Electrochemical immunosensor for detecting organophosphorus pesticide as well as preparation method and application of electrochemical immunosensor

Abstract

The invention provides an electrochemical immunosensor for detecting organophosphorus pesticide as well as a preparation method and application of the electrochemical immunosensor, and belongs to the technical field of pesticide detection. According to the electrochemical immunosensor, PANI / Co3O4 is used as a substrate material, an organophosphorus broad-spectrum antibody (Abs) is used as a recognition element to carry out specific recognition on diethoxy thiophosphate organophosphorus pesticides, and the Abs can form a six-channel interface on the electrochemical immunosensor, so that the electrochemical immunosensor can carry out detection according to specific recognition of each pesticide; therefore, the purpose of detecting various ethyoxyl thiophosphate organic phosphorus pesticide residues on one instrument is achieved, simultaneous detection of various pesticide residues is achieved, and the purpose of rapid screening is achieved. According to the method, specific detection values can be given in a detection range, qualitative and quantitative analysis is realized, and meanwhile, the reaction time can be greatly shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide detection, and particularly to an electrochemical immunosensor for detecting organophosphorus pesticides, a preparation method thereof, and an application thereof. Background Art

[0002] Among the currently used pesticides, organophosphorus pesticides are the most widely used and the most toxic. Organophosphorus pesticides are a type of insecticide with the ability to inhibit cholinesterase activity, and have the characteristics of high efficiency, broad spectrum, and low cost. In recent years, they have been widely used in agriculture and planting, so they often cause problems such as pesticide residues in food and environmental pollution. Diethoxy thiophosphate organophosphorus pesticides are a large class of organophosphorus pesticides containing the thiodiethyl phosphate functional group, which are widely used in agricultural production. However, overuse has led to excessive residues in agricultural products, which is an important hidden danger affecting the safety of agricultural products and poses a great threat to the health of consumers. Therefore, detecting the residues of such organophosphorus pesticides in agricultural products such as fruits and vegetables is of great significance for monitoring the rational use of pesticides and ensuring human health. Traditional pesticide detection equipment has problems such as high personnel requirements, expensive equipment, and long time consumption, which is not conducive to the rapid detection of pesticide residues in practical applications; existing electrochemical immunosensors use bioactive agents (such as enzymes, microorganisms, antigens, antibodies, or animal and plant tissues, etc.) as biological sensitive elements, and then form a bioelectrochemical analysis system with appropriate physical or chemical signal transducer devices to detect residual substances, realizing the efficient, rapid, and portable detection of pesticide residues. However, this method can only detect a certain type of pesticide residue and cannot achieve the simultaneous detection of multiple pesticide residues. At the same time, its accuracy and sensitivity also need to be further improved.

[0003] Therefore, how to achieve the simultaneous detection of multiple pesticide residues by an electrochemical immunosensor and improve its detection accuracy and sensitivity has become an urgent technical problem in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide an electrochemical immunosensor for detecting organophosphorus pesticides, a preparation method thereof, and an application thereof. The electrochemical immunosensor prepared by the preparation method provided by the present invention realizes the simultaneous detection of multiple pesticide residues, and has high accuracy and sensitivity, and a short screening time, achieving the purpose of rapid screening.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method for an electrochemical immunosensor for detecting organophosphorus pesticides, comprising the following steps:

[0007] (1) Using a solution containing PANI / Co 3 O 4The dispersion of composite nanoparticles is used to modify the SPE film, and then it is activated with Gluta fixative to obtain the activated SPE film;

[0008] (2) The activated SPE film obtained in the step (1) is incubated for the first time with Abs diluent, and then incubated for the second time with BSA solution to obtain an electrochemical immunosensor for detecting organophosphorus pesticides.

[0009] Preferably, in the step (1), PANI / Co 3 O 4 The preparation method of the composite nanoparticles includes: mixing Co 3 O 4 nanoparticles, water and hydrochloric acid, followed by ultrasonic dispersion, then adding aniline and stirring magnetically, then adding ammonium persulfate for polymerization reaction, and finally filtering, washing, drying and grinding in sequence to obtain PANI / Co 3 O 4 composite nanoparticles.

[0010] Preferably, the mass ratio of the Co 3 O 4 nanoparticles, the volume of water, the volume of hydrochloric acid, the volume of aniline and the mass of ammonium persulfate is (0.2 - 1) g:(40 - 60) mL:(4 - 6) mL:(1 - 2) mL:(4 - 5) g.

[0011] Preferably, in the step (1), the preparation method of the dispersion containing PANI / Co 3 O 4 composite nanoparticles is: mixing chitosan solution and PANI / Co 3 O 4 composite nanoparticles and then performing ultrasonic dispersion to obtain a dispersion containing PANI / Co 3 O 4 composite nanoparticles.

[0012] Preferably, in the step (1), the dosage of the dispersion containing PANI / Co 3 O 4 composite nanoparticles is 20 - 70 μL / cm 2 .

[0013] Preferably, in the step (1), the mass concentration of the Gluta fixative is 0.2 - 0.3%, and the dosage of the Gluta fixative is 120 - 200 μL / cm 2 ; the activation temperature is room temperature, and the activation time is 1.5 - 3 h.

[0014] Preferably, in the step (2), the concentration of the Abs diluent is 2-10 μg / mL, and the dosage of the Abs diluent is 20-70 μL / cm 2 ; the temperature of the first incubation is 35-40 °C, and the time of the first incubation is 30-120 min.

[0015] Preferably, in the step (2), the mass concentration of the BSA solution is 1-4%, and the dosage of the BSA solution is 120-200 μL / cm 2 ; the time of the second incubation is 0.5-2 h.

[0016] The present invention provides an electrochemical immunosensor for detecting organophosphorus pesticides prepared by the preparation method described in the above technical solution.

[0017] The present invention provides the application of the electrochemical immunosensor for detecting organophosphorus pesticides described in the above technical solution in the detection of organophosphorus pesticides.

[0018] The present invention provides a preparation method of an electrochemical immunosensor for detecting organophosphorus pesticides, comprising the following steps: (1) modifying an SPE film with a dispersion liquid containing PANI / Co 3 O 4 composite nanoparticles, and then activating with a Gluta fixing solution to obtain an activated SPE film; (2) performing a first incubation on the activated SPE film obtained in the step (1) with an Abs diluent, and then performing a second incubation with a BSA solution to obtain an electrochemical immunosensor for detecting organophosphorus pesticides. The present invention uses PANI / Co 3 O 4 as a substrate material, specifically recognizes diethoxy thiophosphate organophosphorus pesticides by using an organophosphorus broad-spectrum antibody (Abs) as a recognition element, and Abs can form a six-channel interface in the electrochemical immunosensor, enabling the electrochemical immunosensor to detect according to the specific recognition of each pesticide, thereby achieving the purpose of detecting the pesticide residues of multiple ethoxy thiophosphate organophosphorus on one instrument, realizing the simultaneous detection of multiple pesticide residues, and thus achieving the purpose of rapid screening. At the same time, the existing technology for pesticide detection is a preliminary screening, which can only judge whether there is or not, belonging to qualitative analysis, while the electrochemical immunosensor provided by the present invention can also give specific detection values within the detection range, realizing qualitative and quantitative analysis, with high accuracy and sensitivity, short screening time. Compared with traditional large-scale instruments, the present invention can greatly shorten the reaction time while realizing qualitative and quantitative detection. The results of the examples show that the electrochemical immunosensor provided by the present invention can realize the detection of multiple pesticide residues, with high accuracy and sensitivity, short screening time, and good reproducibility and stability of the electrochemical immunosensor, which is convenient for long-term storage. Brief Description of the Drawings

[0019] Figure 1 Schematic diagram of the preparation process of the electrochemical immunosensor provided by the present invention;

[0020] Figure 2 Co 3 O 4 SEM image of the nanoparticles used in Example 1 of the present invention;

[0021] Figure 3 SEM image of the PANI / Co 3 O 4 composite nanoparticles prepared in Example 1 of the present invention;

[0022] Figure 4 Chemical element position distribution of the PANI / Co 3 O 4 composite nanoparticles prepared in Example 1 of the present invention;

[0023] Figure 5 Chemical element composition of the PANI / Co 3 O 4 composite nanoparticles prepared in Example 1 of the present invention;

[0024] Figure 6 Electrochemical characterization of the Co 3 O 4 nanoparticles, PANI and PANI / Co 3 O 4 composite nanoparticles used in Example 1 of the present invention;

[0025] Figure 7 Electrochemical characterization of the products obtained at different stages in Example 1 and Application Example 1 of the present invention;

[0026] Figure 8 Variation curves of the conductivity of the electrochemical immunosensor with the change of the types and concentrations of OPs in Application Examples 1 to 20;

[0027] Figure 9 Variation curves of the conductivity of the electrochemical immunosensor with the change of the types of OPs and the first incubation time in Application Examples 1, 6, 11, 16 and 21 to 36;

[0028] Figure 10 Variation curves of the conductivity of the electrochemical immunosensor with the change of the types of OPs and the incubation time in Application Examples 1, 6, 11, 16 and 37 to 52;

[0029] Figure 11 Relationship between the concentration (C) of parathion and the corresponding peak value of the electrochemical signal (ΔI);

[0030] Figure 12 It is the relationship between the concentration (C) of quinalphos and the peak value of the corresponding electrochemical signal (ΔI);

[0031] Figure 13 It is the relationship between the concentration (C) of coumaphos and the peak value of the corresponding electrochemical signal (ΔI);

[0032] Figure 14 It is the relationship between the concentration (C) of triazophos and the peak value of the corresponding electrochemical signal (ΔI);

[0033] Figure 15 It is the specificity and anti-interference ability of the electrochemical immunosensor for detecting organophosphorus pesticides provided in Example 1 to different types of substances;

[0034] Figure 16 It is the reproducibility of the electrochemical immunosensor for detecting organophosphorus pesticides provided in Example 1 to different types of substances. Detailed implementation mode

[0035] The present invention provides a preparation method of an electrochemical immunosensor for detecting organophosphorus pesticides, including the following steps:

[0036] (1) Modify the SPE film with a dispersion liquid containing PANI / Co 3 O 4 composite nanoparticles, and then activate it with a Gluta fixing solution to obtain an activated SPE film;

[0037] (2) Perform the first incubation on the activated SPE film obtained in step (1) with an Abs diluent, and then perform the second incubation with a BSA solution to obtain an electrochemical immunosensor for detecting organophosphorus pesticides.

[0038] In the present invention, unless otherwise specified, the raw materials used are all commercially available products well-known to those skilled in the art.

[0039] The present invention modifies the SPE film with a dispersion liquid containing PANI / Co 3 O 4 composite nanoparticles, and then activates it with a Gluta fixing solution to obtain an activated SPE film.

[0040] In the present invention, the SPE film is preferably a super-functional polyethylene film. The present invention has no special limitation on the specific source and thickness of the SPE film, and a commercially available SPE film well-known to those skilled in the art can be used.

[0041] In the present invention, the SPE film is preferably cleaned and dried before use. In the present invention, the detergent used for cleaning is preferably anhydrous ethanol. The present invention has no special limitation on the specific operations of the cleaning and drying, which are determined according to the common technical knowledge of those skilled in the art, as long as the impurities can be removed. By cleaning and drying, the present invention can remove the impurities on the surface of the SPE film.

[0042] In the present invention, the 3 O 4 preparation method of the PANI / Co 3 O 4 composite nanoparticles preferably includes mixing Co 3 O 4 nanoparticles, water and hydrochloric acid, followed by ultrasonic dispersion, then adding aniline for magnetic stirring, then adding ammonium persulfate for polymerization reaction, and finally performing filtration washing, drying and grinding in sequence to obtain PANI / Co

[0043] The present invention has no special limitation on the power and time of the ultrasonic dispersion, which are determined according to the common technical knowledge of those skilled in the art, as long as the components can be mixed evenly. As an embodiment of the present invention, the time of the ultrasonic dispersion can be 0.5 - 2 h, and can also be 1 - 1.5 h.

[0044] In the present invention, the time of the magnetic stirring is preferably 0.5 - 1 h. The present invention has no special limitation on the rate of the magnetic stirring, which is determined according to the common technical knowledge of those skilled in the art, as long as aniline can be mixed evenly with other components.

[0045] In the present invention, the time of the polymerization reaction is preferably 8 - 15 h, more preferably 10 - 12 h. By controlling the time of the polymerization reaction, the present invention can make the raw materials fully mixed and composite with Co 3 O 4 nanoparticles, so as to form PANI / Co 3 O 4 composite nanoparticles.

[0046] In the present invention, the particle size of the Co 3 O 4 nanoparticles is preferably 80 - 95 nm; the water is preferably deionized water; the concentration of the hydrochloric acid is preferably 1 - 3 mol / L, more preferably 2 mol / L. In the present invention, the Co 3 O 4The mass ratio of the nanoparticles, the volume of water, the volume of hydrochloric acid, the volume of aniline, and the mass of ammonium persulfate is preferably (0.2 - 1) g : (40 - 60) mL : (4 - 6) mL : (1 - 2) mL : (4 - 5) g, more preferably (0.5 - 0.9) g : (45 - 55) mL : (4 - 6) mL : (1.4 - 1.8) mL : (4.4 - 4.6) g, and even more preferably 0.9 g : 50 mL : 5 mL : 1.6 mL : 4.56 g. By controlling the dosages of the respective components in the present invention, the proportions of the components in the PANI / Co 3 O 4 composite nanoparticles can be controlled, thereby obtaining the desired PANI / Co 3 O 4 composite nanoparticles.

[0047] In the present invention, the filtration and washing are preferably carried out successively with hydrochloric acid, deionized water, and ethanol. There are no special limitations on the concentration of the hydrochloric acid and the specific operation of the filtration and washing in the present invention, as long as the impurities can be removed until the filtrate is colorless. In the present invention, the drying is preferably vacuum drying; the temperature of the vacuum drying is preferably 50 - 80 °C; the time of the vacuum drying is preferably 20 - 30 h; the vacuum drying is preferably carried out in a full-automatic silicone oil circulation system freeze dryer. By vacuum drying in the present invention, the detergent can be removed without affecting the structure of the product. As an embodiment of the present invention, the temperature of the vacuum drying can be 60 - 70 °C; the time of the vacuum drying can be 22 - 28 h, or can also be 24 - 26 h. There are no special limitations on the specific operation of the grinding in the present invention, as long as the particle size of the PANI / Co 3 O 4 composite nanoparticles meets the requirements. In the present invention, the particle size of the PANI / Co 3 O 4 composite nanoparticles is preferably 80 - 95 nm.

[0048] In the present invention, the preparation method of the dispersion liquid containing the PANI / Co 3 O 4 composite nanoparticles is preferably: mixing the chitosan solution and the PANI / Co 3 O 4 composite nanoparticles and then performing ultrasonic dispersion to obtain a dispersion liquid containing the PANI / Co 3 O 4Dispersion of composite nanoparticles. In the present invention, the preparation method of the chitosan solution is preferably: 100 - 150 mg of CS chitosan powder, 500 μL of acetic acid and 50 mL of ultrapure water are mixed and dissolved by ultrasonic treatment to obtain a chitosan solution. More preferably: 125 mg of CS chitosan powder, 500 μL of acetic acid and 50 mL of ultrapure water are mixed and dissolved by ultrasonic treatment to obtain a chitosan solution; the PANI / Co 3 O 4 The mass ratio of the composite nanoparticles to the volume of the chitosan solution is preferably 1 - 4 mg:1 mL, more preferably 2 - 3 mg:1 mL. By controlling the preparation method and dosage relationship of the dispersion containing PANI / Co 3 O 4 in the present invention, the dosage of PANI / Co 3 O 4 composite nanoparticles can be better controlled.

[0049] In the present invention, the dosage of the dispersion containing PANI / Co 3 O 4 composite nanoparticles is preferably 20 - 70 μL / cm 2 , more preferably 30 - 50 μL / cm 2 , further preferably 35 - 45 μL / cm 2 . In the present invention, the dispersion containing PANI / Co 3 O 4 composite nanoparticles is preferably dried at 30 - 35 °C. By controlling the dosage of the dispersion, the dosage of PANI / Co 3 O 4 composite nanoparticles can be further controlled.

[0050] In the present invention, the coating method of the Gluta fixative is preferably drop coating. The present invention has no special limitation on the specific operation of the drop coating, which is determined according to the common technical knowledge of those skilled in the art, as long as the dosage of the Gluta fixative meets the requirements.

[0051] In the present invention, the mass concentration of the Gluta fixative is preferably 0.2 - 0.3%, more preferably 0.22 - 0.28%, further preferably 0.25%; the dosage of the Gluta fixative is preferably 120 - 200 μL / cm 2 , more preferably 140 - 180 μL / cm 2 , further preferably 150 - 165 μL / cm 2; The activation temperature is preferably room temperature; the activation time is preferably 1.5 to 3 h, more preferably 2 to 2.5 h. By using Gluta fixative for activation, the present invention can firmly fix PANI / Co 3 O 4 composite nanoparticles on the surface of the SPE film.

[0052] Preferably, after the activation is completed, the activated product is washed and dried in sequence. In the present invention, the detergent used for washing is preferably an immunocleaning solution, more preferably deionized water. The present invention has no special limitation on the drying temperature and time, which are determined according to the common technical knowledge of those skilled in the art as long as the product can be dried. By washing and drying, the present invention can remove impurities.

[0053] After obtaining the activated SPE film, the present invention uses the coated Abs diluent to perform the first incubation on the activated SPE film, and then uses the BSA (bovine serum albumin) solution to perform the second incubation to obtain an electrochemical immunosensor for detecting organophosphorus pesticides.

[0054] In the present invention, the Abs is preferably a commercially available broad-spectrum antibody for organophosphorus pesticides. The present invention has no special limitation on the specific source of the Abs, and a commercially available broad-spectrum antibody for organophosphorus pesticides well-known to those skilled in the art can be used. As an embodiment of the present invention, the Abs is preferably the Abs produced by Beijing Boao Long Biotechnology Co., Ltd.

[0055] In the present invention, the coating method of the Abs diluent is preferably drop coating. The present invention has no special limitation on the specific operation of the drop coating, which is determined according to the common technical knowledge of those skilled in the art as long as the dosage of the Abs diluent can meet the requirements.

[0056] In the present invention, the concentration of the Abs diluent is preferably 2 to 10 μg / mL, more preferably 4 to 8 μg / mL, and further preferably 6 μg / mL; the dosage of the Abs diluent is preferably 20 to 70 μL / cm 2 , more preferably 30 to 50 μL / cm 2 , and further preferably 35 to 45 μL / cm 2 ; the temperature of the first incubation is preferably 35 to 40 °C, more preferably 32 °C; the time of the first incubation is preferably 30 to 120 min, more preferably 60 to 90 min. By using the broad-spectrum antibody for organophosphorus pesticides (Abs) as the recognition element to specifically recognize diethoxy thiophosphate organophosphorus pesticides, the present invention can improve the recognition efficiency of organophosphorus pesticides. By controlling the dosage and incubation time of the Abs, the detection accuracy can be further improved.

[0057] The present invention preferably washes the product of the first incubation after the end of the first incubation; the detergent used for the washing is preferably an immuno-washing solution, more preferably deionized water. By washing, the present invention can remove the unreacted Abs.

[0058] In the present invention, the mass concentration of the BSA solution is preferably 1-4%, more preferably 2-3%; the dosage of the BSA solution is preferably 120-200 μL / cm 2 , more preferably 140-180 μL / cm 2 , further preferably 150-165 μL / cm 2 ; the time of the second incubation is preferably 0.5-2 h, more preferably 1-1.5 h. By incubating with BSA, the present invention can block the non-specific adsorption sites on the electrode surface.

[0059] The present invention preferably washes the product of the second incubation after the end of the second incubation. In the present invention, the detergent used for the washing is preferably an immuno-washing solution, more preferably deionized water. By washing, the present invention can remove the remaining BSA.

[0060] In the present invention, the storage temperature of the electrochemical immunosensor for detecting organophosphorus pesticides is preferably ≤5°C, more preferably 4°C.

[0061] The present invention uses PANI / Co 3 O 4 as the base material, and uses an organophosphorus broad-spectrum antibody (Abs) as the recognition element to specifically recognize diethoxy thiophosphate organophosphorus pesticides. And the Abs can form a six-channel interface on the electrochemical immunosensor, enabling the electrochemical immunosensor to detect according to the specific recognition of each pesticide, thereby achieving the purpose of detecting the pesticide residues of multiple ethoxy thiophosphate organophosphorus pesticides on one instrument, realizing the simultaneous detection of multiple pesticide residues, and thus achieving the purpose of rapid screening. At the same time, the prior art for pesticide detection is a preliminary screening, which can only judge whether there is or not, belonging to qualitative analysis. While the electrochemical immunosensor provided by the present invention can also give specific detection values within the detection range, realizing qualitative and quantitative analysis, with high accuracy and sensitivity, short screening time. Compared with traditional large-scale instruments, the present invention can greatly shorten the reaction time while realizing qualitative and quantitative detection.

[0062] The present invention also provides an electrochemical immunosensor for detecting organophosphorus pesticides prepared by the preparation method described in the above technical solution.

[0063] The electrochemical immunosensor for detecting organophosphorus pesticides provided by the present invention has a six-channel interface.

[0064] The present invention also provides the application of the electrochemical immunosensor for detecting organophosphorus pesticides described in the above technical solution in the detection of organophosphorus pesticides.

[0065] In the present invention, the preferred application method is: dropwise coat the sample to be tested on the surface of the electrochemical immunosensor for detecting organophosphorus pesticides, then incubate, then wash, and finally perform electrochemical analysis to obtain the concentration of organophosphorus in the sample to be tested.

[0066] The present invention has no special limitation on the specific source of the sample to be tested, and any sample to be tested containing organophosphorus well-known in the art can be used.

[0067] In the present invention, the dosage of the sample to be tested is preferably 20 - 70 μL / cm 2 , more preferably 30 - 50 μL / cm 2 , further preferably 35 - 45 μL / cm 2 .

[0068] The present invention has no special limitation on the specific operation of the dropwise coating, which is determined according to the common technical knowledge of those skilled in the art, as long as the dosage of the sample to be tested meets the requirements.

[0069] In the present invention, the incubation temperature is preferably 35 - 40 °C, more preferably 32 °C; the incubation time is preferably 30 - 60 min, more preferably 40 - 50 min, and further preferably 45 min. By controlling the incubation parameters, the present invention can make the organophosphorus in the sample to be tested fully contact with the electrochemical immunosensor.

[0070] In the present invention, the detergent used for washing is preferably an immunological washing solution, more preferably deionized water. By washing, the present invention can remove residual impurities.

[0071] In the present invention, the electrochemical analysis preferably uses a CHI1240C electrochemical workstation for electrochemical analysis. The present invention has no special limitation on the specific source of the CHI1240C electrochemical workstation, and a commercially available CHI1240C electrochemical workstation well-known to those skilled in the art can be used.

[0072] In the present invention, the operation of the electrochemical analysis preferably includes: detecting using a three-electrode system, where the working electrode is the SPE film in the electrochemical immunosensor for detecting organophosphorus pesticides, the reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum electrode. Immerse the ends of the counter electrode and the reference electrode into a solution containing [Fe(CN)6] 3- or [Fe(CN)6] 4-In a mixed solution of [Fe(CN)₆] and KCl, cyclic voltammetry (CV) was used for analysis to obtain the content of organophosphorus pesticides in the sample to be tested. In the present invention, the measurement parameters of the cyclic voltammetry are preferably: scanning potential -0.45 to 0.85 V, scanning rate 0.1 V / s, sampling interval 1 mV, and standing time 2 s.

[0073] In the present invention, the reference electrode preferably maintains a constant potential through a saturated chloride ion solution; the saturated chloride ion solution is preferably a 3 mol / L KCl solution.

[0074] In the present invention, the concentration of [Fe(CN)₆] in the mixed solution 3- or [Fe(CN)₆] 4- is preferably 5 mmol / L; the concentration of KCl in the mixed solution is preferably 0.1 mol / L.

[0075] In the present invention, the measurement parameters of the cyclic voltammetry are preferably: scanning potential -0.1 to 0.8 V, scanning rate 50 mV / s, sampling interval 1 mV, and standing time 2 s.

[0076] Compared with the existing rapid detection technologies, the electrochemical detection method of the electrochemical immunosensor provided by the present invention is that the existing technology is a preliminary screening, which can only judge whether there is or not, and it is a qualitative analysis. While the present invention can give specific detection values within the detection range to achieve qualitative and quantitative analysis. Compared with traditional large instruments, the present invention can greatly shorten the reaction time while realizing qualitative and quantitative detection.

[0077] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0078] Example 1

[0079] A preparation method of an electrochemical immunosensor for detecting organophosphorus pesticides comprises the following steps:

[0080] (1) Clean a circular SPE film with a diameter of 4 mm using absolute ethanol. After drying, drop 5 μL of a solution containing PANI / Co 3 O 4A dispersion of composite nanoparticles was used to modify the SPE film, which was dried at 32 °C, then 20 μL of Gluta fixative was drop-coated for activation, and finally washed with deionized water. After drying, the activated SPE film was obtained; the SPE film was a commercially available ultra-functional polyethylene film; the mass concentration of the Gluta fixative was 0.25%; the activation temperature was room temperature and the activation time was 2 h;

[0081] (2) 5 μL of Abs diluent was drop-coated on the activated surface of the activated SPE film obtained in step (1) for the first incubation. After washing away the unreacted Abs with deionized water, 20 μL of BSA solution was continued to be drop-coated for the second incubation. Finally, the excess BSA was washed with deionized water, and after drying, an electrochemical immunosensor for detecting organophosphorus pesticides was obtained, and then stored at a temperature of 4 °C; the concentration of the Abs diluent was 6 μg / mL; the Abs was from Beijing BoaoLong Biotechnology Co., Ltd.; the temperature of the first incubation was 32 °C and the time of the first incubation was 60 min; the mass concentration of the BSA solution was 2%; the time of the second incubation was 1 h;

[0082] In step (1), PANI / Co 3 O 4 The preparation method of the composite nanoparticles was as follows: Co 3 O 4 nanoparticles, deionized water and hydrochloric acid were mixed and then ultrasonically dispersed for 1 h, then aniline was added and magnetically stirred for 0.5 h, then ammonium persulfate was added for polymerization reaction for 10 h, and finally filtration washing, vacuum drying and grinding were carried out in sequence to obtain PANI / Co 3 O 4 composite nanoparticles; the concentration of the hydrochloric acid was 2 mol / L; the particle size of the Co 3 O 4 nanoparticles was 80 - 95 nm; the mass ratio of the Co 3 O 4 nanoparticles, the volume of water, the volume of hydrochloric acid, the volume of aniline and the mass of ammonium persulfate was 0.9 g:50 mL:5 mL:1.6 mL:4.56 g; the filtration washing was carried out by filtering with hydrochloric acid, deionized water and ethanol in sequence; the drying was vacuum drying, the temperature of the vacuum drying was 60 °C, the time of the vacuum drying was 24 h, and the vacuum drying was carried out in a full-automatic silicone oil circulation system freeze dryer;

[0083] The preparation method of the dispersion containing PANI / Co 3 O 4 composite nanoparticles was as follows: 1 mL of chitosan solution and 2 mg of PANI / Co 3 O 4The composite nanoparticles are ultrasonically dispersed after mixing to obtain a dispersion containing PANI / Co 3 O 4 composite nanoparticles; the preparation method of the chitosan solution is: 125 mg of CS chitosan powder, 500 μL of acetic acid and 50 mL of ultrapure water are mixed and ultrasonically dissolved to obtain a chitosan solution.

[0084] Example 2

[0085] In step (2), the concentration of the Abs diluent is 2 μg / mL, and other conditions are the same as those in Example 1.

[0086] Example 3

[0087] In step (2), the concentration of the Abs diluent is 4 μg / mL, and other conditions are the same as those in Example 1.

[0088] Example 4

[0089] In step (2), the concentration of the Abs diluent is 6 μg / mL, and other conditions are the same as those in Example 1.

[0090] Example 5

[0091] In step (2), the concentration of the Abs diluent is 8 μg / mL, and other conditions are the same as those in Example 1.

[0092] Example 6

[0093] In step (2), the concentration of the Abs diluent is 10 μg / mL, and other conditions are the same as those in Example 1.

[0094] Example 7

[0095] In step (2), the time of the first incubation is 15 min, and other conditions are the same as those in Example 1.

[0096] Example 8

[0097] In step (2), the time of the first incubation is 30 min, and other conditions are the same as those in Example 1.

[0098] Example 9

[0099] In step (2), the time of the first incubation is 45 min, and other conditions are the same as those in Example 1.

[0100] Example 10

[0101] In step (2), the time of the first incubation is 75 min, and other conditions are the same as those in Example 1.

[0102] Example 11

[0103] In step (2), the time of the second incubation is 15 min, and other conditions are the same as those in Example 1.

[0104] Example 12

[0105] In step (2), the time of the second incubation is 30 min, and other conditions are the same as those in Example 1.

[0106] Example 13

[0107] In step (2), the time of the second incubation is 45 min, and other conditions are the same as those in Example 1.

[0108] Example 14

[0109] In step (2), the time of the second incubation is 75 min, and other conditions are the same as those in Example 1.

[0110] Figure 2 The Co 3 O 4 nanoparticles used in Example 1 of the present invention. As Figure 2 can be seen, the Co 3 O 4 nanoparticles have a flat, smooth spherical or ellipsoidal structure.

[0111] Figure 3 The SEM image of the PANI / Co 3 O 4 composite nanoparticles prepared in Example 1 of the present invention. As Figure 3 can be seen, the surface of the composite nanoparticles is relatively rough and has obvious wrinkles. From the comparison between Figure 2 and Figure 3 it shows that the specific surface area of the PANI / Co 3 O 4 composite nanoparticles generated after PANI coating increases significantly.

[0112] Figure 4 and Figure 5 The EDS characterization of the PANI / Co 3 O 4 composite nanoparticles prepared in Example 1 of the present invention, where Figure 4 is the chemical element position distribution of the PANI / Co 3 O 4 composite nanoparticles; Figure 5 is the chemical element composition of the PANI / Co 3 O 4 composite nanoparticles. As Figure 4 and Figure 5 can be seen, PANI / Co3 O 4 The components of the composite nanoparticles include carbon (C), nitrogen (N), oxygen (O), cobalt (Co), etc., which is in line with expectations, verifying the effective preparation of PANI / Co 3 O 4 composite nanoparticles. At the same time, it can be seen that PANI / Co 3 O 4 composite nanoparticles are core-shell structured composite nanoparticles with Co 3 O 4 as the core, PANI as the shell, and a large surface area.

[0113] Figure 6 The Co 3 O 4 nanoparticles, PANI, and PANI / Co 3 O 4 composite nanoparticles used in Example 1 of the present invention are electrochemically characterized. It can be seen from Figure 6 that the peak current value of the Co 3 O 4 nanoparticles is 49.93 μA, the peak current value of PANI is 64.16 μA, while the peak current value of the composite nanoparticles with PANI / Co 3 O 4 as the substrate material is 66.45 μA. This shows that the combination of PANI and Co 3 O 4 nanoparticles has good conductivity and can effectively increase the peak current value of the electrode.

[0114] Application Example

[0115] An electrochemical immunosensor for detecting organophosphorus pesticides is used to perform electrochemical analysis on the sample to be tested. The specific method is as follows:

[0116] 1) Drop the sample to be tested on the surface of the electrochemical immunosensor for detecting organophosphorus pesticides, then incubate, and then wash with deionized water and dry to obtain a working electrode; the dosage of the sample to be tested is 5 μL / cm 2 ; the incubation temperature is 32 °C and the incubation time is 45 min; the sample to be tested is an OPs (i.e., organophosphorus pesticide) solution;

[0117] 2) Use a CHI1240C electrochemical workstation to perform electrochemical analysis. A three-electrode system is used for detection. Among them, the working electrode is the working electrode obtained in step 1), the reference electrode is an Ag / AgCl electrode, and the reference electrode maintains a constant potential through a saturated chloride ion solution. The saturated chloride ion solution is a 3.0 mol / L KCl solution, and the counter electrode is a platinum electrode with good conductivity and stability;

[0118] 3) Immerse the ends of the counter electrode and reference electrode in step 2) into a mixed solution containing 5 mmol / L [Fe(CN)6] 3- and 0.1 mol / L KCl, and analyze it by cyclic voltammetry (CV). The measurement parameters of cyclic voltammetry are set as follows: scanning potential -0.1 to 0.8 V, scanning rate 50 mV / s, sampling interval 1 mV, standing time 2 s; measure the CV response value of each electrochemical immunosensor after BSA blocking, and denote it as I 1 , after cleaning and drying the electrode surface, collect the CV response peak of the electrochemical immunosensor electrode after adding OPs, and denote it as I 2 , calculate the ΔI value (ΔI = I 1 - I 2 ), and then analyze the relationship between ΔI and the concentration of OPs.

[0119] The schematic diagram of the preparation process of the electrochemical immunosensor provided by the present invention is as Figure 1 shown. As Figure 1 can be seen, the present invention coats PANI / Co 3 O 4 composite nanoparticles on the circular SPE film as the substrate material, and then introduces the organophosphorus broad-spectrum antibody (Abs). Abs can form a six-channel interface in the electrochemical immunosensor. Subsequently, BSA is added for incubation to block the non-specific adsorption sites on the electrode surface. In this way, the formed electrochemical immunosensor can detect the antigen Ag (i.e., organophosphorus pesticide) in the sample to be tested, and perform detection according to the specific recognition of each pesticide, thus achieving the purpose of detecting the pesticide residues of multiple ethoxy thiophosphate organophosphorus on one instrument, realizing the simultaneous detection of multiple pesticide residues, and thus achieving the purpose of rapid screening.

[0120] Application Example 1

[0121] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 1. The OPs is quinalphos, and the concentration of the OPs solution is 6 μg / mL. Other conditions are the same as those in the application example.

[0122] Figure 7 This is the electrochemical characterization of the products obtained at different stages in Example 1 and Application Example 1 of the present invention. As Figure 7 can be seen, the peak current value of the bare SPE film electrode is relatively high, which is due to the relatively fast electron transfer rate on the surface of the bare SPE electrode. When PANI / Co 3 O 4After the composite nanoparticles, the redox peak current increased significantly, indicating that the prepared composite nanoparticles were successfully modified on the surface of the SPE and had good conductivity. With the modification of 0.25% Gluta fixative, as a cross-linking agent, it successfully connected with the PANI / Co 3 O 4 composite nanoparticles, resulting in an obvious decrease in the peak current value. Further modification with Abs, the protein molecules adsorbed on the electrode, hindered the electron transfer, leading to a decrease in the peak current value. When BSA was added to the surface of the electrochemical immunosensor to block the non-specific binding sites, the peak current of the electrochemical immunosensor continued to decrease. Finally, after the modification of OPs, it specifically bound to the antibody, and the formed antigen-antibody complex further blocked more pore channels on the surface of the modified electrode, increasing the resistance through the membrane and resulting in a further decrease in the response current. The above results indicate that the electrochemical immunosensor for detecting organophosphorus pesticides was successfully modified step by step.

[0123] Application Examples 2 - 5

[0124] The concentrations of the OPs solution were 2 μg / mL, 4 μg / mL, 8 μg / mL, and 10 μg / mL in sequence, and other conditions were the same as those in Application Example 1, and the obtained ones were Application Examples 2 - 5 in sequence.

[0125] Application Example 6

[0126] The OPs was coumaphos, and other conditions were the same as those in Application Example 1.

[0127] Application Examples 7 - 10

[0128] The concentrations of the OPs solution were 2 μg / mL, 4 μg / mL, 8 μg / mL, and 10 μg / mL in sequence, and other conditions were the same as those in Application Example 6, and the obtained ones were Application Examples 7 - 10 in sequence.

[0129] Application Example 11

[0130] The OPs was triazophos, the concentration of the OPs solution was 8 μg / mL, and other conditions were the same as those in Application Example 1.

[0131] Application Examples 12 - 15

[0132] The concentrations of the OPs solution were 2 μg / mL, 4 μg / mL, 6 μg / mL, and 10 μg / mL in sequence, and other conditions were the same as those in Application Example 11, and the obtained ones were Application Examples 12 - 15 in sequence.

[0133] Application Example 16

[0134] The OPs was parathion, the concentration of the OPs solution was 4 μg / mL, and other conditions were the same as those in Application Example 1.

[0135] Application Examples 17 - 20

[0136] The concentrations of the OPs solutions are 2 μg / mL, 6 μg / mL, 8 μg / mL, and 10 μg / mL in sequence. Other conditions are the same as those in Application Example 16, and Application Examples 17 - 20 are obtained in sequence.

[0137] Figure 8 It is the change curve of the conductivity of the electrochemical immunosensor with the change of the type and concentration of OPs in Application Examples 1 - 20. From Figure 8 It can be seen that the coupling efficiency of Abs and OPs significantly affects the charge transfer efficiency and performance of the sensor interface. Although increasing the number of OPs on the sensor surface can improve the detection sensitivity, it will also lead to an increase in cost, and excessive OPs coupling may reduce the conductivity of the sensor. It was observed in the experiment that as the concentration of OPs increased, the peak current also gradually increased. The ΔI value changed the most when the antibody concentration was 6 μg / mL for quinalphos and coumaphos, 8 μg / mL for triazophos, and 4 μg / mL for parathion, and the average deviation was the smallest at this time, indicating that the electrochemical immunosensor had the best stability under these conditions.

[0138] Application Example 21

[0139] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 7, and other conditions are the same as those in Application Example 1.

[0140] Application Example 22

[0141] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 8, and other conditions are the same as those in Application Example 1.

[0142] Application Example 23

[0143] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 9, and other conditions are the same as those in Application Example 1.

[0144] Application Example 24

[0145] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 10, and other conditions are the same as those in Application Example 1.

[0146] Application Example 25

[0147] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 7, and other conditions are the same as those in Application Example 6.

[0148] Application Example 26

[0149] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 8, and other conditions are the same as those in Application Example 6.

[0150] Application Example 27

[0151] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 9, and other conditions are the same as those in Application Example 6.

[0152] Application Example 28

[0153] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 10, and other conditions are the same as those in Application Example 6.

[0154] Application Example 29

[0155] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 7, and other conditions are the same as those in Application Example 11.

[0156] Application Example 30

[0157] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 8, and other conditions are the same as those in Application Example 11.

[0158] Application Example 31

[0159] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 9, and other conditions are the same as those in Application Example 11.

[0160] Application Example 32

[0161] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 10, and other conditions are the same as those in Application Example 11.

[0162] Application Example 33

[0163] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 7, and other conditions are the same as those in Application Example 16.

[0164] Application Example 34

[0165] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 8, and other conditions are the same as those in Application Example 16.

[0166] Application Example 35

[0167] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 9, and other conditions are the same as those in Application Example 16.

[0168] Application Example 36

[0169] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 10, and other conditions are the same as those in Application Example 16.

[0170] Figure 9 It is the change curve of the conductivity of the electrochemical immunosensor with the change of the types of OPs and the first incubation time in Application Examples 1, 6, 11, 16 and 21 - 36. From Figure 9 It can be seen that with the extension of the first incubation time, the change amount of the current response shows an upward trend. The change amount of ΔI of quinalphos is the largest at the incubation time of 45 min, and that of coumaphos, triazophos and parathion is the largest at 60 min (see Figure 9 ). With the increase of the first incubation time, more Abs are immobilized on the electrode surface, thus increasing the chance of specific binding with OPs, resulting in an enhanced current response. When the first incubation time exceeds the optimal time, due to the too long deposition time, the activity of Abs is affected, and the binding with OPs is restricted, ultimately leading to a decrease in the value of ΔI.

[0171] Application Examples 37 - 40

[0172] The incubation times in step 1) are 15 min, 30 min, 60 min and 75 min in sequence, and other conditions are the same as those in Application Example 1, and Application Examples 37 - 40 are obtained in sequence.

[0173] Application Examples 41 - 44

[0174] The incubation times in step 1) are 15 min, 30 min, 60 min and 75 min in sequence, and other conditions are the same as those in Application Example 6, and Application Examples 41 - 44 are obtained in sequence.

[0175] Application Examples 45 - 48

[0176] The incubation times in step 1) are 15 min, 30 min, 60 min and 75 min in sequence, and other conditions are the same as those in Application Example 11, and Application Examples 45 - 48 are obtained in sequence.

[0177] Application Examples 49 - 52

[0178] In step 1), the incubation times are 15 min, 30 min, 60 min, and 75 min in sequence. Other conditions are the same as those in Application Example 16, and Application Examples 49 - 54 are obtained accordingly.

[0179] Figure 10 They are the change curves of the conductivity of the electrochemical immunosensor with the change of the types of OPs and the incubation time in Application Examples 1, 6, 11, 16, and 37 - 52. Figure 10 It can be seen that the formation of the immune complex by the combination of OPs and Abs is a process that progresses with time. During the incubation period of 15 - 45 min, the observed ΔI value increases with the extension of the incubation time, indicating that the intensity of the immune reaction is positively correlated with the incubation time. However, when the incubation time reaches 45 min, the current change curve begins to show a downward trend, indicating that the immune reaction may be approaching equilibrium. Therefore, 45 min is used as the incubation time for the experiment. The above phenomenon is because in the initial stage, the Abs on the electrode can quickly and specifically bind to the target OPs and form an immune complex on the electrode surface, increasing the difficulty of electron transfer and making the current stronger. With the extension of the incubation time, the Abs highly bind to the OPs in the detection system, and the electrode surface gradually reaches the saturated state of OPs, resulting in a decrease in the change of the reaction current. Based on these observations, 45 min of incubation time is used as the standard time for the sensor immune reaction.

[0180] Figure 11 It is the relationship between the concentration (C) of parathion and the corresponding peak value of the electrochemical signal (ΔI); Figure 12 It is the relationship between the concentration (C) of quinalphos and the corresponding peak value of the electrochemical signal (ΔI); Figure 13 It is the relationship between the concentration (C) of coumaphos and the corresponding peak value of the electrochemical signal (ΔI); Figure 14 It is the relationship between the concentration (C) of triazophos and the corresponding peak value of the electrochemical signal (ΔI). Figures 11 - 14 It can be seen that with the increase of the concentration of the target pesticide molecule, the number of OPs - Abs complexes attached to the electrode surface increases, resulting in the blockage of the pore channels on the electrode surface, which seriously hinders the electron transfer process on the electrode surface and thus causes a decrease in the response current. Among them, Figure 11 The linear regression equation in it is: Y (i.e., ΔI) = 0.32207lgC OPs +5.79994, Figure 12 The linear regression equation in it is: Y (i.e., ΔI) = 0.44837lgC OPs +4.92187, Figure 13 The linear regression equation in it is: Y (i.e., ΔI) = 0.44482lgC OPs +5.46849, Figure 14The linear regression equation in it is: Y (i.e., ΔI) = 0.32508lgC OPs +5.34975, Figures 11 - 14 The linear correlation coefficients (R 2 ) are 0.99522, 0.98135, 0.99444, and 0.99754 respectively, indicating a good linear relationship between ΔI and the logarithm of the OPs mass concentration. The detection ranges of parathion, quinalphos, coumaphos, and triazophos are 0.1 - 10 4 ng / mL, 0.1 - 10 4 ng / mL, 0.1 - 10 4 ng / mL, and 1 - 10 4 ng / mL respectively. According to LOD = 3L / b, the detection limits of this electrochemical immunosensor for parathion, quinalphos, coumaphos, and triazophos are 0.416 ng / mL, 0.298 ng / mL, 0.300 ng / mL, and 0.411 ng / mL respectively.

[0181] Figure 15 Figure shows the specificity and anti - interference ability of the electrochemical immunosensor for detecting organophosphorus pesticides provided in Example 1 to different types of substances; among them, a is the blank control without the test solution, b is the carbamate pesticide - carbofuran, c is the pyrethroid pesticide - deltamethrin, d is EPN, e is the OPs mixed standard solution (parathion, coumaphos, quinalphos, and triazophos), f is the mixture of OPs (parathion, coumaphos, quinalphos, triazophos) and non - OPs (carbofuran and pyrethroids), g is parathion, h is coumaphos, i is triazophos, j is quinalphos. From Figure 15 it can be seen that in the absence of specific targets, the value of ΔI is very small, indicating that it is difficult for the antibody to bind to these interfering pesticide molecules. The values of ΔI in Figures d, e, and f are all large, and the result values are close, indicating that the electrochemical immunosensor provided by the present invention has good specificity and certain anti - interference ability.

[0182] Application Example 53

[0183] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 1, OPs is quinalphos, the concentration of the OPs solution is 4 ng / mL, and other conditions are the same as those in the application example.

[0184] Application Example 54

[0185] The electrochemical immunosensor for detecting organophosphorus pesticides is the electrochemical immunosensor for detecting organophosphorus pesticides prepared in Example 1, OPs is triazophos, the concentration of the OPs solution is 8 ng / mL, and other conditions are the same as those in the application example.

[0186] Application Example 55

[0187] The electrochemical immunosensor for detecting organophosphorus pesticides is the one prepared in Example 1 for detecting organophosphorus pesticides. OPs is coumaphos, and the concentration of the OPs solution is 1 μg / mL. Other conditions are the same as those in the application example.

[0188] Application Example 56

[0189] The electrochemical immunosensor for detecting organophosphorus pesticides is the one prepared in Example 1 for detecting organophosphorus pesticides. OPs is parathion, and the concentration of the OPs solution is 100 ng / mL. Other conditions are the same as those in the application example.

[0190] To investigate the reproducibility of the electrochemical immunosensor provided by the present invention, the operations of Application Examples 53 to 56 were repeated 6 times respectively under the same conditions, and the obtained results are as Figure 16 shown, Figure 16 where a is Application Example 53, b is Application Example 54, c is Application Example 55, and d is Application Example 56. It can be Figure 16 seen that when OPs is quinalphos, the relative standard deviation (Rlative SandardDeviation, RSD) is 2.09%, when OPs is triazophos, the relative standard deviation is 1.4%, when OPs is coumaphos, the relative standard deviation is 1.87%, and when OPs is parathion, the relative standard deviation is 1.82%. The relative standard deviation of the test results for the same organophosphorus pesticide is within 2.1%, indicating that the electrochemical immunosensor provided by the present invention has good reproducibility.

[0191] To investigate the stability of the electrochemical immunosensor provided by the present invention, 20 electrochemical immunosensors prepared in Example 1 were taken and evenly divided into 5 groups. The first group directly tested the current values of parathion, coumaphos, quinalphos, and triazophos according to the operations in Application Examples 53 to 56 respectively. The second group tested the current values of parathion, coumaphos, quinalphos, and triazophos according to the operations in Application Examples 53 to 56 respectively after being stored sealed at 4°C for 1 day. The third group tested the current values of parathion, coumaphos, quinalphos, and triazophos according to the operations in Application Examples 53 to 56 respectively after being stored sealed at 4°C for 3 days. The fourth group tested the current values of parathion, coumaphos, quinalphos, and triazophos according to the operations in Application Examples 53 to 56 respectively after being stored sealed at 4°C for 7 days. The fifth group tested the current values of parathion, coumaphos, quinalphos, and triazophos according to the operations in Application Examples 53 to 56 respectively after being stored sealed at 4°C for 15 days. The results showed that after being stored for 1 day, 3 days, 7 days, and 15 days respectively, the obtained results were 93.34% - 97.48%, 87.46 - 95.82%, 90.67 - 96.57%, and 84.37 - 96.77% of the initial current respectively, indicating that the electrochemical immunosensor prepared by the present invention has good stability.

[0192] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical immunosensor for detecting organophosphorus pesticides, comprising the following steps: (1) The SPE film is modified with a dispersion containing PANI / Co3O4 composite nanoparticles, and then activated with a Gluta fixative to obtain an activated SPE film; (2) The activated SPE film obtained in step (1) is incubated for the first time with an Abs diluent, and then incubated for the second time with a BSA solution to obtain an electrochemical immunosensor for detecting organophosphorus pesticides.

2. The preparation method according to claim 1, characterized in that: The preparation method of the PANI / Co3O4 composite nanoparticles in step (1) comprises: mixing the Co3O4 nanoparticles, water and hydrochloric acid and performing ultrasonic dispersion, then adding aniline for magnetic stirring, then adding ammonium persulfate for polymerization, and finally filtering, washing, drying and grinding in sequence to obtain the PANI / Co3O4 composite nanoparticles.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the Co3O4 nanoparticles, the volume of water, the volume of hydrochloric acid, the volume of aniline and the mass ratio of ammonium persulfate is (0.2-1) g: (40-60) mL: (4-6) mL: (1-2) mL: (4-5) g.

4. The preparation method according to claim 1, characterized in that: The preparation method of the dispersion containing PANI / Co3O4 composite nanoparticles in step (1) is: chitosan solution and PANI / Co3O4 composite nanoparticles are mixed and then ultrasonically dispersed to obtain a dispersion containing PANI / Co3O4 composite nanoparticles.

5. The preparation method according to claim 1, characterized in that: The amount of the dispersion containing PANI / Co3O4 composite nanoparticles in step (1) is 20-70 μL / cm 2 .

6. The preparation method according to claim 1, characterized in that: The mass concentration of the Gluta fixative in step (1) is 0.2-0.3%, and the amount of the Gluta fixative is 120-200 μL / cm 2 ; The activation temperature is room temperature, and the activation time is 1.5 to 3 hours.

7. The preparation method according to claim 1, characterized in that: The concentration of the Abs diluent in step (2) is 2-10 μg / mL, and the amount of the Abs diluent is 20-70 μL / cm 2 ; The temperature of the first incubation is 35-40°C, and the time of the first incubation is 30-120 minutes.

8. The preparation method according to claim 1, characterized in that: The mass concentration of the BSA solution in step (2) is 1-4%, and the amount of the BSA solution is 120-200 μL / cm 2 ; The second incubation time is 0.5 to 2 hours.

9. An electrochemical immunosensor for detecting organophosphorus pesticides prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the electrochemical immunosensor for detecting organophosphorus pesticides according to claim 9 in detecting organophosphorus pesticides.

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