Doctor of Philosophy
Dr. Zhifeng Ding
Scanning electrochemical microscopy (SECM) employs a biased ultramicroelectrode (few nm to 25 µm diameter) as a probe that is scanned over a sample to electrochemically characterize its physical properties and chemical reactivity with high temporal and spatial resolutions. In this dissertation, SECM was used to investigate the membrane responses of single live human bladder cancer (T24) cells to obtain insights into their topography, physiology and pathology.
First, the membrane-impermeable ferrocene carboxylate was used as the SECM redox mediator to investigate the geometry and topography of these cells along with 3D finite elemental analysis (FEA) simulations. The use of 3D simulation models now allows characterization of asymmetric samples, previously restricted to a 2D axially symmetric geometry.
Upon exposure to toxic and non-essential Cd2+ (mM range), the membrane permeability to the hydrophobic mediator, ferrocenemethanol, in T24 cells increased within minutes. Time-lapsed SECM was able to measure and quantify these changes in membrane permeability. Expanding on this work, T24 cells were exposed to low exposure (25 µM, up to 6 hours) or 1 hour of acute Cd2+ concentrations. Specifically, the permeation of hydrophilic redox mediators: ferrocene carboxylate, ferrocene dicarboxylate, and hexaamineruthenium(III), into the Cd2+-treated T24 cells confirmed membrane integrity loss. In conjunction with FEA, the membrane permeability coefficients were quantified. A correlation between cellular loss and membrane integrity was confirmed using MTT cell proliferation assays.
Exposure to the trace essential Zn2+ (0-75 µM, 24 hours) did not significantly affect the cell membranes of T24 cells. Higher Zn2+-treatment led to cytotoxicity, where membrane integrity loss (increased permeation) and cellular death were observed (100-400 µM). The above discoveries suggested the Zn2+-induced apoptosis in these cells. This was confirmed by several apoptotic indicators, such as the externalization of phosphatidylserine and activation of caspases 3 and 7.
Finally, to improve the spatial resolution of SECM, a fabrication method of nanometer-sized probes was successfully developed. This led to the reduction of Pt electrode disk diameters from 25 µm to 25 nm. Visualization of reactivity features of nanometer-sized samples using SECM is anticipated with this advancement.
Li, Michelle S. M., "Scanning Electrochemical Microscopy of Human Bladder Cancer (T24) Cells" (2017). Electronic Thesis and Dissertation Repository. 4906.
Available for download on Monday, April 30, 2018