Intracellular recording of cardiomyocyte action potentials with nanopatterned volcano-shaped microelectrodes

Micro-nanotechnology based multielectrode arrays have led to remarkable progress in the field of transmembrane voltage recordings of excitable cells. However, electrode geometries alone have failed to produce a cell-electrode interface that is sufficiently robust and stable over extended periods of time to perform high-quality electrophysiological recordings. This thesis addresses the current limitations of micro-nanoelectrode arrays with a particular focus on whether patterning protruding microelectrodes with nanometer-wide biomimetic self-assembled monolayers that fuse with the cell membrane, significantly improves the quality of the electrophysiological recordings when compared to state-of-the-art technology.

The first step involved developing a novel nanofabrication process exploiting the sputtering of photoresist sidewalls during Ar+ ion beam etching to rapidly manufacture sub-100-nm complex multimaterial nanostructures at the wafer scale.

Ion beam etching redeposition was subsequently used to manufacture a novel nanopatterned volcano-shaped microelectrode (nanovolcano) array integrating most of the recent technological advances from the literature into an original structure. Electrophysiological studies on neonatal rat cardiomyocyte monolayers demonstrated that nanovolcanoes enabled in vitro electroporation-free intracellular recordings of cardiomyocyte action potentials. The recordings lasted more than one continuous hour with amplitudes as high as 20 mV from nearly 30 % of the device's channels.

The second phase investigated whether the use of electroporation improved the performance of nanovolcano arrays in terms of action potential amplitudes, recording durations, and yields. Experiments with neonatal rat cardiomyocyte monolayers grown on nanovolcano arrays showed that electroporation increased the efficiency of nanovolcano recordings as it enabled on-demand multiple registration of intracellular action potentials with amplitudes as high as 62 mV and parallel recordings in up to approximately 76 % of the available channels over consecutive days. The performance of nanovolcanoes showed no dependence on the presence of functional nanopatterns, indicating that the tip geometry itself was instrumental for establishing the tight seal at the cell-electrode interface. These results suggest that nanovolcanoes could prove useful not only for basic research but also for comprehensive drug testing in cardiac research.

Preliminary studies performed with hippocampal neonatal rat neurons demonstrated that nanovolcano arrays permit the recording of non-attenuated neuronal action potentials in addition to, potentially, subthreshold activity featuring a high signal-to-noise ratio. These results expand the function of nanovolcano arrays to other kinds of electrogenic cells and open new avenues for the field of neuroscience.

During an exploratory phase, a nanovolcano electrode was integrated at the tip of a suspended cantilever (nanovolcano probe) to conduct combined-force electrophysiological recordings. Proof-of-concept experiments on neonatal rat cardiomyocytes demonstrated that extracellular field potentials and contraction displacement curves could be recorded simultaneously. These features render the nanovolcano probe especially suited for mechanobiological studies aiming at linking mechanical stimuli to the electrophysiological responses of single cells.