Fluorescent biosensor for simultaneous detection of C. sakazakii and S. typhimurium using DNA-templated metal nanoclusters and Exonuclease III-assisted target recycling amplification.

Sensitive and simultaneous detection of multiple foodborne pathogens has become an urgent need in the food industry. This work presents a signal-on fluorescence biosensor using DNA-templated metal nanoclusters as signal nanotags and an Exonuclease III (Exo III)-assisted target-recycling (EATR) strategy for the detection of Cronobacter sakazakii (C. sakazakii) and Salmonella typhimurium (S. typhimurium). The detection system consists of carboxylated polypyrrole nanoparticles functionalized with hairpin DNA sequences HP1 and HP2 (HP1-cPPy-HP2 NPs), the target ompA gene (tDNA1) of C. sakazakii and invA gene (tDNA2) of S. typhimurium, DNA-templated gold and silver nanocluster nanotags (sDNA1-AuNCs and sDNA2-AgNCs), and Exo III. In the presence of the targets, HP1 and HP2 on cPPy surface are opened through hybridization, forming tDNA1-HP1-cPPy-HP2-tDNA2 complex. These opened HP1 and HP2 then hybridize with their respective sDNA1 (sDNA1-AuNCs) and sDNA2 (sDNA2-AgNCs), leading to the increased fluorescence intensity of both AuNCs and AgNCs in the detection system. Furthermore, with EATR amplification strategy, the targets can be released from the complex through cleavage by Exo III and proceed to the next cycle of HP1/HP2 opening. As a result, the fluorescence signals of AuNCs and AgNCs can be significantly increased. Under optimal conditions, the fluorescence biosensor shows wide linear ranges for C. sakazakii (1.64 ∼ 1.64 × 105 CFU mL-1) and S. typhimurium (2.56 ∼ 2.56 × 105 CFU mL-1), with LODs of 1.4 CFU mL-1 and 2.2 CFU mL-1, respectively. The biosensor was used for the detection of C. sakazakii and S. typhimurium in real samples, with recovery ranges of 88.8% to 110.6% and 90.4% to 114.4%, respectively.