Self-Healing Bilayer Hydrogel Solid-State Electrochemical Platform: Time-Resolved In Situ Dynamic Monitoring of Escherichia coli Activity

Achieving in situ and time-resolved monitoring of microbial metabolites without disrupting the microbial growth environment remains a key challenge in electrochemical biosensing. Herein, we propose a self-healing bilayer hydrogel-based solid-state electrochemical sensing platform for the in situ, time-resolved analysis of purine metabolites produced byEscherichia coli(E. coli). This platform integrates an upper Agar culture module and a lower borax-crosslinked poly(vinyl alcohol) (PVA) detection module, forming a contiguous structure that allows metabolites (e.g., guanine, xanthine, hypoxanthine) to migrate across the solid–solid interface for sensitive electrochemical detection. The detection layer exhibits excellent ionic conductivity; when coupled with its robust structural self-healing capacity, the platform achieved a detection limit of 0.05 µM for guanine. ForE. colidetection, a linear response range of 1.1 × 106to 9.5 × 106CFU·mL−1(R2= 0.9974) was obtained, and relative standard deviations (RSDs) of less than 2.34% even after two weeks of storage. Leveraging this integrated design, the platform enables continuous, label-free tracking of bacterial metabolic dynamics throughout all growth phases. Notably, it detects metabolic transition points earlier than traditional plate counting methods and accurately evaluates antibiotic inhibition trends, with results consistent with colony-forming unit (CFU) analysis. This integrated culture–detection architecture thus provides a versatile strategy for functional microbial analysis and rapid antimicrobial susceptibility testing.