Smart-Sandwich: A Thin Flexible Sensing Device Based on an Agarose–Chitosan–Agarose (ACA) Triple-Layer Biofilm for Onsite Monitoring of <i>Escherichia coli</i>

Portable, cost-effective, and durable thin-film sensors are essential for real-time <i>E. coli</i> detection, ensuring safe drinking water and public health protection. In this work, we developed a solid-state, flexible sensing device using an agarose-chitosan-agarose (ACA) sandwich biofilm for the selective colorimetric detection of <i>E. coli</i> in water. The chitosan in ACA biofilm functions as an artificial enzyme, exhibiting peroxidase-like activity, which catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂). Upon incubating the ACA biofilm with <i>E. coli</i>, the agarose layers undergo enzymatic degradation by the β-galactosidase enzyme produced by <i>E. coli</i>. The degradation takes place due to the cleavage of β-1,4-glycosidic bonds. This exposes the underlying chitosan layer which enhances the catalytic activity, triggering a visible color change due to TMB oxidation within 30 min. The device achieves a highly sensitive detection limit of 6.8 CFU/mL, with excellent accuracy in real samples, further supported by android-based, smartphone-assisted detection. The developed solid-state, flexible ACA biofilm offers a novel, rapid, and reliable solution for onsite <i>E. coli</i> detection, combining sensitivity, stability, and ease of use.