“Two Birds One Stone” Strategy for Efficient Preparation of Gold Nanoclusters-Labeled Immunoprobes and Sensitivity Boosting of Lateral Flow Immunoassays

• A “two birds one stone” strategy is proposed to efficiently prepare bright MNCs-based immunoprobes for FLFIAs. • Y 3+ ions significantly enhance the fluorescence intensity of MTU-AuNCs by over 9-fold. • Y 3+ -AuNCs-based immunoprobes achieve a coupling efficiency of 92% within 5 min. • High-sensitive detection of ZEN and OTA with LODs of 0.001 ng/mL and 0.005 ng/mL, respectively. • The Ab-Y 3+ -AuNCs-FLFIA exhibits good reliability for detecting ZEN and OTA in wheat samples. Fluorescent lateral flow immunoassay (FLFIA) is highly suitable for portable quantitative detection but faces challenges of inefficient immunoprobe preparation. Herein, we propose a “two birds one stone” strategy leveraging yttrium(III) ions (Y 3+ ) to enhance the fluorescence of 6-methyl-2-thiouracil protected gold nanoclusters (MTU-AuNCs) and simultaneously streamline the conjugation of AuNCs with antibodies (Abs). We found that Y 3+ remarkably improved the fluorescence intensity of MTU-AuNCs by over 9-fold, with quantum yield increasing from 5.81% to 19.23% through suppressing the proton transfer phase in the concerted proton coupled electron transfer process. Moreover, the Ab-Y 3+ -AuNCs immunoprobes were efficiently prepared within 5 min with a coupling efficiency of 92%, avoiding the tedious covalent conjugation process. Based on this strategy, a FLFIA was established for simultaneous detection of zearalenone (ZEN) and ochratoxin A (OTA), achieving limits of detection of 0.001 ng/mL for ZEN and 0.005 ng/mL for OTA. Compared with the conventional gold nanoparticle-based lateral flow immunoassays (AuNPs-LFIAs), the detection sensitivity for ZEN and OTA improved by 80-fold and 18-fold, respectively. The recoveries ranging from 94.8% to 100.4% were obtained in the detection of wheat samples. This study provides a facile and efficient approach for preparing high-performance fluorescent immunoprobes, significantly advancing the sensitivity and practicality of FLFIA for rapid on-site detection of food hazardous substances.