Highly sensitive amperometric immunosensor for the detection of Escherichia coli

With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH₂-anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole (Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl β-d-galactopyranoside (PAPG), the bacterial enzyme, β-d-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC) electrode at 0.22 V. The operational procedure consists in first adding the test sample containing the bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte (being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates an improved detection limit of more than 10 times less bacteria detected than other immunosensing techniques without the need for multi step pretreatments of the test sample and/or incubation as found in some of the existing methods.