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

Foodborne contamination by pathogenic Escherichia coli is a major safety concern, particularly in dairy products, and necessitates the rapid, sensitive, and selective detection of these pathogens directly in complex matrices. In this work, we present an electric field-assisted, phage-based electrochemical biosensor for the 15-minute detection of the model bacterium E. coli BL21 in PBS buffer and milk. Indium tin oxide (ITO) electrodes were functionalized with (3-aminopropyl)triethoxysilane and glutaraldehyde to allow covalent attachment of T4 bacteriophages. During immobilization, an electric field was applied to promote the effective orientation of the virions and increase the accessibility of tail receptor-binding proteins for bacterial capture. After blocking residual sites with bovine serum albumin, differential pulse voltammetry was used as the transduction method. Electric field-assisted phage layers produced approximately threefold higher signal than non-oriented phage layers and about tenfold higher signal than phage-free controls at 10⁴ CFU/mL, confirming the benefit of controlled phage orientation. In PBS, the biosensor enabled quantitative detection of E. coli from 10 to 10⁷ CFU/mL with a limit of detection of 62 CFU/mL. The response to the non-host Staphylococcus aureus was up to almost 30-fold lower than that to E. coli at the same concentration, demonstrating high specificity. In milk, the sensor retained a linear calibration and achieved a limit of detection of 12 CFU/mL after 15 minutes of incubation, without any pre-enrichment of the sample. The biosensor retained its response after at least two weeks of storage at 4 °C. Results indicate that electric field-assisted phage interfaces on ITO provide a simple, rapid, and selective platform for E. coli detection in both model solutions and complex food samples.