Gold nanocluster and application thereof in organophosphorus pesticide residue detection

Abstract

The invention discloses a gold nanocluster and application of the gold nanocluster in organophosphorus pesticide residue detection, and the gold nanocluster comprises a gold core center structure; the first layer of ligand structure is formed by combining 6-aza-2-thiothymine on the surface of the gold core through a gold-sulfur bond; l-arginine is combined with 6-aza-2-thiothymine through a hydrogen bond to form a second layer of ligand structure, and L-arginine is combined with 6-aza-2-thiothymine through a hydrogen bond; trimethylphenyl ammonium iodide is combined with L-arginine through electrostatic acting force, and trimethylphenyl ammonium iodide molecules form a third-layer ligand structure through pi-pi acting force. The gold nanocluster has high sensitivity to sulfhydryl compounds and pH, the detection limit of the adopted detection method to chlorpyrifos is 0.02 microgram / L, the sensitivity is high, and the detection method is easy to operate, economical and rapid.

Description

Technical Field

[0001] The present application belongs to the technical field of pesticide residue detection, and specifically relates to a gold nanoclusters (AuNCs for short) and a preparation method thereof, as well as an application of AuNCs as a fluorescent tracer in combination with acetylcholinesterase in the detection of organophosphorus pesticide residues. Background Art

[0002] Organophosphorus pesticides, as a class of organic compounds mainly containing phosphorus (a few contain sulfur and nitrogen), are mostly in liquid form and easily soluble in organic solvents. This type of pesticide has shown significant pest and disease control effects in the agricultural field, effectively increasing crop yields, and has therefore been widely used. However, its toxicity to non-target organisms cannot be ignored. As a neurotoxin, organophosphorus pesticides can accumulate in environmental media such as soil and water through the ecological circulation system, thereby exacerbating their toxic effects.

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

[0004] At present, the detection methods of organophosphorus pesticides are mainly divided into two categories: chromatography and rapid detection methods. Chromatography, including gas chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography and liquid chromatography-tandem mass spectrometry, is the current authoritative means of pesticide detection. However, these methods require expensive instruments, time-consuming and labor-intensive operations, and require high professional and technical requirements, making them difficult to popularize. In contrast, rapid detection methods, such as enzyme inhibition and immunoassay, are simpler and faster. Among them, the enzyme inhibition method utilizes the toxicological properties of organophosphorus pesticides and has become a widely used rapid detection method. However, the sensitivity of the traditional enzyme inhibition method still needs to be improved.

[0005] In recent years, fluorescence detection has gradually become a hot research direction in the field of rapid detection of pesticide residues due to its advantages such as low detection limit and high cost performance. Therefore, combining enzyme inhibition method with fluorescence detection method is expected to create a highly sensitive detection method for organophosphorus pesticides. This innovative method is expected to provide more accurate and reliable technical support for pesticide residue detection and make important contributions to protecting the lives, health and environmental safety of the people. Summary of the invention

[0006] The purpose of the present application is to provide a gold nanocluster for use as a fluorescent tracer in the detection of organophosphorus pesticide residues. The gold nanocluster has a gold core center structure and a three-layer ligand structure, is highly sensitive to thiol compounds and pH, has a low detection limit for chlorpyrifos, has high sensitivity and is simple to operate.

[0007] To achieve the above objectives, this application provides the following technical solutions: In a first aspect, the present application provides a gold nanocluster, wherein the gold nanocluster comprises: Gold core center structure; 6-aza-2-thiothymine is bound to the gold core surface through gold-sulfur bonds to form the first layer of ligand structure; L-arginine combines with 6-aza-2-thiothymine through hydrogen bonds to form the second layer of ligand structure; The third layer of ligand structure is formed by electrostatic force between tritolyl ammonium iodide and L-arginine and π-π force between tritolyl ammonium iodide molecules.

[0008] In a second aspect, the present application proposes a method for preparing gold nanoclusters, wherein the preparation method comprises: Step 1: Dissolve 6-aza-2-thiothymine in NaOH solution, add it to HAuCl4 solution under stirring, and when the solution turns light yellow, add dilute hydrochloric acid to adjust the pH to 9-10, continue stirring for at least 1 h, 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 conditions, 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.

[0009] As a specific solution in the technical solution of the present application, the stirring speed in step 1 is 500 ~ 700 rpm / min.

[0010] As a specific solution in the technical solution of the present application, in step 1, a 30 KDa ultrafiltration tube is used to purify the reaction product to obtain L1-AuNCs.

[0011] As a specific solution in the technical solution of the present application, NaOH is added to the L1-AuNCs solution prepared in step 1 to adjust the pH to 11-12.

[0012] As a specific solution in the technical solution of the present application, the storage temperature of 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 is 2°C~6°C.

[0013] As a specific solution in the technical solution of the present application, before adding tritolyl ammonium iodide to the L2-AuNCs prepared in step 2, the L2-AuNCs solution is dispersed and dissolved in ultrapure water.

[0014] In a third aspect, the present application proposes an application of gold nanoclusters, wherein the gold nanoclusters are used as fluorescent tracers in the detection of organophosphorus pesticide residues.

[0015] As a specific solution in the technical solution of the present application, the method for detecting organophosphorus pesticide residues using 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, then adding thioacetylcholine solution and reacting for at least 30 min, finally adding the L3-AuNCs solution prepared in step 3 and letting it stand for 4-6 min, the fluorescence intensity of the reaction solution was measured using a fluorescence spectrometer.

[0016] As a specific solution of the technical solution of the present application, the sample solution to be tested in step (1) includes but is not limited to chlorpyrifos solution, vegetable and fruit sample extraction solution.

[0017] Compared with the prior art, the beneficial effects of the present application are as follows: the L3-AuNCs provided in the present application include a gold core center structure and a three-layer ligand structure. The multi-layer ligand structure is subject to the strong constraint state and low-frequency acoustic vibration of the metal core, thereby suppressing the non-radiative attenuation of L3-AuNCs and achieving a high absolute quantum yield of 28.34%. Moreover, L3-AuNCs are highly sensitive to thiol compounds and pH. The detection method used has a detection limit of 0.02 μg / L for chlorpyrifos, has high sensitivity, and is simple to operate, economical and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the gold core and ligand structure of the gold nanoclusters proposed in the examples of the present application; Figure 2 The preparation route of the gold nanoclusters proposed in the examples of this application; Figure 3 This is a graph showing the fluorescence intensity of the gold nanoclusters proposed in the examples of the present application as a function of pH; Figure 4 This is a curve diagram showing the fluorescence intensity of the gold nanoclusters proposed in the examples of the present application as a function of thiocholine; Figure 5 This is a transmission electron micrograph of the gold nanoclusters proposed in the examples of the present application and after being incubated with thiocholine and acetic acid; Figure 6 The gold nanoclusters-based organophosphorus pesticide fluorescence detection method proposed in the embodiments of the present application; Figure 7 This is a fluorescence detection method for organophosphorus pesticides based on gold nanoclusters proposed in the embodiments of the present application. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be further clearly and completely described below. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1: Structure of gold nanoclusters like Figure 1 As shown, the structure of the gold nanoclusters (abbreviated as L3-AuNCs) provided in this embodiment includes: The gold core is located in the center; the first layer of ligand structure is formed by 6-aza-2-thiothymine binding to the gold core surface through gold-sulfur bonds; the second layer of ligand structure is formed by L-arginine binding to 6-aza-2-thiothymine through hydrogen bonds; the third layer of ligand structure is formed by tritolyl ammonium iodide binding to L-arginine through electrostatic forces and tritolyl ammonium iodide molecules through π-π forces. The multi-layer ligand structure is strongly constrained by the metal core and low-frequency acoustic vibration, thereby suppressing the non-radiative decay of L3-AuNCs and achieving a high absolute quantum yield of 28.34%.

[0021] Example 2: Preparation method of gold nanoclusters The synthesis process of gold nanoclusters (abbreviated as: L3-AuNCs) is as follows Figure 2 As shown, the specific method includes the following steps: ① Dissolve 6-aza-2-thiothymine in 0.2 M NaOH to a concentration of 80 mM, take 5 mL of the above 6-aza-2-thiothymine solution and quickly add it to 5 mL of 10 mg / mL HAuCl4 solution under stirring at room temperature (600 rpm / min). After the mixture turns light yellow due to the reduction of Au(III) ions by thiolate, add 0.1 M dilute hydrochloric acid to adjust the pH of the above reaction system to 9.5. During this process, the yellow color deepens slightly. After stirring the reaction at room temperature for 1 h, the obtained product is purified using a 30 KDa ultrafiltration tube to obtain L1-AuNCs. The purified solution is diluted to 10 mL with ultrapure water, and then the pH is adjusted to 11.2 with 0.2 M NaOH and stored at 4 °C for future use.

[0022] ② Under stirring conditions, 1.2 mL of freshly prepared 40 mM L-arginine solution was added to 20 mL of 2-fold diluted L1-AuNCs. The mixed solution was stirred and reacted for 24 h in a 40 °C water bath. The obtained product was stored at 4 °C for 2 days to stabilize its physicochemical properties, thereby obtaining L2-AuNCs.

[0023] ③ Disperse 1 mL of L2-AuNCs solution in 8 mL of ultrapure water, stir evenly, add 5 mg of tritolyl ammonium iodide, and stir at room temperature in the dark for 1 h to obtain L3-AuNCs. Store it at 4 °C for subsequent experiments.

[0024] Example 3: Fluorescence intensity of L3-AuNCs changes with thiocholine and pH L3-AuNCs were added to different concentrations of thiocholine (solubility 0-1.25mM) and acetic acid aqueous solution (pH=5-13), mixed and allowed to stand for 5 min, and the fluorescence intensity of L3-AuNCs was measured using a fluorescence spectrometer. The excitation wavelength was set at 360 nm and the emission wavelength was measured at 530 nm. The change trend of fluorescence intensity corresponding to thiocholine and acetic acid is shown in the figure. Figure 3 , Figure 4 As shown. As the content of thiocholine in the thiocholine solution increases, the fluorescence intensity weakens. In order to verify this result, the transmission electron microscopy images of L3-AuNCs and thiocholine coexist in this example are shown in Figure 5As shown, L3-AuNCs present a particle size of about 1.5 nm, but after incubation with thiocholine, the particle size increases significantly to 50 nm, which is much larger than the particle size of AuNCs (usually <2 nm). This is due to the presence of +1 valent gold around the gold core, and the thiocholine thiol compound can reduce the monovalent gold ions near the gold core to generate zero-valent gold, and further generate nano-gold particles with AuNCs as the gold core, while destroying the structure of L3-AuNCs, resulting in fluorescence quenching and reduced fluorescence intensity. When the pH of the acetic acid aqueous solution is 11, the fluorescence intensity is the strongest, and when pH>11 or pH<11, the fluorescence intensity becomes weaker. Similarly, in order to verify this result, the transmission electron microscopy images of L3-AuNCs coexisting with acetic acid in this example are shown below Figure 5 As shown in the figure, when L3-AuNCs are incubated with acetic acid, acetic acid destroys the original intermolecular forces of L3-AuNCs, and AuNCs aggregate, resulting in fluorescence quenching.

[0025] Example 4: The operating steps of the fluorescence detection method of organophosphorus pesticides based on L3-AuNCs are as follows: In this example, chlorpyrifos solution was used as the detection solution. Chlorpyrifos was purchased from the market and prepared into chlorpyrifos solutions of different concentrations. 20 μL of acetylcholinesterase solution (10 mU / mL) was reacted with 50 μL of chlorpyrifos solutions of different concentrations for 30 min, and then 20 μL of 0.25 mM thioacetylcholine solution was added for 30 min. Finally, 10 μL of L3-AuNCs was added, and the fluorescence intensity was measured by fluorescence spectrometer after 5 min.

[0026] Example 5: Fluorescence detection method based on L3-AuNCs for detection of pakchoi samples In this embodiment, the prepared pakchoi sample solution is used as the detection solution, and the steps are as follows: the pakchoi sample is homogenized using a wall-breaking food processor, 5 g of pakchoi homogenate is accurately weighed into a 50 mL centrifuge tube, 10 mL of 50% methanol-PBS buffer is added to the added sample, shaken for 5 min, ultrasonicated for 10 min, and centrifuged at 4000 rpm for 5 min. 50 μL of the supernatant is taken to replace the chlorpyrifos solution in Example 4, and the detection task is performed according to the steps in Example 4. The method is highly accurate through the recovery rate experiment of the actual sample.

[0027] In Example 4 and Example 5, gold nanoclusters (L3-AuNCs) were used as fluorescent tracers to detect the residual amounts of organophosphorus pesticides in chlorpyrifos and Chinese cabbage. The mechanism is as follows: Figure 6As shown in the figure, when there is no organophosphorus pesticide in the reaction system, acetylcholinesterase hydrolyzes thioacetylcholine to generate thiocholine and acetic acid. The thiol compound in thiocholine reduces the monovalent gold ions near the gold core to generate zero-valent gold, and at the same time destroys the structure of L3-AuNCs, resulting in fluorescence quenching and low fluorescence intensity of the reaction system; acetic acid destroys the weak interaction between ligands in the structure of L3-AuNCs, causing L3-AuNCs to agglomerate, 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.

[0028] Example 6: Sensitivity of the fluorescence detection method for organophosphorus pesticides based on L3-AuNCs The 1000 mg / L chlorpyrifos standard solution was diluted with buffer solution twice in concentration gradient to form a series of standard solutions with concentration gradients ranging from 2000 to 0.03 µg / L. The buffer solution was used as a negative control. The corresponding relationship between the fluorescence intensity and the chlorpyrifos concentration is shown in the following figure: Figure 7 As shown in the figure, the minimum detection limit of chlorpyrifos was 0.02 µg / L based on the 3-fold signal-to-noise ratio.

[0029] It should be specially noted in the present application that L3-AuNCs prepared from the same batch were stored in a 4°C refrigerator. Under the condition that acetylcholinesterase (AChE) and acetylthiocholine (ATCH) were prepared and used immediately, the sensitivity of the L3-AuNCs test method was tested at intervals within 3 months. There was no significant difference in the sensitivity, which proved that the method had good stability.

[0030] Although the embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A gold nanocluster, characterized in that: The gold nanoclusters include: Gold core center structure; 6-aza-2-thiothymine is bound to the gold core surface through gold-sulfur bonds to form the first layer of ligand structure; L-arginine combines with 6-aza-2-thiothymine through hydrogen bonds to form the second layer of ligand structure; Tritolyl ammonium iodide binds to L-arginine through electrostatic force, and the third layer of ligand structure is formed by π-π force between tritolyl ammonium iodide molecules.

2. A method for preparing gold nanoclusters as claimed in claim 1, characterized in that: The preparation method comprises: Step 1: Dissolve 6-aza-2-thiothymine in NaOH solution, add it to HAuCl4 solution under stirring, and when the solution turns light yellow, add dilute hydrochloric acid to adjust the pH to 9-10, continue stirring for at least 1 h, 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 conditions, 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.

3. The preparation method according to claim 2, characterized in that: In step 1, the stirring speed is 500-700 rpm / min.

4. The preparation method according to claim 2, characterized in that: Step 1: Purify the reaction product using a 30 KDa ultrafiltration tube to obtain L1-AuNCs.

5. The preparation method according to claim 2, characterized in that: NaOH was added to the L1-AuNCs solution prepared in step 1 to adjust the pH to 11-12.

6. The preparation method according to claim 2, 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.

7. The preparation method according to claim 2, 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.

8. A use of the gold nanoclusters as claimed in claim 1, characterized in that: The gold nanoclusters are used as fluorescent tracers in detecting organophosphorus pesticide residues.

9. The use according to claim 8, characterized in that: The method for detecting organophosphorus pesticide residues using 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.

10. The use according to claim 9, characterized in that: The sample solution to be tested in step (1) includes but is not limited to chlorpyrifos solution and vegetable and fruit sample extraction solution.

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