Mediated amperometry as a prospective method for the investigation of electroporation

Pulsed electric field effects induced in a membrane, as well as intracellular structures, depend on cell type, field and media parameters. To achieve desired outcomes, membranes should be permeabilized in a controlled manner, and thus efficiency of electroporation should be investigated in advance. Here, we present a framework for using mediated amperometry as a prospective method for the investigation of electroporation and its effects on cellular machinery. Whole-cell sensors with single mediator systems comprised of hydrophilic or lipophilic mediators were successfully employed to investigate membrane permeability as well as cellular responses. Exposure of yeast cells to single electric field pulse (τ = 300 µs, E = 16 kV/cm) resulted in up to tenfold increase of current strength mediated with hydrophilic mediators. Exposure to PEF resulted in decrease of menadione mediated current strength (from 138 ± 15 to 32 ± 15 nA), which could be completely compensated by supplementing electrolyte with NADH.

Mediated amperometry reveals two distinct modes of yeast responses to glucose

Mediated amperometry was exploited to monitor intracellular redox activity without cell disruption. Continuous measurements of menadione-mediated glucose currents at carbon paste electrodes with various immobilized intact wild type yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Clavispora lusitaniae, Wickerhamomyces anomalus, Pichia guilliermondii, Kluyveromyces lactis var. lactis, Debaryomyces hansenii, Candida zeymolaydes and Candida tropicalis) revealed two distinct and previously unreported modes of development of the currents during the first 2 to 3 min. after subjection to glucose. A correlation among the values of the currents and the capacities of wild type yeasts to secrete various substances was observed.