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

Abstract

Heavy metal cytotoxicity has become a mainstay in scientific research due to the vast range of detrimental effects on living organisms. Cd 2+ 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 Cd 2+ . Hydrophilic and charged sensing agents should be impermeable to the cells. However, when T24 cells were treated with 25 μM Cd 2+ (t ≤ 6 h), the membrane permeability to these sensing agents can be induced to approximately 7.0 × 10 −5 m/s. For acute mM Cd 2+ exposure, the membrane permeability can be induced to 2.0 × 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 Cd 2+ 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.

Correlating Live Cell Viability with Membrane Permeability Disruption Induced by Trivalent Chromium

“…The use of impermeable redox mediators when performing SECM on biological samples can indicate significant disruption in normal cell membrane performance. 63,66 Therefore, the use of FcCOO – as the SECM mediator can provide good indication of when Cr(III) no longer behaves as a trace essential metal, but rather as a toxin.…”

“…The diffusion coefficients in both domains were set to 7.6 × 10 –10 and 5.7 × 10 –10 m 2 /s for FcMeOH and FcCOO – , respectively. 63,73,75 The simulation was performed in two stages. The first stage allowed the two domains to reach equilibrium (over 10 min), which provides an initial concentration distribution for the UME approach.…”

Tracking Live‐Cell Response to Hexavalent Chromium Toxicity by using Scanning Electrochemical Microscopy

“…The other constants considered are the Faraday constant ( F =96,485 C/mol) and the diffusion coefficient of the species ( D ). The diffusion coefficient for FcMeOH in both domains was set to 7.6×10 −10 m 2 /s and 5.7×10 −10 m 2 /s for FcCOOH …”

In Situ and Operando Techniques for Investigating Electron Transfer in Biological Systems

“…modified conductive colloid AFM‐SECM probes with polystyrene sulfonate and poly(3,4‐ethylenedioxythiophene) to use them for single‐cell force measurements in mouse fibroblasts and investigate single‐cell interactions under different applied electric potentials. This approach has been used as a bioanalytical technique for monitoring cellular homeostasis through the evaluation of living cell membrane permeability to a series of compounds such as toxic heavy metals [128–130] . AFM‐SECM is also useful for the study of living single‐cell microorganisms such as bacteria, providing insights into metabolic exchange between two different bacteria [131] or even furnishing information on the efficiency of charge transfer from yeast cells under anaerobic conditions [132] .…”