Bio-barcodes Immunoassay For Detection Of Organophosphorus Pesticides Residue In Agricultural Products By Iterative Cycles Of DNA/RNA Hybridization

The quality and safety of agricultural products is related to national health and economy of social development for China.Although pesticides play an important role in preventing insect and regulating the crop growth,the illegally use of pesticides leads residues and poses a significant risk to environment and even human health.Hence,more sensitive and specific methods for detecting organophosphorus pesticides residue are urgently needed,which is of great significance to the development of society and human health.This study was based on bio-barcode immunoassay,coupled with iterative cycles of DNA-RNA hybridization and dissociation of fluorophores by Ribonuclease H.Notably,sandwich assay is not applicable for detection of pesticides and other small molecules,because they contain only one antibody binding site.This strategy utilizes a competitive immunoassay to replace the antibody sandwich.The detection procedure can be divided into immunochemical and detection reaction,and the detected fluorescence signal intensity would be indirectly converted into pesticide residue concentration.The main research contents and results are as follows:The AuNPs probe was prepared based on the gold nanoparticles and labelled with a large amount of single-stranded DNA?ssDNA?and mAbs,which were acted as a carrier for signal amplification.The AuNPs probe was characterized by transmission electron microscopy?TEM?.To maximize the fluorescence signal,the reaction conditions?immunochemical reaction and detection reaction?were further optimized during the experimental protocol.The immunochemical reaction of this method such as the pH and concentration of the phosphate-buffered saline solution,the immunoreagent dilutions of the probe and the hapten,and methanol concentration were optimized.The detection reaction such as DTT concentration,ionic strength,pH,Mg²⁺concentration and temperature were optimized.Under the established optimized assay conditions,the competitive immunoassay showed a wide linear range of0.01?100 ng/mL with a limit of detection?LOD?of 0.0032 ng/mL.Matrix effect?ME?remains a great challenge for quantitative analysis of pesticide residues in various matrices.The matrix effects were reduced to acceptable levels when the samples were diluted either 20 fold with buffer.To further evaluate the analytical accuracy of the developed bio-barcode immunoassay method,the recovery test was performed.Accuracy?expressed as spiked recovery%?and coefficient of variation?CV?were ranged from 73.4%to 116%and 7.04%to 17.4%,respectively.Through comparing with LC-MS/MS,the two methods exhibited good correlation coefficients.In sum,the proposed bio-barcode amplification immunoassay could be a reliable technique for detecting triazophos residues in agriculture products and surface water.Then,a bio-barcode immunoassay for multi residue analysis of three organophosphorus pesticides based on single residue detection was established.The antibody concentration of the probes of parathion and chlorpyrifos were optimized.The cross-reactivity of fluorophore was evaluated in the multi-residue system,and it was found that FAM,Cy3 and Cy5 had high fluorescence intensity and low wavelength overlapping.The specificity of modified AuNP probes and OVA-hapten and three labeled RNA were evaluated.The results suggested that the present immunoassay had excellent specificity for multi residue analysis.The immunoreagent dilutions of three probe and the hapten were optimized.Under the established optimized assay conditions,the method had good linearity for triazophos,parathion and chlorpyrifos in the range of 0.01?100 ng/mL?0.05?100 ng/mL?0.1?500ng/mL,respectively.The limits of detection were 2.42×10^-2 ng/mL?8.19×10^-3 ng/mL?7.62×10^-2 ng/mL,respectively.The recovery test was performed in selected samples and the recovery and CV were ranged from 73.4%to 116%and 7.04%to17.4%,respectively.The results were 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.