Identifying fungicide difenoconazole as illegal growth regulator in vegetable: Computer-aided hapten similarity to enhance immunoassay sensitivity

Difenoconazole, a fungicide with broad-spectrum properties, has recently been found to have been used illegally used as a plant growth regulator in Brassica campestris, with the intent of inducing thick stems and dark green leaves. However, analysts have encountered challenges in implementing a rapid surveillance screening approach for this purpose. In this study, a novel hapten was designed to improve the analytical performance of difenoconazole immunoassay. Specifically, the triazole of the original hapten was replaced with a benzene ring, guided by molecular simulation. This led to the development of a very sensitive antibody and the subsequent development of a competitive indirect enzyme linked immunosorbent assay (ciELISA) for the detection of difenoconazole in vegetable samples. The assay exhibited a working range of 0.16 ng mL−1 to 9.64 ng mL−1, with a detection limit of 0.05 ng mL−1. Upon analysis of blind samples, a strong correlation was observed between the ciELISA and HPLC-MS/MS methods. As a result, the proposed technique may prove to be an excellent tool for the rapid detection of difenoconazole overuse and adulteration in vegetables.