Probing Cd2+-Stressed Live Cell Membrane Permeability with Various Redox Mediators in Scanning Electrochemical Microscopy

Heavy metal cytotoxicity has become a mainstay in scientific research due to the vast range of detrimental effects on living organisms. Cd2+ in particular can activate a wide range of physiological pathways that can cause significant increases in oxidative stress, cancer formation, or cellular death. This is partly due to its ability to bioaccumulate in the body with a long half-life for excretion. Here, we used hydrophilic redox mediators, ferrocenecarboxylate, 1'1-ferrocene dicarboxylate and hexaamineruthenium, as molecular probes in scanning electrochemical microscopy (SECM) to determine the membrane permeability in single live human bladder cancer (T24) cells affected by Cd2+. Hydrophilic and charged sensing agents should be impermeable to the cells. However, when T24 cells were treated with 25 mu M Cd2+ (t <= 6 h), the membrane permeability to these sensing agents can be induced to approximately 7.0 x 10(-5) m/s. For acute mM Cd2+ exposure, the membrane permeability can be induced to 2.0 X 10(-4) m/s within an hour. This increase in membrane permeability is indicative of the loss in membrane integrity, where species can now enter the cell through nonspecific diffusion channels, pore formation, or structural collapse of the membrane. Using the MTT proliferation assay, we were able to confirm that the low dosage Cd2+ treatment had very minor effect on cellular viability, while the acute treatments decreased viability to 68%. Our SECM results coupled with the MTT assays show the sufficient permeation of these sensors and compromised cell viability. These findings by means of SECM demonstrate that hydrophilic redox mediators are ideal in sensing the effects of toxins, which may induce permeability through detrimental effects on the membrane integrity of the cells.