Method for rapidly detecting pesticide residues in fruits and vegetables

Abstract

The invention provides a method for rapidly detecting pesticide residues in fruits and vegetables, and belongs to the field of pesticide analysis and detection. The method comprises the following steps: mixing a copper nanoparticle / reduced graphene oxide / chitosan composite material, acetylcholin esterase, choline oxidase and a cross-linking agent glutaraldehyde solution for reaction to obtain a double-enzyme functionalized nano composite material; dispensing a chitosan solution on the surface of a working electrode of a screen-printed electrode, drying to form a film, and covalently modifying the surface of the electrode with the bienzyme functionalized nano composite material through a cross-linking agent glutaraldehyde to obtain a sensing electrode; according to the method, acetylcholine is taken as a substrate, a sensing electrode is used, a chronoamperometry is adopted, inhibition ratios of pesticide standard substance solutions with different known concentrations to electrode response current are detected, and a standard curve between the pesticide concentration and the current inhibition ratio is established; and calculating the pesticide residue concentration in the fruit and vegetable sample to be detected according to the current inhibition ratio of the liquid to be detected and the standard curve. Therefore, the sensitivity and accuracy of pesticide residue detection in fruits and vegetables are improved.

Description

Technical Field

[0001] This application relates to the field of pesticide analysis and detection technology, and in particular to a method for rapid detection of pesticide residues in fruits and vegetables. Background Technology

[0002] With increasing public concern about food safety, rapid detection technologies for pesticide residues in fruits and vegetables have become a crucial link in ensuring agricultural product safety. Currently, pesticide residue detection mainly relies on large-scale laboratory instrumental analysis methods, such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). While these methods offer high accuracy and precision, they also have inherent limitations, including expensive equipment, complex operation, lengthy processing times, and stringent sample pretreatment requirements, making them unsuitable for applications demanding high timeliness and widespread applicability.

[0003] To address the demand for rapid detection, detection methods based on biosensing principles, particularly electrochemical biosensors, have been widely studied due to their ease of operation, low cost, and suitability for miniaturization and field deployment. Among these, sensors using acetylcholinesterase as the recognition element, which quantify activity by detecting the degree of inhibition by organophosphates and carbamates, represent one of the most mainstream technical routes. However, existing rapid detection technologies still face several technical bottlenecks, hindering further improvements in detection performance and large-scale practical applications. The core issues are concentrated in two aspects: insufficient sensitivity and the need to improve accuracy. Firstly, regarding sensitivity, traditional single-enzyme (acetylcholinesterase) systems typically rely on the direct electrochemical oxidation of enzyme catalytic products (such as thiocholine) to generate signals. Their limited electrochemical activity results in weak signal amplification, insignificant response to trace pesticides, and difficulty in meeting increasingly stringent detection limits. Secondly, regarding accuracy, various electroactive substances (such as ascorbic acid and polyphenols) in complex fruit and vegetable sample matrices are prone to redox reactions on the electrode surface, severely interfering with the detection signal. Simultaneously, if the enzyme immobilization method on the electrode surface is not stable enough, it can easily lead to enzyme activity loss or fluctuations in sensor performance. These factors directly affect the reliability, repeatability, and anti-interference ability of the detection results. Therefore, improving the detection sensitivity and accuracy of pesticide residues in fruits and vegetables is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] This application provides a method for rapid detection of pesticide residues in fruits and vegetables to solve the following technical problem: how to improve the sensitivity and accuracy of detection methods for pesticide residues in fruits and vegetables based on biosensor principles.

[0005] This application provides a method for rapid detection of pesticide residues in fruits and vegetables, the method comprising the following steps:

[0006] S1. Copper nanoparticles / reduced graphene oxide / chitosan composite material, acetylcholinesterase, choline oxidase and crosslinking agent glutaraldehyde solution are mixed and reacted to obtain dual-enzyme functionalized nanocomposite material.

[0007] S2. Chitosan solution is drop-coated onto the working electrode surface of the screen-printed electrode and dried to form a film. Then, the dual-enzyme functionalized nanocomposite material is covalently modified onto the electrode surface using the crosslinking agent glutaraldehyde to obtain the sensing electrode.

[0008] S3. Using acetylcholine as a substrate, and employing the aforementioned sensing electrode, the inhibition rate of the electrode response current of pesticide standard solutions of different known concentrations was detected by the chronoamperometry method, and a standard curve between pesticide concentration and current inhibition rate was established.

[0009] S4. The fruit and vegetable samples to be tested are pretreated to obtain the test solution, and then the current inhibition rate of the test solution is measured. Based on the current inhibition rate of the test solution and the standard curve, the pesticide residue concentration in the fruit and vegetable samples to be tested is calculated.

[0010] Optionally, in step S1, the ratio of the activity units of the acetylcholinesterase to the choline oxidase is (1-3):1.

[0011] The mass ratio of the copper nanoparticle / reduced graphene oxide / chitosan composite material to the total mass of the acetylcholinesterase and the choline oxidase is (50-200):1.

[0012] The final mass concentration of the crosslinking agent glutaraldehyde solution in the reaction system is 0.5-2%.

[0013] Optionally, in step S1, the preparation method of the copper nanoparticle / reduced graphene oxide / chitosan composite material includes the following steps:

[0014] S101. Mix the graphene oxide dispersion, chitosan acetate solution and copper salt solution, and adjust the pH to 4.0-5.0 to obtain the precursor mixture.

[0015] S102. The precursor mixture is irradiated with ultraviolet light to simultaneously reduce and recombine graphene oxide and copper ions using photochemical reduction. After post-treatment, the copper nanoparticle / reduced graphene oxide / chitosan composite material with a three-dimensional porous composite structure is obtained.

[0016] Optionally, the mass concentration of the graphene oxide dispersion is 0.5–2 mg / mL;

[0017] The chitosan acetate solution has a mass-volume concentration of 0.5–2 wt%.

[0018] The concentration of the copper salt solution is 0.01–0.1 M;

[0019] The volume ratio of the graphene oxide dispersion, the chitosan acetate solution, and the copper salt solution is 5:(5-15):1.

[0020] Optionally, the wavelength of the ultraviolet light irradiation is 254–365 nm, and the vertical irradiation time is 1–4 h.

[0021] Optionally, in step S1, the amount of acetylcholinesterase used is 5-20 U / mL and the amount of choline oxidase used is 2-15 U / mL, based on the volume of the reaction system.

[0022] Optionally, in step S2, the drop volume of the chitosan solution is 3–10 µL, and the thickness of the dried film is 1–5 µm.

[0023] Optionally, in step S2, the mass concentration of the crosslinking agent glutaraldehyde is 1-3%, and the crosslinking time is 10-30 min.

[0024] Optionally, in step S3, the concentration of the acetylcholine substrate in the detection system is 0.5–2.0 mM.

[0025] Optionally, in steps S3 and S4, the operating potential of the chronoamperometry is +0.35 to +0.45V relative to the integrated Ag / AgCl reference electrode on the screen-printed electrode.

[0026] The technical solutions provided in this application have the following advantages compared with the prior art:

[0027] This application provides a method for rapid detection of pesticide residues in fruits and vegetables. Through an integrated material design and detection strategy, the sensitivity and accuracy of pesticide residue detection in fruits and vegetables are systematically improved from three levels: signal generation, signal transmission and signal measurement.

[0028] First, at the signal generation level, this application creatively employs a dual-enzyme cascade system of acetylcholinesterase and choline oxidase. Acetylcholinesterase is a key component in recognizing pesticides and generating an initial inhibitory effect, while choline oxidase acts as a signal transducer and amplifier. In the presence of pesticides, the inhibited activity of acetylcholinesterase leads to a reduction in its catalytic product choline, which in turn reduces the substrate of choline oxidase, ultimately resulting in a decrease in the production of its final product, hydrogen peroxide. This design transforms the inhibitory effect of a single pesticide molecule on acetylcholinesterase into a significant change in hydrogen peroxide production through a two-stage enzymatic reaction, achieving a cascaded amplification of the chemical signal for trace amounts of pesticides. This is the core principle for improving detection sensitivity.

[0029] Secondly, at the signal transduction and conversion level, this application constructs a sensing interface of dual-enzyme functionalized copper nanoparticles / reduced graphene oxide / chitosan nanocomposite material. This composite material, serving as both an enzyme carrier and a site for electrochemical reactions, offers multiple structural advantages: the three-dimensional chitosan network provides a biocompatible environment for enzyme immobilization, helping to maintain enzyme activity; the uniformly loaded copper nanoparticles exhibit highly efficient electrocatalytic activity towards hydrogen peroxide, the end product of the dual-enzyme reaction, efficiently converting changes in hydrogen peroxide chemical concentration into a strong electrochemical oxidation current signal; and the highly conductive network of reduced graphene oxide ensures that the electrons generated by catalysis are rapidly collected and conducted to the electrode. This synergy between "enzyme-induced amplification" and "nanomaterial electrocatalytic amplification" achieves dual amplification of the initial inhibition signal, thereby significantly improving detection sensitivity.

[0030] Finally, at the signal measurement and quantification level, this application employs a scientific analytical method combining chronoamperometry with standard curve quantification. Chronoamperometry measures steady-state current under a fixed optimized potential, exhibiting stability and strong anti-interference capabilities. By establishing a "pesticide concentration-current inhibition rate" standard curve using pesticide standards of known concentrations beforehand, the current decrease (inhibition rate) caused by the test sample is compared with this curve to calculate the specific concentration of pesticide residues. This quantitative method, based on relative change values ​​(inhibition rate) rather than absolute current values, effectively reduces systematic errors caused by batch differences in sensors or minor environmental fluctuations, and strictly adheres to the standards of quantitative analytical chemistry, fundamentally ensuring the accuracy and reliability of the detection results.

[0031] In summary, this application utilizes a combination of chemical amplification via a dual-enzyme cascade, electrocatalytic amplification at the interface of nanocomposite materials, and scientific quantification via a standard curve method. These three interconnected elements together constitute a complete detection system with high sensitivity and high accuracy. Attached Figure Description

[0032] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0033] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a flowchart illustrating a method for rapidly detecting pesticide residues in fruits and vegetables, provided as an embodiment of this application. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Figure 1 This is a flowchart illustrating a method for rapidly detecting pesticide residues in fruits and vegetables, provided as an embodiment of this application.

[0037] like Figure 1 As shown in the embodiments of this application, a method for rapid detection of pesticide residues in fruits and vegetables is provided, the method comprising the following steps:

[0038] S1. Copper nanoparticles / reduced graphene oxide / chitosan composite material, acetylcholinesterase, choline oxidase and crosslinking agent glutaraldehyde solution are mixed and reacted to obtain dual-enzyme functionalized nanocomposite material.

[0039] S2. Chitosan solution is drop-coated onto the working electrode surface of the screen-printed electrode and dried to form a film. Then, the dual-enzyme functionalized nanocomposite material is covalently modified onto the electrode surface using the crosslinking agent glutaraldehyde to obtain the sensing electrode.

[0040] S3. Using acetylcholine as a substrate, and employing the aforementioned sensing electrode, the inhibition rate of the electrode response current of pesticide standard solutions of different known concentrations was detected by the chronoamperometry method, and a standard curve between pesticide concentration and current inhibition rate was established.

[0041] S4. The fruit and vegetable samples to be tested are pretreated to obtain the test solution, and then the current inhibition rate of the test solution is measured. Based on the current inhibition rate of the test solution and the standard curve, the pesticide residue concentration in the fruit and vegetable samples to be tested is calculated.

[0042] This method provides a rapid pesticide residue detection approach based on dual-enzyme cascade amplification and nanomaterial-enhanced electrochemical signals. Its core lies in constructing a highly sensitive and stable biosensor interface and, through a clever signal conversion mechanism, transforming the trace pesticide inhibitory effect into a significant and easily measurable electrical signal change. This method creatively employs an acetylcholinesterase-choline oxidase dual-enzyme cascade reaction, combined with a three-dimensional porous nanocomposite material of copper nanoparticles / reduced graphene oxide / chitosan exhibiting highly efficient electrocatalytic activity towards hydrogen peroxide, achieving secondary signal amplification and thus significantly improving detection sensitivity and accuracy.

[0043] Specifically, the method involves four key steps: The first step is to construct a dual-enzyme functionalized nanocomposite material, which aims to prepare the core functional unit of the sensor. Acetylcholinesterase and choline oxidase are simultaneously covalently immobilized on a pre-synthesized three-dimensional porous copper nanoparticle / reduced graphene oxide / chitosan composite material using glutaraldehyde as a crosslinking agent. Acetylcholinesterase acts as a "molecular switch" specifically recognizing organophosphorus and carbamate pesticides, and its activity is inhibited by these pesticides. Choline oxidase is responsible for converting the previous product into hydrogen peroxide. This nanocomposite material not only provides a large specific surface area and good biocompatibility for enzyme immobilization, but the synergistic effect of its core components, copper nanoparticles and reduced graphene oxide, also endows it with excellent electrocatalytic activity for hydrogen peroxide, laying the foundation for the efficient conversion of biochemical signals into electrical signals.

[0044] The second step is to fabricate the sensing electrode, essentially building a robust "signal conversion station." On the working area of ​​the portable screen-printed electrode, a chitosan solution is first drop-coated to form a pre-modification layer, increasing the electrode surface's affinity and active sites. Then, glutaraldehyde is used again for cross-linking to firmly modify the dual-enzyme functionalized nanocomposite material obtained in the first step onto the electrode, thus constructing a complete and stable disposable electrochemical biosensor. The use of screen-printed electrodes is a crucial prerequisite for achieving portability and rapid detection in this approach.

[0045] The third step is to establish a standard curve, i.e., to plot a precise "quantitative scale." Using the prepared sensing electrode, detection is performed by chronoamperometry in a buffer solution containing the substrate acetylcholine. In the absence of pesticides, the two-enzyme cascade reaction proceeds smoothly, producing stable hydrogen peroxide, which is oxidized at the electrode to generate a high-intensity background anolyte current. When pesticides are added, acetylcholinesterase activity is inhibited, leading to a decrease in hydrogen peroxide production and a drop in the measured current value. By measuring the current inhibition rate caused by a series of pesticide standards at known concentrations, a quantitative standard curve between "pesticide concentration and current inhibition rate" can be established, providing a calibration model for subsequent sample analysis.

[0046] The fourth step is sample testing and judgment, which is to complete the actual "on-site rapid testing". After the fruit and vegetable samples to be tested are extracted and pretreated according to standard methods, the current inhibition rate of the extract on the sensing electrode is measured under exactly the same conditions. This value is then substituted into the standard curve established in the third step, and the pesticide residue concentration in the sample can be directly calculated. This allows for comparison with national standards to determine whether the residue exceeds the limit.

[0047] The significant advantages of this approach lie in the perfect combination of speed and accuracy. Regarding speed, the core materials are synthesized using a one-pot UV reduction method, simplifying sensor preparation. The rapid dual-enzyme catalysis and electrochemical detection process, combined with portable electrodes and equipment, allows the entire process from sample processing to result acquisition to be completed within 30 minutes, greatly meeting the needs of rapid on-site screening. In terms of accuracy, the dual signal amplification mechanism of the dual-enzyme cascade and nanomaterial electrocatalysis endows the method with extremely high sensitivity, achieving a detection limit down to trace levels. Based on the specific inhibition of acetylcholinesterase, the method exhibits good selectivity. Covalent cross-linking immobilization with glutaraldehyde ensures high sensor stability and reproducibility. The use of a standard curve method for scientific quantification ensures objective and reliable results.

[0048] In summary, this method deeply integrates innovative material preparation technology, efficient biocatalytic design, and a portable electrochemical detection platform, successfully addressing the comprehensive needs for high sensitivity, high accuracy, rapid portability, and low-cost operation in the detection of pesticide residues in fruits and vegetables. It is an advanced analytical technology with great market application prospects.

[0049] In some embodiments, in step S1, the ratio of the active units of the acetylcholinesterase to the choline oxidase is (1-3):1.

[0050] The mass ratio of the copper nanoparticle / reduced graphene oxide / chitosan composite material to the total mass of the acetylcholinesterase and the choline oxidase is (50-200):1.

[0051] The final mass concentration of the crosslinking agent glutaraldehyde solution in the reaction system is 0.5-2%.

[0052] In some embodiments, in step S1, the amount of acetylcholinesterase used is 5-20 U / mL based on the volume of the reaction system, and the amount of choline oxidase used is 2-15 U / mL.

[0053] The ratio of acetylcholinesterase to choline oxidase activity units was set to (1-3):1. This ensures that acetylcholinesterase, as the first-stage reaction and pesticide inhibition target, has sufficient or slightly high catalytic capacity, which can continuously provide sufficient substrate (choline) for the second-stage choline oxidase, thereby ensuring that the entire cascade reaction proceeds efficiently and smoothly and obtains a strong initial signal.

[0054] The ratio of the mass of the copper nanoparticle / reduced graphene oxide / chitosan composite material to the total mass of the two enzymes is (50-200):1. This ratio makes full use of the huge specific surface area of ​​the nanocomposite material, providing ample space for enzyme immobilization, so that enzyme molecules can be distributed at a suitable density. This maximizes the loading of active enzyme molecules while avoiding steric hindrance and mass transfer limitation between enzyme molecules due to over-dense loading, which is conducive to maintaining high enzyme activity.

[0055] The final mass concentration of the crosslinking agent glutaraldehyde in the reaction system is 0.5-2%. This concentration range can form appropriate covalent crosslinks between the composite material carrier and the enzyme molecules, achieving firm fixation of the enzyme and effectively preventing enzyme leakage during use. At the same time, it avoids excessive modification and inactivation of the enzyme active site caused by excessive crosslinking agent concentration.

[0056] In some embodiments, step S1, the preparation method of the copper nanoparticle / reduced graphene oxide / chitosan composite material includes the following steps:

[0057] S101. Mix the graphene oxide dispersion, chitosan acetate solution and copper salt solution, and adjust the pH to 4.0-5.0 to obtain the precursor mixture.

[0058] S102. The precursor mixture is irradiated with ultraviolet light to simultaneously reduce and recombine graphene oxide and copper ions using photochemical reduction. After post-treatment, the copper nanoparticle / reduced graphene oxide / chitosan composite material with a three-dimensional porous composite structure is obtained.

[0059] In some embodiments, the mass concentration of the graphene oxide dispersion is 0.5–2 mg / mL;

[0060] The chitosan acetate solution has a mass-volume concentration of 0.5–2 wt%.

[0061] The concentration of the copper salt solution is 0.01–0.1 M;

[0062] The volume ratio of the graphene oxide dispersion, the chitosan acetate solution, and the copper salt solution is 5:(5-15):1.

[0063] In some embodiments, the wavelength of the ultraviolet light irradiation is 254–365 nm, and the vertical irradiation time is 1–4 h.

[0064] In the aforementioned rapid detection method, the preparation of the core sensing material is fundamental to its performance. The preparation process mainly includes two key steps: the preparation of the precursor mixture (S101) and ultraviolet light reduction and recombination (S102).

[0065] Step S101 aims to construct a reaction system with a uniform molecular distribution. In this step, the graphene oxide dispersion serves as a precursor for the two-dimensional conductive framework of the composite material. Its abundant oxygen-containing functional groups not only ensure good dispersibility but also provide sites for the subsequent adsorption and reduction of copper ions. The chitosan acetate solution plays a dual role: firstly, as a biopolymer template and stabilizer, its long-chain molecules interweave between the graphene oxide sheets to prevent aggregation and lay the foundation for the formation of a three-dimensional porous network; secondly, the amino groups on its chains can coordinate with copper ions, promoting the uniform distribution of metal ions. The added copper salt solution provides an ion source for the synthesis of copper nanoparticles.

[0066] Adjusting the pH of the mixture to a slightly acidic environment of 4.0–5.0 is crucial, as this condition optimizes several processes: it ensures that the chitosan molecular chains are fully extended to perform their dispersion and template functions, promotes the stable coordination of copper ions and prevents their premature hydrolysis and precipitation, and also helps maintain the stable dispersion of graphene oxide.

[0067] The concentrations and proportions of each material were carefully designed: the graphene oxide concentration was in the range of 0.5–2 mg / mL, which could form a uniform dispersion system with suitable viscosity, providing sufficient raw materials for constructing an effective conductive network; the chitosan concentration was between 0.5–2 wt%, and its volume ratio with the graphene oxide dispersion was (5–15):5. This ratio ensured that sufficient chitosan molecules could fully encapsulate the graphene sheets and form a continuous three-dimensional network prototype, giving the final material abundant porosity; the copper salt solution concentration was controlled at 0.01–0.1 M and added at a small volume ratio, aiming to achieve efficient and controllable loading of copper nanoparticles, ensuring the generation of fine, uniform particles that are tightly anchored on the framework, forming high-density active sites.

[0068] Step S102 is a one-step reduction and recombination process driven by ultraviolet light, which is the core innovation of this method. The precursor mixture is vertically irradiated under ultraviolet light with a wavelength of 254–365 nm for 1–4 hours. Under ultraviolet irradiation, chitosan molecular chains generate free radicals with reducing capabilities, thereby initiating a chain reaction. These free radicals have reducing properties and can participate in the reduction of metal ions or initiate grafting reactions, making them green reducing agents. During this process, the oxygen-containing functional groups on the surface of graphene oxide are reduced, significantly improving its conductivity and transforming it into reduced graphene oxide. At the same time, copper ions in the solution accept photogenerated electrons or are reduced by excited organic matter, nucleating in situ and growing into copper nanoparticles. Driven by light energy, the interaction between chitosan chains is enhanced, and they synergistically self-assemble with the newly generated reduced graphene oxide and copper nanoparticles, ultimately solidifying into a structurally stable three-dimensional porous composite. After irradiation, pure solid target composite materials can be obtained through post-processing such as centrifugation, washing, and drying.

[0069] The "three-dimensional porous nanocomposite material of copper nanoparticles / reduced graphene oxide / chitosan" constructed in this application is a multi-level, integrated system whose three-dimensional structure is achieved through careful component design and a controllable ultraviolet light reduction process. The successful construction and application of this structure covers multiple synergistic levels from molecular self-assembly to macroscopic functional realization.

[0070] In terms of structural construction, this process aims to simultaneously achieve in-situ generation of copper nanoparticles, reduction of graphene oxide, and self-assembly and solidification of the chitosan network in a mild one-pot photochemical reduction process, forming a three-dimensional porous framework with suitable pore size, interconnected structure, and sufficient mechanical stability. To achieve this goal, precise control of precursor ratios, pH value, and UV irradiation conditions is required. The obtained three-dimensional porous structure possesses extremely high specific surface area and abundant interfaces, providing ample anchoring sites for subsequent high-capacity immobilization of enzyme molecules.

[0071] In terms of functional integration, ensuring that the newly generated copper nanoparticles are uniformly and firmly loaded onto the reduced graphene oxide sheets, while simultaneously ensuring that both are effectively encapsulated and stabilized by the chitosan three-dimensional network, forming a stable "point (CuNPs)-line (rGO)-surface (CS network)" composite, is the core of guaranteeing material performance. The in-situ photoreduction strategy of this application promotes the formation of strong chemical bonds or tight physical contacts between the copper nanoparticles and the reduced graphene oxide, while the chitosan network acts like a "three-dimensional scaffold" to encapsulate and fix them as a whole. This configuration not only ensures efficient electron transport between the active components but also significantly improves the electrical conductivity stability and long-term service life of the composite material.

[0072] In terms of mass transfer and signal transduction, the design of three-dimensional porous structures directly addresses the challenge of achieving efficient mass flow and electron transfer at solid-state sensing interfaces. Interconnected channels provide pathways for substrates (acetylcholine, H₂O₂) and product molecules in solution to rapidly diffuse to every active site deep within the material; simultaneously, the reduced graphene oxide is interconnected in three-dimensional space, forming a high-speed electron conduction network that runs throughout the entire structure. This synergy between "unobstructed mass transfer channels" and "high-speed electron pathways" significantly accelerates the reaction kinetics of the entire sensing interface.

[0073] In terms of compatibility with biological enzymes, the three-dimensional porous structure provides a crucial biocompatible microenvironment. The excellent biocompatibility of chitosan itself, combined with its constructed hydrophilic and flexible three-dimensional network, creates a near-native living space for immobilized acetylcholinesterase and choline oxidase, which is essential for maintaining the high catalytic activity of the enzyme proteins.

[0074] In summary, this three-dimensional porous composite structure, serving as the core framework and performance amplifier of the sensing material, integrates the electrocatalytic activity of copper nanoparticles, the high conductivity of reduced graphene oxide, and the biocompatibility and structural characteristics of chitosan through multi-component and multi-scale synergistic design and integration, resulting in a synergistic enhancement effect. This is directly reflected in the highly efficient electrocatalysis of hydrogen peroxide and the dual amplification of pesticide inhibition signals, thus supporting the comprehensive excellent performance of the proposed method, characterized by high sensitivity, high accuracy, rapid response, and good stability. The resulting copper nanoparticle / reduced graphene oxide / chitosan three-dimensional porous nanocomposite material exhibits highly efficient electrocatalytic activity for hydrogen peroxide, which is a direct result of the synergistic effect of its multi-component and multi-scale structure.

[0075] First, uniformly dispersed copper nanoparticles provide a high density of intrinsic catalytic active sites, forming the core of the direct catalytic electrochemical oxidation of hydrogen peroxide. Second, interconnected reduced graphene oxide constitutes a highly conductive three-dimensional network throughout the material, acting like an electron highway that rapidly collects and conducts electrons released from the reaction sites to the external circuit, crucial for obtaining a high-response current signal. Third, the copper nanoparticles formed through in-situ photoreduction are tightly bonded to the reduced graphene oxide sheets, forming a strongly coupled heterojunction. This interfacial interaction not only stabilizes the nanoparticles but may also optimize their electronic structure, resulting in a synergistically enhanced catalytic effect. Finally, the hydrophilic three-dimensional porous framework formed by chitosan provides the entire composite material with a large specific surface area and interconnected channels, ensuring efficient diffusion of electrolyte and hydrogen peroxide molecules to every deep active site, while facilitating the expulsion of product gases and guaranteeing efficient mass transfer in the reaction.

[0076] In summary, this composite material combines high activity, high conductivity, and excellent mass transfer structure, which together ensure a rapid, sensitive, and stable electrochemical response to hydrogen peroxide, laying a solid material foundation for the construction of highly sensitive biosensors.

[0077] In some embodiments, in step S2, the drop volume of the chitosan solution is 3 to 10 µL, and the thickness of the dried film is 1 to 5 µm.

[0078] The drop volume of the chitosan solution is 3–10 µL, which allows for the formation of a pre-modified film of uniform thickness (approximately 1–5 µm) on the working area of ​​the screen-printed electrode. This film not only increases the hydrophilicity and roughness of the electrode surface, enhancing the adhesion of subsequent functional materials, but its abundant amino functional groups also provide reaction sites for covalent cross-linking.

[0079] In some embodiments, in step S2, the mass concentration of the crosslinking agent glutaraldehyde is 1-3%, and the crosslinking time is 10-30 min.

[0080] Crosslinking is performed using a glutaraldehyde solution with a mass concentration of 1–3% for 10–30 minutes. These conditions are sufficient to form a stable covalent bond between the pre-modified chitosan layer and the dual-enzyme functionalized nanocomposite material, ensuring that the sensing layer is firmly anchored to the electrode surface and guaranteeing the mechanical stability and reproducibility of the electrochemical response of the sensor during use.

[0081] In some embodiments, in step S3, the concentration of the acetylcholine substrate in the detection system is 0.5–2.0 mM.

[0082] The concentration of acetylcholine substrate in the detection system was set at 0.5–2.0 mM. This concentration range allows the enzymatic reaction to be at or near its maximum reaction rate, thereby generating a high and stable background current signal. This provides a significant baseline for detecting pesticide inhibition effects and helps improve detection sensitivity.

[0083] In some embodiments, in steps S3 and S4, the operating potential of the chronoamperometry is +0.35 to +0.45 V relative to the integrated Ag / AgCl reference electrode on the screen-printed electrode.

[0084] The operating potential for the chronoamperometry method was set to +0.35 to +0.45 V (relative to the integrated Ag / AgCl reference electrode). This potential window was optimized for the optimal oxidation potential of hydrogen peroxide on the copper nanoparticle / reduced graphene oxide composite catalyst. At this potential, the hydrogen peroxide generated by the cascade reaction can be efficiently and specifically oxidized, producing a strong current response. Simultaneously, it effectively avoids interference currents caused by the oxidation of most common reducing substances in fruit and vegetable extracts (such as ascorbic acid and polyphenols) at this potential, thus ensuring high selectivity and accuracy of the detection method.

[0085] In summary, the method for rapid detection of pesticide residues in fruits and vegetables provided in this application integrates material innovation, principle innovation, and process innovation, demonstrating significant advantages in many aspects.

[0086] Its core advantages are primarily reflected in its ultra-high detection sensitivity and accuracy. The method creatively employs an acetylcholinesterase-choline oxidase dual-enzyme cascade reaction system, amplifying a single pesticide inhibition event into the generation and detection of multiple hydrogen peroxide molecules through two-stage biocatalysis, achieving initial chemical amplification of the signal. Furthermore, a self-designed and synthesized three-dimensional porous nanocomposite material of copper nanoparticles / reduced graphene oxide / chitosan is used as the sensing interface. The highly dispersed copper nanoparticles and highly conductive reduced graphene oxide form a strongly coupled heterojunction, exhibiting excellent electrocatalytic activity towards hydrogen peroxide, achieving secondary electrochemical amplification of the signal. This dual amplification mechanism, combined with a strategy of detection at an optimized potential to avoid interference from common electroactive substances, enables the method to achieve precise and reliable quantification of trace pesticide residues.

[0087] Secondly, this method boasts outstanding speed and ease of operation. Starting from the source, the core sensing material is synthesized using a UV-driven one-pot simultaneous reduction process, a green and concise procedure that avoids cumbersome preparation steps. Sensor assembly can be completed through simple drop-coating and cross-linking, facilitating standardization and mass production. In actual detection, based on screen-printed electrodes and a portable electrochemical workstation, the entire detection process, including sample pretreatment, pesticide inhibition reaction, and signal measurement, can be completed within 30 minutes. Compared to traditional laboratory methods relying on large instruments, this significantly shortens the detection time, making it ideal for scenarios with high timeliness requirements, such as on-site market screening and production base self-inspection.

[0088] Furthermore, this method offers excellent practicality and cost-effectiveness. The entire process design fully considers the feasibility of practical applications. The screen-printed electrodes used are inexpensive and disposable, avoiding the cumbersome polishing and regeneration steps required for traditional electrodes and reducing cross-contamination. In the core material, abundant and inexpensive copper nanoparticles replace expensive precious metals (such as gold and platinum), significantly reducing sensor manufacturing costs while ensuring high performance. In addition, the enzyme immobilization strategy through glutaraldehyde covalent cross-linking significantly improves the stability and lifespan of the sensor, enhancing the method's practical potential.

[0089] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards / industry standards / the disclosure herein; if there are no corresponding national standards / industry standards / the disclosure herein, they are performed according to generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer.

[0090] Example 1

[0091] (1) Preparation of three-dimensional porous CuNPs / rGO / CS composite material:

[0092] 5.0 mL of a 1.0 mg / mL graphene oxide (GO) aqueous dispersion was placed in a quartz beaker. Under magnetic stirring, 10.0 mL of a 1.0 wt% chitosan (CS) acetic acid solution was slowly added. Subsequently, 1.0 mL of a 0.05 M copper sulfate aqueous solution was added. The pH of the mixture was adjusted to 4.5 with dilute ammonia, and stirring was continued for 30 min to obtain a homogeneous blue-brown precursor mixture. This mixture was placed 10 cm directly below a 36 W, 365 nm UV lamp and vertically irradiated for 3 h at room temperature and in air. During irradiation, the solution color gradually changed from blue-brown to gray-black. After irradiation, the reaction solution was transferred to a centrifuge tube and centrifuged at 12000 rpm for 15 min to collect the solid product. The product was washed three times each with deionized water and anhydrous ethanol. Finally, the product was dried overnight in a vacuum drying oven at 60°C, and after grinding, a copper nanoparticle / reduced graphene oxide / chitosan (CuNPs / rGO / CS) composite powder with a three-dimensional porous structure was obtained.

[0093] (2) Preparation of dual-enzyme functionalized nanocomposites:

[0094] Accurately weigh 5.0 mg of the CuNPs / rGO / CS composite material prepared in step (1) and disperse it in 960 μL of 0.01 M phosphate buffer (PBS, pH 7.4). Sonicate for 30 min to form a uniform black dispersion. Add 10 μL of acetylcholinesterase (AChE) solution (enzyme activity 800 U / mL) and 10 μL of choline oxidase (ChOx) solution (enzyme activity 400 U / mL) to the dispersion sequentially. Gently shake the mixture at 4 °C for 1 h to allow the enzyme molecules to fully adsorb onto the surface of the composite material. Then, add 20 μL of 25% glutaraldehyde solution to bring the final mass concentration in the system to 0.5%. Place the mixture in a 4 °C refrigerator for crosslinking for 2 h. After the reaction is complete, centrifuge the mixture at 4 °C and 10,000 rpm for 10 min, discarding the supernatant to remove unfixed enzymes and excess crosslinking agent. The precipitate was washed twice with PBS buffer at pH 7.4 and then redispersed in 1.0 mL of the same buffer to obtain a stock solution of the dual-enzyme functionalized nanocomposite material, which was stored at 4°C in the dark.

[0095] (3) Construction of sensing electrodes:

[0096] A three-electrode integrated screen-printed electrode (SPE) was used, with a carbon electrode as the working electrode, a carbon electrode as the counter electrode, and a printed Ag / AgCl electrode as the reference electrode. Using a micropipette, 8 μL of a 1.0 wt% chitosan acetate solution was precisely measured and vertically drop-coated onto the center of the working electrode area. The electrode was then dried in a 40°C oven for 20 min, forming a uniform, transparent hydrophilic film of approximately 3 μm thickness on the electrode surface. The dried electrode was then immersed in a 2.0% glutaraldehyde solution and crosslinked at room temperature for 15 min. After removal, the electrode was gently rinsed with ultrapure water and dried with nitrogen. Then, 6 μL of the dual-enzyme functionalized nanocomposite material stock solution prepared in step 2 was drop-added to the glutaraldehyde-activated electrode surface, ensuring complete coverage of the working area. The electrode was incubated in a 4°C humidified chamber for 3 h to allow the composite material to be firmly fixed to the electrode surface via covalent bonds. After incubation, the electrode surface was gently rinsed three times with 0.01M PBS (pH 7.4) buffer to remove physically adsorbed material, resulting in the AChE-ChOx / (CuNPs / rGO / CS) / SPE sensing electrode, which was then stored in a humid environment at 4°C for later use.

[0097] (4) Establishment of the standard curve (taking chlorpyrifos as an example):

[0098] Using chlorpyrifos standard, a standard stock solution with a concentration of 100 mg / L was prepared with anhydrous ethanol, and then serially diluted with 0.01 M PBS (pH 7.4) to prepare a series of standard working solutions with concentrations of 1.0, 2.5, 5.0, 10.0, 25.0 and 50.0 ng / mL.

[0099] The sensing electrode prepared in step (3) was connected to a portable electrochemical workstation and inserted into a detection cell containing 5.0 mL of 0.01 M PBS (pH 7.4) as the supporting electrolyte. Chromoamperometry (it) was used for detection, with the working potential set to +0.40 V (relative to the integrated Ag / AgCl reference electrode on the screen-printed electrode). Stirring was started, and after the background current stabilized (60 s), 50 μL of 100 mM acetylcholine (ACh) chloride stock solution was injected into the detection cell, bringing the final concentration in the system to 1.0 mM. The stable oxidation current value generated at this point was recorded, and the average value was taken over 60 s, recorded as the initial current I0.

[0100] Subsequently, 50 μL of a standard working solution of chlorpyrifos at a certain concentration was added to the above system, and stirring was continued. The mixture was incubated at room temperature for 10 min to allow the pesticide molecules to fully interact with acetylcholinesterase. After incubation, the stable oxidation current value was recorded again for 60 s at the same potential, and the average value was recorded as the inhibition current I. The current inhibition rate corresponding to this concentration of chlorpyrifos was calculated using the formula: inhibition rate (%) = [(I0-I) / I0] × 100%. After each detection, a new sensing electrode was replaced, and the above operation was repeated. Each concentration was measured in parallel three times, and the average inhibition rate was recorded.

[0101] Linear regression analysis was performed with the logarithm of chlorpyrifos concentration (lgC, where C is ng / mL) as the abscissa (X) and the corresponding average current inhibition rate (Y, %) as the ordinate. The standard curve equation obtained in this embodiment is Y = 32.15X + 18.76, with a correlation coefficient R0. 2 =0.995, and the linear detection range is 1.0–50.0 ng / mL. Based on a signal-to-noise ratio of 3 (S / N=3), the limit of detection (LOD) for chlorpyrifos using this method is 0.3 ng / mL.

[0102] (5) Detection and spiked recovery experiment of actual spinach samples

[0103] Take commercially available fresh spinach samples and pre-treat them according to the standard of "Rapid Detection of Organophosphorus and Carbamate Pesticide Residues in Vegetables": Weigh 2.0g of chopped spinach leaves into a centrifuge tube, add 10mL of extraction buffer at pH 8.0, vortex for 2min, let stand, and then filter the supernatant through a 0.22μm microporous membrane. The resulting filtrate is used as the blank sample to be tested.

[0104] Take 5.0 mL of 0.01 M PBS (pH 7.4) into the detection cell, and measure the initial current after adding the ACh substrate as described in step 4. Then add 50 μL of blank sample test solution, incubate for 10 min, and measure the current to calculate the inhibition rate. After substituting into the standard curve equation, the calculated concentration is lower than the detection limit, indicating that no chlorpyrifos residue was detected in the spinach sample.

[0105] To evaluate the accuracy and resistance to matrix interference of the method, a spiked recovery experiment was conducted: two identical blank sample test solutions were prepared by adding an appropriate amount of chlorpyrifos standard stock solution to each, resulting in spiked simulated samples with low (5.0 ng / mL) and high (20.0 ng / mL) concentrations. The inhibition rate was determined and the recovery rate was calculated using the same method. The results showed that the average recovery rate at the low-concentration spiked level was 98.2%, and the average recovery rate at the high-concentration spiked level was 102.5%. The relative standard deviation (RSD) of the three parallel determinations was less than 5.0%. These results indicate that the method has good accuracy and precision and is suitable for the rapid and reliable detection of trace organophosphorus pesticide residues in complex fruit and vegetable matrices.

[0106] Example 2

[0107] (1) Preparation of three-dimensional porous CuNPs / rGO / CS composite material:

[0108] Take 5.0 mL of a 1.5 mg / mL graphene oxide (GO) aqueous dispersion and add 7.5 mL of a 1.5 wt% chitosan (CS) acetic acid solution while stirring. Then, add 1.0 mL of a 0.08 M copper nitrate aqueous solution. Adjust the pH of the mixture to 4.2 with dilute acetic acid and stir to obtain the precursor mixture. Irradiate the mixture vertically under a 254 nm UV lamp for 2 h. After the reaction, centrifuge, wash, and vacuum dry at 50 °C to obtain the composite material powder.

[0109] (2) Preparation of dual-enzyme functionalized nanocomposites:

[0110] Accurately weigh 5.0 mg of the composite material prepared in step (1) and disperse it in 970 μL PBS (pH 7.0). Add 15 μL of AChE solution (enzyme activity 600 U / mL) and 15 μL of ChOx solution (enzyme activity 600 U / mL) sequentially. After mixing and adsorption for 1 h, add 50 μL of 10% glutaraldehyde solution (final concentration approximately 0.5%) and crosslink at 4 °C for 3 h. After centrifugation and washing, redisperse in 1.0 mL PBS to obtain the stock solution.

[0111] (3) Construction of sensing electrodes:

[0112] 5 μL of 1.5 wt% chitosan solution was dropped onto the SPE working electrode and dried at 35 °C to form a film with a thickness of 2 μm on the electrode surface. The film was then crosslinked with 1.5% glutaraldehyde solution for 20 min. 8 μL of the stock solution from step (2) was used to modify the electrode surface and incubated at 4 °C for 4 h to obtain the sensing electrode.

[0113] (4) Establishment of the standard curve (taking methyl parathion as an example):

[0114] A series of methyl parathion standard solutions were prepared (concentrations: 0.5, 1.0, 5.0, 10.0, 50.0 ng / mL). At a working potential of +0.38 V, ACh substrate was added to 5.0 mL of PBS to a final concentration of 0.8 mM, and I0 was measured. After incubation with 50 μL of standard solution for 12 min, I was measured, and the inhibition rate was calculated. A standard curve was fitted with the logarithm of concentration on the x-axis and the inhibition rate on the y-axis, yielding the equation Y = 28.94X + 22.05, R0. 2 =0.997, the linear range is 0.5~50.0ng / mL, and the calculated LOD is 0.2ng / mL.

[0115] (5) Testing of actual blueberry samples:

[0116] Blueberry samples were homogenized and extracted and filtered using standard methods. Methyl parathion residues were not detected using the above method. Spiking recovery experiments were performed (spiking levels: 2.0 ng / mL and 10.0 ng / mL), and the average recoveries were 96.5% and 103.8%, respectively, with RSD < 4.5%.

[0117] Example 3

[0118] (1) Preparation of three-dimensional porous CuNPs / rGO / CS composite material:

[0119] Take 5.0 mL of GO dispersion with a mass concentration of 0.8 mg / mL and add 12.5 mL of CS solution with a mass-to-volume concentration of 0.8 wt% (volume ratio 5:12.5). Add 1.0 mL of 0.02 M copper acetate solution. Adjust the pH to 4.8 and stir to obtain the precursor solution. Irradiate vertically under 365 nm ultraviolet light for 1.5 h. Post-treatment is the same as in Example 1 to obtain the composite material.

[0120] (2) Preparation of dual-enzyme functionalized nanocomposites:

[0121] Weigh 5.0 mg of the composite material and disperse it in 965 μL PBS (pH 7.2). Add 20 μL of AChE solution (enzyme activity 500 U / mL) and 5 μL of ChOx solution (enzyme activity 1000 U / mL). After mixing, add 30 μL of 20% glutaraldehyde solution (final concentration approximately 0.60%) and crosslink overnight at 4°C. The resulting solution is the stock solution.

[0122] (3) Construction of sensing electrodes:

[0123] 10 μL of 0.8 wt% chitosan solution was dropped onto SPE and dried to form a film with a thickness of 4 μm on the electrode surface. Crosslinked with 3.0% glutaraldehyde for 10 min. 5 μL of the stock solution from step (2) was used for modification and incubated at 4 °C for 2 h to obtain the sensing electrode.

[0124] (4) Establishment of the standard curve (taking dichlorvos as an example):

[0125] A series of standard solutions of dichlorvos were prepared (concentrations: 2.0, 5.0, 10.0, 25.0, 100.0 ng / mL). At a relatively high working potential of +0.45 V, ACh substrate was added to the detection cell to a final concentration of 1.5 mM, and I0 was measured. After incubation with 50 μL of standard solution for 8 min, I was measured and the inhibition rate was calculated. The fitted standard curve equation was Y = 25.60X + 15.30, R0 2 =0.998, linear range was 2.0 to 100.0 ng / mL, LOD was 0.7 ng / mL.

[0126] (5) Testing of actual cucumber samples:

[0127] Cucumber samples were taken and processed according to standard methods to obtain the test solution. No dichlorvos residue was detected after testing. Spiking recovery experiments were performed (spiking levels: 10.0 ng / mL and 50.0 ng / mL), and the average recoveries were 101.2% and 97.8%, respectively, with RSD < 5.0%.

[0128] The detection data of Examples 1 to 3 are summarized in Table 1.

[0129] Table 1. Detection data from Examples 1-3

[0130] Example Detection object Linear detection range (ng / mL) <![CDATA[Coefficient of correlation (R 2 )]]> Limit of detection (LOD, ng / mL) Spike level (ng / mL) Spike recovery rate (%) Relative standard deviation (RSD) 1 Chlorpyrifos 1.0～50.0 0.995 0.3 5.0、20.0 98.2、102.5 ＜5.0% 2 Methyl parathion 0.5～50.0 0.997 0.2 2.0、10.0 96.5、103.8 ＜4.5% 3 Dichlorvos 2.0～100.0 0.998 0.7 10.0、50.0 101.2、97.8 ＜5.0%

[0131] As shown in Table 1, the linear detection range covers the national standard residue limits for various organophosphorus pesticides, meeting the needs for trace detection; the correlation coefficient R... 2 All values ​​were ≥0.995, indicating an excellent linear relationship between concentration and inhibition rate and high quantitative accuracy; the detection limit was as low as 0.2–0.7 ng / mL, demonstrating the high sensitivity of the method; the spiked recoveries were all in the range of 96.5–103.8%, with RSD <5.0%, proving that the method has good accuracy and precision and strong resistance to matrix interference.

[0132] The range descriptions used herein, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1 to 6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "including" and "contains" as used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships mentioned herein, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained through commercial purchase or prepared using existing methods.

[0133] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for rapid detection of pesticide residues in fruits and vegetables, characterized in that, The method includes the following steps: S1. Copper nanoparticles / reduced graphene oxide / chitosan composite material, acetylcholinesterase, choline oxidase and crosslinking agent glutaraldehyde solution are mixed and reacted to obtain dual-enzyme functionalized nanocomposite material. S2. Chitosan solution is drop-coated onto the working electrode surface of the screen-printed electrode and dried to form a film. Then, the dual-enzyme functionalized nanocomposite material is covalently modified onto the electrode surface using the crosslinking agent glutaraldehyde to obtain the sensing electrode. S3. Using acetylcholine as a substrate, and employing the aforementioned sensing electrode, the inhibition rate of the electrode response current of pesticide standard solutions of different known concentrations was detected by the chronoamperometry method, and a standard curve between pesticide concentration and current inhibition rate was established. S4. The fruit and vegetable samples to be tested are pretreated to obtain the test solution, and then the current inhibition rate of the test solution is measured. Based on the current inhibition rate of the test solution and the standard curve, the pesticide residue concentration in the fruit and vegetable samples to be tested is calculated.

2. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 1, characterized in that, In step S1, the ratio of the activity units of the acetylcholinesterase to the choline oxidase is (1-3):

1. The mass ratio of the copper nanoparticle / reduced graphene oxide / chitosan composite material to the total mass of the acetylcholinesterase and the choline oxidase is (50-200):

1. The final mass concentration of the crosslinking agent glutaraldehyde solution in the reaction system is 0.5% to 2%.

3. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 2, characterized in that, In step S1, the preparation method of the copper nanoparticle / reduced graphene oxide / chitosan composite material includes the following steps: S101. Mix the graphene oxide dispersion, chitosan acetate solution and copper salt solution, and adjust the pH to 4.0-5.0 to obtain the precursor mixture. S102. The precursor mixture is irradiated with ultraviolet light to simultaneously reduce and recombine graphene oxide and copper ions using photochemical reduction. After post-treatment, the copper nanoparticle / reduced graphene oxide / chitosan composite material with a three-dimensional porous composite structure is obtained.

4. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 3, characterized in that, The mass concentration of the graphene oxide dispersion is 0.5–2 mg / mL; The chitosan acetate solution has a mass-volume concentration of 0.5–2 wt%. The concentration of the copper salt solution is 0.01–0.1 M; The volume ratio of the graphene oxide dispersion, the chitosan acetate solution, and the copper salt solution is 5:(5-15):

1.

5. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 3, characterized in that, The wavelength of the ultraviolet light irradiation is 254–365 nm, and the vertical irradiation time is 1–4 h.

6. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 2, characterized in that, In step S1, the amount of acetylcholinesterase used is 5-20 U / mL and the amount of choline oxidase used is 2-15 U / mL, based on the volume of the reaction system.

7. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 1, characterized in that, In step S2, the drop volume of the chitosan solution is 3-10 µL, and the thickness of the dried film is 1-5 µm.

8. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 1, characterized in that, In step S2, the mass concentration of the crosslinking agent glutaraldehyde is 1-3%, and the crosslinking time is 10-30 min.

9. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 1, characterized in that, In step S3, the concentration of the acetylcholine substrate in the detection system is 0.5–2.0 mM.

10. The method for rapid detection of pesticide residues in fruits and vegetables according to claim 1, characterized in that, In steps S3 and S4, the operating potential of the chronoamperometry is +0.35 to +0.45V relative to the integrated Ag / AgCl reference electrode on the screen-printed electrode.

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