Soft-probe-scanning electrochemical microscopy reveals electrochemical surface reactivity of E. coli biofilms

•In situ and label-free investigation of the electrochemical surface reactivity of E. coli biofilms.•E. coli biofilms reduce the oxidized form of ferrocene methanol.•Soft microelectrodes in contact mode brush over the biofilm keeping the working distance constant.•Soft-Probe-SECM in feedback mode distinguishes biofilms with ampicillin-resistant from ampicillin-susceptible E. Coli cells.•The effect of antibiotic treatment on E. coli biofilms can be visualized in real time. Investigating and understanding dynamic biofilm growth mechanisms is challenging, often because state-of-the-art optical characterization tools provide limited information. Micrometric electrochemical imaging of Escherichia coli biofilms using Soft-Probe-Scanning Electrochemical Microscopy (Soft-Probe-SECM) is herein presented as a complementary technique. A soft microelectrode is scanned over biofilms in a gentle contact mode, which is essential to provide a constant working distance. The on-film reduction of an electro-active compound, here the oxidized form of ferrocene methanol, is used to create in situ biofilm metabolic activity maps by applying the feedback mode of SECM. SECM approach curves of identically grown biofilms suggest that the SECM-based detection of metabolic activity is surface-confined. The analysis could therefore be carried out on entire biofilms as well as on tape-stripped biofilm surface layers. The method is further capable of distinguishing between biofilms containing E. coli cells either with or without ampicillin-resistance. Finally, the SECM detection of the degradation of an E. coli biofilm in the presence of different gentamicin concentrations is presented.