Monitoring passive transport of redox mediators across a confluent cell monolayer with single-cell resolution by means of scanning electrochemical microscopy

Abstract

Scanning electrochemical microscopy (SECM) was used for time-resolved studies of passive transport of redox active substances across cell monolayers with sub-cellular resolution. Platinum disk ultramicroelectrodes (UMEs) with effective electrode radii in the sub μm range were operated in the constant-height mode. Monolayers of normal rat kidney (NRK) or Madin Darby canine kidney strain II (MDCK II) epithelial cells served as model cell lines and were grown to confluence on permeable membrane filters to investigate the transport of redox mediators across epithelial barriers. A novel electrochemical cell was developed ensuring reliable mechanical fixation of the growth substrate and preventing membrane vibrations during the SECM operation. The detection of different redox mediators was enabled by potential switching at the UME. This concept proved to be an attractive alternative to the use of dual-microelectrodes or micropipette-microelectrode combinations. The various cell lines were investigated regarding their barrier functions with respect to different redox mediators. Para- and/or trans-cellular transport mechanisms could be distinguished using either hydrophilic ruthenium hexamine chloride or lipophilic ferrocene methanol as redox mediators.