Rapid electrochemical detection of Escherichia coli in milk using an oriented phage-based biosensor

Foodborne contamination by pathogenic Escherichia coli (E. coli) is a major safety concern, particularly in dairy products, and necessitates the rapid selective detection in complex matrices. We present an electric-field-assisted phage-based electrochemical biosensor for the 15-min detection of the model bacterium E. coli in phosphate-buffered saline (PBS) and in non-fat milk. Indium tin oxide (ITO) electrodes are functionalized with (3-aminopropyl)triethoxysilane and glutaraldehyde to allow covalent attachment of T4 bacteriophages. During phage immobilization, an electric field is applied to orient virions, thereby increasing the accessibility of tail receptor-binding proteins for bacterial capture. Differential pulse voltammetry with the external redox probe Fe(CN)₆^3-/4- is used for detection. Electric field-assisted orientation of the phage layer produces about threefold higher electrochemical signal than non-oriented phage layers and about tenfold higher signal than phage-free controls at 10⁴ CFU/mL. The developed biosensor enables quantitative detection of E. coli from 10 to 10⁷ CFU/mL in PBS with a limit of detection of 62 CFU/mL. The response to the non-host Staphylococcus aureus is up to almost 30-fold lower than that to E. coli at the same concentration, demonstrating high specificity. The sensor retains a linear calibration and achieves a limit of detection of 12 CFU/mL in milk, without any pre-enrichment of the sample. The biosensor maintains its response after two weeks of storage at 4 °C. Results indicate that an electric-field-assisted phage film on ITO enables simple, rapid, and selective E. coli detection in both model solutions and complex food matrices.