Date of Graduation
2016
Document Type
Dissertation
Degree Type
PhD
College
Statler College of Engineering and Mineral Resources
Department
Chemical and Biomedical Engineering
Committee Chair
Cerasela Dinu
Committee Co-Chair
Rakesh Gupta
Committee Member
Yon Rojanasakul
Committee Member
Charter Stinespring
Committee Member
Todd Stueckle
Abstract
The majority of current techniques to assess the toxicity of analytes (i.e., nanomaterials, drugs and toxins) in vitro rely on the application of affinity or catalytic recognition elements (i.e., biosensors), their activity, sensitivity and selectivity, as well as the processing power of micro- and opto-detection devices. For instance, water-soluble tetrazolium (WST-1) assay is among the most common techniques used to measure the viability of cells following exposure. Specifically, in this assay a catalytic biosensor (tetrazolium salt) is reduced by cellular NAD(P)H-dependent oxidoreductase or mitochondrial dehydrogenases resulting in colorimetric changes quantified using a UV-Vis spectrophotometer (detection element). Similarly, imaging of cellular components mainly relies on the application of affinity biosensors that bind to selected cellular organelles or proteins and subsequently emit a fluorescent signal upon detection using fluorescence microscopy.;Although, the majority of these assays and techniques have high sensitivity, stability and reliability, however; inaccurate measurements and false positives may occur especially when detecting the toxicity of nanoparticles, such as carbon nanotubes (CNTs). In principle, the high surface area of CNTs as well as the presence of metal impurities and functional groups on their external surfaces decrease the availability and the activity of biosensors, as well as interfere with the detection ability of micro-and optical processors. Therefore, there is a critical need for sensitive bio-sensing tools to provide comprehensive measurements of the cellular behavior post exposure to analytes (e.g., nanomaterials, drugs and toxins) without relying on the applications of affinity or enzymatic biosensors.;In this work a combinatorial approach had been employed to evaluate the cellular behavior and fate in real-time using an Electric Cell Impedance Sensing (ECIS) platform. In principle, the ECIS system relies on the natural sensitivity of cells as a primary transducer to provide high throughput, real-time measurements of the cell behavior and fate upon exposure to different analytes. Previous studies have employed the ECIS system to quantify changes in cell morphology, cell adhesion, cell migration, chemotaxis, and wound healing capability, in response to different drugs, toxins and chemotherapeutic agents or just for standard behavior quantification. Herein, we have extended on these studies by employing the ECIS system to evaluate the cellular behavior of human lung (immortalized and non small lung cancer) cells upon exposure to different concentrations and types of nanoparticles (carbon nanotubes, nanodiamons and anti-cancer drugs). The ECIS real-time measurements were further complemented and correlated with different cellular and microscopical assays (Western blots, flow cytometry, optical and florescence imaging) to derive structure-function relationships and thus unravel the cellular mechanisms associated with the any cytotoxic or apoptotic events experimented by the cells upon analytes exposure.
Recommended Citation
Eldawud, Reem, "Combinatorial Real-Time High Throughput Analyses of Cell Behavior Upon Exposure to Analytes" (2016). Graduate Theses, Dissertations, and Problem Reports. 5535.
https://researchrepository.wvu.edu/etd/5535