Hydrogel-Integrated MIM-SERS Platforms for <i>In-Situ</i>, Spatiotemporal Profiling of <i>E. coli</i> Biofilm Responses to Chemo-Physical Perturbation

Abstract

Monitoring biofilm responses to antimicrobial and physical interventions requires in situ, minimally perturbative sensing in tissue-like environments. Here, we present a hydrogel-integrated surface-enhanced Raman spectroscopy (SERS) platform in which Escherichia coli is cultured beneath an LB–agarose layer on a nanolaminated metal–insulator–metal (MIM) substrate, enabling label-free, spatiotemporal readouts under chemo-physical perturbations. Time-resolved spectra (785 nm) were acquired at 1, 11, 24, 24&#42;h (immediately post-intervention), and 36 h over 4 mm² maps (~400 spectra per map), with electronic Raman scattering (ERS) used as an internal standard to reduce instrumental drift. Interventions introduced at 24 h included ampicillin (AMP), femtosecond (fs) laser-induced nanobubbles (950 nm), or their combination. Spectra consistently exhibited a band near 445 cm⁻¹, tentatively associated with carbohydrate-rich EPS components and a feature near 732 cm⁻¹ that is adenine-associated with a substantial hydrogel contribution. Principal component analysis (PCA) and linear discriminant analysis (LDA) discriminated timepoints and treatments with high apparent accuracy (leave-one-out cross-validation, LOOCV >95%), while 2D maps visualized treatment-dependent heterogeneity. Immediate global intensity increases at 24&#42;h were consistent with transient hotspot re-exposure of plasmonic hotspots after fs excitation. Overall, this study provides a method-focused validation of hydrogel-integrated MIM-SERS for real-time biofilm profiling and motivates replicate-level validation, orthogonal biological readouts, and translation toward flexible device formats.