Peptide nucleic acid-based biosensor for detection of E. coli with differential pulse voltammetry.

AIMS: This study aimed to develop a rapid and highly sensitive electrochemical biosensor for the sequence-specific detection of Escherichia coli 16S rRNA using a peptide nucleic acid (PNA) probe immobilized on a graphene - NH/gold-nanoparticle (GNH - AuNP) composite. MATERIALS & METHODS: An SH-terminated PNA probe was covalently attached to a GNH - AuNP nanocomposite layer deposited on gold screen-printed electrodes via 3-mercaptopropionic acid (MPA). The electrode assembly and hybridization processes were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and X-ray photoelectron spectroscopy (XPS). Quantification was achieved using differential pulse voltammetry (DPV) with hexaammineruthenium(III) (RuHex) as an electrostatic redox indicator. RESULTS: Hybridization of the target 16S rRNA sequence produced a distinct "signal-on" DPV response proportional to target concentration. The biosensor exhibited excellent linearity from 1 to 1000 pM (R2 = 0.9984), with a limit of detection (LOD) of 0.21 pM and a limit of quantification (LOQ) of 0.70 pM. The neutral-backbone PNA probe improved hybridization kinetics and selectivity. CONCLUSIONS: The PNA/GNH - AuNP interface enabled fast, stable, and highly sensitive E. coli detection. Its disposable electrode format and portable electrochemical readout make it suitable for on-site diagnostic and food-safety monitoring applications.