Enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate as well as preparation and application of enzyme biosensor
An enzyme biosensor was constructed by combining a glassy carbon electrode modified with graphene and gold nanoparticles with acetylcholinesterase, which solved the problems of complex and costly detection of dimethoate in the existing technology, and realized rapid and accurate detection of dimethoate, which is suitable for on-site screening of agricultural products.
Patent Information
- Application Number
- CN202511015991.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2026-01-09
AI Technical Summary
Existing technologies make it difficult to achieve rapid, accurate, and on-site detection of the organophosphorus pesticide dimethoate. Traditional instrumental analysis methods are complex and costly, and are not suitable for grassroots or on-site screening.
A glassy carbon electrode modified with graphene and gold nanoparticles was used to construct an enzyme biosensor in combination with acetylcholinesterase. The residue of dimethoate was detected by electrochemical methods, and the electrochemical response signal was reduced by the inhibition of acetylcholinesterase activity by dimethoate.
It enables rapid, accurate, and on-site detection of dimethoate with high sensitivity, a detection limit of 5.87 × 10⁻⁴ mM, and a correlation coefficient of 9.91 × 10⁻¹. It exhibits good reproducibility and stability and is suitable for safety screening of agricultural products.
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Figure CN121298848A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of electrochemical sensors, in particular to an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate and preparation and application thereof. BACKGROUND
[0002] In modern agriculture, organophosphorus pesticides are widely used in the planting process of agricultural products such as fruits and vegetables due to their high insecticidal efficiency and wide application range. However, such pesticides are prone to remain on the surface or inside of agricultural products, and if not detected and treated in time, they can easily enter the human body through the food chain, causing serious health threats. Among them, dimethoate, as a typical high-toxicity organophosphorus insecticide, has neurotoxicity and potential carcinogenicity, and has been listed as a limited or banned object in many countries. According to the joint report of the Food and Agriculture Organization of the United Nations and the World Health Organization, organophosphorus poisoning causes more than 3 million poisoning events and about 200,000 deaths per year, which brings heavy burden to public health and social economy.
[0003] Traditional organophosphorus detection methods mainly rely on instrument analysis techniques such as high performance liquid chromatography, gas chromatography and mass spectrometry, which have high sensitivity and accurate results, but the instruments are expensive, the sample pretreatment is complex, and the detection period is long, which is not suitable for rapid screening at the grassroots or on-site. With the increasing demand for food safety supervision from "farmland to dining table", the development of rapid detection methods with high sensitivity, high selectivity, simple operation and suitability for on-site detection has become a current research hotspot. SUMMARY
[0004] The present application aims at providing an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate and preparation and application thereof to solve the above-mentioned defects in the prior art. The present application develops a high-selectivity and high-sensitivity electrochemical enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate, which facilitates rapid, accurate and on-site safety screening of agricultural products.
[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides a preparation method of an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate, comprising the following steps: (1) Preparation of GR / GCE Take the glassy carbon electrode (GCE), and sequentially polish the GCE on a polishing cloth with 0.3 µm and 0.05 µm aluminum oxide powder, and then ultrasonically clean the polished GCE in methanol and ultrapure water, each for 0.33 h, to obtain a pretreated glassy carbon electrode (GCE) for standby; take 1.2 mg of graphene (GR), disperse it in 1 mL of N-methylpyrrolidone, and ultrasonically treat it for 0.33 h at an ultrasonic power of 300 W to obtain a GR dispersion; evenly drop 10 μL of the GR dispersion on the surface of the pretreated glassy carbon electrode (GCE), and dry it at 40°C in air for 2 h to obtain a graphene modified glassy carbon electrode (GR / GCE); (2) Preparation of AuNPs / GR / GCE Immerse the GR / GCE modified electrode in a mixed solution containing 5 mM tetrachloroauric acid and 500 mM sulfuric acid, and perform electrochemical deposition by cyclic voltammetry (CV), with the potential scanning range set to -400 ~ 800 mV and the scanning rate set to 50 mV / s, and perform a total of 15 scanning cycles. After deposition is completed, a gold nanoparticle loaded graphene modified glassy carbon electrode (AuNPs / GR / GCE) is obtained. (3) Preparation of AChE / AuNPs / GR / GCE Drop 5 μL of a 10 U / mL acetylcholinesterase solution on the surface of the AuNPs / GR / GCE, and dry it at room temperature to obtain an acetylcholinesterase biosensor based on a gold nanoparticle / graphene modified glassy carbon electrode (AChE / AuNPs / GR / GCE), and store it in a 4°C refrigerator for standby.
[0006] In a second aspect, the present application provides an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate, which is prepared by the above preparation method.
[0007] In a third aspect, the present application provides an application of an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate in the detection of dimethoate content in agricultural products. When the enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate is used to detect dimethoate content by DPV method, the dimethoate concentration is in the range of 2.18×10 -6 ~ 2.18×10 -2 mM, the logarithm of the dimethoate concentration and the corresponding enzyme inhibition rate show a good linear relationship, I R = 101.45 × 10 -1 logC + 336.28 × 10 -1 , the correlation coefficient is 9.91 × 10 -1 , I R is the enzyme inhibition rate, C is the concentration, the unit is mM, and the lowest detection limit is 5.87 × 10-4 mM, signal-to-noise ratio S / N = 3, and the limit of quantification is 9.18 × 10 -4 mM, signal-to-noise ratio S / N = 10.
[0008] Compared with the prior art, the application has the beneficial technical effects that: the application utilizes graphene (GR) and gold nanoparticles (AuNPs) to form AuNPs / GR / GCE, significantly reduces the electrochemical impedance of the glassy carbon electrode, increases the effective specific surface area and the electron transfer rate of the surface, provides rich binding sites for the immobilization of enzyme molecules, improves the stability of the enzyme, and reduces the degradation or inactivation of the enzyme in the use process; acetylcholinesterase is taken as a recognition probe, AChE / AuNPs / GR / GCE is constructed on the AuNPs / GR / GCE through a self-assembly method, and the organophosphorus pesticide dimethoate can hinder the hydrolysis of iodized acetylthiocholine to generate thiocholine, so that the electrochemical response signal is reduced, the dimethoate has a good electrochemical response signal, and thus the rapid, accurate and on-site safe screening of the organophosphorus pesticide dimethoate is realized. BRIEF DESCRIPTION OF DRAWINGS
[0009] Figure 1 is a construction principle schematic diagram of an enzyme biosensor for rapid detection of the organophosphorus pesticide dimethoate in the embodiments of the application.
[0010] Figure 2 is a material physical and chemical structure characterization and electrochemical characterization diagram in the embodiments of the application, wherein (A)-(D) are respectively a Raman spectrum, an X-ray diffraction spectrum, a Fourier transform infrared spectrum and a scanning electron microscope (SEM) image of GR, (E) is an SEM image of AuNPs / GR / GCE, (F) is a cyclic voltammetry (CV) spectrum of AuNPs / GR / GCE, (G)-(I) are respectively Nyquist diagrams of GCE, GR / GCE and AuNPs / GR / GCE, and (J)-(L) are respectively Bode diagrams of GCE, GR / GCE and AuNPs / GR / GCE.
[0011] Figure 3 is an electrode interface characterization diagram of AuNPs / GR / GCE in the embodiments of the application, wherein (A) is a Q-t curve of GCE, GR / GCE and AuNPs / GR / GCE, (B) is a Q-t curve of GCE, GR / GCE and AuNPs / GR / GCE, (C) is a schematic diagram of the increase of the effective specific surface area of GCE→AuNPs / GR / GCE, (D)-(H) are CV curves of AChE / AuNPs / GR / GCE, the relationship between the peak current (I 1 / 2 ) and the scan rate (v), I P and v P and v 1 / 2Relationship between logv and the peak current, Relationship between logv and the peak potential (E P Relationship between logv and the peak potential (E P Relationship between logv and the peak potential (E
[0012] Figure 4 Fig. 6 is the electrochemical characterization of AChE / AuNPs / GR / GCE for pyrethrum and the results of condition optimization in the embodiments of the present application, wherein (A) is the schematic diagram of AChE / AuNPs / GR / GCE for detecting pyrethrum, (B) is the DPV curve of AChE / AuNPs / GR / GCE for detecting pyrethrum, (C)-(H) are the column charts of AChE / AuNPs / GR / GCE for detecting pyrethrum under the conditions of different GR solution concentrations, AuNPs deposition circles, pH, ATCHI concentration, AChE modification amount, and inhibition time, respectively, and (I)-(N) are the CV curves of AChE / AuNPs / GR / GCE for detecting pyrethrum under the conditions of different GR dispersion concentrations, AuNPs deposition circles, pH, ATCHI concentration, AChE modification amount, and inhibition time, respectively.
[0013] Figure 5 Fig. 7 is the performance evaluation results of the AChE / AuNPs / GR / GCE enzyme biosensor in the embodiments of the present application, wherein (A) is the DPV curve of detecting pyrethrum in the range of 2.18 × 10 -6 ~ 2.18 × 10 -2 mM, (B) is the relationship between the inhibition rate and the logarithm of the concentration of pyrethrum in the DPV detection, (C)-(E) are the reproducibility, stability, and specificity test results of the AChE / AuNPs / GR / GCE enzyme biosensor, and (F) is the schematic diagram of the electrode used in the performance evaluation test of the present application. DETAILED DESCRIPTION
[0014] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.
[0015] In the present application, the raw materials involved are all commercially available products well known in the art, unless otherwise specified.
[0016] EMBODIMENT As Figure 1As shown, the embodiment provides a preparation method of an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate, comprising the following steps: (1) Preparation of GR / GCE Take a glassy carbon electrode (GCE), and polish the GCE on a polishing cloth with 0.3 μm and 0.05 μm aluminum oxide powder in sequence for step-by-step fine polishing. Then, place the polished GCE in methanol and ultrapure water in sequence for ultrasonic cleaning, with each cleaning lasting 0.33 h. Thus, a pretreated glassy carbon electrode (GCE) is obtained and reserved for later use. Take 1.2 mg of graphene (GR), and disperse the GR in 1 mL of N-methylpyrrolidone by ultrasonic treatment for 0.33 h at an ultrasonic power of 300 W. Thus, a GR dispersion liquid is obtained. Then, drop 10 μL of the GR dispersion liquid uniformly on the surface of the pretreated glassy carbon electrode (GCE), and place the GCE in air at 40℃ for 2 h for drying. Thus, a graphene modified glassy carbon electrode (GR / GCE) is prepared. (2) Preparation of AuNPs / GR / GCE Immerse the GR / GCE modified electrode in a mixed solution containing 5 mM tetrachloroauric acid and 500 mM sulfuric acid, and perform electrochemical deposition by cyclic voltammetry (CV). The potential scanning range is set to -400 ~ 800 mV, and the scanning rate is 50 mV / s. Perform 15 scanning cycles in total. After deposition is completed, a gold nanoparticle loaded graphene modified glassy carbon electrode (AuNPs / GR / GCE) is obtained. (3) Preparation of AChE / AuNPs / GR / GCE Drop 5 μL of a 10 U / mL acetylcholinesterase solution on the surface of the AuNPs / GR / GCE, and air dry at room temperature. Thus, an acetylcholinesterase biosensor based on a gold nanoparticle / graphene modified glassy carbon electrode (AChE / AuNPs / GR / GCE) is obtained, and the biosensor is stored in a 4℃ refrigerator for later use.
[0017] As shown in Figure 2 (A)-(E), after the AChE / AuNPs / GR / GCE modified electrode is constructed, the materials involved in the preparation process are characterized by Raman spectrum, X-ray diffraction spectrum, Fourier transform infrared spectrum, and scanning electron microscope chart. Figure 2 (F)-(L), electrochemical measurements are performed on the GCE, GR / GCE, and AuNPs / GR / GCE. Figure 2 The test results show that the GR / GCE and AuNPs / GR / GCE modified electrodes are successfully prepared.
[0018] The electrochemical measurements include cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The measurement sensitivity of the CV method is 10 -4, the potential range was -0.4 V ~ 0.8 V, and the scanning rate was 0.10 V / s. The frequency parameter of EIS was 1 Hz ~ 1×10 -6 Hz, the static time was 2 s, and the voltage amplitude was 0.005 V. The sensitivity of DPV was 10 -4 , the potential range was 0.4 V ~ -0.4V, the static time was 2 s, the voltage amplitude was 0.05 V, the pulse period was 0.5 s, and the sampling width was 0.0167 s. The test base solution was a mixed solution containing 2 mM [Fe(CN)6] 3- / 4- and 0.1M KCl.
[0019] As shown in Figure 3 (A)-(C), we used the chronocoulometry method to determine the specific surface area of GCE, GR / GCE, and AuNPs / GR / GCE. Through calculation, the effective specific surface areas of GCE, GR / GCE, and AuNPs / GR / GCE were 19.13 × 10 -3 cm 2 , 21.47 ×10 -2 cm 2 , and 46.38 × 10 -2 cm 2 , respectively. As shown in Figure 3 (D)-(K), through sweep speed analysis, a higher electron transfer rate constant k s = 8.19 × 10 1 cm / s was obtained, indicating that the transfer speed of electrons between the electrode and the substances in the solution was very fast, which proved that the modified electrode had very excellent electrocatalytic activity. The electron transfer number n was about 8.48 × 10 -2 , which was very close to the theoretical single electron transfer of the [Fe(CN)6] 3- / 4- system, which verified the correctness of the reaction mechanism. The electron transfer coefficient a was 0.54, indicating that the energy barriers of the forward and reverse electron transfers were relatively symmetrical.
[0020] AChE can catalyze the generation of thiocholine from iodized thioacetylcholine, and thiocholine has redox activity and can be catalyzed by the AuNPs / GR modified on the electrode surface to generate an electrochemical signal that can be detected.
[0021] As shown in Figure 4 (A)-(B), when no dimethoate was added, the current response was high, and when the concentration of dimethoate was 2.18 × 10 - 4The current response was significantly reduced after the mM dimethoate solution, indicating that dimethoate inhibited the redox reaction of the decomposition of iodized thioacetylcholine into thiocholine on the electrode surface, thereby reducing the response current specifically. As shown in Figure 4 (C) - (N), by optimizing the GR dispersion liquid concentration, AuNPs deposition circle number, solution environment pH, iodized thioacetylcholine concentration, acetylcholinesterase modification amount, inhibition time, the AChE / AuNPs / GR / GCE sensor under the optimal conditions was obtained.
[0022] Under the optimal experimental conditions, the dimethoate standard solution of different concentrations was determined by DPV method. To prepare the dimethoate standard solution for quantitative analysis, a certain volume of dimethoate anhydrous methanol stock solution was diluted in phosphate buffer solution (PBS). The prepared standard solution concentrations were 0.22 × 10 -2 mM, 0.44 × 10 -2 mM, 2.18 × 10 -2 mM, 4.36 ×10 -2 mM, 2.18 × 10 -1 mM, 43.61 × 10 -2 mM, 218.04 × 10 -2 mM, 436.08 × 10 -2 mM and 218.04 × 10 -1 mM.
[0023] As shown in Figure 5 (A) - (B), in the range of 2.18 × 10 -6 ~ 2.18×10 -2 mM dimethoate concentration, the logarithm of dimethoate concentration and its corresponding enzyme inhibition rate showed a good linear relationship, I R = 101.45 × 10 -1 logC + 336.28 ×10 -1 , the correlation coefficient was 9.91 × 10 -1 , I R is the enzyme inhibition rate, C is the concentration, unit mM. The lowest detection limit was calculated to be 5.87 × 10 -4 mM, the signal-to-noise ratio S / N = 3, the limit of quantification was 9.18 × 10 -4 mM, the signal-to-noise ratio S / N = 10.
[0024] As shown in Figure 5(C) - (E) shown, reproducibility, stability, specificity test of AChE / AuNPs / GR / GCE enzyme biosensor. Five modified electrodes were prepared in parallel, and the concentration of dimethoate 2.18 × 10 -4 mM dimethoate-containing solution, Figure 5 (C) The results show that the RSD is 0.71%, indicating that the electrochemical platform based on AChE / AuNPs / GR composite material has good reproducibility. At room temperature 25℃, the electrode was sealed and stored for 5 days, 10 days, 15 days, 20 days and 25 days respectively, and the concentration of dimethoate 2.18 × 10 -4 mM dimethoate-containing solution, Figure 5 (D) as shown, the detection results of the electrochemical sensor are 98.50% ~ 99.80% of the initial performance, indicating that the electrochemical sensor has good stability. The specificity of AChE / AuNPs / GR / GCE was evaluated by detecting different component interference items, and the detection items in the interference dimethoate residue detection included dimethoate, L-methionine + dimethoate, glycine + dimethoate (Glycine + Dimethoate), ascorbic acid + dimethoate (Ascorbic acid + Dimethoate), starch soluble + dimethoate (Starch soluble + Dimethoate), glucose + dimethoate (Glucose + Dimethoate), L-methionine, glycine, ascorbic acid, starch soluble, glucose. The signal change when the interferent and dimethoate residue exist is compared, as shown in Figure 5 (E) shown, the DPV response signal of the sensor after incubation of the interference item group has almost no difference, and the RSD is less than 1.46%. It is indicated that the AChE / AuNPs / GR / GCE based on enzyme inhibition principle has excellent specificity for dimethoate residue detection. As shown in Figure 5 (F) shown, the electrodes used in the performance evaluation test of the application include: AChE / AuNPs / GR / GCE working electrode, platinum wire auxiliary electrode (Pt), saturated calomel reference electrode (SCE).
[0025] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement within the technical solutions and concepts of the present application shall be included in the protection scope of the present application.
Claims
1. A method for preparing an enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate, characterized in that, Comprising the following steps: (1) Preparation of GR / GCE Take GCE, and polish the GCE with 0.3 µm and 0.05 µm alumina powder on a polishing cloth in stages, and then clean the polished GCE in methanol and ultrapure water for 0.33 h each time, to obtain a pretreated GCE for standby use; take 1.2 mg of GR, disperse it in 1 mL of N-methyl pyrrolidone, and ultrasonically treat it for 0.33 h at an ultrasonic power of 300 W, to obtain a GR dispersion; evenly drop 10 μL of the GR dispersion on the surface of the pretreated GCE, and dry it at 40℃ in air for 2 h, to obtain a GR / GCE; (2) Preparation of AuNPs / GR / GCE Immerse the GR / GCE modified electrode in a mixed solution containing 5 mM tetrachloroauric acid and 500 mM sulfuric acid, and perform electrochemical deposition by CV method, with the potential scanning range set to -400 ~ 800 mV and the scanning rate set to 50 mV / s, for a total of 15 scanning cycles; after deposition is completed, an AuNPs / GR / GCE is obtained. (3) Preparation of AChE / AuNPs / GR / GCE Drop 5 μL of 10 U / mL acetylcholinesterase solution on the surface of the AuNPs / GR / GCE, and dry it at room temperature, to obtain an AChE / AuNPs / GR / GCE, which is stored in a 4℃ refrigerator for standby use.
2. An enzyme biosensor for rapid detection of organophosphorus pesticide dimethoate, characterized in that, Prepared by the preparation method of claim 1.
3. The use of the enzyme biosensor for quick detection of organophosphorus pesticide dimethoate in the detection of the content of organophosphorus pesticide dimethoate in agricultural products, wherein the enzyme biosensor for quick detection of organophosphorus pesticide dimethoate is used to detect the content of dimethoate by DPV method, the concentration of dimethoate is in the range of 2.18 x 10 -6 ~ 2.18 x 10 -2 mM, the logarithm of the concentration of dimethoate and the corresponding enzyme inhibition rate present a good linear relationship, I R = 101.45 x 10 -1 logC + 336.28 x 10 -1 , the correlation coefficient is 9.91 x 10 -1 , I R is the enzyme inhibition rate, C is the concentration, the unit is mM, the minimum detection limit is 5.87 x 10 -4 mM, the signal-to-noise ratio S / N = 3, the quantitative limit is 9.18 x 10 -4 mM, the signal-to-noise ratio S / N = 10.