Carneiro da Cunha Martorelli, V.

bioRxiv2025

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.

biophysics↗

bioRxiv2023

Electrical Signalling in Three Dimensional Bacterial Biofilms Using an Agent Based Fire-Diffuse-Fire Model

Agent based models were used to describe electrical signalling in bacterial biofilms in three dimensions. Specifically, wavefronts of potassium ions in E. coli biofilms subjected to stress from blue light were modelled from experimental data. Electrical signalling only occurs when the biofilms grow beyond a threshold size, which we have shown to vary with the K+ ion diffusivity and the K+ ion threshold concentration which triggered firing in the fire-diffuse-fire model. The transport of the propagating wavefronts shows super-diffusive scaling on time. K+ ion diffusivity is the main factor that affects the wavefront velocity. The K+ ion diffusivity and the firing threshold also affect the anomalous exponent for the propagation of the wavefront determining whether the wavefront is sub-diffusive or super-diffusive. The geometry of the biofilm and its relation to the mean square displacement (MSD) of the wavefront as a function of time was investigated for spherical, cylindrical, cubical and mushroom-like structures. The MSD varied significantly with geometry; an additional regime to the kinetics occurred when the potassium wavefront leaves the biofilm. Adding cylindrical defects to the biofilm, which are known to occur in E. coli biofilms, the wavefront MSD also had an extra kinetic regime for the propagation through the defect.

biophysics↗