 46 au. After adding 1 mM N,N-diisopropylethanolamine (DIPEA-OH) as a co-reactant and adjusting the pH of the system to 11.5, the measured ECL signal was 604 au. Therefore, DIPEA-OH enhances Ru(bpy)3 2+ The ECL signal is 14 times stronger.

[0042] The gold nanocluster probe of this invention was used to modify a glassy carbon electrode (MTD-AuNCs) with 4 μL of the sample, which was then detected in PBS at pH 11.5 and in 1 mM DIPEA-OH at pH 11.5. The ECL signal measured in pure PBS was 159 au. After adding 1 mM DIPEA-OH as a co-reactant and adjusting the pH of the system to 11.5, the measured ECL signal was 12798 au. Therefore, DIPEA-OH enhanced the ECL signal of MTD-AuNCs by 80 times.

[0043] As can be seen from the above comparison, although the co-reactant N,N-diisopropylethanolamine is existing technology, its signal enhancement efficiency for known probe detection is 14 times; while when used in the probe of this invention, the signal enhancement efficiency reaches 80 times, far exceeding the known effects and achieving unexpected technical results.

[0044] The above description is merely a typical embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

A method for using a 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe, comprising a multifunctional gold nanocluster obtained by using 2-mercapto-1,3,4-thiadiazole ligand as a reducing agent or a protecting agent, characterized in that: Quaternary ammonium salts were used as co-reactants during the detection.

2. The method of using the 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe as described in claim 1, characterized in that: The quaternary ammonium salt is N,N-diisopropylethanolamine.

3. The method of using the 2-mercapto-1,3,4-thiadiazole-gold nanocluster electrochemiluminescent probe as described in claim 1, characterized in that: 2-Mercapto-1,3,4-thiadiazole ligands were modified onto a glassy carbon electrode and used as the working electrode, with phosphate buffer containing N,N-diisopropylethanolamine as the co-reactant.

Images