Activity-Specificity Balanced Au@Pt Nanozyme Immunoassay via Dual-Au Deposition for Enhanced Mycotoxin Detection

Mycotoxins persistently threaten global food and herbal medicine safety, demanding the urgent development of detection methods that integrate high sensitivity with strong specificity. Conventional nanozyme-based immunoassays, however, are constrained by the inherent trade-off between sensitivity and specificity, limiting their performance. In this study, we propose a dual-gold enhanced Au@Pt nanozyme-linked immunosorbent assay (Au@Pt/Au-ELISA), which leverages a rationally engineered sequential optimization paradigm to fundamentally overcome the intrinsic trade-off between sensitivity and specificity. The method innovatively integrates mild gold deposition for background suppression with targeted catalytic site amplification, thereby maximizing the catalytic potential of Au@Pt nanozymes while preserving antibody recognition. The dual-stage design significantly enhances nanozyme activity, reducing the usage of antibody probes to 14.3% and 33.3% of that in the original scheme for zearalenone (ZEN) and aflatoxin B 1 (AFB 1 ) detection, respectively. As a secondary outcome, the assay maintains excellent analytical sensitivity, achieving half-maximal inhibitory concentrations of 42 pg/mL for ZEN and 28 pg/mL for AFB 1 , corresponding to 7.2-fold and 2.0-fold enhancements over conventional ELISA, respectively. Importantly, the method exhibits high quantitative accuracy in complex matrices, with ZEN recoveries of 109.6-119.5% in coix seeds and AFB 1 recoveries of 100.2-113.2% in lotus seeds. Limited cross-reactivity toward related mycotoxins confirms the high immunospecificity of the assay, which shows strong agreement with LC-MS/MS results (R > 0.99). By harmonizing catalytic activity with immunospecificity, this cost-effective platform not only provides a refined design strategy for nanozyme-based immunoassays but also establishes a solid technical foundation for high-performance mycotoxin screening in complex food and herbal medicine matrices. TOC: This study presents a dual-step gold deposition strategy that effectively overcomes the trade-off between probe activity and specificity in Au@Pt nanozyme-based immunoassays, enabling ultrasensitive, highly specific, and broad-spectrum detection of multiple mycotoxins. • Dual-Au enhanced Au@Pt nanozyme immunoassay breaks sensitivity-specificity trade-off. • Gold deposition reduces probe use to 14.3% of that in the unenhanced system. • Boosts sensitivity for ZEN/AFB 1 up to 7.2-fold via dual catalytic enhancement. • Robust matrix tolerance demonstrated in complex food samples. • Excellent consistency with LC-MS/MS confirms accuracy in real sample detection.