A gold nanocluster with polypeptide as ligand and detection method of methyl parathion

Abstract

The application provides a kind of gold nanoclusters with polypeptide as ligand and detection method of methyl parathion, the sequence of polypeptide in the gold nanoclusters with polypeptide as ligand is H (S, D) GGFFYCC;The gold nanoclusters with polypeptide as ligand can be used for double signal detection of methyl parathion and detection of methyl parathion in fruits and vegetables.Fluorescence signal detection: gold nanoclusters are added to the test solution, 37 DEG C reaction 30-60 min, detect the fluorescence change at 700 nm.Colorimetric signal: in pH=8.0 buffer solution, different concentrations of methyl parathion, acetylcholinesterase, choline oxidase and acetylcholine are added, 37 DEG C preheating 30-60 min.pH=4.0 buffer solution, gold nanoclusters and TMB are added, 37 DEG C reaction 10 min, 652 nm is measured absorbance.The detection method of the application has high detection sensitivity, strong anti-interference ability, low detection limit, high correlation coefficient of detection curve, good reproducibility in fruit and vegetable detection recovery experiment, and has good practical value in the detection of methyl parathion.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and relates to a gold nanocluster and its preparation method, particularly to a gold nanocluster prepared with a polypeptide as a ligand and a method for detecting methyl parathion using the gold nanocluster. Background Technology

[0002] Pesticides are indispensable chemical substances in agricultural production, used to protect crops from insects, weeds, fungi, and other pests, thereby increasing crop yields. Organophosphorus pesticides (OPs), due to their high efficiency and broad spectrum, are among the most widely used pesticides. However, excessive or illegal use of pesticides can lead to pesticide residue problems in crops, soil, and water sources. Traditional methods for pesticide residue detection include high-performance liquid chromatography (HPLC), gas chromatography (GC), and mass spectrometry (MS), but due to technical limitations, they cannot provide rapid and accurate detection of actual samples. Therefore, developing simple, sensitive, portable, and efficient methods for pesticide residue detection is of great significance. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a gold nanocluster prepared using polypeptides as ligands and a method for detecting the organophosphorus pesticide methyl parathion using the gold nanocluster.

[0004] This invention provides a gold nanocluster prepared using a polypeptide as a ligand, which is prepared by the following method:

[0005] Prepare a 2 mM peptide solution and a 4 mM chloroauric acid solution;

[0006] Synthesis of AuNCs: 2.4 mM peptide solution and 4 mM chloroauric acid solution were added to the reaction vessel in a volume ratio of 1:1; NaOH solution was then immediately added until the pH reached 13, yielding gold nanoclusters with peptide as ligand.

[0007] The peptides were designed using a modular strategy, and the peptide sequences are HGGFFYCC(HF); SGGFFYCC(SF); DGGFFYCC(DF); where YCC is a gold cluster stabilizing reduction module, and the terminal amino acid residues H, S, and D of the peptide are used to construct a simulated hydrolase active site.

[0008] The gold nanoclusters with polypeptide ligands prepared in this invention are applied to the detection of methyl parathion.

[0009] The gold nanoclusters prepared by this invention with polypeptide as ligand are applied to the detection of methyl parathion in fruits and vegetables.

[0010] This invention provides a dual-signal detection method for methyl parathion, characterized by comprising the following steps:

[0011] Fluorescence signal: Prepare a certain concentration of methyl parathion (PM), take 30 μL and 100 μL of AuNCs, add water to make up to 250 μL of total reaction system, and after hydrolysis reaction at 37℃ for 30-60 min, detect fluorescence change and observe fluorescence intensity at 700 nm.

[0012] Colorimetric signal: A certain concentration of methyl parathion, acetylcholinesterase, choline oxidase, and acetylcholine were added to a pH 8.0 buffer solution, and the mixture was pre-reacted at 37°C for 30-60 min. Then, a pH 4.0 buffer solution, gold nanoclusters with polypeptide ligands prepared by the method described in claim 1, and 3,3',5,5'-tetramethylbenzidine (TMB) were added, and the mixture was further reacted at 37°C for 10 min. The absorbance at a wavelength of 652 nm was then measured.

[0013] The beneficial effects of this invention are:

[0014] This invention employs a modular strategy to design and synthesize polypeptide sequences, and uses the polypeptides as ligands to prepare gold nanoclusters. By utilizing the bifunctional enzymatic properties of gold nanoclusters, combined with the fluorescence changes of the gold nanoclusters themselves, quantitative analysis and detection of methyl parathion can be achieved.

[0015] The method for detecting methyl parathion provided by this invention has certain specificity. Compared with traditional chromatographic analysis methods, it does not require expensive experimental equipment and has the advantages of being simple and rapid. Dual-signal detection has self-calibration capabilities, which can improve the detection accuracy of methyl parathion. Furthermore, the fluorescence signal detection method is specific for a class of pesticides that can hydrolyze to produce p-nitrophenol. In addition, this method has high detection sensitivity, strong anti-interference ability, low detection limit, high correlation coefficient of the detection curve, and good reproducibility in fruit and vegetable detection and recovery experiments. Therefore, this method has great practical value in the detection of methyl parathion. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the fluorescence properties of gold nanoclusters with polypeptide ligands according to the present invention;

[0017] Figure 2 This is a schematic diagram of the enzymatic reaction kinetics of the gold nanocluster hydrolase activity of the present invention;

[0018] Figure 3 This is a schematic diagram of the enzymatic reaction kinetics of the gold nanoclusters peroxidase activity of the present invention;

[0019] Figure 4 This is a schematic diagram illustrating the detection of PM fluorescence intensity using fluorescence signals according to the present invention.

[0020] Figure 5 This is a schematic diagram illustrating the linear relationship of PM detection by fluorescence signal according to the present invention;

[0021] Figure 6 This is a schematic diagram of the colorimetric signal detection of PM fluorescence intensity according to the present invention;

[0022] Figure 7 This is a schematic diagram illustrating the linear relationship of PM detection in colorimetric signal detection according to the present invention;

[0023] Figure 8 This is a schematic diagram illustrating the selectivity and anti-interference capability of fluorescence signal detection in this invention;

[0024] Figure 9 This is a schematic diagram illustrating the selectivity and anti-interference capability of colorimetric signal detection in this invention; Detailed Implementation

[0025] This invention provides a gold nanocluster prepared using a polypeptide as a ligand, which is prepared by the following method:

[0026] Preparation of polypeptide solutions:

[0027] Take a certain amount of polypeptide powder and prepare a polypeptide solution using Watson's distilled water;

[0028] Preparation of chloroauric acid solution:

[0029] Take 800 μL of 50 mM chloroauric acid stock solution and add it to 9200 μL of Watson's distilled water to prepare a 4 mM chloroauric acid solution.

[0030] Synthesis of AuNCs:

[0031] Take a 2 mL EP tube, first add 200 μL of 2 mM HF peptide solution and 100 μL of 2.8 mM SF and DF peptide solutions respectively, then add 200 μL of 4 mM chloroauric acid solution, and immediately add 20 μL of 2 M NaOH solution. Mix well and use pH test paper to check that the pH of the mixed solution is 13, thus obtaining gold nanoclusters with peptides as ligands.

[0032] The fluorescence properties of the gold nanoclusters prepared in this embodiment with peptide ligands are as follows: Figure 1 As shown, the excitation wavelength is 515nm and the emission wavelength is 700nm.

[0033] Enzymatic reaction kinetics determination of AuNCs:

[0034] a. Enzymatic kinetics determination of AuNCs hydrolase activity: Simulated hydrolase reactions were performed using different concentrations of the hydrolase substrate, p-nitrophenyl acetate (pNPA). 100 μL and 30 μL of AuNCs solutions of different concentrations of pNPA were added, and water was added to bring the total reaction volume to 250 μL. The reaction temperature was 37℃, and the reaction time was 5 min. After the reaction, the absorbance of the product p-nitrophenol (pNP) was measured at 400 nm. The Km was calculated to be 5.64 mM, and the Vmax was 9.32 × 10⁻⁶ mM. -3 mM·s -1 It possesses good hydrolytic enzyme activity and can be used for detection.

[0035] b. Kinetic determination of AuNCs peroxidase reaction: First, the concentration of TMB was fixed at 0.5 mM and the volume was 10 μL. 10 μL of H₂O₂ was added at concentrations of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. 10 μL of AuNCs was added, for a total reaction volume of 200 μL. The reaction conditions were: pH = 4, 45℃, 5 min. The absorbance at 652 nm was measured, and Km was calculated to be 61.23 mM and Vmax to be 16.78 × 10⁻⁶. -5 mM·s -1 The concentration of H₂O₂ was fixed at 50 mM, and the volume was 10 μL. 10 μL of TMB was added at concentrations of 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM. 10 μL of AuNCs was added, bringing the total reaction volume to 200 μL. The reaction conditions were: pH = 4, 45℃, 5 min. The absorbance at 652 nm was measured, and Km was calculated to be 0.41 mM and Vmax to be 14.97 × 10⁻⁶. -5 mM·s -1 It possesses good peroxidase activity and can be used for detection.

[0036] This invention applies the prepared gold nanoclusters with polypeptide ligands to the dual-signal detection of methyl parathion (PM). The detection method and detection limit are as follows:

[0037] Fluorescence signal: Prepare a certain concentration of methyl parathion (PM), take 30 μL and 100 μL of AuNCs, add water to make up to 250 μL of total reaction system, and after hydrolysis reaction at 37℃ for 30-60 min, detect fluorescence change and observe fluorescence intensity at 700 nm.

[0038] Colorimetric signal: A certain concentration of methyl parathion, acetylcholinesterase, choline oxidase, and acetylcholine were added to a pH 8.0 buffer solution and reacted at 37°C for 30-60 min beforehand. Then, a pH 4.0 buffer solution, gold nanoclusters, and 3,3',5,5'-tetramethylbenzidine (TMB) were added, and the reaction was continued at 37°C for 10 min. The absorbance at 652 nm was then measured.

[0039] Selective experiments and anti-interference experiments:

[0040] Prepare Ba separately 2+ Mg 2+ Cu 2+ Na + 5 mM each of glutamic acid, alanine, phenylalanine, glucose, glutathione, casein, and bovine serum albumin.

[0041] a. Selectivity experiment: The blank group contains no PM; the group containing PM serves as the positive control group; for the other groups, 10 μL of the above substance is added to the reaction system, but no pesticides are added. Detection is performed according to the detection method of this invention; the fluorescence and colorimetric signals are only affected by PM.

[0042] b. Anti-interference experiment: The blank group did not add PM; the group with added PM served as the positive control group; for the other groups, 10 μL of the above substances were added to the reaction system, along with the same concentration of PM as the positive control group. When the detection method of this invention was performed, the addition of the above substances did not affect the detection process of the fluorescence signal and colorimetric signal.

[0043] Dual-signal detection of real samples:

[0044] A dual-signal detection method was applied to detect PM in bok choy. The surface of the bok choy was rinsed with distilled water to remove soil and then air-dried for 2 hours. Different concentrations of PM were prepared, and equal weights of bok choy were used to apply the PM to the surface of each bok choy. After air-drying for one day, the bok choy was chopped and soaked in 1 mL of methanol. The mixture was stirred in a beaker for 6 hours, then centrifuged at 12000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm filter. The detection concentration of PM in the sample was determined using the method of this invention.

[0045] The fluorescence signal detection method for PM constructed in this invention has a linear range of 6-540 ng / mL and a limit of detection (LOD) of 4 ng / mL (ppb). Actual sample recovery experiments showed a recovery rate of 96.4%-101.6% and an RSD of <5%. The colorimetric signal detection method for PM has a linear range of 8-480 ng / mL and a LOD of 1.08 ng / mL (ppb). Actual sample recovery experiments showed a recovery rate of 98.7%-103% and an RSD of <5%. The detection method of this invention has good accuracy and plays an important role in addressing the problem of PM residues in fruits and vegetables.

[0046]

Claims

1. A method for preparing gold nanoclusters using polypeptides as ligands, characterized in that: A 2 mM peptide solution and a 4 mM chloroauric acid solution were added to the reaction vessel at a volume ratio of 1:

1. NaOH solution was then immediately added until the pH reached 13, resulting in gold nanoclusters with peptide as ligand, which simultaneously possessed hydrolytic and peroxidase activities. The sequences of the peptides are HGGFFYCC, SGGFFYCC, and DGGFFYCC.

2. A gold nanocluster with a polypeptide as a ligand, characterized in that: It is prepared by the preparation method described in claim 1.

3. The application of a gold nanocluster with a polypeptide as a ligand according to claim 2, characterized in that: For non-diagnostic or therapeutic purposes, it is used for the detection of methyl parathion.

4. The application of a gold nanocluster with a polypeptide as a ligand according to claim 2, characterized in that: It is applied to the detection of methyl parathion in fruits and vegetables.

5. A dual-signal detection method for methyl parathion for non-diagnostic or therapeutic purposes, characterized in that: Includes the following steps: Fluorescence signal: The gold nanoclusters with peptide ligands prepared by the preparation method described in claim 1 were added to the sample solution to be tested. After reacting at 37°C for 30-60 min, the fluorescence change was detected and the fluorescence intensity at 700 nm was observed. Colorimetric signal: Different concentrations of methyl parathion, acetylcholinesterase, choline oxidase and acetylcholine were added to a pH 8.0 buffer solution and reacted at 37°C for 30-60 min beforehand; then a pH 4.0 buffer solution, gold nanoclusters with polypeptide ligands prepared by the method described in claim 1, and 3,3',5,5'-tetramethylbenzidine were added, and the reaction was continued at 37°C for 10 min before measuring the absorbance at a wavelength of 652 nm.

Images