Electrochemical immunosensor for the detection of organochlorine pesticide residues in fruits and vegetables

Abstract

The application provides a kind of electrochemical immune sensing detection method for fruit and vegetable organochlorine pesticide multi-residue, belongs to the technical field of food safety detection, comprising: using liquid pulse discharge plasma synchronous reduction method to prepare g-C3N4-COOH / AuNPs heterojunction composite material with lattice coupling interface and modify on the surface of silk screen printing carbon electrode, through click chemistry reaction, azide modified organochlorine antigen is covalently fixed on the electrode surface, and the labeled immune probe loaded with different electrochemical signal substances and different organochlorine pesticide specific antibodies is used for competitive immune reaction, the differential pulse voltammetry response curve in the whole potential interval is collected, a plurality of characteristic parameters are extracted, and the concentration of multiple organochlorine pesticides is simultaneously solved by using partial least squares multivariate correction model.The application solves the problems of low interface electron transport efficiency, uncontrollable antigen fixation orientation, serious interference of complex matrix and difficult multi-residue signal resolution in the prior art.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to an electrochemical immunosensing method for detecting multiple residues of organochlorine pesticides in fruits and vegetables. Background Technology

[0002] Organochlorine pesticides are persistent organic pollutants that are extremely difficult to degrade in the natural environment. They remain in water and soil and enter the human body through the food chain, causing serious damage to the liver and nervous system. Although my country has banned the use of organochlorine pesticides such as DDT and HCH in food production, the residue levels of organochlorine pesticides in crops and the environment remain high. There is an urgent need to establish rapid, sensitive, and accurate detection methods to ensure food safety.

[0003] Currently, the main methods for detecting organochlorine pesticide residues include chromatography and chromatography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA), and electrochemical immunosensing (ECIS). Chromatography and GC-MS (such as gas chromatography, gas chromatography-tandem mass spectrometry, and liquid chromatography-tandem mass spectrometry) offer high sensitivity and accuracy, making them the standard methods for organochlorine pesticide residue detection. However, they suffer from drawbacks such as expensive equipment, complex operation, long detection cycles, and the need for specialized technical personnel, making them unsuitable for rapid on-site detection. ELISA is relatively simple to operate, but it typically only detects a single pesticide, making simultaneous analysis of multiple residues difficult. ECIS offers advantages such as low cost, simple operation, high sensitivity, and strong specificity, and has been widely researched and applied in clinical testing and biological detection, demonstrating promising prospects for on-site detection.

[0004] However, existing electrochemical immunosensors still have the following technical problems when applied to the detection of multiple organochlorine pesticide residues in fruits and vegetables: First, the graphitic carbon nitride and gold nanoparticles in the electrode modification materials are mostly prepared by physical mixing or chemical reduction blending, and the two only have weak physical adsorption or electrostatic binding, resulting in low interfacial electron transport efficiency, which directly affects the sensitivity of the sensor; Second, the fixation of antigens on the electrode surface is mostly carried out by random covalent coupling of amide bonds activated by EDC / NHS, and the orientation of the antigen determinants is uncontrollable, some recognition sites are buried, and the recognition efficiency is low; Third, the signal readout method is singular, and the single-point peak current method is mostly used for quantification, which is greatly affected by baseline drift and fruit and vegetable matrix effects, making it difficult to achieve accurate detection in complex fruit and vegetable matrices; Fourth, there is a lack of effective signal discrimination methods for simultaneous detection of multiple residues, making it difficult to achieve parallel quantitative detection of multiple organochlorine pesticides on the same sensing interface.

[0005] Therefore, there is an urgent need to develop an electrochemical immunosensing detection method that can simultaneously solve the above problems, so as to achieve highly sensitive, highly specific, and interference-resistant on-site simultaneous detection of multiple organochlorine pesticide residues in fruits and vegetables. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide an electrochemical immunosensing method for detecting multiple residues of organochlorine pesticides in fruits and vegetables. This method effectively solves the problems of low interface electron transmission efficiency, uncontrollable antigen fixation orientation, severe interference from complex matrices, and difficulty in distinguishing multiple residue signals in existing technologies, and achieves simultaneous detection of multiple residues with high sensitivity, high specificity, and strong anti-interference capabilities.

[0007] To achieve the above objectives, this invention provides an electrochemical immunosensing method for detecting multiple residues of organochlorine pesticides in fruits and vegetables, comprising the following steps: S1. Using a liquid-phase pulsed discharge plasma synchronous reduction method, gold nanoparticles are directionally nucleated and epitaxially grown on the surface of carboxyl functionalized graphitic carbon nitride to form a g-C3N4-COOH / AuNPs heterojunction composite material with a lattice coupling interface; the heterojunction composite material is then modified onto the surface of a screen-printed carbon electrode to obtain the composite electrode AG-SPCE. S2. The composite electrode AG-SPCE is functionalized with alkyne groups on its surface. The azide-modified organochlorine antigen is covalently fixed onto the surface of AG-SPCE by click chemistry reaction. After sealing, an immunosensing electrode is obtained. S3. Prepare labeled immunoprobes, wherein the labeled immunoprobes are loaded with specific antibodies against electrochemical signaling substances and organochlorine pesticides, and the characteristic potentials of each electrochemical signaling substance do not overlap. S4. Place the immunosensing electrode in the sample solution to be tested, add the labeled immunoprobe to perform a competitive immunoreaction; collect the differential pulse voltammetric response curve in the full potential range, extract multiple characteristic parameters, and simultaneously calculate the concentrations of multiple organochlorine pesticides using a partial least squares multivariate correction model to complete the simultaneous detection of multiple organochlorine pesticide residues in fruits and vegetables.

[0008] Preferably, in step S1, the liquid-phase pulsed discharge plasma synchronous reduction method uses chloroauric acid as the gold source, a tungsten rod as the anode and a stainless steel mesh as the cathode, and applies a high-voltage pulse power supply for discharge treatment under a nitrogen atmosphere. The pulse voltage is 15~25kV, the pulse frequency is 50~200Hz, and the discharge treatment time is 10~30 minutes; the mass ratio of the carboxyl functionalized graphitic carbon nitride to chloroauric acid is 5:1~10:1.

[0009] Preferably, in step S1, the carboxyl-functionalized graphitic carbon nitride is prepared by the following method: using melamine as a precursor, heating to 500-600°C at a heating rate of 2-5°C / min, holding at that temperature for 2-4 hours, and naturally cooling to obtain graphitic carbon nitride; dispersing the graphitic carbon nitride in a mixed acid of concentrated nitric acid and concentrated sulfuric acid, refluxing at 60-80°C for 4-8 hours, and obtaining the carboxyl-functionalized product after washing, centrifugation, and drying.

[0010] Preferably, in step S2, the surface alkyne functionalization modification is performed by immersing AG-SPCE in a PBS solution containing 4-ethynylbenzoic acid and EDC / NHS, and introducing terminal alkyne groups on the electrode surface through an amide condensation reaction; the azidated organochlorine antigen is prepared by reacting N-hydroxysuccinimide-azidoacetate with the free amino group of the organochlorine antigen.

[0011] Preferably, in step S2, the click chemical reaction is a copper-catalyzed azide-alkyne cycloaddition reaction, the reaction solution contains azide-modified organochlorine antigen, copper sulfate, sodium ascorbate and THPTA, the reaction temperature is 25~37℃, and the reaction time is 1~3 hours; after fixation, the electrode surface is sealed with bovine serum albumin solution for 30~60 minutes.

[0012] Preferably, in step S3, the labeled immunoprobe uses gold nanoparticles prepared by sodium citrate reduction as a carrier. After adjusting the pH of the gold nanoparticle sol to 8.5-9.0, it is sequentially bound to specific antibodies and electrochemical signaling substances, and then obtained after blocking with bovine serum albumin and centrifugation and washing.

[0013] Preferably, in step S4, the differential pulse voltammetry has a scanning range of -1.2V to +1.0V vs. Ag / AgCl, a scanning rate of 10 to 100mV / s, a pulse amplitude of 20 to 80mV, and a pulse width of 20 to 100ms; the multiple characteristic parameters include the characteristic peak current, peak half-width, and the ratio of characteristic peak current to background current for each electrochemical signal substance.

[0014] Preferably, the partial least squares multivariate calibration model is constructed in the following way: a mixed standard of organochlorine pesticides with different concentration ratios is prepared as a training set, the characteristic parameters corresponding to each standard are extracted as the independent variable matrix, the concentration values ​​of each organochlorine pesticide are used as the dependent variable matrix, the optimal number of principal components is determined through cross-validation, and a calibration model is established.

[0015] Preferably, the sample solution to be tested is obtained by the following pretreatment: after homogenizing the fruit and vegetable sample to be tested, add a 1:1 volume ratio of n-hexane-acetone mixed solvent for extraction, perform ultrasonic-assisted extraction for 10-30 minutes, centrifuge to collect the supernatant, then purify the supernatant by solid-phase extraction and dry it, and redissolve it with buffer solution to obtain the sample solution to be tested.

[0016] Preferably, the labeled immune probe is of at least two types, and the corresponding electrochemical signaling substances are neutral red and ferrocene, respectively, with a characteristic potential difference of 1.0V between the two signaling substances.

[0017] Compared with the prior art, the present invention discloses at least the following technical effects: (1) The present invention enables gold nanoparticles to be nucleated and epitaxially grown in situ on the surface of carboxyl functionalized graphitic carbon nitride by liquid phase pulse discharge plasma synchronous reduction method, forming a heterojunction composite material with lattice coupling interface. Strong metal-carrier interaction is generated between gold nanoparticles and graphitic carbon nitride, forming a solid lattice epitaxial interface, which effectively reduces the interface electron transport barrier and greatly improves the charge transfer efficiency of the electrode interface, thereby significantly improving the detection sensitivity of the sensor.

[0018] (2) By introducing alkyne groups on the electrode surface and azide groups on the organochlorine antigen, this invention utilizes the high selectivity and mild conditions of click chemistry to covalently fix the antigen on the electrode surface in an oriented and upright manner, so that all the antigen determinants of the antigen are fully exposed to the solution phase. This effectively solves the technical problems of uncontrollable antigen orientation and buried recognition sites in traditional random coupling methods, and greatly improves the immune recognition efficiency and sensor response performance.

[0019] (3) This invention extracts multiple characteristic parameters, including characteristic peak current, peak half-width and peak current to background current ratio, by acquiring differential pulse voltammetric response curves in the full potential range. Combined with partial least squares multivariate correction model for data analysis, it makes full use of the electrochemical information of the full waveform, effectively eliminates background interference and baseline drift caused by complex matrices of fruits and vegetables, and achieves accurate differentiation of characteristic signals of different probes. It solves the problems of inaccurate quantification and difficulty in distinguishing multiple residual signals in complex matrices by traditional single-point peak current method, and realizes the simultaneous detection of multiple organochlorine pesticides with high sensitivity, high specificity and anti-interference. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart of an electrochemical immunosensing method for detecting multiple residues of organochlorine pesticides in fruits and vegetables according to the present invention.

[0022] Figure 2 The AG-SPCE composite electrode of this invention and the bare SPCE are used at 5 mmol / L [Fe(CN)6]. 3- / 4- Comparison of cyclic voltammetry in solution.

[0023] Figure 3 This is a cyclic voltammetry characterization diagram of the sensor layer-by-layer assembly process of the present invention.

[0024] Figure 4 This is a schematic diagram illustrating the separation of characteristic potentials of the NR and Fc dual probes in the DPV scanning of this invention.

[0025] Figure 5 This is a scatter plot showing the correlation between the method of this invention and the GC-MS / MS method in actual sample detection. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] like Figure 1 As shown, this invention provides an electrochemical immunosensing method for detecting multiple residues of organochlorine pesticides in fruits and vegetables, comprising the following steps: S1. Using a liquid-phase pulsed discharge plasma synchronous reduction method, gold nanoparticles are directionally nucleated and epitaxially grown on the surface of carboxyl functionalized graphitic carbon nitride to form a g-C3N4-COOH / AuNPs heterojunction composite material with a lattice coupling interface; the heterojunction composite material is then modified onto the surface of a screen-printed carbon electrode to obtain the composite electrode AG-SPCE.

[0029] Specifically, this step first prepares carboxyl-functionalized graphitic carbon nitride using melamine as a precursor. Melamine is placed in a muffle furnace and heated to 500-600°C at a rate of 2-5°C / min. After holding at this temperature for 2-4 hours, it is naturally cooled to obtain graphitic carbon nitride. The graphitic carbon nitride is then dispersed in a mixed acid of concentrated nitric acid and concentrated sulfuric acid and refluxed at 60-80°C for 4-8 hours. After washing, centrifugation, and drying, the carboxyl-functionalized product is obtained. Subsequently, using chloroauric acid as the gold source, the mass ratio of carboxyl-functionalized graphitic carbon nitride to chloroauric acid is 5:1. The two components were dispersed in pure water at a ratio of ~10:1, stirred evenly, and then transferred to a plasma reactor. A tungsten rod was used as the anode and a stainless steel mesh as the cathode. A high-voltage pulse power supply was applied under a nitrogen atmosphere, with the pulse voltage controlled at 15~25kV and the pulse frequency at 50~200Hz. The product was discharged at room temperature for 10~30 minutes. After centrifugation, washing, and vacuum drying, a heterojunction composite material was obtained. Finally, the screen-printed carbon electrode was cleaned, and an aqueous dispersion of the heterojunction composite material was drop-coated onto the surface of the working electrode. After drying at room temperature, the composite electrode AG-SPCE was obtained.

[0030] Among them, the high-energy electrons and plasma active species generated during the pulsed discharge process can convert Au 3+ In-situ restoration to Au 0 The defect sites on the surface of g-C3N4-COOH nanosheets can serve as directional nucleation sites for gold nuclei, guiding the epitaxial growth of gold nanoparticles along the crystal plane orientation. This forms a lattice-coupled heterojunction interface between the two phases and generates strong metal-carrier interactions, effectively reducing the interfacial electron transport barrier. This avoids the problem of weak adsorption bonding and high interfacial resistance in traditional physical mixing methods, thus significantly improving the conductivity and interfacial electron transport efficiency of the prepared composite electrode, which can greatly enhance the sensitivity of the detection method.

[0031] S2. The composite electrode AG-SPCE is modified with surface alkyne functionalization. The azide-modified organochlorine antigen is covalently fixed to the surface of AG-SPCE by click chemistry reaction. After sealing, an immunosensing electrode is obtained.

[0032] Specifically, in this step, AG-SPCE is first immersed in PBS buffer containing 4-ethynylbenzoic acid and EDC / NHS. Terminal alkyne groups are introduced onto the electrode surface through amide condensation reaction. After reacting at room temperature for 2-4 hours, the electrode is thoroughly washed with PBS. Simultaneously, azido-modified organochlorine antigen is prepared by dissolving the organochlorine antigen in PBS buffer at pH 7.4, adding N-hydroxysuccinimide-azidoacetate, and stirring at room temperature in the dark for 4-6 hours. After removing unreacted reagents using a desalting column, the azido-modified organochlorine antigen is obtained. Then, the alkyne-functionalized AG-SPCE is immersed in a reaction solution containing azido-modified organochlorine antigen, copper sulfate, sodium ascorbate, and THPTA ligand. The reaction temperature is controlled at 25-37°C, and the reaction is carried out for 1-3 hours. After the reaction, the electrode is washed, and bovine serum albumin solution is added to block the non-specific binding sites on the electrode surface. After blocking for 30-60 minutes, the electrode is washed again to obtain the immunosensing electrode.

[0033] In the above process, EDC / NHS can activate the carboxyl groups on the surface of g-C3N4-COOH, causing them to undergo amide condensation with the amino group of 4-ethynylbenzoic acid, uniformly introducing terminal alkyne groups onto the electrode surface; after the free amino group of the antigen molecule reacts with NHS-azidoacetate, azido groups can be introduced at specific sites; in the copper-catalyzed azido-alkyne cycloaddition reaction, the azido group and the alkyne group undergo directional cycloaddition to generate a triazole ring, so that the antigen molecule is covalently linked to the electrode surface with a fixed orientation, and all antigen determinants face the solution phase, avoiding the problem of recognition site burying caused by the antigen lying flat or lying on its side in traditional random amide coupling. Therefore, this step can significantly increase the amount of antigen immobilization, ensure sufficient exposure of antigen recognition sites, effectively improve the efficiency of immune recognition, broaden the detection linear range, and enhance the intensity of the detection response signal.

[0034] S3. Prepare labeled immunoprobes, wherein the labeled immunoprobes are loaded with specific antibodies against electrochemical signaling substances and organochlorine pesticides, and the characteristic potentials of each electrochemical signaling substance do not overlap.

[0035] Specifically, this step first uses the sodium citrate reduction method to prepare gold nanoparticle sol. The chloroauric acid solution is heated to boiling, and sodium citrate solution is added under vigorous stirring. After boiling for 10-20 minutes, the mixture is refluxed and stirred for 15-30 minutes. After natural cooling, a gold nanoparticle sol with uniform particle size is obtained. The pH of the gold nanoparticle sol is then adjusted to 8.5-9.0, and the specific antibody corresponding to the organochlorine pesticide is added. The mixture is incubated at room temperature with shaking for 1-2 hours. Then, the corresponding electrochemical signal substance is added and incubation continues. Finally, bovine serum albumin solution is added to block the remaining active sites. After centrifugation and washing, the precipitate is resuspended in PBS buffer to obtain the labeled immune probe.

[0036] The labeled immunoprobes include at least two types, with corresponding electrochemical signaling substances of neutral red and ferrocene, respectively. The characteristic potentials of the two signaling substances have a sufficient difference, and their signal windows do not overlap. Gold nanoparticles have a large specific surface area, allowing them to bind a large number of antibody molecules and electrochemical signaling substances via electrostatic interactions with Au-S bonds, while also amplifying the signal. Adjusting the pH to a weakly alkaline state allows the antibody molecules to bind more stably to the surface of the gold nanoparticles. Using different electrochemical signaling substances with significantly different characteristic potentials to label probes corresponding to different pesticides ensures that the signal peaks do not interfere with each other during simultaneous detection of multiple components, avoiding signal crosstalk.

[0037] Furthermore, the labeled immunoprobe antibodies and signal molecules prepared in this step have high loading efficiency, stable detection signal intensity, and no signal crosstalk in the multi-probe combination, providing a reliable signal basis for the simultaneous quantification of various organochlorine pesticides.

[0038] S4. Place the immunosensing electrode in the sample solution to be tested, add the labeled immunoprobe to perform a competitive immunoreaction; collect the differential pulse voltammetric response curve in the full potential range, extract multiple characteristic parameters, and simultaneously calculate the concentrations of multiple organochlorine pesticides using a partial least squares multivariate correction model to complete the simultaneous detection of multiple organochlorine pesticide residues in fruits and vegetables.

[0039] Specifically, the sample solution to be tested is pretreated. After homogenizing the fruit and vegetable samples to be tested, a 1:1 (v / v) hexane-acetone mixed solvent is added for extraction. The extraction is performed with ultrasonic assistance for 10-30 minutes. The supernatant is collected by centrifugation, purified by solid-phase extraction, dried, and reconstituted with buffer solution. During detection, the immunosensing electrode is immersed in the sample solution to be tested, and labeled immunoprobes are added simultaneously. The electrode is subjected to a competitive reaction at 25-37℃ for 30-60 minutes. After the reaction, the electrode is washed with PBS buffer. The electrode is scanned across the entire potential range using differential pulse voltammetry. The scanning range is -1.2V to +1.0V vs. Ag / AgCl, the scanning rate is 10-100mV / s, the pulse amplitude is 20-80mV, and the pulse width is 20-100ms. A complete current response curve is acquired. Multiple characteristic parameters, including the characteristic peak current, peak half-width, and the ratio of characteristic peak current to background current, are extracted from the response curve for each electrochemical signal substance.

[0040] Then, a mixed standard of organochlorine pesticides with different concentration ratios is prepared in advance as a training set. The characteristic parameters corresponding to each standard are extracted as the independent variable matrix, and the concentration values ​​of each organochlorine pesticide are used as the dependent variable matrix. The optimal number of principal components is determined through cross-validation, and a partial least squares multivariate calibration model is constructed. During actual testing, the characteristic parameters of the sample to be tested are input into the calibration model, and the concentrations of multiple organochlorine pesticides can be calculated simultaneously.

[0041] The detection employs a competitive immunoassay mode, where free organochlorine pesticides in the sample compete with antigens immobilized on the electrode surface for binding to antibodies on labeled immunoassay probes. The higher the pesticide concentration in the sample, the fewer labeled probes bind to the electrode surface, resulting in a lower corresponding electrochemical signal peak current. Traditional single-point peak current quantification uses only the peak current as a single parameter, which is susceptible to matrix interference, leading to baseline drift and peak shape distortion. However, multi-feature parameters combined with a partial least squares correction model can automatically identify and compensate for interference such as baseline shift and peak shape changes caused by the matrix through multi-dimensional data analysis, achieving accurate quantification in complex matrices.

[0042] Furthermore, the multivariate correction method in this step can effectively eliminate the interference of endogenous substances in fruit and vegetable matrices, significantly improve the accuracy and reproducibility of detection results in complex matrices, achieve simultaneous and accurate quantification of multiple organochlorine pesticides, and ensure that the detection results are consistent with the laboratory calibration verification method.

[0043] Based on the above, the following will further describe the specific implementation methods.

[0044] Example 1 This embodiment describes the preparation and characterization of the g-C3N4-COOH / AuNPs heterojunction composite material, as well as the preparation and electrochemical performance testing of the composite electrode AG-SPCE.

[0045] The preparation process of carboxyl-functionalized graphitic carbon nitride is as follows: Using melamine as a precursor, 10.0 g of melamine was placed in a covered alumina crucible and placed in a muffle furnace. The temperature was increased to 550°C at a heating rate of 3°C / min and held for 3 hours. After natural cooling to room temperature, pale yellow solid graphitic carbon nitride was obtained. 1.0 g of the obtained g-C3N4 was dispersed in 100 mL of a mixture of concentrated nitric acid and concentrated sulfuric acid (volume ratio 1:3) and refluxed at 70°C for 6 hours. After the reaction, the mixture was slowly poured into 500 mL of ice water and allowed to stand overnight. The precipitate was collected by centrifugation at 8000 rpm for 10 minutes and repeatedly washed with pure water until the pH of the supernatant was approximately 7. The supernatant was then vacuum dried at 60°C for 12 hours to obtain carboxyl-functionalized graphitic carbon nitride g-C3N4-COOH.

[0046] The process for preparing heterojunction composite materials by liquid-phase pulsed discharge plasma synchronous reduction method is as follows: Take 50 mg of the above g-C3N4-COOH, disperse it in 50 mL of pure water, ultrasonically disperse for 30 minutes, and add chloroauric acid aqueous solution (containing Au). 3+5 mg of g-C3N4-COOH was mixed with HAuCl4 at a mass ratio of 10:1 and magnetically stirred until homogeneous before being transferred to a plasma reactor. A tungsten rod (2 mm diameter, 99.95% purity) was used as the anode, and a stainless steel mesh (100 mesh) as the cathode, with an electrode spacing of 10 mm. After sealing the reactor, high-purity nitrogen (99.999%) was introduced for 30 minutes to remove dissolved oxygen. A nitrogen atmosphere was maintained, and a high-voltage pulsed power supply was applied with a pulse voltage of 20 kV, a pulse frequency of 100 Hz, a pulse width of 3 μs, and a duty cycle of 30%. Discharge was performed at room temperature for 20 minutes, with the solution temperature maintained at 25 ± 2 °C during the discharge process using circulating cooling water. After discharge, the product was collected by centrifugation at 12000 rpm for 15 minutes, washed three times sequentially with pure water and anhydrous ethanol, and then vacuum-dried at 50 °C for 12 hours to obtain the g-C3N4-COOH / AuNPs heterojunction composite material.

[0047] The preparation process of the composite electrode AG-SPCE is as follows: The screen-printed carbon electrode (SPCE, working electrode diameter 3mm) was sequentially ultrasonically cleaned with pure water, anhydrous ethanol, and pure water for 5 minutes each, and then dried with nitrogen. 2.0 mg of the above heterojunction composite material was taken, dispersed in 2.0 mL of pure water, and ultrasonically dispersed for 30 minutes. 10 μL of the dispersion was drop-coated onto the working electrode surface of the SPCE and allowed to air dry at room temperature to obtain the composite electrode AG-SPCE.

[0048] Transmission electron microscopy (TEM) observation of the obtained material showed that gold nanoparticles were uniformly distributed on the surface of g-C3N4 nanosheets without obvious agglomeration. Approximately 200 particles were counted, with an average particle size of 7.2 nm and a narrow particle size distribution (PDI = 0.08). High-resolution TEM observation showed that the interplanar spacing of the gold nanoparticles was 0.235 nm, corresponding to the Au(111) crystal plane, while the interplanar spacing of g-C3N4 was 0.248 nm, corresponding to the (002) plane of the triazine ring structure. The lattice fringes at the interface between the two phases were continuous, with no obvious interface defects or amorphous layers. The lattice mismatch was approximately 5.2%, confirming the existence of an epitaxial growth relationship between the Au(111) crystal plane and the g-C3N4 triazine ring, forming a heterojunction structure with a coherent interface. In X-ray photoelectron spectroscopy analysis, the binding energy of Au 4f7 / 2 was 83.6 eV, which was 0.4 eV negatively shifted from the standard binding energy of elemental Au (84.0 eV), confirming the existence of strong metal-support interaction. In addition to the characteristic peak of g-C3N4, the X-ray diffraction pattern showed four new diffraction peaks at 2θ = 38.2°, 44.4°, 64.6°, and 77.6°, corresponding to the (111), (200), (220), and (311) crystal planes of face-centered cubic Au, respectively. In Fourier transform infrared spectroscopy, g-C3N4-COOH showed a peak at 1710 cm⁻¹. -1 A C=O stretching vibration peak appears at 3420 cm⁻¹.-1 The appearance of an OH stretching vibration peak confirms the successful introduction of the carboxyl group.

[0049] Subsequently, an electrochemical workstation was used, with AG-SPCE as the working electrode, Ag / AgCl (saturated KCl) as the reference electrode, and platinum wire as the counter electrode, at 5 mmol / L [Fe(CN)6] 3- / 4- Cyclic voltammetry and electrochemical impedance spectroscopy were performed in the solution (containing 0.1 mol / L KCl). Figure 2 As shown, cyclic voltammetry results indicate that the peak current of the bare SPCE is 52.3 μA, and the potential difference between the oxidation and reduction peaks is 112 mV. After modification with the g-C3N4-COOH / AuNPs heterojunction, the peak current significantly increases to 78.6 μA, and the potential difference decreases to 68 mV. Electrochemical impedance spectroscopy (EIS) was performed using an equivalent circuit R(Q(RW)). The charge transfer resistance of the bare SPCE was 185.6 Ω, while that of the AG-SPCE was significantly reduced to 96.3 Ω, a 48.1% reduction compared to the bare electrode. This confirms that the heterojunction composite material possesses excellent conductivity and interfacial electron transfer efficiency.

[0050] Example 2 This embodiment describes the preparation and characterization of an immunosensing electrode, specifically including: The process of alkynyl functionalization modification of the electrode surface is as follows: The AG-SPCE electrode prepared in Example 1 was immersed in PBS solution (0.01mol / L, pH 6.0) containing 10mmol / L 4-ethynylbenzoic acid and 20mmol / L EDC / NHS, and reacted at room temperature for 3 hours. After the reaction, it was thoroughly washed 3 times with PBS (0.01mol / L, pH 7.4) for 5 minutes each time.

[0051] The azidation modification process of organochlorine antigens is as follows: Endosulfan-bovine serum albumin conjugate (Ed-BSA) was dissolved in PBS (0.01 mol / L, pH 7.4) to prepare a 1 mg / mL solution. A 30-fold molar excess of N-hydroxysuccinimide-azidoacetate (dissolved in DMSO, concentration 10 mmol / L) was added. The mixture was stirred at room temperature in the dark for 5 hours. After the reaction, unreacted reagents and byproducts were removed using a desalting column (molecular weight cutoff 7 kDa). The azidated endosulfan antigen (N3-Ed-BSA) was collected, lyophilized, and stored at -20℃ for later use. Following the same method, azidated hexachlorocyclohexane (HCH) antigen (N3-BHC-BSA) was prepared using the HCH-bovine serum albumin conjugate.

[0052] The chemical fixation process is as follows: The alkyne-functionalized AG-SPCE electrode was immersed in a reaction solution containing N3-Ed-BSA (20 μg / mL), CuSO4 (2 mmol / L), sodium ascorbate (20 mmol / L), and THPTA (200 μmol / L) (0.01 mol / L PBS, pH 7.4) and reacted at 37°C for 2 hours. After the reaction, the electrode was thoroughly washed three times with PBS containing 0.02% Tween-20 for 5 minutes each time. A 1% (w / v) bovine serum albumin solution (dissolved in PBS, pH 7.4) was added to the electrode surface, and the electrode was blocked at room temperature for 45 minutes. After blocking, the electrode was thoroughly washed three times with PBS for 5 minutes each time to obtain the immunosensing electrode.

[0053] The layer-by-layer assembly process of the sensor was characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical parameters of each modification step are shown in Table 1. Figure 3 As shown, the cyclic voltammetry test results show that the peak current of each modification step exhibits regular changes, and the electrochemical impedance spectroscopy fitting results also show regular changes, confirming the successful assembly of each layer of material.

[0054] Table 1 Electrochemical parameters of sensor layer-by-layer assembly

[0055] The amount of antigen immobilized on the electrode surface was determined using surface plasmon resonance (SPR) technology. Alkyne-functionalized gold chips were immobilized with Ed-BSA using both click chemistry and EDC / NHS random coupling methods. Protein adsorption was calculated based on changes in the SPR angle. The antigen immobilization amount using click chemistry was 15.6 ng / mm². 2 The antigen fixation level of the EDC / NHS random conjugation method is only 8.3 ng / mm. 2 The antigen immobilization amount by the click chemistry method was approximately 88% higher than that by the EDC / NHS method. Atomic force microscopy morphological observation showed that the antigen immobilized by the click chemistry method was uniformly distributed in a granular form on the electrode surface with an average height of approximately 4.5 nm, while the antigen immobilized by the EDC / NHS method had an average height of only about 1.8 nm and was randomly lying flat or on its side. This confirmed that the click chemistry method achieved directional upright immobilization of the antigen, allowing the antigenic determinants to be fully exposed to the solution phase.

[0056] Example 3 This embodiment describes the preparation and characterization of labeled immune probes, specifically including: The preparation process of gold nanoparticle sol is as follows: AuNPs sol was prepared by sodium citrate reduction method. 100 mL of 0.01% (w / v) chloroauric acid solution was added to a 250 mL round-bottom flask and heated to a vigorous boil. Under vigorous stirring, 2 mL of 1% (w / v) sodium citrate solution was rapidly added, and boiling continued for 15 minutes. The solution color gradually changed from pale yellow to wine red. The mixture was then refluxed and stirred for another 20 minutes and allowed to cool naturally to room temperature to obtain the AuNPs sol. UV-Vis spectrophotometry showed that AuNPs had a characteristic plasmon resonance absorption peak at 520 nm. Transmission electron microscopy showed that the AuNPs were spherical with a particle size of approximately 15 nm. Dynamic light scattering analysis determined the hydrated particle size to be 15.2 nm, and the Zeta potential was -35.6 mV.

[0057] The preparation process of NR-labeled endothelium-sulfuron immunoprobe is as follows: Take 40 mL of the above AuNPs sol, adjust the pH to 8.5 with 0.1 mol / L K2CO3, and add 0.5 mL of endothelium-sulfuron monoclonal antibody solution (1 mg / mL, dissolved in 0.01 mol / L PBS, pH 7.4) while stirring. Incubate at room temperature with shaking for 1.5 hours, then add neutral red solution (1 mmol / L, 0.5 mL), and continue incubation for 1 hour. Finally, add 0.5 mL of 10% (w / v) bovine serum albumin solution and block for 30 minutes. Centrifuge the reaction solution at 12000 rpm for 20 minutes at 4℃, discard the supernatant, resuspend the precipitate in PBS, repeat centrifugation and washing twice, and finally resuspend in 2 mL of PBS to obtain the NR-AuNPs@Ed-Ab labeled immunoprobe, which is stored at 4℃ for later use. Using the same method, Fc-AuNPs@BHC-Ab labeled immune probes were prepared using hexachlorocyclohexane (HCH) monoclonal antibody and ferrocene (dissolved in ethanol, concentration 1 mmol / L).

[0058] UV-Vis absorption spectroscopy characterization of the labeled immunoprobes revealed a double peak at 520 nm and 530 nm for NR-AuNPs@Ed-Ab, with the shoulder peak at 530 nm attributed to the characteristic absorption of NR molecules. Similarly, Fc-AuNPs@BHC-Ab exhibited a double peak at 520 nm and 440 nm, with the absorption peak at 440 nm attributed to the characteristic absorption of ferrocene. Dynamic light scattering measurements showed that the hydrated particle size of AuNPs increased from 15.2 nm to 38.7 nm for NR-AuNPs@Ed-Ab, and the Zeta potential changed from -35.6 mV to -18.4 mV. Probe loading was quantitatively determined using UV-Vis spectroscopy. Based on the molar absorptivity of NR at 530 nm, each AuNP in NR-AuNPs@Ed-Ab was calculated to load an average of approximately 52 NR molecules. Based on the molar absorptivity of Fc at 440 nm, each AuNP in Fc-AuNPs@BHC-Ab was calculated to load an average of approximately 48 Fc molecules. Antibody loading was determined using the BCA protein quantification method, with each AuNP loading an average of approximately 8–10 antibody molecules.

[0059] Example 4 This embodiment describes the establishment of an electrochemical immunosensing detection and multivariate correction model, specifically including: The Ed-Ag / AG-SPCE immunosensing electrode prepared in Example 2 was immersed in a solution containing different concentrations of endosulfan standard (concentrations of 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ppt, solvent: 0.01 mol / L PBS, pH 7.4, containing 0.1% Tween-20). At the same time, the NR-AuNPs@Ed-Ab probe prepared in Example 3 (diluted to 2 nmol / L) was added, and the reaction was carried out at 37°C for 45 minutes. After the reaction, the electrode was washed three times with PBS containing 0.02% Tween-20 for 5 minutes each time. An electrochemical workstation was used with an immunosensing electrode as the working electrode, Ag / AgCl (saturated KCl) as the reference electrode, and platinum wire as the counter electrode. Differential pulse voltammetry was performed in 5 mL of 0.01 mol / L PBS (pH 7.4). The scan range was -1.0 V to +0.8 V vs. Ag / AgCl, the scan rate was 50 mV / s, the pulse amplitude was 50 mV, and the pulse width was 50 ms. Complete current response curves were acquired across the entire potential range. Following the same method, a standard curve for HCH was established using a BHC-Ag / AG-SPCE electrode and an Fc-AuNPs@BHC-Ab probe.

[0060] Differential pulse voltammetry results showed that NR exhibited a characteristic reduction peak at -0.6V, with the peak current gradually decreasing with increasing endothelium concentration; HCH exhibited a characteristic oxidation peak at +0.4V, with the peak current gradually decreasing with increasing HCH concentration. Figure 4 As shown, the characteristic potential difference between the two probes reaches 1.0V, and the peak intervals do not overlap, enabling simultaneous resolution detection of two components. The linear regression equations and detection limits for the two pesticides are shown in Table 2.

[0061] Table 2. Parameters of the single-component standard curves for endosulfan and hexachlorocyclohexane (HCH).

[0062] The process of establishing the multi-residue simultaneous detection and PLS calibration model is as follows: Mixed standards containing endosulfan and hexachlorocyclohexane (HCH) were prepared with concentration ratios of 1:1, 1:2, 1:5, 2:1, and 5:1, covering a range of 0.5–500 ppt, resulting in 57 mixed standard samples. Ed-Ag / AG-SPCE and BHC-Ag / AG-SPCE immunosensing electrodes (fixed on different working electrodes of the same SPCE array) were simultaneously immersed in the mixed standard solution. Two probes, NR-AuNPs@Ed-Ab and Fc-AuNPs@BHC-Ab (each diluted to 2 nmol / L), were added simultaneously. The mixture was subjected to a competitive reaction at 37°C for 45 minutes, followed by washing and differential pulse voltammetry scanning. Six characteristic parameters were extracted from each DPV response curve: the reduction peak current of NR at -0.6V, the oxidation peak current of Fc at +0.4V, the half width at half maximum (WWHM) of the NR peak, the half width at half maximum (WWHM) of the Fc peak, the ratio of the NR peak current to the background current, and the ratio of the Fc peak current to the background current.

[0063] Using 57 mixed standard samples as the training set, the independent variable matrix X (57×6) consists of 6 feature parameters, and the dependent variable matrix Y (57×2) consists of the concentration values ​​of endosulfan and hexachlorocyclohexane. After mean centering preprocessing, a calibration model was established using partial least squares method. The optimal number of principal components was determined to be 3 through 5-fold cross-validation. The model fitting and validation results are shown in Table 3.

[0064] Table 3 Fitting and Validation Results of the PLS Correction Model

[0065] Example 5 This embodiment demonstrates the pretreatment of fruit and vegetable samples and the verification of actual sample testing, specifically including: Take approximately 100g each of commercially available spinach, apple, strawberry, lettuce, and tomato samples. Remove inedible parts and homogenize them into a uniform paste using a homogenizer. Accurately weigh 5.0g of the homogenized sample into a 50mL centrifuge tube, add 10mL of a hexane-acetone mixed solvent (volume ratio 1:1), vortex for 2 minutes, and then perform ultrasonic-assisted extraction for 20 minutes (ultrasonic power 200W, frequency 40kHz). Centrifuge the extract at 8000rpm for 10 minutes, transfer the supernatant to another centrifuge tube, and repeat the extraction once more. Combine the two supernatants. Pass the combined supernatant through a pre-activated Florisil solid-phase extraction column (1g / 6mL, activated sequentially with 5mL of hexane and 5mL of a hexane-acetone mixed solvent) at a flow rate of approximately 1mL / min. Collect the eluent, elute with 5mL of the hexane-acetone mixed solvent, and combine the collected eluents. The collected solution was dried under nitrogen at 40°C. The residue was reconstituted with 2 mL of PBS (0.01 mol / L, pH 7.4) containing 0.1% Tween-20. After filtration through a 0.22 μm filter membrane, the sample solution to be tested was obtained.

[0066] Endosulfan and hexachlorocyclohexane (HCH) standards were added to homogenized samples of spinach, apple, strawberry, lettuce, and tomato at concentrations of 1 ppt, 5 ppt, 10 ppt, 50 ppt, and 100 ppt (five replicates were prepared for each concentration level). The test solutions were prepared according to the pretreatment method described above. Detection was performed using the method of this invention, the single-point peak current standard curve method, and GC-MS / MS, respectively. The recoveries and relative standard deviations were calculated. The recoveries of 5 ppt spikes in the five matrices are shown in Tables 4 and 5.

[0067] Table 4. Spiking recoveries of different methods in five fruit and vegetable matrices (with 5 ppt of endosulfan spiked).

[0068] Table 5. Spiked recoveries of different methods in five fruit and vegetable matrices (5 ppt of hexachlorocyclohexane spiked).

[0069] Within the spiked concentration range of 1–100 ppt, the recovery rate of the method of the present invention in five fruit and vegetable matrices was 91.8%–96.2%, with an RSD of 3.8%–5.5%. Linear regression analysis of the detection results of the method of the present invention with those of GC-MS / MS, as shown in Table 6, revealed a high correlation between the two methods, with a relative deviation within ±6%, indicating that the method of the present invention has analytical accuracy comparable to that of GC-MS / MS. Figure 5 As shown, the scatter plots of the detection values ​​of endosulfan and hexachlorocyclohexane (HCH) in the actual fruit and vegetable samples are evenly distributed on both sides of the fitted straight line, and the linear correlation coefficient R0 is [value missing]. 2Both values ​​were above 0.993, indicating good consistency between the two detection methods.

[0070] Table 6 shows the linear correlation between the detection results of the method of the present invention and the results of GC-MS / MS.

[0071] Where x is the value measured by GC-MS / MS method, and y is the value measured by the method of the present invention, in ppt.

[0072] Example 6 This embodiment is for sensor performance evaluation. In the intra-batch reproducibility test, five Ed-Ag / AG-SPCE sensors prepared in the same batch were used to detect a 5ppt endothelium standard, and the RSD of the measured value was 3.8%. In the inter-batch reproducibility test, sensors prepared in different batches (5 batches, 3 sensors per batch) were used to detect a 5ppt endothelium standard, and the inter-batch RSD was 5.2%. In the long-term stability test, the Ed-Ag / AG-SPCE sensors were sealed and stored at 4°C. They were taken out on days 0, 3, 7, 14, 21, 28, and 35, and the 5ppt endothelium standard was detected. The signal response remained above 95.6% of the initial value for the first 21 days, decreased to 90.3% of the initial value on day 28, and decreased to 85.2% of the initial value on day 35. Assuming the signal decays to 90% of the initial value, the effective storage period of the sensor at 4°C is approximately 30 days. In the operational stability test, the same sensor was used 6 times consecutively (after washing and regeneration with PBS after each use), and the RSD for detecting 5 ppt of endosulfan was 6.3%.

[0073] In the method specificity evaluation, the interference of common coexisting substances on the detection of endosulfan (5 ppt) by the method of this invention was investigated. The concentration of the interfering substances was set to 100 times the concentration of endosulfan (500 ppt). The interference evaluation results of common coexisting substances are shown in Table 7.

[0074] Table 7. Interference assessment of common coexisting substances on endosulfan detection

[0075] Except for chlorophyll, which showed a signal change of -8.2% at a 100-fold concentration (still within the acceptable range of ±10%), the other coexisting substances did not cause significant interference, indicating that the method of this invention has good specificity. Further investigation was conducted on the interference of structural analogs HCH, DDT, and dieldrin at 10-fold and 100-fold concentrations. At a 10-fold concentration, the signal change rate of the three analogs was +0.5% to +1.2%, and at a 100-fold concentration, it was +1.8% to +3.5%, both less than ±4%, indicating that the method of this invention has good selectivity for the target pesticide.

[0076] Comparative Example 1 This comparative example demonstrates the preparation of a g-C3N4-COOH / AuNPs composite electrode using a conventional physical mixing method. AuNPs sol was prepared using the sodium citrate reduction method (same as in Example 3). g-C3N4-COOH (same as in Example 1) and AuNPs were physically mixed at a mass ratio of 10:1. After ultrasonic dispersion for 30 minutes, 10 μL was drop-coated onto the surface of the SPCE working electrode and air-dried at room temperature to obtain the physical mixing composite electrode. The remaining steps (random coupling and fixation of antigen EDC / NHS, single-point peak current detection) were performed according to conventional methods. Electrochemical impedance spectroscopy results showed that the charge transfer resistance of the physical mixing electrode was 238.7 Ω, while the charge transfer resistance of the AG-SPCE of this invention was 96.3 Ω, a reduction of 59.7%. Using endothelium as the target analyte, the detection limit of the physical mixing electrode was 3.2 ppt, while the detection limit of the electrode of this invention was 0.15 ppt, a reduction of 95.3%. The results show that the lattice epitaxial heterojunction interface constructed by the liquid phase pulsed discharge plasma synchronous reduction method is significantly better than the traditional physical mixing method in terms of reducing interface resistance and improving sensitivity.

[0077] Comparative Example 2 This comparative example demonstrates antigen fixation using the EDC / NHS random covalent coupling method. The antigen is fixed using the traditional EDC / NHS method, where activated carboxyl groups are then linked to OP-Ag amino groups to form amide bonds, replacing the click chemidirectional fixation method of this invention. The specific steps are as follows: AG-SPCE is activated for 30 minutes by immersing it in a PBS solution (pH 6.0) containing 10 mmol / L EDC and 20 mmol / L NHS. After washing, it is immersed in Ed-BSA solution (20 μg / mL, PBS, pH 7.4) and reacted overnight at 4°C. After BSA blocking, an immunosensing electrode is obtained. The electrode material and signal reading method are the same as in this invention. Using endothelium as the target, the detection limit of the EDC / NHS sensor is 0.85 ppt, and the linear range is 1–1000 ppt (R0). 2 =0.982); the detection limit of the click chemistry method of this invention is 0.15 ppt, and the linear range is 0.5~1000 ppt (R 2 =0.997). SPR assay for antigen fixation, EDC / NHS method: 8.3 ng / mm. 2 The click chemical method yielded 15.6 ng / mm 2 The antigen fixation rate was increased by approximately 88%. AFM morphology observation showed that the antigens fixed by click chemistry were uniformly upright with an average height of approximately 4.5 nm, while the antigens fixed by EDC / NHS were randomly lying flat or on their sides with an average height of only 1.8 nm. These results indicate that click chemistry-based directional fixation can effectively improve antigen fixation volume and immune recognition efficiency.

[0078] Comparative Example 3 This comparative example uses the single-point peak current standard curve method for quantification. The traditional single-point peak current standard curve method (reading only the peak current at -0.6V for NR and plotting a univariate standard curve of I~lgC) replaces the PLS multivariate correction model of this invention. The electrode materials and fixation method are the same as in this invention. In the PBS buffer system, the detection limit of the univariate method for endosulfan is 0.18 ppt, which is not significantly different from the 0.15 ppt of this invention. However, in spinach matrix spiked samples (5 ppt spiked), the univariate method showed a large deviation, with a relative error of -18.5% to +22.3% and an RSD of 14.2% (n=5); while the relative error of the PLS method of this invention in the same matrix is ​​only -4.2% to +5.8%, with an RSD of 4.8% (n=5). In strawberry matrix, the recoveries of the univariate method spiked ...

[0079] Comparative Example 4 This comparative example uses a dual-probe system of NR and PB. Prussian blue (characteristic potential +0.2V) was used instead of the ferrocene (characteristic potential +0.4V) of this invention, while the other conditions were exactly the same as in this invention. DPV scanning results showed that NR peaked at -0.6V and PB peaked at +0.2V, with a potential difference of 0.8V. When the concentrations of the two pesticides were high, there was partial baseline overlap between the tail of the reduction peak of NR (approximately -0.2 to -0.1V) and the oxidation peak of PB, which was particularly noticeable in spinach matrix, resulting in an integral area error of 8.5% to 12.3% for the PB peak current. In contrast, Fc peaked at +0.4V, with a potential difference of 1.0V from NR, and the baselines were completely separated without any overlap interference. In spinach-based spiked samples, the recovery rate of hexachlorocyclohexane (HCH) in the NR+Fc system of this invention was 88.2%–109.7% (RSD = 5.1%), while the recovery rate in the NR+PB system was 73.8%–128.5% (RSD = 11.2%). The results indicate that increasing the potential difference between the two probes helps eliminate signal crosstalk and improves detection reliability.

[0080] Comparative Example 5 This comparative example uses GC-MS / MS to detect the same fruit and vegetable samples. The detection limits of GC-MS / MS for endosulfan and hexachlorocyclohexane are approximately 0.5 μg / kg (0.5 ppt), which is on the same order of magnitude as the detection limit of this invention (0.15 ppt). GC-MS / MS is characterized by high accuracy and good reproducibility, but it is expensive, complex to operate, and takes approximately 2-3 hours per sample. The detection limits of this invention are comparable, but the instrument cost is low, the operation is simple, and the testing time for a single sample is approximately 60 minutes, making it suitable for rapid on-site screening.

[0081] Therefore, the above-mentioned electrochemical immunosensing detection method for multiple residues of organochlorine pesticides in fruits and vegetables effectively solves the problems of low interface electron transmission efficiency, uncontrollable antigen fixation orientation, severe interference from complex matrices, and difficulty in distinguishing multiple residue signals in the existing technology, and realizes the simultaneous detection of multiple residues with high sensitivity, high specificity, and anti-interference.

[0082] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0083] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. An electrochemical immunosensing method for detecting multiple residues of organochlorine pesticides in fruits and vegetables, characterized in that, Includes the following steps: S1. Using a liquid-phase pulsed discharge plasma synchronous reduction method, gold nanoparticles are directionally nucleated and epitaxially grown on the surface of carboxyl functionalized graphitic carbon nitride to form a g-C3N4-COOH / AuNPs heterojunction composite material with a lattice coupling interface; the heterojunction composite material is then modified onto the surface of a screen-printed carbon electrode to obtain the composite electrode AG-SPCE. S2. The composite electrode AG-SPCE is functionalized with alkyne groups on its surface. The azide-modified organochlorine antigen is covalently fixed onto the surface of AG-SPCE by click chemistry reaction. After sealing, an immunosensing electrode is obtained. S3. Prepare labeled immunoprobes, wherein the labeled immunoprobes are loaded with specific antibodies against electrochemical signaling substances and organochlorine pesticides, and the characteristic potentials of each electrochemical signaling substance do not overlap. S4. Place the immunosensing electrode in the sample solution to be tested, add the labeled immunoprobe to perform a competitive immunoreaction; collect the differential pulse voltammetric response curve in the full potential range, extract multiple characteristic parameters, and simultaneously calculate the concentrations of multiple organochlorine pesticides using a partial least squares multivariate correction model to complete the simultaneous detection of multiple organochlorine pesticide residues in fruits and vegetables.

2. The detection method according to claim 1, characterized in that, In step S1, the liquid-phase pulsed discharge plasma synchronous reduction method uses chloroauric acid as the gold source, a tungsten rod as the anode and a stainless steel mesh as the cathode. A high-voltage pulse power supply is applied under a nitrogen atmosphere for discharge treatment. The pulse voltage is 15~25kV, the pulse frequency is 50~200Hz, and the discharge treatment time is 10~30 minutes. The mass ratio of the carboxyl-functionalized graphitic carbon nitride to chloroauric acid is 5:1~10:

1.

3. The detection method according to claim 1, characterized in that, In step S1, the carboxyl-functionalized graphitic carbon nitride is prepared by the following method: using melamine as a precursor, the temperature is raised to 500-600°C at a heating rate of 2-5°C / min, held for 2-4 hours, and then naturally cooled to obtain graphitic carbon nitride; the graphitic carbon nitride is dispersed in a mixed acid of concentrated nitric acid and concentrated sulfuric acid, refluxed at 60-80°C for 4-8 hours, and then washed, centrifuged, and dried to obtain the carboxyl-functionalized product.

4. The detection method according to claim 1, characterized in that, In step S2, the surface alkyne functionalization modification is performed by immersing AG-SPCE in a PBS solution containing 4-ethynylbenzoic acid and EDC / NHS, and introducing terminal alkyne groups on the electrode surface through an amide condensation reaction; the azidated organochlorine antigen is prepared by reacting N-hydroxysuccinimide-azidoacetate with the free amino group of the organochlorine antigen.

5. The detection method according to claim 1, characterized in that, In step S2, the click chemical reaction is a copper-catalyzed azide-alkyne cycloaddition reaction. The reaction solution contains azide-modified organochlorine antigen, copper sulfate, sodium ascorbate and THPTA. The reaction temperature is 25~37℃ and the reaction time is 1~3 hours. After fixation, the electrode surface is sealed with bovine serum albumin solution for 30~60 minutes.

6. The detection method according to claim 1, characterized in that, In step S3, the labeled immunoprobe uses gold nanoparticles prepared by sodium citrate reduction as a carrier. After adjusting the pH of the gold nanoparticle sol to 8.5-9.0, it is sequentially bound to specific antibodies and electrochemical signaling substances, and then obtained after blocking with bovine serum albumin and centrifugation and washing.

7. The detection method according to claim 1, characterized in that, In step S4, the differential pulse voltammetry has a scanning range of -1.2V to +1.0V vs. Ag / AgCl, a scanning rate of 10 to 100mV / s, a pulse amplitude of 20 to 80mV, and a pulse width of 20 to 100ms. The multiple characteristic parameters include the characteristic peak current, peak half-width, and the ratio of characteristic peak current to background current for each electrochemical signal substance.

8. The detection method according to claim 7, characterized in that, The partial least squares multivariate calibration model is constructed as follows: mixed standards of organochlorine pesticides with different concentration ratios are prepared as training sets, the feature parameters corresponding to each standard are extracted as independent variable matrices, the concentration values ​​of each organochlorine pesticide are used as dependent variable matrices, the optimal number of principal components is determined through cross-validation, and a calibration model is established.

9. The detection method according to claim 1, characterized in that, The sample solution to be tested was obtained through the following pretreatment: after homogenizing the fruit and vegetable sample to be tested, a mixed solvent of n-hexane-acetone with a volume ratio of 1:1 was added for extraction, and ultrasonic extraction was performed for 10-30 minutes. The supernatant was collected by centrifugation, and then the supernatant was purified by solid-phase extraction and dried. It was then reconstituted with buffer solution to obtain the sample solution to be tested.

10. The detection method according to claim 1, characterized in that, The labeled immune probes are of at least two types, and the corresponding electrochemical signaling substances are neutral red and ferrocene, respectively, with a characteristic potential difference of 1.0V between the two signaling substances.

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