Dual-electrochemical-signal biosensor using phage-modified ZIF-8 to discriminate viable and nonviable bacteria

Foodborne pathogen contamination represents a significant global public health challenge. It should be noted that both active pathogens and dead pathogens pose health risks through toxin release. However, conventional detection techniques, such as plate counting methods, immunoassays, and molecular biology-based approaches, generally struggle to perform simple, rapid, and simultaneous discrimination between live and dead bacteria. Therefore, new detection technologies should be developed to meet the demand for on-site and simultaneous quantification of live and dead bacterial counts, ensuring food safety.

This work developed a phage-based biosensor using dual electrochemical signals for simultaneous total and viable E. coli O157:H7 quantification. A deactivated phage capture probe immobilized on an electrode bound total bacteria, generating an impedance (EIS) signal proportional to total count. A viable phage signal probe, conjugated with ferrocene-doped ZIF-8, captured target bacteria forming a sandwich complex. Subsequent gold-sputtering enhanced signal transduction and phosphate-triggered ZIF cleavage released electroactive reporters produced a differential pulse voltammetry (DPV) signal proportional to viable bacteria. Dead bacteria were quantified by subtracting the viable (DPV) signal from the total (EIS) signal. This dual-signal approach enabled detection on a single device. The method achieved a linear range of 10-10⁷ CFU/mL and a limit of detection of 7 CFU/mL. Satisfactory performance was demonstrated in lettuce, milk, and pork samples.

This integrated strategy overcomes key limitations in food safety inspections by enabling single-device, simultaneous quantification of total and viable bacterial loads within complex matrices like lettuce, milk, and pork. It establishes a promising field-deployable paradigm for rapid pathogen viability assessment, overcoming reliance on laboratory methods and addressing a critical technological void in real-time food safety surveillance across supply chains.