Characterization of cellular dielectrophoretic (DEP) behaviors, when cells are exposed to an alternating current (AC) electric field of varying frequency, is fundamentally important to many applications using dielectrophoresis. However, to date, that characterization has been performed with monotonically increasing or decreasing frequency, not with successive increases and decreases, even though cells might behave differently with those frequency modulations due to the nonlinear cellular electrodynamic responses reported in previous works. In this report, we present a method to trace the behaviors of numerous cells simultaneously at the single-cell level in a simple, robust manner using dielectrophoretic tweezers-based force spectroscopy. Using this method, the behaviors of more than 150 cells were traced in a single environment at the same time, while a modulated DEP force acted upon them, resulting in characterization of nonlinear DEP cellular behaviors and generation of different cross-over frequencies in living cells by modulating the DEP force. This study demonstrated that living cells can have non-linear di-polarized responses depending on the modulation direction of the applied frequency as well as providing a simple and reliable platform from which to measure a cellular cross-over frequency and characterize its nonlinear property.

Electroporation is pivotal in bioelectrochemistry for cellular manipulation, with prominent applications in drug delivery and cell membrane studies. A comprehensive understanding of pore generation requires an in-depth analysis of the critical pore size and the corresponding energy barrier at the onset of cell rupture. However, many studies have been limited to basic models such as artificial membranes or theoretical simulations. Challenging this paradigm, our study pioneers using a microfluidic electroporation chip array. This tool subjects live breast cancer cell species to a diverse spectrum of alternating current electric field conditions, driving electroporation-induced cell rupture. We conclusively determined the rupture voltages across varying applied voltage loading rates, enabling an unprecedented characterization of electric cell rupture dynamics encompassing critical pore radius and energy barrier. Further bolstering our investigation, we probed cells subjected to cholesterol depletion via methyl-β-cyclodextrin and revealed a strong correlation with electroporation. This work not only elucidates the dynamics of electric rupture in live cell membranes but also sets a robust foundation for future explorations into the mechanisms and energetics of live cell electroporation.