Development Of Plasma Enzyme-linked Immunoassays Based On Gold Nanorods For Organophosphorus Pesticides

Organophosphorus pesticides?OPTs?are synthetic esters containing carbon–phosphorus bonds that have been widely used in agricultural production due to their high insecticidal and herbicidal activities.They can phosphorylate acetylcholinesterase?ChE?in the body andinhibits catabolism of acetylcholine?ACh?,which in turn,ACh accumulates in large amounts in the body.It causes symptoms of central nervous system poisoning including cholinergic muscarinic,nicotinicand other serious toxic effects on the body.Therefore,it is a research hotspot to develop a simple,rapid and ultra-sensitive multi-residue detection technology of organophosphorus pesticides in recent years.At present,immunological detection technology has gradually become a widely used method for pesticide residue detection due to its advantages of simple,rapid,ultra-sensitive,low-cost and suitable for multi-sample detection.In this study,three organophosphorus pesticides?methyl parathion,fenitrothion and fenthion?were used as research targets.Using a broad-specificity monoclonal antibody,an improved seed-mediated growth method was adopted for the preparation of monodisperse gold nanorods?AuNRs?with longitudinal localized surface plasmon resonance?LLSPR?around 750 nm.The magnetic beads probe was prepared by activating the carboxyl magnetic beads through the EDC method and covalently binding with the detection antigen.Three detection methods were developed based on specific reactions between antigenic antibodies,including:indirect competition ELISA?ic-ELSIA?,TMB²⁺etched AuNRs mediated multicolor based plasma ELISA?AuNRs-p-ELISA?and TMB²⁺etched AuNRs mediated immunomagnetic beadmulticolor based plasma ELISA?AuNRs-IMBs-p-ELISA?.The three methods can meet the requirements of the quantitative detection of three organophosphorus pesticides in fruits and vegetables.Five parameters of the developed ic-ELISA were optimized,three standard curves for methyl parathion,fenitrothionand and fenthion were obtained.The linear regression equations of the standard curve were y₁=36.947x-135.75,y₂=38.275x-145.75 and y₃=32.275x-123.04,respectively;the correlation coefficient were R₁²=0.9935,R₂²=0.9982 and R₃²=0.9979;the IC₅₀ of three organophosphorous pesticides including methyl parathion,fenitrothion and fenthion were 106.53,130.11 and 229.84 ng/mL respectively;the detection limits were 8.81,11.73 and 13.25 ng/mL;the average recoveries of the three pesticides in cabbage,pear,and rice were 103.14%,92.96%,74.70%;105.02%,77.44%,63.50%and 79.83%,98.87%,76.13%respectively.A naked eye semi-quantitative detection method was developed based on this principle:TMB²⁺can quantitatively and efficiently etch AuNRs and the LLSPR of AuNRs were blue-shifted,which causes changes in the color of solution.Three parameters of the method were optimized to obtain three standard curves for methyl parathion,fenitrothion and fenthion.The linear regression equations and correlation coefficients for the three organophosphorus pesticides were:y₁=0.0188x+3.0082,R2₁=0.9822,y₂=0.0215x+2.5467,R2₂=0.9526 and y₃=0.0189x+3.4365,R2₃=0.9874;the IC₅₀ values were 71.61,45.99,and 208.00 ng/mL,respectively;the detection limits were 2.42,0.95 and 6.52 ng/mL;the average recoveries of methyl parathion in cabbage,pear,and rice were 90.55,102.29,and 69.11%,respectively;the average recoveries of fenitrothion in cabbage,pear,and rice samples were 108.91%,91.51%,and 76.57%;the average recoveries of fenthion in cabbage,pear,and rice samples were 78.66%,90.94%,and 69.50%respectively.Immuno-bead probes were prepared by covalently coupling the carboxyl magnetic beads and the detection antigen,and a semi-quantitative detection method was developed based on these immuno-bead probes and AuNRs.The main parameters were optimized,and the linear regression equations and correlation coefficients for methyl parathion,fenitrothion and fenthion were y₁=-49.464x+316.22,R2₁=0.9684,y₂=-55.412x+355.5,R2₂=0.9769 and y₃=-53.762x+350.08,R2₃=0.9895.The IC₅₀ of the three organophosphorus pesticides were 37.45,61.86 and 68.80ng/mL,respectively;the detection limits were 1.31,3.10 and 3.15 ng/mL,respectively.the average recoveries of methyl parathion in cabbage,pear,and rice were 104.02%,113.33%and79.67%,respectively;the average recoveries of fenitrothion in cabbage,pear,and rice samples were 104.19%,91.05%and 70.19%;the average recoveries of fenthion in cabbage,pear,and rice samples were 100.86%,88.65%and 70.76%,respectively.The developed three detection methods for methyl parathion,fenitrothion and fenthioncan meet the requirements of China's and EU countries'demand of maximum residue limits?MRL?of OPTs in water and foods.