| Chlorpyrifos[O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)phosphorothioate] had been promoted to be an efficient insecticides by the Dow Chemical Company in 1965. Chlorpyrifos is widely used in agricultural production. Chlorpyrifos residues in grain and other crops are frequent problems, especially the common cabbage, celery, spinach, lettuce and other leafy vegetables. In the application process, they will inevitably go into the atmosphere, soil, rivers and lakes, causing environmental pollution and ecological destruction, and chlorpyrifos are becoming increasingly serious in environmental samples. Organophosphorus pesticide residues in food, water and the environment, which are very important for the maintenance of people’s health. So we have established a new sensitive and rapid chemiluminescent immunoassay method to detect chlorpyrifos residues in soil, drinking water, fruits and vegetables. Forming a complete antigen with isoluminol and chlorpyrifos, a coupled complex with magnetic particles coupled chlorpyrifos antibody. In experiment markers were purified and identified, as well as the evaluation of the optical properties and immunological properties. On this basis, a new analysis method of homogeneous chemiluminescent immunoassay for the detection of organophosphorus pesticide chlorpyrifos has been established.1. First organophosphorus pesticide chlorpyrifos with 3-mercaptopropionic acid are heated to reflux under an alkaline condition, thereby introducing a carboxyl group(-COOH) as chlorpyrifos hapten,which reacts with isoluminol in methylene chloride solution and forms complete antigen. The purified products were analysisde and identificted by UV spectroscopy, infrared spectroscopy, thin layer chromatography. The results show that, chlorpyrifos hapten in UV absorption maximum absorption peak has undergone significant changes after the transformation and appears a strong absorption peak1711.90cm-1 in the infrared spectrum;The most obvious change of the chlorpyrifos-luminous markers is absorption peak red-shifted and weakened in 1738.40cm C=O.The purified product in the thin plate only presents a point, which can prove the transformation process is successful.2. There are four magnetic particles that are GoldMag-CS magnetic particles, Iso-thiocyanate end of the magnetic particles, the Amino-terminal magnetic particles, Carboxyl-terminal magnetic particles can choose to make antibody-magnetic particles coupled complex in the four magnetic particles. The coupling rate of gold magnetic particles is 73.47%, which is the highest. So I choose the gold magnetic particles couple chlorpyrifos antibody.3. According a series of evaluation for chemiluminescent markers to determine various aspects indicators can be good use for the actual detection or not. Which comprise optical properties and fluorescence optical properties, stability and immunological properties. The results show that, under the conditions of the optimum pH value of 9.5, 0.5mmol/L H2O2 excited light-emitting reaction, the marker can reach the maximum luminous at 6s intensity. In the reaction system that has the chlorpyrifos, chlorpyrifos chemiluminescent markers, the Antibodies-gold GoldMag particles conjugates, the chlorpyrifos markers are able to strongly compete binding to homologous antibodies with the (not marker) chlorpyrifos, which indicat that this marker can be used for detection of chlorpyrifos residue.4. According the optimal of light conditions and immune conditions, a new method for analysis of organophosphorus pesticide chlorpyrifos homogeneous chemiluminescent immunoassay has been established. The linear ranges in 5ng/mL～90.0ng/mL, the correlation coefficient is 0.9902, the detection limit is 2.50ng/mL,the recovery of sample is 98.72%～101.64%, coefficient of variation is less than 12% and between the coefficient of variation is less than 11%. The results showed that the proposed multi-marker for the detection of chlorpyrifos in homogeneous chemiluminescence immunoassay method is feasible. |
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