The Bio-barcode Immunoassay For Organophosphorus Pesticide Residues Detection Based On Nanozyme Catalysis

The quality and safety of agricultural products is a major issue of people’s health in the world,with an increasing more and more attention.Pesticide residues are one of the most important hazard factors.Establishment of high-sensitivity and rapid detection methods for pesticide residues in agricultural products is an important means to find problems,with scientific supervision and practical significance.This thesis is based on the principle of colloidal gold nano-signal amplification combined with DNA bio-barcode recognition and nanozyme technology,to study the bio-barcode immunoassay method for detecting pesticide residues and achieve high sensitivity and rapid detection of pesticide residues.In this paper,the platinum nanoparticles（Pt）are synthsised as nanozyme.A competitive bio-barcode immunoassay is described for the trace detection of parathion in water,pear,cabbage,and rice samples.It is based on amplification by Pt acting as nanozyme.Gold nanoparticles（AuNPs）were modified with（a）monoclonal antibodies（mAbs）against parathion,and（b）thiolated single-stranded DNA（ssDNA）oligonucleotides.Magnetic nanoparticles（MNPs）were functionalized with ovalbumin coupled with parathion hapten.Parathion and its hapten compete with mAbs on the surface of the AuNPs.Subsequently,the Pt probe,which was functionalized with the complementary thiolated ssDNA,was added to the reaction mixture for the detection of parathion.The immunoassay has a limit of detection（LOD）is 2.04 ng?kg^-1.The immunoassay was performed on water,pear,cabbage and rice samples to verify the feasibility of the method.The recoveries and relative standard deviations（RSDs）ranged from 91.06%～114.44%and 3.59%～15.75%,respectively.The method correlates well with gas chromatography-tandem mass spectrometry（GC-MS/MS）.Compared with chemiluminescence enzyme immunoassay（CLEIA）,the LOD improve an order of magnitude.This developed approach has promise for detecting and screening pesticide residues in various matrices.To improve the stability of nanozyme,the good stability bimetallic nanozyme（Au@Pt）was proposed in this paper.A competitive sensitive bio-barcode immunoassay based on Au@Pt catalysis has been designed for the detection of the pesticide parathion.The immunoassay exhibited LOD and IC₅₀are 2.13 ng?kg^-1 and 1.75μg?kg^-1,respectively.Compared with the HRP based on bio-barcode immunoassay(LOD:20.82 ng?kg^-1,IC₅₀:5.83μg?kg^-1),The the LOD of Au@Pt catalysis bio-barcode immunoassay improve an order of magnitude than the HRP catalysis bio-barcode immunoassay.These results indicate that the bio-barcode immunoassay method based on Au@Pt nanozyme is a more sensitive and stable analysis method for pesticide residue detection.The bimetallic nanozyme-based bio-barcode immunoassay was performed on rice,pear,apple,and cabbage samples to verify the feasibility of the method.The recoveries and RSDs ranged from 73.12%～116.29%and 5.59%～10.87%,respectively.The method was found to correlate well with data obtained by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.In conclusion,this method exhibits potential as a sensitive alternative method for the detection of a variety of pesticides,ensuring the safety of fruits and vegetables in agriculture.To improve high-throughput testing of pesticides,the nanozyme Au@Pt catalysis bio-barcode immunoassay based on amino modified 96 well microplate for parathion,triazophos and chlorpyrifos residue detection.The three nanozyme Au@Pt probes are functionalized on amino modified microplate to catalysed Amplex?Red solution.The working concentration of probes in bio-barcode immunoassay are optimized.Under optimized conditions,the three pesticides had good linearity in the range of0.01～40μg?kg^-1.The IC₅₀ of parathion,triazophos and chlorpyrifos are 0.74μg?kg^-1,0.65μg?kg^-1 and1.06μg?kg^-1,respectively.The LODs are 9.88 ng?kg^-1 for parathion,3.91 ng?kg^-1 for triazophos and 1.47ng?kg^-1 for chlorpyrifos.The range of recovery is 1.26%～112.66%（RSD:2.92%～14.52%）in this immunoassay in samples（cabbage,apple,pear and rice）.To sum up,the highly sensitive detection method for parathion,triazophos,and chlorpyrifos in the bio-barcode immunoassay method based on nanozyme Au@Pt catalysis.It is reliable for a variety of pesticides detection in agro-products.