Quantitative Assessment of Molecular Transport through Sub-3 nm Silica Nanochannels by Scanning Electrochemical Microscopy.

Scanning electrochemical microscopy (SECM) has been proved to be a powerful technique to study molecular transport across ionic channels in biomembranes and artificial nanoporous membranes. In this work SECM was used to study the dynamics of molecular transport across the ultrathin silica nanoporous membrane consisting of sub-3 nm diameter perpendicular channels. We focused on the quantitative assessment of permselectivity and permeability of the membrane and the effect of radial electrical double layer (EDL) on them. By SECM imaging, it was phenomenologically observed that the membrane with negatively charged surface exhibited permselectivity to anionic molecule, for instance hexacyanoruthenate(II) (Ru(CN)64-). And the permselective transport of Ru(CN)64- was obviously more favored at a higher concentration of KCl. Precise membrane permeability to Ru(CN)64- was quantitatively determined by overlapping experimental SECM approach curves with the ones generated by finite element simulations. The high permeability up to 35 µm s-1 was ascribed to the straight channel structure and ultrahigh channel density of 4 × 1012 cm-2. Moreover, the permeability was varied from 35 µm s-1 to 2.5 µm s-1 when decreasing the concentration of KCl from 1.0 to 0.01 M, corroborating the electrostatic origin of membrane permselectivity. On the other hand, the simulated concentration profiles at both sides of the membrane suggested that the molecular transport across the membrane was mainly driven by the large transmembrane concentration gradient while the tip-induced transport was relatively negligible. These results help to quantitatively understand the molecular transport selectivity and dynamics across nanoporous membranes and to rationally design artificial molecular sieving membranes.

Analysing single live cells by scanning electrochemical microscopy.

Single live cell analysis methods provide information on the characteristics of individual cells, yielding not only bulk population averages but also their heterogeneity. Scanning electrochemical microscopy (SECM) offers single live cell activities along its topography with high accuracy probe tip positioning. Both intracellular and extracellular processes can be electrochemically examined through the use of SECM. This non-invasive technique allows for high resolution mapping of electrochemical measurements in or around the cell sample of interest. Reactive oxygen species and reactive nitrogen species can be determined in a non-invasive label-free method and utilized as a probe for cellular pathology and physiology. Membrane permeability and rate of membrane species transport can be quantified in SECM. The cell response to external stressors can be monitored and modelled. SECM is able to offer nanoscale mapping and low concentration detection, providing a powerful bioanalytical tool for live cell studies. Herein we present an overview of recent progress in the imaging and characterization of single live cells using SECM.

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

Cr(III) is often regarded as a trace essential micronutrient that can be found in many dietary supplements due to its participation in blood glucose regulation. However, increased levels of exposure have been linked to adverse health effects in living organisms. Herein, scanning electrochemical microscopy (SECM) was used to detect variation in membrane permeability of single cells (T24) resulting from exposure to a trivalent Cr-salt, CrCl3. By employing electrochemical mediators, ferrocenemethanol (FcMeOH) and ferrocenecarboxylic acid (FcCOO-), initially semipermeable and impermeable, respectively, complementary information was obtained. Three-dimensional COMSOL finite element analysis simulations were successfully used to quantify the permeability coefficients of each mediator by matching experimental and simulated results. Depending on the concentration of Cr(III) administered, three regions of membrane response were detected. Following exposure to low concentrations (up to 500 µM Cr(III)), their permeability coefficients were comparable to that of control cells, 80 µm/s for FcMeOH and 0 µm/s for FcCOO-. This was confirmed for both mediators. As the incubation concentrations were increased, the ability of FcMeOH to permeate the membrane decreased to a minimum of 17 µm/s at 7500 µM Cr(III), while FcCOO- remained impermeable. At the highest examined concentrations, both mediators were found to demonstrate increased membrane permeability. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability studies were also conducted on Cr(III)-treated T24 cells to correlate the SECM findings with the toxicity effects of the metal. The viability experiments revealed a similar concentration-dependent trend to the SECM cell membrane permeability study.

The effects of long duration chronic exposure to hexavalent chromium on single live cells interrogated by scanning electrochemical microscopy.

Chromium is a useful heavy metal which has been employed in numerous industry and house applications. However, there are several known health risks associated with its uses. Cr (VI) is a toxic heavy metal format which serves no essential biological role in humans. It has been associated with oxidative stress, cytotoxicity, and carcinogenicity. Contamination of groundwater or soil due to improper handling lead to long term environmental damage. This study explores the effects of long duration chronic exposure to Cr (VI) on live human cells. Herein, scanning electrochemical microscopy (SECM) depth scan imaging was employed to monitor the membrane permeability of single live human bladder cancer (T24) cells following incubation with various Cr (VI) concentration stimuli. SECM was used to provide insights into the long duration effects on membrane homeostasis of individual cells exposed to constant levels of Cr (VI). Further investigation of total population viability was performed by MTT assay. Dependent on the exposure time, transition between three distinct trends was observed. At short incubation times (≤1-3 h) with low concentrations of Cr (VI) (0-10 µM), membrane permeability was largely unaffected. As time increased a decrease in membrane permeability coefficient was observed, reaching a minimum at 3-6 h. Following this a dramatic increase in membrane permeability was observed as cell viability decreased. Higher concentrations were also found to accelerate the timeframe at which these trends occurred. These findings further demonstrate the strength of SECM as a bioanalytical technique for monitoring cellular homeostasis.

Tracking live cell response to cadmium (II) concentrations by scanning electrochemical microscopy.

The biological chemistry of toxic heavy metals, such as Cd (II), has become an active area of research due to connections with increased oxidative stress, cytotoxicity, and human/animal carcinogenicity. In this study, scanning electrochemical microscopy (SECM) was used as a noninvasive technique to monitor membrane permeability of single live human bladder cancer cells (T24) subjected to exposure of Cd (II) at various concentrations. The addition of a membrane permeable redox mediator, ferrocenemethanol (FcMeOH), in combination with depth scan imaging provided probe approach curves (PACs) to reveal changes in membrane homeostasis. To demonstrate the strength of SECM as a bioanalytical technique for cell physiology and pathology, we tested responses of live cells after 1h incubations with various concentrations of Cd (II). For the first time, a trend in membrane permeability of Cd (II) treated live T24 cells was discovered. Dependent on the incubation concentration, the trend displayed an initial decrease in membrane permeability coefficient from 75µm/s for control cells to 25µm/s for cells incubated with 75µM Cd (II). This was followed by an eventual return to the permeability coefficient of control cells (75µm/s) with further increases in Cd (II) exposure. The cells were found to respond at as little as 10µM Cd (II) concentrations. This work further demonstrates the use of SECM as a bioanalytical technique to monitor cell physiology and topography. A greater insight into the complex mechanisms behind Cd (II) toxicity is anticipated.

Mapping Cd²âº-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 µM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented.

A time course study of cadmium effect on membrane permeability of single human bladder cancer cells using scanning electrochemical microscopy.

Cd(2+) is carcinogenic to both humans and experimental animals. We present quantitative time-course imaging of Cd(2+)-induced variation in the membrane permeability of single live human bladder cancer cells (T24) to ferrocenemethanol using scanning electrochemical microscopy (SECM). High temporal resolution combined with non-invasive nature renders a time-lapse SECM depth scan, a promising method to quantitatively investigate the effectiveness, kinetics, and mechanism of metal ions based on the responses of single live cells in real time. Under unstimulated conditions, T24 cells have constant membrane permeability to ferrocenemethanol of approximately 5.0×10(-5) m/s. When cadmium is added in-situ to T24 cells, the membrane permeability increases up to 3.5×10(-4) m/s, allowing more flux of ferrocenemethanol to the ultramicroelectrode tip. This suggests an increased spreading between the phospholipid heads in the cytoplasmic membrane. Membrane permeability might be used as a measure to probe cell status in practical intoxication cases. The methodology reported here can be applied to many other metals and their interactions with extracellular biomolecules, leading insights into cell physiology and pathobiology.