Microfluidic Electrochemical/Electro-optical Arrays and Liquid Chromatography Mass Spectrometry for Metabolic Genetic Toxicity Screening

A significant research effort is being granted towards the high-throughput screening of toxicities at early stages of drug development for safer & better healthcare economics. This thesis focuses on the development and validation of high-throughput, microfluidic arrays for detection of DNA damage by reactive metabolites (Metabolic genetic toxicity). In addition to vital human metabolic enzymes and DNA, these arrays feature a specific ruthenium redox polymer ([Ru(bpy)2(PVP)10]2+ {PVP = poly(4-vinylpyridine}) to generate electrochemical or electrochemiluminescent (ECL) output. Molecular information related to reactive metabolite-induced genetic toxicity is elucidated by employing liquid chromatography mass spectrometry (LC-MS/MS). Chapter one clearly addresses the purpose and significance of microfluidic metabolic genetic toxicity screening platform with reliable molecular information for simple, yet high throughput detection at the early stages of drug development. Chapter two describes the first prototypic electrochemical microfluidic array to detect reactive metabolites generated by cytochrome P450 enzymes. Chapter three presents the successful development and validation of a multiplexed microfluidic platform for screening reactive metabolites bioactivated by multiple enzyme pathways as in natural human metabolism. Chapter four reports an ECL, fluidic chip for high-throughput screening of DNA damage with metabolite-DNA adduct structure elucidation by LC-MS/MS for the first time. The chip facilitates 64 measurements in a single run. Charter five concludes the thesis with a presentation of a simple, fluidic platform to detect genetic toxicity caused by common pollutants at specific human organs. These organ-specific genetic toxicity responses are complemented by LC-MS/MS analysis of appropriate metabolite-DNA adducts and are validated by standard bioassays