Measurement of analyte concentrations and gradients near 2D cell cultures and analogs using electrochemical microelectrode arrays: fast transients and physiological applications
thesisposted on 12.10.2021, 15:21 by Jose F. Rivera-MirandaJose F. Rivera-Miranda
This PhD research relates to the design, fabrication, characterization, and optimization of on-chip electrochemical microelectrode arrays (MEAs) for measurement of transient concentrations and gradients, focusing on fast transients and physiological applications. In particular, this work presents the determination of kinetic mechanisms taking place at an active interface (either physiological or non-physiological) in contact with a liquid phase using the MEA device to simultaneously estimate the concentration and gradient of the analyte of interest at the surface of the active interface. The design approach of the MEA device and the corresponding measurement methodology to acquire reliable concentration information is discussed. The ability of the MEA device to measure fast (i.e., in sub-second time scale) transient gradients is demonstrated experimentally using a controllable diffusion-reaction system which mimics the consumption of hydrogen peroxide by a 2D cell culture. The proposed MEA device and measurement methodology meet effectively most of the requirements for physiological applications and as a demonstration of this, two physiological applications are presented. In one application, the MEA device was tailored to measure the hydrogen peroxide uptake rate of human astrocytes and glioblastoma multiforme cells in 2D cell culture as a function of hydrogen peroxide concentration at the cell surface; the results allowed to quantitatively determine the uptake kinetics mechanisms which are well-described by linear and Michaelis-Menten expressions, in agreement with the literature. In the other application, further customization of the MEA device was realized to study the glucose uptake kinetics of human bronchial epithelial and small cell lung cancer cells, these latter with and without DDX5 gene knockdown; the results allowed to distinguish mechanistic differences in the glucose uptake kinetics among the three cell lines. These results were complemented with measurements of glycolytic and respiration rates to obtain a bigger picture of the glucose metabolism of the three cell lines. Finally, additional applications, both physiological and non-physiological, are proposed for the developed MEA device.