Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing –NH2 functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes–SiO2/thionine (CHIT–MWNTs–SiO2@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT–MWNTs–SiO2@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT–MWNTs, and CHIT–MWNTs–SiO2@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12 × 102–4.12 × 105 colony-forming units (CFU)/ml, and the total assay time was less than 45 min.