Gold nanoclusters and application thereof in detection of organophosphorus pesticide residues

Abstract

The application discloses gold nanoclusters and application thereof in detection of organophosphorus pesticide residues, wherein the gold nanoclusters comprise a gold core center structure, a first layer of ligand structure formed by 6-azido-2-thiothymine combined on the surface of the gold core through a gold-sulfur bond, a second layer of ligand structure formed by L-arginine combined with the 6-azido-2-thiothymine through a hydrogen bond, and a third layer of ligand structure formed by tri-methylphenyl ammonium iodide combined with the L-arginine through electrostatic force and by tri-methylphenyl ammonium iodide molecules combined through a pi-pi interaction force. The gold nanoclusters have high sensitivity to mercapto compounds and pH, a detection limit of chlorpyrifos of 0.02 micrograms per liter is achieved by using the detection method, the detection method has high sensitivity, and the detection method is simple, economical and rapid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pesticide residue detection, and particularly relates to a gold nanocluster (AuNCs) and a preparation method thereof, and application of the gold nanocluster as a fluorescent tracer combined with acetylcholinesterase in detection of organophosphorus pesticide residues. BACKGROUND

[0002] As a kind of organic compounds mainly containing phosphorus elements (a few containing sulfur and nitrogen elements), organophosphorus pesticides are mostly in liquid form and are easily dissolved in organic solvents. This kind of pesticide has shown significant pest control effect in the field of agriculture, effectively improving the yield of crops, and therefore has been widely used. However, its toxicity to non-target organisms cannot be ignored. As a neurotoxin, organophosphorus pesticides can accumulate in the environment through the ecological cycle system, such as soil and water, and thus intensify their toxic effects.

[0003] Studies have shown that organophosphorus pesticides can not only cause reproductive health problems such as infertility, miscarriage, and premature birth, but also increase the risk of uterine cancer. More seriously, this kind of pesticide can penetrate the blood-brain barrier and cause adverse effects on the nervous system development of newborns, leading to serious consequences such as cognitive impairment and motor impairment. Therefore, while ensuring agricultural production, we must strengthen the control of pesticide use and establish a sensitive, accurate, convenient and efficient method for detecting organophosphorus pesticides to ensure the life and health of the people and the safety of the environment.

[0004] Currently, the detection methods of organophosphorus pesticides mainly include two categories: chromatography and rapid detection method. Chromatography, including gas chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography, and liquid chromatography-tandem mass spectrometry, is the current authoritative pesticide detection method. However, these methods require expensive instruments, are time-consuming and laborious to operate, and have high professional technical requirements, making it difficult to popularize. In contrast, rapid detection methods such as enzyme inhibition method and immunoassay are more convenient and fast. Among them, the enzyme inhibition method utilizes the toxicological characteristics of organophosphorus pesticides and has become a widely used rapid detection method. However, the traditional enzyme inhibition method still needs to be improved in terms of sensitivity.

[0005] In recent years, fluorescence detection method has gradually become a hot research direction in the field of rapid detection of pesticide residues due to its low detection limit and high cost performance. Therefore, combining enzyme inhibition method with fluorescence detection method is expected to create a high-sensitivity organophosphorus pesticide detection method. This innovative method is expected to provide more accurate and reliable technical support for pesticide residue detection and contribute to the protection of people's life and health and environmental safety. SUMMARY

[0006] The application aims to provide an application of gold nanoclusters as a fluorescent tracer in detection of organophosphorus pesticide residues, the gold nanoclusters have a gold core center structure and a three-layer ligand structure, have high sensitivity to mercapto compounds and pH, have a low detection limit for chlorpyrifos, and are high in sensitivity and simple to operate.

[0007] To achieve the above-mentioned purpose, the application provides the following technical scheme:

[0008] In a first aspect, the application provides a gold nanocluster, wherein the gold nanocluster comprises:

[0009] a gold core center structure;

[0010] a first layer of ligand structure formed by 6-azido-2-thiothymine combined on the surface of the gold core through a gold-sulfur bond;

[0011] a second layer of ligand structure formed by L-arginine combined with 6-azido-2-thiothymine through a hydrogen bond;

[0012] a third layer of ligand structure formed by tri-methyl-phenyl ammonium iodide through electrostatic force and by intermolecular π-π force between tri-methyl-phenyl ammonium iodide molecules.

[0013] In a second aspect, the application provides a preparation method of a gold nanocluster, wherein the preparation method comprises:

[0014] Step one, 6-azido-2-thiothymine is dissolved in a NaOH solution, and then added to a HAuCl4 solution under stirring, after the color of the solution presents a light yellow color, dilute hydrochloric acid is added to adjust the pH to 9-10, and then the stirring is continued for at least 1 h, and then L1-AuNCs are obtained by filtration and purification;

[0015] Step two, under stirring, L-arginine solution is added to the L1-AuNCs solution prepared in step one, and then L2-AuNCs are prepared by continuing to stir under the condition of water bath heating at 40-45°C for at least 24 h.

[0016] Step three, tri-methyl-phenyl ammonium iodide is added to the L2-AuNCs solution prepared in step two, and then L3-AuNCs are obtained by stirring at room temperature and avoiding light for at least 1 h.

[0017] As a specific scheme in the technical scheme of the application, the stirring speed in step one is 500-700 rpm / min.

[0018] As a specific scheme in the technical scheme of the application, a 30 KDa ultrafiltration tube is used to purify the reaction product in step one to obtain L1-AuNCs.

[0019] As a specific scheme in the technical scheme of the present application, the L1-AuNCs solution prepared in step one is added with NaOH to adjust the pH to 11-12.

[0020] As a specific scheme in the technical scheme of the present application, the L1-AuNCs solution prepared in step one, the L2-AuNCs solution prepared in step two and the L3-AuNCs solution prepared in step three are stored at a temperature of 2-6℃.

[0021] As a specific scheme in the technical scheme of the present application, the L2-AuNCs prepared in step two is dispersed and dissolved in ultrapure water before adding tri-methyl-phenyl ammonium iodide.

[0022] In a third aspect, the present application provides an application of gold nanoclusters, wherein the gold nanoclusters are used as fluorescent tracers in detection of organophosphorus pesticide residues.

[0023] As a specific scheme in the technical scheme of the present application, the method for detecting organophosphorus pesticide residues by using the gold nanoclusters comprises the following steps:

[0024] (1) preparing a sample liquid to be detected;

[0025] (2) adding an acetylcholinesterase solution to the sample liquid and reacting for at least 30 min, then adding a thioacetylcholine solution and reacting for at least 30 min, and finally adding the L3-AuNCs solution prepared in step three and standing for 4-6 min, and then using a fluorescence spectrometer to measure the fluorescence intensity of the reaction liquid.

[0026] As a specific scheme in the technical scheme of the present application, the sample liquid to be detected in step (1) includes but is not limited to chlorpyrifos solution, vegetable and fruit sample extraction solution.

[0027] Compared with the prior art, the present application has the following beneficial effects: the L3-AuNCs provided by the present application comprises a gold core center structure and a three-layer ligand structure, the multi-layer ligand structure is in a strong constraint state and low-frequency acoustic vibration by the metal core, thereby suppressing the non-radiative decay of the L3-AuNCs, achieving a high absolute quantum yield of 28.34%, and the L3-AuNCs has high sensitivity to mercapto compounds and pH, and the detection method adopted has a detection limit of 0.02 μg / L for chlorpyrifos, high sensitivity, simple, economical and fast operation. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The gold core and ligand structure of the gold nanoclusters proposed in the embodiments of the present application are shown in the following figure:

[0029] Figure 2 The preparation route of the gold nanoclusters proposed in the embodiments of the present application is shown in the following figure:

[0030] Figure 3 This is a graph showing the fluorescence intensity of the gold nanoclusters as a function of pH, as proposed in the examples of the present application;

[0031] Figure 4 This is a graph showing the fluorescence intensity of the gold nanoclusters proposed in the examples of the present application as a function of thiocholine;

[0032] Figure 5 This is a transmission electron micrograph of the gold nanoclusters proposed in the examples of the present application and after incubation with thiocholine and acetic acid;

[0033] Figure 6 The gold nanoclusters-based fluorescence detection method for organophosphorus pesticides proposed in the embodiments of this application;

[0034] Figure 7 This is a fluorescence detection method for organophosphorus pesticides based on gold nanoclusters proposed in the examples of this application. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be further clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1: Structure of gold nanoclusters

[0037] like Figure 1 As shown, the structure of the gold nanoclusters (abbreviated as L3-AuNCs) provided in this embodiment includes:

[0038] The L3-AuNCs consist of a gold core at the center; a first layer of ligands formed by 6-aza-2-thiothymine bound to the gold core via gold-sulfur bonds; a second layer of ligands formed by L-arginine hydrogen-bonded to 6-aza-2-thiothymine; and a third layer of ligands formed by electrostatic interactions between tritolyl ammonium iodide and L-arginine, and π-π interactions between tritolyl ammonium iodide molecules. This multilayered ligand structure, driven by the strong confinement of the metal core and low-frequency acoustic vibrations, suppresses the nonradiative decay of L3-AuNCs, achieving a high absolute quantum yield of 28.34%.

[0039] Example 2: Preparation method of gold nanoclusters

[0040] The synthesis process of gold nanoclusters (abbreviated as L3-AuNCs) is as follows: Figure 2 As shown, the specific method includes the following steps:

[0041] ① 6-Aza-2-thiothymine was dissolved in 0.2 M NaOH to a concentration of 80 mM, 5 mL of the above 6-Aza-2-thiothymine solution was quickly added to 5 mL of 10 mg / mL HAuCl4 solution under stirring (600 rpm / min) at room temperature. After the thiolate reduced Au(III) ions to make the mixture appear light yellow, 0.1 M dilute hydrochloric acid was added to adjust the pH of the above reaction system to 9.5. During this process, the yellow color slightly deepened. After continuing to stir the reaction at room temperature for 1 h, the product was purified using a 30 KDa ultrafiltration tube to obtain L1-AuNCs. The purified solution was diluted to 10 mL with ultrapure water, and then adjusted to pH 11.2 with 0.2 M NaOH and stored at 4 °C for standby.

[0042] ② Under stirring, 1.2 mL of freshly prepared 40 mM L-arginine solution was added to 20 mL of 2-fold diluted L1-AuNCs, and the mixed solution was continued to be stirred under the condition of 40 °C water bath heating for 24 h. The obtained product was stored at 4 °C for 2 days to stabilize its physical and chemical properties, and L2-AuNCs were obtained.

[0043] ③ 1 mL of L2-AuNCs solution was dispersed in 8 mL of ultrapure water, and 5 mg of mesityl iodide was added thereto after stirring uniformly, and L3-AuNCs were obtained by stirring at room temperature for 1 h in the dark. It was stored at 4 °C for subsequent experiments.

[0044] Example 3: Trend of fluorescence intensity of L3-AuNCs with variation of thiocholine and pH

[0045] L3-AuNCs were added to different concentrations of thiocholine (solubility of 0-1.25 mM) and aqueous acetic acid (pH=5-13), and after mixing and standing for 5 min, the fluorescence intensity of L3-AuNCs was measured using a fluorescence spectrometer, with the excitation wavelength set to 360 nm and the emission wavelength measured to 530 nm. The variation trend of fluorescence intensity with thiocholine and acetic acid is shown in Figure 3 、 Figure 4 With the increase of thiocholine content in the thiocholine solution, the fluorescence intensity becomes weaker. In order to verify this result, the transmission electron microscopy image of L3-AuNCs coexisting with thiocholine is shown in Figure 5As shown, L3-AuNCs presented a particle size of about 1.5 nm, but the particle size was significantly increased to 50 nm after co-incubation with thiocyanate, which was much larger than the particle size of AuNCs (usually <2 nm). This was because there was +1 valence gold around the gold core, and the thiocyanate mercapto compound could reduce the monovalent gold ions near the gold core to generate zero-valent gold, further taking the AuNCs as the gold core to generate gold nanoparticles, while destroying the structure of L3-AuNCs, leading to fluorescence quenching and reduced fluorescence intensity. When the pH of the acetic acid aqueous solution was 11, the fluorescence intensity was the strongest, and when the pH was >11 or <11, the fluorescence intensity became weaker. Similarly, in order to verify this result, the transmission electron microscopy image of L3-AuNCs coexisting with acetic acid in this embodiment is as shown in Figure 5 As shown, when L3-AuNCs were co-incubated with acetic acid, the acetic acid would destroy the original intermolecular force of L3-AuNCs, and the AuNCs would aggregate, leading to fluorescence quenching.

[0046] Example 4: The operation steps of the fluorescent detection method of organophosphorus pesticides based on L3-AuNCs are as follows:

[0047] In this embodiment, chlorpyrifos solution was used as the detection liquid, and chlorpyrifos was purchased from the market and prepared into chlorpyrifos solutions with different concentrations. 20 μL of acetylcholinesterase solution (10 mU / mL) was reacted with 50 μL of chlorpyrifos solution with different concentrations for 30 min, then 20 μL of 0.25 mM thioacetylcholine solution was added and reacted for 30 min, finally, 10 μL of L3-AuNCs was added, and the fluorescence intensity was measured by a fluorescence spectrometer after 5 min.

[0048] Example 5: Fluorescent detection method based on L3-AuNCs for Chinese cabbage sample detection

[0049] In this embodiment, the prepared Chinese cabbage sample solution was used as the detection liquid, and the steps were as follows: the Chinese cabbage sample was homogenized using a wall-breaking food processor, 5 g of Chinese cabbage homogenate was accurately weighed into a 50 mL centrifuge tube, 10 mL of 50% methanol-PBS buffer was added to the sample, shaken for 5 min, ultrasonicated for 10 min, and centrifuged at 4000 rpm for 5 min. The supernatant 50 μL was used to replace the chlorpyrifos solution in Example 4, and the detection task was performed according to the steps in Example 4. The recovery rate experiment of the actual sample determined that this method had high accuracy.

[0050] Example 4 and Example 5 used gold nanoclusters (L3-AuNCs) as fluorescent tracers to detect the amount of organophosphorus pesticide residues in chlorpyrifos and Chinese cabbage, and the mechanism was as shown in Figure 6As shown, when there is no organophosphorus pesticide in the reaction system, acetylcholinesterase hydrolyzes thioacetylcholine to generate thiocholine and acetic acid, and the thiol compound in thiocholine reduces the monovalent gold ion near the gold core to generate zero-valent gold, while destroying the structure of L3-AuNCs, resulting in fluorescence quenching, and the fluorescence intensity of the reaction system is low; acetic acid destroys the weak interaction between ligands in the structure of L3-AuNCs, causing L3-AuNCs to aggregate, resulting in fluorescence quenching. When there is an organophosphorus pesticide in the reaction system, the activity of acetylcholinesterase is inhibited, and it cannot catalyze the hydrolysis of thioacetylcholine, and the fluorescence intensity of the reaction system is high.

[0051] Example 6: Sensitivity of the L3-AuNCs-based fluorescence detection method for organophosphorus pesticides

[0052] The 1000 mg / L chlorpyrifos standard solution was diluted with buffer 2 times to obtain a series of concentration gradient standard solutions of 2000-0.03 μg / L. The buffer was used as a negative control, and the corresponding relationship between the fluorescence intensity and the concentration of chlorpyrifos was as shown in Figure 7 As shown, according to the calculation of 3 times the signal-to-noise ratio, the minimum detection limit of chlorpyrifos was 0.02 μg / L.

[0053] It needs to be particularly pointed out that the L3-AuNCs prepared in the same batch were stored in a 4°C refrigerator, and under the conditions of acetylcholinesterase (abbreviated as: AChE) and acetylthiocholine (abbreviated as: ATCH) being prepared and used, the sensitivity of the L3-AuNCs test method was tested at intervals within 3 months, and the sensitivity did not produce significant differences, proving that the method has good stability.

[0054] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for detecting chlorpyrifos solution using gold nanoclusters as fluorescent tracers, characterized in that: The preparation method of the gold nanoclusters comprises: Step 1: Dissolve 6-aza-2-thiothymine in NaOH solution and add it to HAuCl4 solution under stirring. After the solution turns light yellow, add dilute hydrochloric acid to adjust the pH to 9-10. Continue stirring for at least 1 hour and then filter and purify to obtain L1-AuNCs. Step 2: Add L-arginine solution to the L1-AuNCs solution prepared in step 1 under stirring, and continue stirring in a water bath at 40°C to 45°C for at least 24 h to obtain L2-AuNCs; Step 3: Add tritolyl ammonium iodide to the L2-AuNCs solution prepared in step 2, and stir at room temperature in the dark for at least 1 h to obtain L3-AuNCs.

2. The use according to claim 1, characterized in that The stirring speed in step 1 is 500-700 rpm / min.

3. The use according to claim 1, characterized in that In step 1, the reaction product was purified using a 30 KDa ultrafiltration tube to obtain L1-AuNCs.

4. The use according to claim 1, characterized in that The L1-AuNCs solution prepared in step 1 was added with NaOH to adjust the pH to 11-12.

5. The use according to claim 1, characterized in that The storage temperature of the L1-AuNCs solution prepared in step 1, the L2-AuNCs solution prepared in step 2, and the L3-AuNCs solution prepared in step 3 is 2°C~6°C.

6. The use according to claim 1, characterized in that Before the L2-AuNCs solution reacts with tritolyl ammonium iodide in step 3, the L2-AuNCs solution is dispersed and dissolved in ultrapure water.

7. The use according to claim 1, characterized in that The method for detecting chlorpyrifos solution with gold nanoclusters comprises the following steps: (1) Prepare the sample solution to be tested; (2) After adding acetylcholinesterase solution to the sample solution and reacting for at least 30 min, add thioacetylcholine solution and react for at least 30 min. Finally, add the L3-AuNCs solution prepared in step 3 and let it stand for 4-6 min. Then use a fluorescence spectrometer to measure the fluorescence intensity of the reaction solution.

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