Preparation of a high specificity imidacloprid monoclonal antibody and its application in homogeneous fluorescence immunoassay based on hydrogel beads assembling CdSe/ZnS quantum dots

Abstract The risk associated with imidacloprid (IMI) residues necessitates the development of highly sensitive detection methods for ensuring food safety. In this study, IMI-hapten is synthesized via the reaction of IMI with β-mercaptopropionic acid, and used to prepare immunogen, coating antigen, and IMI monoclonal antibody (IMI-mAb). An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) is established, yielding a half-maximal inhibitory concentration (IC 50 ) of 0.34 µg L -1 and a limit of detection (LOD) of 0.04 µg L -1 . To further enhance sensitivity and analytical efficiency, fluorescent hydrogel beads are engineered by embedding CdSe/ZnS quantum dots (QDs) into biocompatible chitosan hydrogel matrices. A homogeneous fluorescence immunoassay is subsequently established, relying on the competitive interaction between an antigen-conjugated quenching probe (AgNPs@IMI-OVA) and an antibody-labeled signal probe, which modulates the fluorescence intensity accordingly. Compared with conventional ELISA, this assay exhibits remarkable superiority, which achieves a LOD of 5.62×10 − 4 µg L -1 (representing over a 70‑fold enhancement), eliminates tedious washing steps, and completes the entire assay within 20 min. Validation using spiked fruit and vegetable samples confirms the method’s high specificity and reliability, demonstrating its great potential for the rapid screening of IMI residues in agro-products.