Experiments using electrical stimulation with electrodes in direct contact with the sample often encounter electrochemical interface effects and non-linear reactions like corrosion. Additionally, variations in electrode-manufacturing precision can lead to changes in electrical properties, impacting the repeatability and reproducibility of experiments. In this study, we examine three different electrode setups employed in recent research using impedance spectroscopy: the SNEX-100 from Microprobes Inc. (Gaithersburg, USA), the ECIS culture plate 8W20idf from Applied BioPhysics Inc. (Troy, USA), and custom-made titanium rod electrodes designed for 6-well plates.

The electrical properties of the electrodes were obtained utilising electrochemical impedance spectroscopy. The impedance spectra were measured in a frequency range from 1 Hz to 10 MHz in a two-electrode setup, i.e. no reference electrode was utilised. The electrodes were immersed in conductivity standards in a thermally stable environment to ensure replicability.
The validity of the data was checked using the LinKK test utilising the open-source software ImpedanceFitter. The system's electrochemical properties were accessed through the software’s fitting capabilities. The knowledge of the electrochemical properties allows numerical simulations to predict the outcome of the experiments. This may, for example, also apply to the electric field as a non-measurable observable. Tracking the input current and using Ohm's law to determine the DC equivalent enabled us to observe shifts in impedance throughout the stimulation process.

The custom-made titanium electrodes demonstrated corrosion, as energy-dispersive X-ray analysis detected a calcium phosphate layer forming on their surfaces. The unusual impedance spectra of the SNEX-100 electrodes pointed to altered geometries likely caused by manufacturing tolerances. For the ECIS culture plates, impedance varied across wells due to the significant impedance of the thin gold traces, rendering them unsuitable for electrical stimulation experiments. All impedance spectra exhibited the characteristic double layer at low frequencies, typical of the electrode-electrolyte interface.

To summarise, impedance spectroscopy allows evaluation of the electrode state at all stages of stimulation. Understanding the specific limitations of each system helps optimise experimental setups, thereby enhancing replicability.