A two-step phage-assisted 3D electrospun fiber-based fluorogenic sensing system for improved specificity and accuracy in E. coli detection.

Current fluorogenic fiber-based biosensors for Escherichia coli detection remain limited by poor sample infiltration, insufficient specificity, and uncertainties in enzyme-based quantification. Here, a two-step fluorogenic biosensing strategy was developed by integrating bacteriophage-assisted lysis with a NaBH₄-expanded three-dimensional (3D) electrospun sensing pad. Phage C-a selectively infected viable E. coli BCRC 13055 cells and released intracellular β-glucuronidase, improving analytical specificity and reducing quantification uncertainties associated with continued enzyme synthesis. The 3D sensing pad markedly enhanced liquid retention and analyte-sensor interactions, resulting in more efficient fluorescence signal generation. The optimized biosensor achieved limits of detection of 108 CFU/mL by fluorescence spectroscopy and 137 CFU/mL by visual image analysis within a total assay time of approximately 4 h. Validation in milk yielded recovery rates of 99.8% and 98.5% for quantitative and qualitative analyses, respectively, while experiments using non-sterilized fruit juices demonstrated minimal interference from indigenous microorganisms. These findings indicate that phage-mediated lysis not only enhances analytical specificity but also improves the reliability of β-glucuronidase-based E. coli quantification. This work presents a practical fluorogenic biosensing platform that integrates engineered fiber architectures with bacteriophage biology for sensitive, selective, and reliable detection of E. coli in food matrices.