Sensitive and simultaneous detection of foodborne pathogens using a magnetic layered double hydroxide-based fluorescent sensor with T7 exonuclease signal amplification

Abstract

The rapid determination of foodborne pathogens is crucial for safeguarding food safety and public health. Layered double hydroxides (LDHs) have emerged as promising materials for fluorescent sensors owing to their large surface area, ease of synthesis, and cost-effectiveness. However, current LDH-based fluorescent sensors are challenged by complex detection protocols, limited sensitivity, and low detection efficiencies. This study presents a novel fluorescent sensor based on magnetic Fe₃O₄@zinc/nickel/aluminum-layered double hydroxide (MLDH) and T7-exonuclease (T7 Exo) for the sensitive and simultaneous detection of Escherichia coli (E. coli) and Vibrio parahaemolyticus (V. parahaemolyticus). MLDH was synthesized via a simple co-precipitation method and employed as a fluorescence quencher for the first time to efficiently quench the 6-carboxyfluorescein (FAM) and 6-carboxy-X-rhodamine (ROX) fluorophores labelled on both single-stranded DNA probes and the target/probe double-stranded DNA duplexes via the inner filter effect. T7 Exo served a dual function in target reporting and signal amplification. The magnetism of MLDH simplifies the detection protocol and provides a cleaner detection background, while the T7 Exo-based signal amplification strategy significantly enhances the sensitivity. This sensor achieves simultaneous detection of E. coli and V. parahaemolyticus characteristic DNA in a single assay, with a broad linear range of 0.01–10 nM and detection limits of 6.8 pM and 6.0 pM, respectively. Moreover, it has been successfully validated in real samples, yielding recoveries from 91.4 % to 114 % and relative standard deviations below 4.88 %. Owing to its high sensitivity, convenience, effectiveness, and accuracy, this sensor holds great potential in simultaneous detection of foodborne pathogens.