A sandwich-type bacteriophage-based amperometric biosensor for the detection of Shiga toxin-producing Escherichia coli serogroups in complex matrices

Abstract

Immuno-based biosensors are a popular tool designed for pathogen screening and detection. The current antibody-based biosensors employ direct, indirect, or sandwich detection approaches; however, instability, cross-reactivity, and high-cost render them unreliable and impractical. To circumvent these drawbacks, here we report a portable sandwich-type bacteriophage-based amperometric biosensor, which is highly-specific to various Shiga toxin-producing Escherichia coli (STEC) serogroups. Environmentally isolated and biotinylated bacteriophages were directly immobilized onto a streptavidin-coated screen-printed carbon electrode (SPCE), which recognized and captured viable target cells. Samples (50 μL) were transferred to these bacteriophage-functionalized SPCEs (12 min, room temp) before sequentially adding a bacteriophage–gold nanoparticle solution (20 μL), H₂O₂ (40 mM), and 1,1′-ferrocenedicarboxylic acid for amperometric tests (100 mV s⁻¹) and analysis (ANOVA and LSD, P < 0.05). The optimum biotin concentration (10 mM) retained 94.47% bacteriophage viability. Non-target bacteria (Listeria monocytogenes and Salmonella Typhimurium) had delta currents below the threshold of a positive detection. With less than 1 h turn-around time, the amperometric biosensor had a detection limit of 10–10² CFU mL⁻¹ for STEC O157, O26, and O179 strains and R² values of 0.97, 0.99, and 0.87, respectively, and a similar detection limit was observed in complex matrices, 10–10² CFU g⁻¹ or mL⁻¹ with R² values of 0.98, 0.95, and 0.76, respectively. The newly developed portable amperometric biosensor was able to rapidly detect viable target cells at low inoculum levels, thus providing an inexpensive and improved alternative to the current immuno- and laboratory-based STEC screening methods.