Electrochemical aptasensor for simultaneous detection of foodborne pathogens based on a double stirring bars-assisted signal amplification strategy

In this work, we demonstrate a double stirring bars-based signal-amplified strategy for the simultaneous electrochemical detection of Vibrio parahaemolyticus (V.P) and Salmonella typhimurium (S.T). Methylene blue (MB) or ferrocene (Fc) labeled functionalized branched-chain DNA hybrid structure (FBCHS) was immobilized on magnetic microspheres and used as encoded signal tags. The encoded signal tags were hybridized with the aptamer-embedded tetrahedral DNA nanostructures (TDNs), which acted as capture probes, on one gold stirring bar (bar-A). The magnetic signal tags on bar-A could capture V.P and S.T, and their complex was later released into the solution. The composites were then digested by exonuclease I that was modified on another gold stirring bar (bar-B). The released free bacteria participated in the next cycle, from bar-A to bar-B, and generated more magnetic tags for signal amplification. After the reaction, the tags were magnetically enriched on a screen-printed carbon electrode (SPCE), generating two distinct voltametric peaks derived from MB and Fc. Their current intensities could reflect the concentration of V.P and S.T in the samples. Under optimal conditions, the double stirring bars-based signal amplification strategy was able to increase the detection sensitivity with a lower detection limit of 4 and 7 CFU/mL for V.P and S.T, respectively, within a detection range of 10–108 CFU/mL. Importantly, the exonuclease I on the stirring bar (bar-B) for signal amplification was able to be reused up to 50 times and avoided its loss if being directly added in solution, which produced cost savings in terms of used enzymes.