Immunodetection method of organophosphorus pesticide multi-residue

An immunodetection method and organophosphorus pesticide technology are applied in immunoglobulins, chemical instruments and methods, biological tests, etc., and can solve the problems of complex preparation steps of test strips, prone to false positives, cumbersome extraction of biological enzymes, etc. Achieve the effects of low preparation cost, improved detection sensitivity and low cost

Inactive Publication Date: 2012-04-11
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0005] After searching the literature, it was found that Chinese patents "Fish esterase reagent for rapid detection of organophosphorus pesticide residues (publication number: CN1396267)", "rapid test kit and manufacturing method for residual organophosphorus pesticides (publication number: CN1293363)", "a kind of energy Quick Test Card for Detecting Organophosphorus and Carbamate Pesticide Residues in Various Agricultural Products (Publication No.: CN101140236), "Preparation and Using Method of Test Paper for Detecting Organophosphorus Pesticide Residues (Publication No.: CN101299029)", among which The principles are based on the fact that organophosphorus pesticides have inhibitory effects on certain enzymes in animals and plants, and the degree of residual organophosphorus pesticides is judged by the degree of hydrolysis (color development) of the substrate of the enzyme. The shortcomings of these methods are: 1. False positives are prone to occur, and the detection specificity is not strong; 2. The process of extracting biological enzymes is relatively cumbersome, and biological enzymes are easily affected by factors such as temperature
The technical scheme disclosed in the patent document "An Immunocolloidal Gold Test Strip for Detecting Organophosphorus Pesticides and Its Preparation Method (Publication No.: CN101042405)" improves the detection specificity, but the preparation steps of the test strip are still very complicated, and only Qualitatively judge the results by color depth

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  • Immunodetection method of organophosphorus pesticide multi-residue
  • Immunodetection method of organophosphorus pesticide multi-residue

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Embodiment 1

[0024] 1. Synthesis of general immunogen and coating agent of organophosphorus pesticides

[0025] exist O,O - After introducing amino acids into the molecular structure of diethylphosphoryl thiochloride, the general hapten DENP containing carboxyl groups can be obtained, and the universal hapten is coupled with the carrier protein by using the combination reaction of carboxyl groups and amino groups in proteins to obtain immunogens and Encapsulated original, synthetic route such as figure 1 shown.

[0026] The specific steps are as follows: Dissolve 52mg DENP (0.2mmol) in 3mL anhydrous DMF, add 46mg NHS (0.4mmol), 78mg EDC (0.4mmol). The reaction was stirred overnight at room temperature (10°C). Dissolve 133mg of bovine serum albumin (BSA) in 15mL of borate buffer (0.2mol / L, pH8.7), then add the activated hapten solution into the BSA solution so that the molar ratio of hapten to BSA is about 100 :1. After reacting for 2 hours at room temperature (10°C), react overnight...

Embodiment 2

[0054] 1. Synthesis of general immunogen and coating agent of organophosphorus pesticides

[0055] exist O,O - After introducing amino acids into the molecular structure of diethylphosphoryl thiochloride, the general hapten DENP containing carboxyl groups can be obtained, and the universal hapten is coupled with the carrier protein by using the combination reaction of carboxyl groups and amino groups in proteins to obtain immunogens and Encapsulated original, synthetic route such as figure 1 shown.

[0056] The specific steps are as follows: Dissolve 52mg DENP (0.2mmol) in 3mL anhydrous DMF, add 46mg NHS (0.4mmol), 78mg EDC (0.4mmol). The reaction was stirred overnight at room temperature (10°C). Dissolve 133mg of bovine serum albumin (BSA) in 15mL of borate buffer (0.2mol / L, pH8.7), then add the activated hapten solution into the BSA solution so that the molar ratio of hapten to BSA is about 100 :1. After reacting for 2 hours at room temperature (10°C), react overnight ...

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Abstract

The invention relates to an immunodetection method of organophosphorus pesticide multi-residue in the technical field of pesticide detection, and the immunodetection method comprises the following steps: synthesizing an organophosphorus generic hapten, and coupling with protein to prepare an immunogen and a coating antigen; using the immunogen to perform animal immunization so as to get an organophosphorus pesticide generic antibody; synthesizing Fe3O4 magnetic nanoparticles and performing surface modification; coupling the Fe3O4 magnetic nanoparticles after surface modification with the organophosphorus pesticide generic antibody to form immune magnetic beads; adding a nitrocellulose membrane on a baseplate of a test strip, coating the immunogen prepared by coupling the organophosphorus generic hapten with the protein on the membrane, closing to form a detection part, and finally determining the optimal coating, the closing conditions and the using quantity of the immune magnetic beads; and performing qualitative and semi-quantitative detection. The detection method is simple and convenient to operate, sensitive and fast; large-scale instruments are not required; and the detection method is suitable for field sampling detection and large-batch detection tasks.

Description

technical field [0001] The invention relates to a method in the technical field of pesticide detection, in particular to an immunological detection method for multi-residue organophosphorus pesticides. Background technique [0002] Organophosphorus pesticides are widely used due to their wide insecticidal spectrum, short residual period, and low price. Improper use of pesticides makes organophosphorus pesticide residues an important problem in food residues in my country. At present, the detection methods for this kind of pesticides mainly include gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), etc. These detection techniques are relatively mature and have high sensitivity, but require expensive instruments, The test takes a long time and requires professional technicians, which is not suitable for large-scale and rapid detection. In recent years, various countries have been researching rapid multi-residue organophosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/52C07K14/765C07K16/44H01F1/11B82Y25/00B82Y40/00
Inventor 朱鸿林沈国清胡寅姜官鑫
Owner SHANGHAI JIAO TONG UNIV
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