Simultaneous and sensitive determination of Escherichia coli O157:H7 and Salmonella Typhimurium using evanescent wave dual-color fluorescence aptasensor based on micro/nano size effect.

The simultaneous and sensitive determination of two common pathogenic bacteria, Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella Typhimurium (S. Typhimurium) was achieved using evanescent wave dual-color fluorescence aptasensor and the fiber nanoprobe through combining the micro/nano size and time-resolved effect. Two fluorescence labeled aptasensors, Cy3-apt-E and Cy5.5-apt-S, were regarded as biorecognition elements and signal reporters for E. coli O157:H7 and S. Typhimurium, which were alternatively excited by evanescent waves originated from 520 nm to 635 nm excitation lights, respectively. The fiber nanoprobe with in-situ etched nanopores was used for distinguishing free aptasensors and aptasensors bound to pathogenic bacteria based on the limited penetrated depth of evanescent wave and the significant size difference of bacteria and nanopore. The E. coli O157:H7 and S. Typhimurium were directly and simultaneously quantitated in less than 35 min without the requirement of the complex immobilization of biorecognition molecules and bacteria enrichment/separation processes. The limits of detection of E. coli O157:H7 and S. Typhimurium were 340 CFU/mL and 180 CFU/mL, respectively. The satisfied recovery rate of real samples testing verified the feasibility and accuracy of the proposed method. Our strategy not only greatly simplifies the detection and identification process of multiple pathogenic bacteria, but also is easy to extend as a universal technology for sensitive determination of other bacteria using their respective biorecognition molecules.