Novel fluorescence immunoassay for the detection of zearalenone using HRP-mediated fluorescence quenching of gold-silver bimetallic nanoclusters

Abstract

In the presented study, a horseradish peroxidase (HRP)-mediated ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) for zearalenone (ZEN) was reported based on fluorescence quenching of gold-silver bimetallic nanoclusters (Au-Ag NCs). HRP-antibody was used as a bridge in this immunoassay, linking the ratiometric fluorescence signal to the ZEN concentration. HRP catalyzed the oxidization of o-phenylenediamine in the presence of H₂O₂, leading to the formation of 2,3-diaminophenazine, which not only delivered a new peak at 580 nm but also quenched Au-Ag NCs fluorescence at 690 nm. Under optimal conditions, the detection limit for the proposed ELISA was 0.017 ng/mL, which was approximately 6.6-fold lower than conventional ELISA. Moreover, analytical performances were evaluated fully including specificity, accuracy, precision, and practicability, and showed that this method provides a potential platform for sensitive and reliable detection of ZEN.

Keywords: Gold-silver bimetallic nanoclusters; Horseradish peroxidase; Ratiometric fluorescence ELISA; Zearalenone.