Synthesis And Charaterization Of Artificial Antigen With Different Coupling Ratio For Chlorpyrifos And Fenitrothion

Organphosphorus pesticides have played a key role in agriculture. However, the extensive use of organphosphorus pesticides in agriculture has raised serious problem in environment and food safety.Conventional methods for detecting organphosphorus pesticides residues included chromatography, mass spectrometry, spectroscopy or coupled techniques such as GC-MS,etal. Although these high sensitivities,these techniques are expensive, time consuming and require highly trained personnel.Furthermore,they are not adapted for in situ and real time detection of pollutants.As a comparison, biosensors provided a way to detect organphosphorus pesticides residues,which are both rapid, sensitive and simple. synthesis of the pesticide hapten and artificial antigen is one of key technique of using immunoassay in pesticide residues analysis. The major contents in this thesis are described as follows :1. Synthesis and characterization of artificial antigen for organophosphorus chlorpyrifos. The hapten of chlorpyrifos was synthesized by using technical grade chlorpyrifos reacted with 3-marcapropanoic acid in the hot alkazid solution. The hapten was washed with water more times to purification.The method is simple and low cost. The hapten was conjugated to bovine serum albumin (BSA) with the active ester method and mixed anhydride method to form artificial antigen. The artificial antigen was then identified by ultraviolet spectrophotometry and IR spectrum. The results showed that the artificial antigen of chlorpyrifos was synthesized successfully. The influence of the factors such as initial molar ratio of reagents and the coupling time were studied in this paper. 1:1, 2:1 and 5:1 as the initial molar ratio of reagents and 1,2,4,6,24h as coupling time were chosen to determine the optimal reaction conditions for synthesis.Experiment results indicated that different coupling time have certain effects to coupling ratio of artificial antigen. With the increasing of coupling time, coupling ratio is from 16 up to 27,and the number of hapten molecule which be conjugated to bovine serum albumin (BSA) reached maximum.after 6 hours. Generally,when coupling ratio get to 10-20:1,immunoenicity of artificial antigen is better.So optimal coupling time was chosen 1h (its coupling ratio is 16:1 ). However,different initial molar ratio of reagents have little effect on the coupling ratio of artificial antigen. When the initial molar ratio were 1:1,2:1,5:1, the coupling ratio were 34:1,36:1,39:1, they are nearly steady, so the optimal reaction condition.is 1:1. The successful synthesis of artificial antigen for organophosphorus chlorpyrifos have solved a key technical problem for using immunoassay in pesticide residues analysis.2. Synthesis and characterization of artificial antigen for the organophosphorus fenitrothion. The molecular structure of fenitrothion is short of activity group, which can be conjugate to bovine serum albumin. Fenitrothion was reduced into amino-fenitrothion which has the activity group by using acetic acid-zinc powder-hydrichloric acid .Its structure was confirmed by IR and GC/MS. Artificial antigens were synthesized by conjugating amino-fenitrothion to bovine serum albumin (BSA) with dizotization method and glutaraldehyde method. The artificial antigen was identified by ultraviolet spectrophotometry and IR spectrum. The result showed that the artificial antigen of fenitrothion was synthesized successfully. The coupling ratios were 37:1and 25:1 respectively. The successful synthesis of artificial antigen for the organophosphorus fenitrothion have solved a key technical problem for using immunoassay in pesticide residues analysis.