AC electro-osmosis in bacterial communities with fluorescence-based electrophysiology measurements using exogeneous fluorophores

Synthetic cationic fluorophores are widely used as probes to measure the membrane potentials of bacterial cells, eukaryotic cells, and organelles (such as mitochondria) in electrophysiology experiments and live/dead assays. We applied an external oscillating electric field to<i>Escherichia coli</i>using microelectrodes and observed that AC electro-osmosis caused fluorescence transients independent of bacterial electrophysiology, which could be mistaken for membrane depolarisation events. The fluorophores migrated within the microfluidic device in vortices, leading to concentration fluctuations manifested as dips in fluorescence. These fluorescent dips were universally present when using cationic fluorophores such as thioflavin-T, propidium iodide, Syto9, and Sytox Green, with or without<i>E. coli</i>present, whenever AC voltages were applied. Furthermore, we also demonstrate that fluorescence dips in dense bacterial communities can arise from AC electro-osmosis rather than ion-channel activity. This cautionary tale highlights how electrical stimulation experiments in microbial communities can yield misleading results if electrokinetic effects are not accounted for. We quantified the relaxation times of fluorophores under AC electro-osmosis, which depended on the community, the cells, and the dye used: PI showed the shortest relaxation time and Syto9 the longest. Removing cells resulted in longer relaxation times, and introducing dense communities did not significantly alter the relaxation times compared with single-cell experiments. Furthermore, fluorescently labelled DNA and fluorescent colloidal beads (30-130 nm) also exhibited fluorescence dips due to AC electro-osmosis, demonstrating that charged molecules and particles readily penetrate and accumulate within these assemblies. To our knowledge, this is the first study to characterise AC electro-osmosis in dense bacterial communities, revealing the high mobility of charged molecules in such systems and suggesting possible applications for enhancing antibiotic delivery.