Title

Electrochemiluminescent arrays utilizing layer-by-layer films of cytochrome P450/DNA/ruthenuim polymer for drug toxicity screening & unraveling direct electrochemistry of cytochrome P450s

Date of Completion

January 2010

Keywords

Chemistry, Analytical|Chemistry, Biochemistry|Chemistry, Pharmaceutical

Degree

Ph.D.

Abstract

This thesis describes development and fundamental studies related to simple, inexpensive, high throughput electrochemiluminescent arrays made of layer-by-layer (LbL) films of cytochrome (cyt) P450, DNA and redox ruthenium polymer ([Ru(bpy)2PVP10]2+, RuPVP) for drug/chemical toxicity screening. A major focus includes unraveling direct electron transfer and biocatalytic kinetics of drug metabolizing cytochrome P450s films, and mimicking natural cytochrome P450 catalytic cycle on electrodes with high catalytic efficiency. ^ Chapter one describes the background and significance of direct electrochemistry of metabolic cyt P450 enzymes with some of the key issues addressed, as well as the need for developing simple electrochemiluminescent arrays for drug/chemical toxicity screening that can complement the existing bioassays in drug development. ^ Chapter two discusses the first report on the use of voltammetric sensors/ECL arrays applied to predict genotoxicity of model N-nitroso compound, N-nitrosopyrrolidine (NPYR), bioactivated by human cyt P450 2E1 in LbL films with DNA and RuPVP. ^ Chapter three reports the application of ECL arrays containing films of DNA, RuPVP and selected human cyt P450 enzymes to elucidate cyt P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). ^ Chapter four describes the successful application of cheap and commercially available liver microsomes as cyt P450 source on the electrochemical sensors to detect comparative toxicity between nitrosamine and styrene compounds, and their evaluation to in vivo toxicity data and reactive metabolite formation. ^ Chapter five reports the first implementation of liver microsomes as LbL films with DNA and RuPVP on ECL arrays to test toxicity of several model nitroso compounds and styrene. Correlation of the toxicity array responses to in vivo toxicity data and to reactive metabolite-DNA adducts formation in the sensor films was observed. ^ Chapter six is on the direct electrochemistry study of cyt P450s films on electrodes that discusses for the first time about the influences of iron heme spin state and amino acids nature in the heme distal region on the direct electron transfer and active ferryloxy formation kinetics of cyt P450s in polyion films. ^ Chapter seven discusses the first ever demonstrated natural cyt P450 catalytic cycle mechanism mimicked electrochemically using LbL films of purified human cytochrome P450 and microsomal cyt P450-reducase on electrodes having high catalytic efficiency. ^