Establishment Of An Immunochip For Multi-pesticides Determination Based On Colloidal Gold Enhancement

With the increasing application of various kinds of pesticides, it is inevitable to use analytical methods for pesticide multi-residue detection. This study has developed an immunochip which could determinate nine pesticides, i.e. triazophos, parathion, methyl-parathion, fenitrothion, fenpropathrin, carbofuran, thiacloprid, chlorothalonil and carbendazim. The established visual immunochip, consisting of seven antigen groups as capture probes coated on the nitrocellulose membrane, used colloidal gold as the label material. By comparison, we selected the indirect-competitive model detection, based on the gold-labled secondary antibody. Different reaction parameters were optimizied, such as chip membrane type, reacting antigen and antibody concentration, dilution time of gold labeled second antibody, blocking buffer, gold enhancement solution, reacting buffer, pH and ionic strength, organic solvent content, and reacting time for each step.Under the optimal condition, the assay sensitivity (IC50), instrument limit of detection (LOD, IC10) and visual limit of detection (vLOD) were respectively0.41ng/mL,0.02ng/mL and1ng/mL for triazophos;10.77ng/mL,0.38ng/mL and100ng/mL for methyl-parathion;2.24ng/mL,0.13ng/mL and10ng/mL for fenpropathrin;27.22ng/mL,3.76ng/mL and lOOng/mL for carbofuran;46.52ng/mL,4.51ng/mL and100ng/mL for thiacloprid;1.17ng/mL,0.20ng/mL and5ng/mL for chlorothalonil;0.61ng/mL,0.07ng/mL and5ng/mL for carbendazim;279.5ng/mL,2.4ng/mL and1000ng/mL for parathion;77.9ng/mL,0.6ng/mL and500ng/mL for fenitrothion. The sensitivity of every target pesticides could be comparable to the classical ELISA method, and the instrument limit of detection also could meet the requirement of the MRLs value (GB2763-2014).Results of method validation tests showed that the immunochip method had an acceptable precision and accuracy.The established method was further used for spike recovery tests on various agricultural samples. After studying the matrix effect and dilution time, the spiked recovery rates of most pesticides was between70.1%and 119.1%, with variable coefficient ranging from4.12%to19.93%. Through comparison with instrumental method, the correlation coefficient between immunochip and mass spectrometer could be as high as0.94, which demonstrated that the established immunochip method could be used for rapid detection of multiple pesticide residues.In this study, we explored a sensitive, cost-effective and integrated immunochip for pesticide multi-residue detection, which laid a good theoretical and practical basis for further development of high-throughput, sensitive, cheap, portable and serialized POCT (point of care testing) technologies and thereof products.