Method for detecting trace residues of organophosphorus pesticides based on dual signal of gold nanoszyme

Abstract

The application is suitable for the technical field of pesticide detection, and provides a method for double-signal detection of trace residues of organophosphorus pesticides based on gold nanoscale enzyme, polypeptide is used as a protective agent to synthesize gold nanoscale enzyme, the double properties of fluorescence and peroxidase-like activity of the gold nanoscale enzyme are used, and the enzyme inhibitor method is combined to establish a detection method, and the specific steps are as follows: step one, synthesizing gold nanoscale enzyme and storing for standby; step two, preparing sodium alginate hydrogel blocks, and physically adsorbing gold nanoscale enzyme into the sodium alginate hydrogel through soaking and storing for standby; step three, establishing an acetylcholinesterase / choline oxidase cascade reaction to generate H2O2; step four, based on the H2O2 generated in step three, and combining the prepared sodium alginate hydrogel in step two, realizing rapid visual quantitative detection of OPs, the double-signal output detection result of the application has self-correction, can resist interference, improves detection accuracy, and expands the practical application range of detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticide detection, and particularly relates to a method for double-signal detection of trace residues of organophosphorus pesticides based on gold nanoscale enzyme. BACKGROUND

[0002] Organophosphorus insecticides (OPs) are the most common pesticides. Due to its low cost, high efficiency and broad spectrum, OPs have been widely used in agricultural and household pest control, and the use amount accounts for about 38% of the world's pesticide use amount. About 2.7 million tons of pesticides are used in China's agriculture every year, and OPs account for about 70% of the total amount of pesticides used in China's agriculture. In addition, the residual OPs enter the human body through crops and water, and can be absorbed through the air, and almost cannot be detected. When OPs are exposed to the human body, even a small amount of OPs can quickly inhibit the activity of choline esterase, leading to the loss of neurotransmitter transmission, thereby causing physical damage or death. Therefore, detection of OPs in crop samples and environmental samples is crucial in the food safety evaluation process.

[0003] So far, the detection of OPs has adopted various laboratory-based traditional analysis methods, such as capillary electrophoresis (CE), thin layer chromatography (TLC), gas-liquid chromatography (GLC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and enzyme-linked immunosorbent assay (ELISA) and other methods, but these methods have some shortcomings, such as the use of expensive instruments, time-consuming process and the requirement of trained personnel. With the improvement of people's safety awareness, the requirements for pesticide detection are gradually increasing, and many new detection methods for organophosphorus pesticides (OPs) have been further developed and applied. Nanomaterial-based sensors play an important role in the trace detection of OPs. Various nanomaterials (such as metals and metal oxides, carbon nanotubes, graphene and graphene oxide, quantum dots, metal organic frameworks and molecularly imprinted polymers, etc.) have their unique properties and play different signal sensing roles in sensors, such as generating optical signals, generating electrochemical signals, enhancing signal responses and colorimetric signals. In addition, the combination of intelligent portable devices makes it possible to realize real-time rapid detection on site. Although these innovative methods successfully solve the problems of expensive instruments, complicated processing steps, strong technical dependence and complex reaction systems, and realize rapid, simple and low-cost detection of OPs with high sensitivity and high selectivity. However, most detection results usually rely on a single output signal, which often accompanies false positive or false negative results. At present, a few double-signal detection methods based on multi-enzyme cascade reaction are constructed by two highly correlated products, and the output signals are still susceptible to interference from natural enzymes or their mimics, and there are few reports on the use of the dual-functionality of a single nanoparticle for double-signal "naked eye" detection of OPs in a single analysis without the need to form composite nanomaterials to eliminate various interference factors unrelated to the analyte. Therefore, in view of the above status, it is urgent to provide a method for detecting trace residues of organophosphorus pesticides based on gold nanoscale enzyme double signal, in order to overcome the shortcomings in current practical applications. Gold nanoscale enzyme, due to its diversified enzymatic properties and luminescent properties, has attracted more and more attention in sensor design. Gold nanoscale enzymes protected by different ligands or gold nanoscale enzymes with different number of gold atoms in the core have great differences in enzymatic and luminescent properties. Therefore, people often change the type of ligand or increase or decrease the number of gold atoms in the gold core to regulate the properties of gold nanoscale enzyme, so that it has corresponding properties and is applied to sensors. SUMMARY

[0004] The purpose of the present application is to provide a method for detecting trace residues of organophosphorus pesticides based on gold nanoscale enzyme double signal, aiming to solve the problems in the above technical background.

[0005] The application is implemented based on a method for detecting trace residues of organophosphorus pesticides by double signals of gold nanoscale enzyme, polypeptide is used as a protective agent to synthesize gold nanoscale enzyme, and a detection method is established based on the dual properties of fluorescence and peroxidase-like activity of gold nanoscale enzyme and combined with an enzyme inhibitor method, and the specific steps are as follows:

[0006] Step one: synthesis of gold nanoscale enzyme and storage for standby use;

[0007] Step two: preparation of sodium alginate hydrogel block, and physical adsorption of gold nanoscale enzyme into sodium alginate hydrogel by soaking for standby use;

[0008] AChE / ChOx cascade reaction is established, the inhibition of OPs on AChE is used to affect the production of hydrogen peroxide (H2O2) as the end product of AChE / ChOx double enzyme cascade reaction;

[0009] Step four: based on the H2O2 produced in step three, the peroxidase-like activity of gold nanoscale enzyme is used to catalyze the oxidation of TMB by H2O2 to form blue oxidation products, and a colorimetric signal is generated; at the same time, Fe 2+ Fenton reaction is used to catalyze H2O2 to produce ·OH, so that the fluorescence of gold nanoscale enzyme is quenched, and a fluorescence signal is generated; and the sodium alginate hydrogel prepared in step two is used to realize rapid visual quantitative detection of OPs.

[0010] As a further scheme of the application: in step one, the specific steps for synthesizing gold nanoscale enzyme are as follows:

[0011] 2mg of polypeptide is weighed into a 1.5mL EP tube, 591μL of pure water is added to make the concentration of the polypeptide aqueous solution 2mM, after being fully dissolved, 295μL of HAuCl4 aqueous solution with a concentration of 4mM is added, the mixed solution is placed in a small oscillator for uniform rotation and stirring, 20μL of NaOH aqueous solution is added to make the pH value of the solution system 13, and the mixed solution is placed in a small oscillator for uniform rotation and stirring, the stirred solution is placed in a 37℃ shaking table for reaction for 10h, then the prepared gold clusters are placed in a dialysis bag with a molecular weight cut-off of 3.5KDa for dialysis, an ultrafiltration centrifuge tube with a molecular weight cut-off of 2KDa is used for centrifugation for 40min, and finally the unreacted small molecules are removed to obtain gold nanoscale enzyme.

[0012] As a further scheme of the application: the dialysis time is 24h.

[0013] As a further scheme of the application: the rotation and stirring treatment is carried out at a speed of 1000rmp.

[0014] As a further scheme of the application: in step two, the preparation steps of sodium alginate hydrogel are as follows:

[0015] Using HAC-AC buffer, a 25mg / mL sodium alginate solution was prepared, and after slowly dissolving at room temperature for one day, 7.5mL of the above solution was evenly spread in a plastic culture dish, and then 7.5mL of CaCl2(10mg / mL) prepared using HAC-AC buffer was introduced. After 1h, a sodium alginate hydrogel was formed.

[0016] As a further aspect of the present application: in step three, an acetylcholinesterase (AChE) / choline oxidase (ChOx) cascade reaction is established, and the specific steps for affecting the production of H2O2, the end product of the AChE / ChOx double enzyme cascade reaction, by using the inhibition of OPs on AChE are as follows:

[0017] Dilute OPs to 0.001-2μg / mL, take 8μL OPs and 2μL acetylcholinesterase to react at 37℃ for 25min to inhibit OPs, further inhibit the entire enzyme cascade reaction, then continue to add 10μL acetylcholine and 10μL choline oxidase to react in 60μL Tris-HCl to generate the end product H2O2;

[0018] Step four: based on the H2O2 generated in step three, the peroxidase-like activity of gold nanoreactase is used to catalyze the oxidation of TMB to form a blue oxidation product, generating a colorimetric signal; at the same time, Fe 2+ Catalyze H2O ２ to generate ·OH, which causes the fluorescence quenching of gold nanoreactase, generating a fluorescence signal; the specific steps for realizing rapid visual and quantitative detection of OPs by combining the sodium alginate hydrogel prepared in step two are as follows:

[0019] The colorimetric signal generation step is: mix Fe 2+ with a concentration of 20mM TMB at a volume ratio of 5:1, and add 6μL to the sodium alginate hydrogel; the fluorescence signal generation step is: add 5μL of Fe 2+ with a concentration of 2mM to the sodium alginate hydrogel; after the above steps are completed, the reaction solution of step three is added to the above gel, respectively, and left to stand for 3min, and finally a mobile device is used to take pictures, and Image J is used for gray scale analysis.

[0020] As a further aspect of the present application: the concentration of acetylcholinesterase is 14U / mL;

[0021] The concentration of acetylcholine is 600mM, and the concentration of choline oxidase is 28U / mL;

[0022] The concentration of Tris-HCl is 100mM, and the pH is 7.4.

[0023] As a further scheme of the present application: the storage temperature of the gold nanosensor and the sodium alginate hydrogel is 4 DEG C.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] The present application can realize mobile phone assisted digital fluorescence-colorimetric detection of OPs, which has not only low "naked eye" detection limit and wide linear detection range, but also meets the requirements of high sensitivity, quantification and on-site rapid visual detection.

[0026] The double signal output detection result has self-correction, which can resist the interference from biological enzymes, enzyme-like substances and other fluorescent signal molecules in more complex actual environment, improve the detection accuracy and expand the practical application range of detection. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a schematic diagram of AChE / ChOx cascade reaction in the embodiment of the present application.

[0028] Figure 2 It is a schematic diagram of the principle of generating double signal output in the embodiment of the present application.

[0029] Figure 3 It is a transmission characterization image of gold nanosensor in the embodiment of the present application.

[0030] Figure 4 It is a result diagram of colorimetric-fluorescent double signal detection of chlorpyrifos in the embodiment of the present application.

[0031] Figure 5 It is a schematic diagram of anti-interference of double signal detection in the embodiment of the present application; wherein a is colorimetric signal and b is fluorescent signal. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The specific implementation of the present application will be described in detail below in combination with specific embodiments.

[0033] Please refer to Figures 1-5The embodiment of the present application synthesizes gold nanoszyme (referred to as CCYK-AuNCs) by using the self-designed polypeptide (CCYGGYRFKYRFK) as a protective agent. Based on the dual properties of the fluorescence and peroxidase-like activity of CCYK-AuNCs, and combined with the enzyme inhibitor method, a simple, instrument-free and efficient double-signal "naked eye" method for detecting OPs is established. The specific steps of the method are as follows:

[0034] Step one: synthesis of gold nanoszyme: take 2 mg of polypeptide (CCYGGYRFKYRFK) into a 1.5 mL EP tube, add 591 μL of pure water (≥18 MΩ / cm, Milli-Q, Millipore) to make the concentration of the polypeptide aqueous solution 2 mM, after fully dissolving, add 295 μL of HAuCl ４ aqueous solution with a concentration of 4 mM, place the mixed solution in a small oscillator, rotate and stir uniformly at a speed of 1000 rpm, add 20 μL of NaOH aqueous solution (2 M) to the uniformly stirred solution to make the pH of the solution system 13, place the mixed solution in a small oscillator, rotate and stir uniformly at a speed of 1000 rpm, place the uniformly stirred solution in a 37℃ shaking table for reaction for 10 h, then place the prepared gold cluster in a dialysis bag with a molecular weight cut-off of 3.5 KDa for dialysis for 24 h, and then use an ultrafiltration centrifuge tube with a molecular weight cut-off of 2 KDa for centrifugation for 40 min to remove unreacted peptides and salts and small molecules, and store the prepared gold nanoszyme at 4℃ for later use;

[0035] Step two: use pH 4 (0.1 M) HAC-AC buffer to prepare a 25 mg / mL sodium alginate solution. Since sodium alginate is difficult to dissolve, slowly dissolve at room temperature for one day, take 7.5 mL of the above solution, evenly spread it in a 9 cm diameter plastic petri dish, then introduce 7.5 mL of CaCl2 (10 mg / mL) prepared using pH 4 (0.1 M) HAC-AC buffer, and place it for 1 h to form a gel. Take the prepared sodium alginate hydrogel and cut it into 8 mm*8 mm square gel blocks. Take the uniform gel blocks with a thickness of 1 mm and evenly soak them in 50 μL of CCYK-AuNCs, store them in the dark, and store them at 4℃ for later use;

[0036] Step three: establish an acetylcholinesterase (AChE) / choline oxidase (ChOx) cascade reaction. The inhibition of OPs on AchE affects the production of H2O2, the end product of the AChE / ChOx double enzyme cascade reaction.

[0037] In the experiment, the most common three kinds of OPs were tested and analyzed, namely chlorpyrifos, dimethoate and methyl parathion;

[0038] The initial concentration of each OPs was 100 μg / mL; each OPs was diluted to 0.001-2 μg / mL, 8 μL of the above OPs was reacted with 2 μL of acetylcholinesterase AChE (14 U / mL) at 37°C for 25 min to produce an inhibitory effect of the OPs on the enzyme, and further inhibit the entire enzyme cascade reaction; then 10 μL of acetylcholine Ach (600 mM) and 10 μL of choline oxidase ChOx (28 U / mL) were added to react in a total volume of 60 μL of Tris-HCl (100 mM, pH 7.4) for 25 min to generate the final product H2O2;

[0039] Step four: based on the H2O2 generated in step three, the peroxidase-like activity of the gold nanoreactor was used to catalyze the oxidation of TMB by H2O2 to form a blue oxidation product, generating a colorimetric signal; at the same time, Fe 2+ The Fenton reaction catalyzes H2O2 to generate ·OH, which causes the fluorescence quenching of the gold nanoreactor, generating a fluorescence signal; the sodium alginate hydrogel prepared in step two is combined to achieve rapid visual and quantitative detection of OPs.

[0040] The colorimetric signal: 6 μL of Fe 2+ (10 mM) and TMB (20 mM) were mixed at a volume ratio of 1:5, and 6 μL was added to the gold cluster-sodium alginate hydrogel; the fluorescence signal: 5 μL of Fe 2+ (2 mM) was added to the gold cluster-sodium alginate hydrogel; the reaction solution of step three was added to the above gel, respectively, after 3 min, a smart phone was used to take a picture, and Image J was used for gray scale analysis;

[0041] In the fluorescence method, the detection limits (LOD) of chlorpyrifos, dimethoate and methyl parathion were 0.2-0.5 ng / mL, and the linear ranges were 0-2000 ng / mL, 0-3000 ng / mL and 0-2000 ng / mL, respectively;

[0042] In the colorimetric method, the detection limits (LOD) of chlorpyrifos, dimethoate and methyl parathion were 0.3-0.5 ppb (ng / mL), and the linear ranges were 0-4000 ng / mL, 0-2500 ng / mL and 0-2000 ng / mL, respectively; the multi-enzyme cascade-double signal detection system was immobilized in the sodium alginate hydrogel, combined with the smart phone photography and ImageJ gray value analysis, and the rapid colorimetric-fluorescence double signal detection of chlorpyrifos was successfully realized; the linear detection range of the double signal sensor was 0-2000 ng / mL, and the naked-eye detection limit was 7 ng / mL;

[0043] Further, substances that can interfere with the colorimetric signal and the fluorescence signal were selected, such as Figure FiveAs shown in a, horseradish peroxidase (HRP) and bovine serum albumin manganese dioxide nanosheet BSA-MnO2, cobalt hydroxide nanoparticles CoOOH with peroxidase-like activity will interfere with the colorimetric signal, and the fluorescence signal is normally output; as shown in b, fluorescent quantum dots (QDs), rhodamine B (RhB) and glutathione (GSH)@Au / Ag interfere with the fluorescence signal, and the colorimetric signal is normally output; the OPs sensing system has strong utilization ability in a complex environment, and enhances the detection application range of the dual-signal sensor. Figure Five

[0044] The mechanism of the application is as follows: the dual-signal detection mechanism involves two signal outputs in the OPs-inhibited enzyme cascade signal pathway:

[0045] (1) The colorimetric signal is derived from the cascade reaction of AchE, ChOx and AuNCs, and after further hydrolysis of acetylcholine ACh to generate H2O2, 3,3,5,5-tetramethylbenzidine (TMB) is oxidized to blue product oxTMB;

[0046] (2) The fluorescence signal is generated by Fe 2+ mediated Fenton reaction to catalyze the intermediate H2O2 to form ·OH, which causes the fluorescence quenching of AuNCs, and a series of gel blocks with significant color difference are generated; the results can be directly captured by a smart phone, and digital gray scale analysis can be performed by using ImageJ software; the rapid detection platform has strong anti-interference ability to other fluorescent molecules, natural enzymes and artificial enzymes, and provides higher accuracy for the analysis of actual fruit and vegetable samples.

[0047] The practical application of the application is as follows:

[0048] Select apples, lychees and horseradish as three representative samples, wipe off the surface soil, weigh 3-5g (accurate to 0.1g) of the skin part of the sample and place it in a petri dish, add 10-15mL of PBS buffer, and the incomplete surface of the sample should not contact the buffer, gently shake for 30-50 times, and stand for 2-4min or more, take 8μL of the above prepared real sample to be tested and 2μL of AChE (14U / mL) to react at 37℃ for 25min, and the remaining reaction steps are the same as those in step three;

[0049] The detection results are shown in the following table, and the colorimetric signal detection method of the three real samples is interfered; since apples are rich in strong reducing molecules such as vitamin C, and lychees and horseradish contain a large amount of peroxidase, they will interfere with the oxidation-reduction reaction of TMB-oxTMB, thereby affecting the colorimetric detection signal; in addition, the OPs in the real sample are detected by using liquid chromatography-mass spectrometry (LC-MS), and the detection results are similar to the fluorescence signal detection results, indicating that the dual-signal detection method is relatively accurate for the detection of OPs.​

[0050]

[0051] Fruit and vegetable sample detection results show that the double signal sensor constructed can quantitatively detect ultramicro OPs in real samples, and the detection results are basically consistent with those of the liquid phase mass spectrometer; when detecting substances that have color signal interference, accurate analysis results can still be obtained, thereby expanding the application range of the sensor in actual detection.

[0052] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting trace residues of organophosphorus pesticides based on dual signals of gold nanoszyme, characterized in that, A polypeptide is used as a protective agent to synthesize a gold nanosensor, and a detection method is established based on the dual properties of fluorescence and peroxidase-like activity of the gold nanosensor and in combination with an enzyme inhibitor method, and the specific steps are as follows: Step one: synthesis of gold nanosensor and storage for standby; Step two: preparation of sodium alginate hydrogel block, and physical adsorption of gold nanosensor into sodium alginate hydrogel by soaking for storage standby; Step three: establishment of acetylcholinesterase / choline oxidase cascade reaction, and use of OPs to affect the production of H2O2, the end product of acetylcholinesterase / choline oxidase double enzyme cascade reaction, by inhibiting acetylcholinesterase; Step four: Based on the H2O2 produced in step three, the peroxidase-like activity of gold nanoszyme was used to catalyze the oxidation of TMB by H2O2 to form blue oxidation products, generating a colorimetric signal; at the same time, Fe 2+ Through Fenton reaction to catalyze H2O2 to produce ·OH, the fluorescence quenching of gold nanoszyme was generated to produce fluorescence signal, and the rapid visual quantitative detection of OPs was realized by combining with the sodium alginate hydrogel prepared in step two; In step one, the specific steps for synthesizing gold nanosensor are as follows: Take 2 mg of polypeptide into a 1.5 mL EP tube, add 591 pure water to make the concentration of the polypeptide aqueous solution 2 mM, after fully dissolving, add 295 mL of HAuCl4 aqueous solution with a concentration of 4 mM, place the mixed solution in a small oscillator for uniform rotation stirring, add 20 mL of NaOH aqueous solution to make the pH value of the solution system 13, and place the mixed solution in a small oscillator for uniform rotation stirring, place the stirred solution in a 37℃ shaking table for reaction for 10 h, then place the prepared gold clusters in a dialysis bag with a molecular weight cut-off of 3.5 KDa for dialysis, centrifuge for 40 min using an ultrafiltration centrifuge tube with a molecular weight cut-off of 2 KDa, and finally remove the unreacted small molecules to obtain the gold nanoscale enzyme; In step two, the preparation steps of sodium alginate hydrogel are as follows: Use HAC-AC buffer to prepare a 25mg / mL sodium alginate solution, and after slowly dissolving at room temperature for one day, take 7.5mL of the above solution and evenly spread it on a plastic culture dish, then introduce 7.5mL of CaCl210mg / mL prepared with HAC-AC buffer, and after placing for 1h, the sodium alginate hydrogel is formed; In step three, the specific steps for establishing enzyme cascade reaction are as follows: OPs were diluted to 0.001-2 mL, 8 OPs were reacted with 2 mM acetylcholinesterase for 25 min at 37°C to inhibit OPs from inhibiting the enzyme cascade, after which 10 mM acetylcholine and 10 mM choline oxidase were added to a total volume of 60 mL Tris-HCl and reacted for 25 min to produce the end product H2O2; The dialysis time is 24h; The rotation speed of the rotation stirring treatment is 1000rmp; The concentration of acetylcholinesterase is 14U / mL; The concentration of acetylcholine is 600mM, and the concentration of choline oxidase is 28U / mL; The concentration of Tris-HCl is 100mM, and the pH is 7.4; The storage temperature of gold nanosensor and sodium alginate hydrogel is 4℃; The sequence of the polypeptide is CCYGGYRFKYRFK. 2.The method for detecting trace residues of organophosphorus pesticides based on gold nanotranszyme double signals according to claim 1, characterized in that, In step four, the specific steps for realizing double signal detection of OPs are as follows: The colorimetric signal generating step is: mixing 6 μL of 10 mM Fe 2+ with 20 mM TMB at a volume ratio of 1:5, and adding 6 μL dropwise to the sodium alginate hydrogel; the fluorescence signal generating step is: mixing 6 μL of 2 mM Fe 2+ with 20 mM TMB at a volume ratio of 1:5, and adding 5 μL dropwise to the sodium alginate hydrogel; The reaction liquid of step three is added dropwise to the above gel, and after the above step is completed, it is placed for 3min, and finally a mobile device is used to take pictures and Image J is used for gray scale analysis.

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