A homogenous fluorescence immunoassay based on a DNA-grafted labeling strategy of magnetic microspheres for multiple neonicotinoids monitoring

Abstract

Given the detrimental impact on non-target pollinators, there is a high demand for monitoring multiple neonicotinoid residues in the environment. In this study, we proposed a novel physical-biological coupled multiplexing system (PBCMS) to achieve a homogeneous and high-performance indirect competitive suspension array technology (ic-SAT) for multiple neonicotinoids monitoring. First, hapten-decorated microspheres (HDMs) were prepared by ligating the streptavidin-modified microspheres with the biotinylated hapten, according to a novel molecularly-defined single-stranded DNA (ssDNA)-grafted labeling approach. Taking thiamethoxam (THX) and imidacloprid (IMI) as models, the binding properties of two anti-neonicotinoid antibodies were profiled by establishing fluorescence monoplex ic-SAT assays. Subsequently, two HDMs (2.8 and 5.0 μm) immobilized with THX and IMI haptens were synthesized and then implemented to fabricate a dualplex ic-SAT assay, while they were distinguished via a facile diameter-resolved decoding process through flow cytometry. Excitingly, the screening capacity of the dualplex ic-SAT assay was significantly expanded by employing antibodies with distinctive binding profiles. It enables the simultaneous monitoring of 7 representative neonicotinoid insecticides (THX, IMI, acetamiprid, clothianidin, thiacloprid, nitenpyram and imidaclothiz) and 4 major metabolites in a single test sample, with IC50 values ranging from 0.02 to 572.70 ng/mL. Furthermore, the dualplex ic-SAT assay was applied to honey, pollen and bee samples, demonstrating satisfactory recovery rates (74.1 %∼106.3 %) and nice agreement with LC-MS/MS analysis. In summary, the PBCMS offers an easy-to-implement, robust and cost-effective way for establishing user-defined ic-SAT, providing a powerful analytical tool to simultaneously monitor multiple neonicotinoid residues in environmental samples.