Probing the inner world of microbial cities: Electrochemical sensors for characterizing biofilm chemical microenvironments

ABSTRACT Biofilms are structurally complex microbial communities whose function is dictated by steep chemical gradients within their microenvironment and the surrounding space outside the biofilm. Although traditional analytical methods provide either bulk or static structural information, they fail to capture real-time metabolic function in situ . Electrochemical sensors provide a powerful solution, offering label-free, quantitative measurements of key chemical species with high spatial and temporal resolution directly within and outside living biofilms. This review highlights recent advances in the design and application of electrochemical sensors for biofilm analysis. We critically discuss the use of core techniques: amperometry for tracking redox-active metabolites (e.g., O 2 , H 2 O 2 , phenazines), potentiometry for monitoring ion activity (e.g., pH, Ca 2+ ), and electrochemical impedance spectroscopy for assessing biofilm volume and interfacial processes. We also explore recent challenges in electrochemical sensor designs and discuss future perspectives on multimodal data collections that combine multiple techniques.