Study On Homogeneous Chemiluminescence Immunoassay Assays Of Organophosphorus Pesticide Chlorpyrifos

Chlorpyrifos [O, O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate] is a broad spectrum organophosphorous insecticide. As acetylcholinesterase inhibitors, organophosphorous pesticides are toxic organic chemicals. The control and detection of organophosphorous pesticide residue in food, water, and environment therefore plays a very important role in maintaining physical health. A new sensitive and rapid chemiluminescence immunoassay method for chlorpyrifos residues had been developped, which was used for the detection of pesticides in soil, drinking water, fruits and vegetables. To chlorpyrifos, Luminol and BSA as a model, the BSA can been marked several luminol and chlorpyrifos, the efficiency of the markers on the impact of luminous intensity. The conjugate were purified, and then identified, then evaluation on a chemiluminescent label oforganophosphorous insecticide chlorpyrifos, and further developed for the determination of organophosphorus pesticide residue in homogeneous chemiluminescence immunoassay method, the result demonstrated the method multilabeled homogeneous chemiluminescence immunoassay is feasible.1. To the new organophosphorus pesticide chlorpyrifos pesticides as our target. Under alkaline conditions, the sixth chlorine atoms of the chlorpyrifos molecule pyridine was replaced through the by nucleophilic substitution of 3 straight chain of carbon atoms 3-mercaptopropionic acid, than carboxyl group (-COOH) was introduced. The extract was dried over Na2SO4 and concentrated. The residue was subjected to column chromatography, and the mobile phase (n-hexane/ethyl acetate 1:2) was collected. Fractions showing only one spot on TLC (Rf 0.39, hexane/tetrahydrofuran/acetic acid 75:25:1) were pooled and concentrated to provide 3.01g of 1 (71.7%), and then solidified on standing:mp 120-125℃; IRvmax (KBr) 2978,1708,1547, the result demonstrated the success of the transformation process was successfully introduced in the chlorpyrifos molecule carboxyl groups.2. There were two marked order to synthetize the chlorpyrifos chemiluminescent markers. The first method:to the chlorpyrifos derivative was coupled to BSA, to form the BSA-chlorpyrifos conjugate, then the BSA-chlorpyrifos was coupled to luminol. The second method:to luminal marker to the BSA, to synthesis luminol-BSA conjugate, then he chlorpyrifos derivative was coupled touminol-BSA conjugate. Two different product of marked was compared by the stability of markers and labeling efficiency, then to choose the best solution. The results show that the first product of the first method, luminol:BSA: chlorpyrifos coupling ratio of 22:1:14. The average relative intensity for the 26343, the stability of conjugate was strong, so choose the first method as the best marker program.3. Through a series evaluation of chemiluminescent markers, to determine marker of success in all aspects of indicators used for the actual detection. First of all markers had SephadexG-25 were purified, and identified by SDS-PAGE electrophoresis. Then chemiluminescent markers was evaluated by fluorescence spectrum, stability and immunological properties. The results showed that under the optimal pH, using the H2O2 chemiluminescence reaction system, the markers reach ed maximum intensity in 6s, luminous detection limit was 9.15 ng/mL. Immune suppression through the preliminary competition reaction, the chlorpyrifos marker and unlabeled chlorpyrifos was strongly competed with homologous, indicating that the markers can be used for the detection of chlorpyrifos residue.4. Under the optimal of light conditions and immune conditions, the homogeneous chemiluminescence immunoassay for organophosphorus pesticide chlorpyrifos residues method was developed, the linear range of 7.0 ng/m mL～100.0 ng/mL, the correlation coefficient was 0.9946, The detection limit is established as 2.35ng/mL, the recovery of sample were 87.7%-104%, coefficient of variation less was than 8% and between the coefficient of variation was less than 20%, the cross-reaction was less than 5%, the results showed that the proposed multi-marker for the detection of pesticides in homogeneous chemiluminescence immunoassay method was feasible.