A new method for detecting four sulfur-containing pesticides based on gold-silver nanoenzyme materials and its application

Abstract

The present invention belongs to the technical field of nano-enzyme material preparation and chemical analysis detection, and relates to a new method for nano-enzyme colorimetric detection of sulfur-containing pesticides. The synthesized Au-Ag NCs have pseudoperoxidase activity, and can catalyze hydrogen peroxide to oxidize TMB to form a blue product under acidic conditions. Based on the electrostatic interaction between Au-Ag NCs and four sulfur-containing pesticides, the exposed Au and Ag single substances are further bonded with S in the pesticide molecules, resulting in the loss of the pseudoenzyme activity of Au-Ag NCs to block the catalytic oxidation process, and the content of sulfur-containing pesticides in the test system can be determined. The present invention has the advantages of good specificity, high sensitivity, fast response speed, etc., and can provide a new means for the content determination of sulfur-containing pesticides.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-mimetic enzyme material preparation and chemical analysis detection, and specifically relates to a method for controllable preparation of a nano-mimetic peroxidase colorimetric sensor and highly sensitive detection of four sulfur-containing pesticides. Background Art

[0002] Thiophanate-methyl, cartap, dimethoate, and bispyribosyl are four sulfur-containing pesticides commonly used in the production and planting of crops. They are systemic, contact, broad-spectrum, and highly toxic. They can quickly prevent and treat pathogenic nematodes, spider mites, mosquitoes, fleas, and other pests and diseases in rice, fruit trees, vegetables, and forests, and can effectively improve crop yield and quality. However, long-term intake of food with excessive pesticide residues poses a potential risk of teratogenicity, carcinogenicity, and mutagenicity, and may even lead to acute / chronic poisoning, which seriously endangers human health. In order to monitor sulfur-containing pesticide residues in food and drug samples, it is of great significance to develop a rapid detection method for sulfur-containing pesticides with high specificity and sensitivity.

[0003] Traditional detection technologies including chromatography, spectroscopy and enzyme inhibition have made great progress in the field of pesticide residue detection, but they often have shortcomings such as complex operation, time-consuming detection and high cost. Therefore, it is still worth further exploring to develop a new sensing method that is easy to operate, sensitive and portable, and low in cost. Nano-enzyme pseudo-colorimetric sensing technology has the advantages of high sensitivity, high selectivity, short response time, and simple operating conditions. It is a new method for detecting pesticide residues. Nano-pseudoperoxidase has received widespread attention due to its simple preparation process and clear color reaction mechanism. Compared with natural peroxidase, nano-pseudoperoxidase has better thermal stability and anti-interference, and can effectively improve the sensitivity and specificity of detecting pesticide residues. Based on the excellent characteristics of nano-pseudoperoxidase, the present invention provides a new method for quantitatively detecting four sulfur-containing pesticides based on a nano-pseudoperoxidase-based colorimetric sensor. Summary of the invention

[0004] In view of the deficiencies in the prior art, one of the objects of the present invention is to provide a new type of nano-pseudoperoxidase and a preparation method thereof; a second object of the present invention is to provide a controllable preparation method of a simple nano-pseudoperoxidase colorimetric sensor; a third object of the present invention is to provide a method for highly sensitive detection of sulfur-containing pesticides in food, which has the advantages of simple operation, rapidity and sensitivity, and can realize rapid and accurate identification of sulfur-containing pesticides in complex matrices such as green tea, apples, cabbage, and wolfberries.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A novel nano-pseudo-peroxidase is a gold-silver bimetallic nanocluster (Au-Ag NCs) with a bowl-shaped hollow structure and a rough surface. The outer layer of the Au-Ag NCs is negatively charged, the average inner diameter is 17.3±0.3nm, and the average outer diameter is 31.6±0.2nm. The aqueous dispersion of the Au-Ag NCs is purple, and the λmax is 537±5nm.

[0007] A preparation method of the novel nano-pseudo-peroxidase is to use trisodium citrate as a reducing agent and a stabilizer to react with silver nitrate and chloroauric acid to synthesize hollow Au-Ag NCs, which specifically includes the following steps:

[0008] (1) Heat the silver nitrate solution to a boil in the dark, quickly add the trisodium citrate aqueous solution while stirring (preferably at a stirring speed of 400 r / min), mix evenly, and boil and react for 1 h while stirring in the dark (preferably at a stirring speed of 200 r / min);

[0009] (2) Slowly add (preferably at a uniform rate of 30 drops / min) chloroauric acid aqueous solution at 90°C with stirring (preferably at a stirring speed of 200 r / min), and continue stirring at 90°C in the dark for 45 min. After the reaction is completed, cool the reaction system to room temperature, filter the obtained solution, and then purify it to obtain Au-Ag NCs.

[0010] Preferably, the molar ratio of silver nitrate, trisodium citrate and chloroauric acid is 0.05:1:0.015.

[0011] The Au-Ag NCs of the present invention can quickly (can exert catalytic effect within 30 seconds) catalyze H2O2 to produce ·OH to oxidize TMB to generate a blue product in an acidic environment, thereby exerting peroxidase activity.

[0012] The present invention uses the novel nano-pseudo-peroxidase as a colorimetric probe, hydrogen peroxide as an enzyme reaction substrate, and 3,3',5,5'-tetramethylbenzidine (TMB) as a colorimetric agent, and the three are added in a specific order in a buffer solution system with appropriate pH to construct a colorimetric sensor. Preferably, the buffer solution is an acetic acid-sodium acetate buffer solution with a pH of 2.8≤≤4.6, and the optimal pH is 3.4.

[0013] A highly sensitive method for detecting four sulfur-containing pesticides in food is based on the electrostatic interaction between the negatively charged Au-Ag NCs on the outer layer and the positively charged sulfur-containing pesticides, exposing the Au and Ag elements in the inner layer of the enzyme-like material, which further bonds with the S in the pesticide molecules, causing the Au-Ag NCs bowl-shaped hollow structure to collapse and lose the enzyme-like activity, which can block the catalytic oxidation process and ultimately achieve the determination of the sulfur-containing pesticide content in the test system; the four sulfur-containing pesticides are methyl thiophanate, cartap, dimethoate, and bispyribac. Specifically, the following steps are included:

[0014] (1) Drawing a standard curve: accurately weigh a methyl thiophanate standard, use anhydrous methanol as a solvent to prepare a methyl thiophanate standard stock solution, and then use ultrapure water to dilute the methyl thiophanate standard stock solution to different concentrations to obtain a methyl thiophanate linear solution, wherein the concentration is in the range of 0.05 to 2 mg / L; add Au-Ag NCs, methyl thiophanate linear solution, hydrogen peroxide, 2.8≤pH≤4.6 acetic acid-sodium acetate buffer solution, and TMB solution to a reaction container in sequence, mix well, react for 3 minutes, and measure the absorbance of the reaction system at 652 nm; draw a standard curve with the concentration of methyl thiophanate as the horizontal coordinate and the absorbance value as the vertical coordinate;

[0015] The standard curves of cartap, dimethoate and bispyribac were drawn in the same way;

[0016] Among them, the linear solution of carbofuran is prepared with ultrapure water as solvent, and the concentration is in the range of 0.05-1 mg / L; the linear solution of dimethoate is prepared by first preparing the stock solution with anhydrous ethanol as solvent, and then diluting it with ultrapure water, and the concentration is in the range of 0.01-2 mg / L; the linear solution of bispyribac is prepared by first preparing the stock solution with anhydrous methanol as solvent, and then diluting it with ultrapure water, and the concentration is in the range of 0.01-1.5 mg / L.

[0017] (2) Sample detection: Pre-treat the sample to prepare a test solution; add Au-AgNCs, the test solution, hydrogen peroxide, acetic acid-sodium acetate buffer solution with a pH of 2.8 ≤ ≤ 4.6, and TMB solution to a reaction vessel in sequence, mix well, react for 3 minutes, and measure the absorbance of the reaction system at 652 nm; substitute the corresponding standard curve equation in step (1) to calculate the contents of methyl thiophanate, cartap, dimethoate, and bispyribac.

[0018] Among them, the pretreatment method described in step (2) is: for solid samples, first crush them, then add pure water to squeeze juice or soak them, filter them, and dilute the filtrate with pure water until it is colorless; for liquid samples, first shake them well and then filter them, and dilute the filtrate with pure water until it is colorless.

[0019] Preferably, in steps (1) and (2), an aqueous dispersion of Au-Ag NCs is added to the reaction container, and the mass ratio of Au-AgNCs to water is 2:1.

[0020] Preferably, in steps (1) and (2), the mass concentration of hydrogen peroxide is 10%.

[0021] Preferably, in steps (1) and (2), the concentration of the acetic acid-sodium acetate buffer solution is 0.2 M and the pH is 3.4.

[0022] Preferably, in steps (1) and (2), a DMSO solution of TMB is added to the reaction container at a concentration of 10 mM.

[0023] Preferably, in steps (1) and (2), the volume ratio of Au-Ag NCs, sulfur-containing pesticide linear solution / test solution, hydrogen peroxide, acetic acid-sodium acetate buffer solution, and TMB is 1:1:1:1:1:1.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] 1. The Au-Ag NCs synthesized in the present invention have good enzyme-like activity and short response time;

[0026] 2. The method of the present invention can detect four kinds of sulfur-containing pesticides at the same time and has a wide range of applications;

[0027] 3. The detection method of the present invention has high sensitivity, short detection time, simple operation, does not involve expensive instruments and reagents, and has low cost;

[0028] 4. The detection method of the present invention can be used to directly determine whether the pesticide residue exceeds the standard with the naked eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the detection principle of the nano-enzyme-mimicking colorimetric sensor of the present invention;

[0030] Figure 2 The Au-Ag NCs in the nano-enzyme-mimicking colorimetric sensor of the present invention and the ultraviolet absorption spectrum of the enzyme-mimicking reaction thereof, the abscissa is the wavelength and the ordinate is the absorbance;

[0031] Figure 3 is the pseudoenzyme activity intensity of Au-Ag NCs in buffer solutions with different pH values, the abscissa is the pH value, and the ordinate is the absorbance value;

[0032] Figure 4 This is the result of the steady-state kinetic experiment of Au-Ag NCs enzyme reaction;

[0033] Figure 5This is the fluorescence spectrum of the Au-Ag NCs-hydrogen peroxide-terephthalic acid system, the abscissa is the wavelength, and the ordinate is the fluorescence intensity;

[0034] Figure 6 The Zeta potential results of Au-Ag NCs and four sulfur-containing pesticides are shown in Figure 1. The horizontal axis is the potential difference, where TM is methyl thiophanate, DT is dimethoate, CT is cartap, and TP is temephos;

[0035] Figure 7 Transmission electron microscopy images and partial magnified images of silver nanoparticles (A, B), Au-Ag NCs (C, D), and a mixed solution of Au-Ag NCs and 10 wt% H2O2 (E, F);

[0036] Figure 8 Transmission electron microscope images of Au-Ag NCs in the nano-enzyme-mimicking colorimetric sensor of the present invention after reacting with four sulfur-containing pesticides (A: methyl thiophanate; B: cartap; C: dimethoate; D: bispyribac);

[0037] Fig. 9 The visualization effect diagrams of the pseudo-enzyme detection system of the present invention before and after the interaction with four sulfur-containing pesticides (A: methyl thiophanate; B: cartap; C: dimethoate; D: bispyribac);

[0038] Fig.10 After constructing the sensor, four sulfur-containing pesticides with known concentrations were added to construct an ultraviolet absorption spectrum of a standard curve (A: methyl thiophanate; B: cartap; C: dimethoate; D: dimethoate), with the horizontal axis being the wavelength and the vertical axis being the absorbance. DETAILED DESCRIPTION

[0039] The applicant will further describe the present invention in conjunction with the embodiments and drawings of the specification below, but the protection scope of the present invention is not limited to these embodiments. Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the technical features of the present invention still fall within the protection scope of the present invention.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified. Among them, the methyl thiophanate standard product was purchased from Sinopharm Group with a purity of 99.8% and a specification of 25 mg; the cartap standard product was purchased from Sinopharm Group with a purity of 99.8% and a specification of 250 mg; the bispyribac standard product was purchased from Sinopharm Group with a purity of 99.8% and a specification of 100 mg; and the dimethoate standard product was purchased from Sinopharm Group with a purity of 99.8% and a specification of 100 mg.

[0041] Example 1: Preparation of Nano-Enzyme Mimicking Materials

[0042] A method for preparing a novel nano-peroxidase comprises the following steps:

[0043] In an oil bath, 50 mL of 0.1 mM silver nitrate aqueous solution was heated and boiled in the dark. 1 mL of 0.1 M sodium citrate trihydrate aqueous solution was quickly added at a speed of 400 r / min on the oil bath magnetic stirrer. After mixing evenly, the speed was adjusted to 200 r / min, and the mixture was stirred and boiled in the dark for 1 h to obtain a yellow transparent nanosilver solution. Then, 1.5 mL of 1 mM chloroauric acid aqueous solution was added at a uniform rate of 30 drops / min under the stirring conditions of 90 ° C and 200 r / min in the oil bath, and the mixture was stirred and reacted in the dark for 45 min at 90 ° C to obtain a purple transparent solution. The obtained solution was filtered with a microporous filter membrane with a pore size of 0.22 μm, and then the solution was purified for 24 hours using a cellulose dialysis bag (1000 Da) to obtain an aqueous dispersion of Au-Ag NCs (the mass ratio of Au-Ag NCs to water was 2:1), which was purple, with a maximum ultraviolet absorption wavelength at 537 nm, and stored in a refrigerator at 4 ° C for use.

[0044] The prepared nanosilver and Au-Ag NCs were scanned by transmission electron microscopy, and the obtained TEM images were as follows: Figure 7 As shown in A, B (silver nanoparticles) and C, D (Au-Ag NCs). From the TEM image, it can be seen that the silver nanoparticles are nearly spherical, with an average particle size of 44.9±0.3nm; Au-Ag NCs are bowl-shaped hollow structures, with an average inner diameter of 17.3±0.3nm, an average outer diameter of 31.6±0.2nm, and a rough surface with a large specific surface area.

[0045] Example 2: Investigation of the pseudoenzyme activity of Au-Ag NCs

[0046] 1. Nano-enzyme-hydrogen peroxide-TMB system

[0047] 1) Investigation of UV absorption behavior

[0048] The Au-Ag NCs of Example 1, ultrapure water, 10 wt% hydrogen peroxide, 0.2 M acetic acid-sodium acetate buffer solution with a pH of 3.4, and 10 mM TMB were added to the EP tube in sequence, wherein the volume ratio of Au-Ag NCs, ultrapure water, hydrogen peroxide, buffer solution, and TMB was 1:1:1:1:1. The mixture was mixed evenly and reacted for 3 minutes before collecting its UV absorption spectrum, as shown in FIG. Figure 2 As shown in “Au-Ag NCs+H2O2+TMB”.

[0049] The same method was used to collect UV absorption spectra of “Au-Ag NCs” (Au-Ag NCs, ultrapure water, DMSO, 0.2M acetic acid-sodium acetate buffer solution with a pH of 3.4, a volume ratio of 1:2:1:1), “Au-Ag NCs+TMB” (Au-Ag NCs, ultrapure water, 0.2M acetic acid-sodium acetate buffer solution with a pH of 3.4, 10mM TMB, a volume ratio of 1:2:1:1), and “H2O2+TMB” (ultrapure water, 10wt% hydrogen peroxide, 0.2M acetic acid-sodium acetate buffer solution with a pH of 3.4, 10mM TMB, a volume ratio of 2:1:1:1).

[0050] In this embodiment and the following embodiments, Au-Ag NCs refers to the Au-Ag NCs aqueous dispersion obtained during synthesis, and the concentration used is: the mass ratio of Au-Ag NCs to water is 2:1; TMB refers to the DMSO solution of TMB.

[0051] Depend on Figure 2 It can be seen that when Au-Ag NCs or H2O2 act alone, TMB does not produce the characteristic ultraviolet absorption peak of its oxidation product; only when Au-Ag NCs and H2O2 act together, the characteristic absorption peaks of oxidized TMB appear at 652nm and 370nm. This confirms that Au-Ag NCs have pseudoperoxidase activity.

[0052] 2) Investigation of the influence of pH

[0053] Prepare 0.2M acetic acid-sodium acetate buffer solutions of different pH values, and collect the UV absorption spectra of the Au-Ag NCs+H2O2+TMB system under different pH conditions according to the method in Example 1), and record the absorbance value at 652nm. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that when the pH of the buffer solution is 3.4, the peroxidase activity of Au-Ag NCs is the strongest.

[0054] 3) Study on steady-state kinetics of enzyme reaction

[0055] The TMB concentration was fixed at 10mM, and multiple H2O2 solutions of different concentrations were prepared. The absorbance values ​​at 652nm were collected at the same reaction time according to the order of adding Au-Ag NCs, ultrapure water, H2O2 solution, 0.2M pH 3.4 acetic acid-sodium acetate buffer solution, and TMB (volume ratio of 1:1:1:1:1) to calculate the reaction rate. Conversely, the H2O2 concentration was fixed at 10wt%, and multiple TMB solutions of different concentrations were prepared. The absorbance values ​​at 652nm were collected in the same way to calculate the reaction rate. The Michaelis curve was drawn, and the results are shown in Figure 2. Figure 4 shown.

[0056] According to the Michaelis-Menten equation, when Au-Ag NCs undergo enzyme-like reaction: when TMB is used as substrate, K m =899.3(μM),V max =0.446(mM·S -1 ); When H2O2 is used as substrate, K m =1672.3(μM),V max =0.575(mM·S -1 ); Compared with other reported pseudoperoxidases, it has stronger activity (K m is the enzyme reaction kinetic constant, V max is the maximum reaction rate of the enzyme; K m The smaller the V max The larger the value, the better the pseudoenzyme activity).

[0057] Table 1: Comparison of parameters of the Michaelis-Menten equation of the Au-Ag NCs of the present invention and other pseudo-peroxidases

[0058]

[0059]

[0060] 2. Nano-enzyme-hydrogen peroxide-terephthalic acid (TA) system

[0061] The Au-Ag NCs of Example 1 were mixed with 10 wt% hydrogen peroxide, and then 0.2 M acetic acid-sodium acetate buffer solution of pH 3.4 was added to react for 3-5 min, followed by 5 mM terephthalic acid solution (solvent: 0.1 M NaOH), and finally 0.2 mM phosphate buffer solution of pH 5.0; wherein the volume ratio of Au-Ag NCs, hydrogen peroxide, acetic acid-sodium acetate buffer solution, terephthalic acid solution, and phosphate buffer solution was 1:1:1:1:1. Mix well, react for 20 min, and collect the fluorescence spectrum of the solution. The fluorescence spectrum collection conditions are: E x =316nm, E m =300-600nm, voltage = 400V, slit width = 10nm.

[0062] The same method was used to collect fluorescence spectra of “TA” (ultrapure water, 0.2M acetic acid-sodium acetate buffer solution with pH 3.4, 5mM terephthalic acid solution, 0.2mM phosphate buffer solution with pH 5.0, with a volume ratio of 2:1:1:1), “TA+Au-Ag NCs” (Au-Ag NCs, ultrapure water, 0.2M acetic acid-sodium acetate buffer solution with pH 3.4, 5mM terephthalic acid solution, 0.2mM phosphate buffer solution with pH 5.0, with a volume ratio of 1:1:1:1:1), and “TA+H2O2” (ultrapure water, 10wt% hydrogen peroxide, 0.2M acetic acid-sodium acetate buffer solution with pH 3.4, 5mM terephthalic acid solution, 0.2mM phosphate buffer solution with pH 5.0, with a volume ratio of 1:1:1:1:1). The results are shown in Figure 6. Figure 5 shown.

[0063] Depend on Figure 5 It can be seen that the product obtained by the reaction of the purple Au-Ag NCs nanoenzyme material with H2O2 in an acidic environment can react with non-fluorescent terephthalic acid to produce fluorescence. Under the condition of Ex = 316nm, the fluorescence characteristic peak of 2-hydroxyterephthalic acid was detected at Em = 425nm using a fluorescence spectrophotometer, confirming the generation of ·OH. The principle of this reaction is that the outer negative charge of Au-AgNCs undergoes electron transfer with H2O2 to generate hydroxyl radicals (·OH), which can react with terephthalic acid to generate stable and unique 2-hydroxyterephthalic acid that emits strong blue fluorescence.

[0064] Example 3: Analysis method establishment

[0065] Accurately weigh the methyl thiophanate standard and use anhydrous methanol as solvent to prepare a 1000 mg / L methyl thiophanate standard stock solution; use ultrapure water to dilute the methyl thiophanate standard stock solution to 10, 5, 3, 1, 0.8, 0.5, and 0.1 mg / L, respectively.

[0066] Accurately weigh the cartap standard and use ultrapure water as solvent to prepare a 1000 mg / L cartap standard stock solution; then use ultrapure water to dilute the stock solution to 10, 5, 3, 1, 0.8, 0.5, and 0.1 mg / L, respectively.

[0067] Accurately weigh the dimethoate standard and use anhydrous ethanol as solvent to prepare a 1000 mg / L dimethoate standard stock solution; use ultrapure water to dilute the dimethoate standard stock solution to 10, 5, 3, 1, 0.8, 0.5, and 0.1 mg / L, respectively.

[0068] Accurately weigh the dimethoate standard and use anhydrous methanol as solvent to prepare a 1000 mg / L dimethoate standard stock solution; use ultrapure water to dilute the dimethoate standard stock solution to 10, 5, 3, 1, 0.8, 0.5, and 0.1 mg / L, respectively.

[0069] The Au-Ag NCs of Example 1 were mixed with 10 wt% hydrogen peroxide, 1 mg / L methyl thiophanate, 1 mg / L dimethoate, 1 mg / L cartap at a volume ratio of 1:1, and the potential difference between each mixed solution and Au-Ag NCs was collected using a Zeta potential meter. The results are shown in FIG. Figure 6 As shown. Figure 6 It can be seen that Au-Ag NCs are negatively charged, and the four sulfur-containing pesticides are positively charged. Electrostatic interactions can occur between Au-Ag NCs and the four sulfur-containing pesticides, and there are favorable reaction conditions.

[0070] The Au-Ag NCs of Example 1 were mixed with 10 wt% hydrogen peroxide, 10 mg / L thiophanate-methyl, 10 mg / L dimethoate, 10 mg / L cartap at a volume ratio of 1:1, and microscopic images of the mixed solutions were taken using a transmission electron microscope. The results are as follows: Figure 7 E, F and Figure 8 As shown. Figure 7 From E and F, we can see that the hollow diameter of Au-Ag NCs becomes larger after the enzyme-like reaction. Figure 8 It can be seen that after the Au-Ag NCs reacted with the four sulfur-containing pesticides, the bowl-shaped hollow structure disintegrated.

[0071] The following pesticide standard solutions were taken respectively: 5, 3, 1, 0.8, 0.5, 0.1, 0 mg / L of thiophanate-methyl, dimethoate, bispyribac, and cartap. Then, Au-Ag NCs, pesticide standard solution, 10 wt% hydrogen peroxide, 0.2 M acetic acid-sodium acetate buffer solution with a pH of 3.4, and 10 mM TMB were added in the order of 1:1:1:1:1 by volume, mixed evenly, and reacted for 3 min. The colorimetric effect was captured by a camera. The results are shown in Figure 2. Fig. 9 As shown (from left to right, the concentration of the pesticide standard solution decreases gradually). Fig. 9 It can be seen that the colorimetric sensing system shows a color change from light to dark according to the concentration of the pesticide standard solution from high to low, which can be clearly observed by naked eyes. The method of colorimetric sensing for identifying sulfur-containing pesticides constructed by the present invention has satisfactory effect.

[0072] The reaction principle is as follows: due to the electrostatic attraction between the outer electrons of Au-Ag NCs and the four sulfur-containing pesticides, the Au and Ag elements in the inner layer of Au-Ag NCs are exposed; the S in the four sulfur-containing pesticides has lone electron pairs, which can combine with the Au and Ag elements in the inner layer of Au-Ag NCs to form bonds; after the sulfur-containing pesticides combine with the Au and Ag elements in the inner layer of Au-Ag NCs to form bonds, the structure of Au-Ag NCs is destroyed and the pseudoenzyme activity is blocked.

[0073] Example 4: Validation of analytical method

[0074] (1) Linearity, limit of quantification, and limit of detection

[0075] Prepare linear solutions of sulfur-containing pesticides: Take the methyl thiophanate standard stock solution in Example 3 and dilute the stock solution with ultrapure water to 2, 1.5, 1, 0.7, 0.5, 0.3, 0.1, and 0.05 mg / L, respectively. Take the cartap standard stock solution in Example 3 and dilute the stock solution with ultrapure water to 1, 0.8, 0.5, 0.3, 0.1, 0.08, and 0.05 mg / L, respectively. Take the dimethoate standard stock solution in Example 3 and dilute the stock solution with ultrapure water to 2, 1.5, 1, 0.8, 0.5, 0.3, 0.1, 0.05, and 0.01 mg / L, respectively. Take the bispyribac standard stock solution in Example 3 and dilute the stock solution with ultrapure water to 1.5, 1, 0.8, 0.5, 0.3, 0.1, 0.05, and 0.01 mg / L, respectively.

[0076] The Au-Ag NCs of Example 1, the linear solution of sulfur-containing pesticides, 10 wt% hydrogen peroxide, 0.2 M acetic acid-sodium acetate buffer solution with a pH of 3.4, and 10 mM TMB were added to the EP tube in sequence, wherein the volume ratio of Au-Ag NCs, the linear solution, hydrogen peroxide, the buffer solution, and TMB was 1:1:1:1:1. Mix well, react for 3 minutes, and collect the UV absorption spectrum of the reaction system ( Fig.10 ), record the absorbance at 652nm; plot the standard curve with the concentration of the linear solution as the abscissa and the absorbance value as the ordinate. The standard curve equations and linear correlation coefficients of each sulfur-containing pesticide are shown in Table 2 below.

[0077] The quantitative limit of the method was calculated according to the formula: LOQ = (10σ) / k, where σ is the standard deviation of the absorbance of the blank control measured 10 times, and k is the slope of the standard curve. The quantitative limits of the four sulfur-containing pesticides were calculated and are shown in Table 2 below.

[0078] The detection limit of the method was calculated according to the formula: LOD = (3σ) / k, where σ is the standard deviation of the absorbance of the blank control measured 10 times, and k is the slope of the standard curve. The detection limits of the four sulfur-containing pesticides were calculated and are shown in Table 2 below.

[0079] Table 2: Linear equations, correlation coefficients, limits of quantification, and limits of detection for four sulfur-containing pesticides

[0080]

[0081] (2) Accuracy

[0082] Green tea, apple, cabbage and wolfberry samples were crushed separately, and appropriate amount of ultrapure water was added to squeeze juice or soak them, filtered, and the filtrate was diluted with ultrapure water until it was colorless.

[0083] The sulfur-containing pesticide standard stock solution of Example 3 was taken and diluted with the above-mentioned food extract to three concentration levels of high (1 mg / L), medium (0.5 mg / L) and low (0.1 mg / L) to obtain a recovery determination solution; a reaction system was constructed according to the method in (1) of this Example (the reaction system was set up in 3 parallel groups), and the absorbance at 652 nm was measured; the absorbance value was substituted into the standard curve equation in (1) to obtain the test concentration of the sulfur-containing pesticide, and the recovery rates of the four sulfur-containing pesticides in the actual samples were calculated according to the formula: spiked recovery rate = (test concentration / sample concentration) * 100%. The results are shown in Table 3 below.

[0084] Table 3

[0085]

[0086]

[0087] As shown in Table 3, the recoveries of the four sulfur-containing pesticides at three concentration levels in apple, wolfberry, green tea and cabbage were all between 95% and 115%, and the RSDs were all less than 5%, which confirmed that the colorimetric sensor of the present invention had a satisfactory detection effect in real samples.

[0088] References

[0089] [1] Hu, L.; Liao, H.; Feng, L.; Wang, M.; Fu, W. Accelerating the Peroxidase-Like Activity of Gold Nanoclusters at Neutral pH for Colorimetric Detection of Heparin and Heparinase Activity. Anal. Chem. 2018, 90, 6247-6252.

[0090] [2]HUANG X,NAN Z.Porous 2D FeS2 nanosheets as a peroxidase mimic forrapid determination of H2O2[J].Talanta,2020,216.

Claims

1. A method for preparing a nano-peroxidase, characterized in that: The nano-pseudo-peroxidase is a gold-silver bimetallic nanocluster Au-Ag NCs, and the Au-Ag NCs has a bowl-shaped hollow structure, a rough surface, and a negatively charged outer layer; The preparation method comprises the following steps: (1) Heat the silver nitrate solution to a boil in the dark, quickly add the trisodium citrate aqueous solution while stirring, mix evenly, and then stir and boil in the dark for 1 h; (2) After slowly adding chloroauric acid aqueous solution at 90°C with stirring, the reaction was continued at 90°C in the dark with stirring for 45 min. After the reaction was completed, the reaction system was cooled to room temperature, the obtained solution was filtered and purified to obtain Au-Ag NCs.

2. The preparation method according to claim 1, characterized in that: The average inner diameter of the Au-Ag NCs is 17.3±0.3 nm, the average outer diameter is 31.6±0.2 nm, the aqueous dispersion of the Au-Ag NCs is purple, and the λmax is 537±5 nm.

3. The preparation method according to claim 1, characterized in that: In step (1), the stirring speed when adding the trisodium citrate aqueous solution is 400 r / min, and the stirring speed during the boiling reaction in the dark is 200 r / min; In step (2), it takes 1-2 min to add the aqueous chloroauric acid solution, and the stirring speed when adding the aqueous chloroauric acid solution is 200 r / min.

4. The preparation method according to claim 1, characterized in that: The molar ratio of silver nitrate, trisodium citrate and chloroauric acid is 0.05:1:0.

015.

5. A colorimetric sensor of nano-pseudo-peroxidase prepared by the preparation method according to claim 1, characterized in that: The nano-pseudo-peroxidase is used as a colorimetric probe, hydrogen peroxide is used as an enzyme reaction substrate, 3,3',5,5'-tetramethylbenzidine is used as a color developer, and an acetic acid-sodium acetate buffer solution with a pH of 2.8≤pH≤4.6 is used as a buffer system.

6. A method for quantitatively detecting four sulfur-containing pesticides in food using the nano-pseudo-peroxidase prepared by the preparation method of claim 1, characterized in that: The four sulfur-containing pesticides are thiophanate-methyl, cartap, dimethoate, and temephos; The method for quantitatively detecting four sulfur-containing pesticides in food comprises the following steps: (1) Drawing a standard curve: accurately weigh the methyl thiophanate standard, prepare the methyl thiophanate standard stock solution with anhydrous methanol, and then dilute the methyl thiophanate standard stock solution with ultrapure water to different concentrations to obtain a methyl thiophanate linear solution, wherein the concentration is in the range of 0.05~2 mg / L; add Au-Ag NCs, methyl thiophanate linear solution, hydrogen peroxide, 2.8≤pH≤4.6 acetic acid-sodium acetate buffer solution, and TMB solution to the reaction container in sequence, mix well, react for 3 minutes, and measure the absorbance of the reaction system at 652nm; draw a standard curve with the concentration of methyl thiophanate as the horizontal axis and the absorbance value as the vertical axis; The standard curves of cartap, dimethoate and bispyribac were drawn in the same way; Among them, the linear solution of cartap is prepared with ultrapure water as solvent, and the concentration is in the range of 0.05~1 mg / L; the linear solution of dimethoate is prepared by first preparing the stock solution with anhydrous ethanol as solvent, and then diluting it with ultrapure water, and the concentration is in the range of 0.01~2 mg / L; the linear solution of bispyribac is prepared by first preparing the stock solution with anhydrous methanol as solvent, and then diluting it with ultrapure water, and the concentration is in the range of 0.01~1.5 mg / L; (2) Sample detection: Pre-treat the sample to prepare the test solution; add Au-AgNCs, the test solution, hydrogen peroxide, acetic acid-sodium acetate buffer solution with a pH of 2.8 ≤ ≤ 4.6, and TMB solution into the reaction container in sequence, mix well, react for 3 minutes, and then measure the absorbance of the reaction system at 652 nm; substitute the corresponding standard curve equation in step (1) to calculate the contents of methyl thiophanate, cartap, dimethoate, and bispyribac.

7. The method according to claim 6, characterized in that In steps (1) and (2), the aqueous dispersion of Au-Ag NCs is added to the reaction container, and the mass ratio of Au-Ag NCs to water is 2:1; In steps (1) and (2), the mass concentration of hydrogen peroxide is 10%; In steps (1) and (2), the concentration of the acetic acid-sodium acetate buffer solution is 0.2 M and the pH is 3.4; In steps (1) and (2), a DMSO solution of TMB at a concentration of 10 mM is added to the reaction vessel.

8. The method according to claim 7, characterized in that In steps (1) and (2), the volume ratio of the Au-Ag NCs aqueous dispersion, the sulfur-containing pesticide linear solution / test solution, hydrogen peroxide, the acetic acid-sodium acetate buffer solution, and the TMB DMSO solution is 1:1:1:1:1.

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