AC electro-osmosis in bacterial biofilms: a cautionary tale for electrophysiology experiments

Synthetic cationic fluorophores are used widely as probes to measure the membrane potentials of bacterial cells, eukaryotic cells and organelles, such as mitochondria. An external oscillating electric field was applied to Escherichia coli cells using microelectrodes and AC electro-osmosis was observed for the fluorophores, independent of the electrophysiology of the bacteria, giving rise to phantom action potentials. The fluorophores migrate around the microfluidic device in vortices modulating their concentration having decreases or dips in fluorescence. We show that the fluorescent dips are universally present when using cationic fluorophores, such as thioflavin-T, propidium iodide, Syto9 and Sytox Green, with or without E. coli cells in the inoculum, when stimulated with AC voltages. This is in contrast to the study of Stratford et al (PNAS, 2019) who claim the existence of action potentials. Furthermore, E. coli biofilms also demonstrated similar phenomena with dips in the fluorescence. We measured the relaxation times of the fluorophores experiencing AC electro-osmosis, which depended on the biofilm, the cells and the fluorophores used. PI had the smallest relaxation time and Syto9 the highest. Removing the cells resulted in longer relaxation times and introducing biofilm did not significantly change the relaxation times compared with the single cell experiments. Furthermore, fluorescently labelled DNA and fluorescent colloidal beads also demonstrate fluorescent dips through AC electro-osmosis, showing that these particles can be driven through biofilms. This is the first study of AC electro-osmosis in bacterial biofilms, indicating a surprisingly high mobility of charged molecules within the extracellular polymeric substance, which could be used to treat biofilms i.e. to increase the kinetics of delivery of antibiotics.