Fluorescence Immunoassay For Multi-reisdue Detection Of Organophosphorus Pesticides Based On Oligonucleotide Signal Amplification

Pesticide residues are concerned by governments and consumers in various countries,and their detection technology has received more and more attention.Therefore,establishing an effective pesticide residue detection method,especially a sensitive,accurate,rapid and simple detection method,is of great significance for protecting ecological and consumer health.This study was based on indirect competitive immunoassay,coupled with colloidal gold and fluorescence-labeled oligonucleotide signal amplification technology.Oligonucleotide signal amplification immunoassay method for the detection of Triazophos single-residues and multiple residues of parathion and chlorpyrifos pesticides have been established successively.The main contents are as follows:A sodium citrate reduction method was selected to produce colloidal gold with uniform particle size,regular shape and good dispersibility,with a concentration of about 9.9 nM.The pesticide monoclonal antibody and the fluorescently labeled oligonucleotide are labeled on the colloidal gold surface under optimized conditions such as stabilizer concentration,aging time,antibody concentration,and oligonucleotide sequence.An indirect competition fluorescent immunoassay method for triazophos based on oligonucleotide signal amplification was established.The analysis conditions of the method such as the working concentration of the probe and the hapten,dithiothreitol（DTT）working conditions and methanol concentration were optimized.The established method has a good linearity in range of 0.01–20μg L^–1.The limit of detection is 6 ngL^–1.The recoveries of this method spiked on water,rice,cucumber,cabbage and apple was 91.6%–102.6%,and RSD was 11.1%–17.4%.Then,ELISA and LC-MS/MS were established for comparison and confirmation.The results demonstrated that indirect competition fluorescent immunoassay method for triazophos based on oligonucleotide signal amplification has good accuracy,precision and high sensitivity.The method can meet the analytical requirements for triazophos pesticide residue in agricultural products and can be used for actual sample analysis.Then,a fluorescence immunoassay method for simultaneous detection of triazophos,parathion and chlorpyrifos pesticides based on oligonucleotide signal amplification was established.Three fluorophores,6-FAM,Cy3,Texas red,with high fluorescence intensity and low wavelength overlapping were selected among seven to label on oligonucleotides.The specificity between the three kinds of probes and haptens was studied.The preparation conditions of the probes of parathion and chlorpyrifos were optimized.The results suggested that the peobes has good specificity and the method had good linearity for triazophos,parathion and chlorpyrifos in the range of 0.01–20μg L^–1,0.05–50μg L^–10.5–1000μg L^–1,respectively.The limits of detection were 0.007μg L^-1,0.009μg L^–1,and 0.087μg L^–1,respectively.The blank samples of tap water,rice,wheat,cucumber,cabbage,and apple were spiked and detected by this method and LC-MS/MS method,respectively.The recoveries were 77.7%to113.6%,and the RSDs were 7.1%to 17.1%which consisted with results of LC-MS/MS.It shows that this method has high sensitivity,good accuracy and reliability,which can meet the multi-residue analysis requirements of triazophos,parathion and chlorpyrifos pesticides in agricultural products,and can also provide reference for other small molecule chemical pollutants multi-residue analysis.