A phage-displayed nanobody-based colorimetric-fluorescent dual-readout immunoassay using Eu-micelles for detecting Bacillus cereus in food.
Ge Guangsheng G, Wang Mengnan M, Zheng Kai K, Fan Xueyi X et al.
Bacillus cereus (B. cereus) is a common foodborne pathogen that contaminates agricultural products and induces emetic or diarrheal food poisoning. We report an europium(Eu)-micelle-mediated colorimetric-fluorescent dual-readout nanobody-based immunoassay for B. cereus detection. Four novel B. cereus-specific nanobodies were isolated for the first time from an immune Bactrian camel phage-displayed nanobody library. A sandwich immunoassay was established using a purified soluble nanobody (Nb23) for target capture and a phage-displayed Nb (phage-Nb23) for detection, leveraging an HRP-conjugated anti-M13 antibody to achieve phage-mediated primary signal amplification. Self-assembled Eu-micelles were introduced post-reaction as an independent fluorescent module. HRP-catalyzed oxidation of TMB generated a 652 nm colorimetric signal while concurrently quenching Eu-micelle red emission at 616 nm through a combination of inner filter effects and interfacial excited-state deactivation. Under optimized conditions, the limits of detection were 3.84 × 104 CFU/mL for the colorimetric channel and 8.57 × 103 CFU/mL for the fluorescent channel, demonstrating a 4.5-fold sensitivity enhancement. Spiked recoveries across diverse food matrices (rice, pork, bean sprouts, and juice) ranged from 92.48% to 116.42%, with relative standard deviations ≤ 5.54%. This dual-readout strategy offers mutual validation and broad applicability for food safety monitoring.