Voltammetric sensors for chemical toxicity and oxidative stress utilizing metallo-polymer

Date of Completion

January 2004

Keywords

Chemistry, Analytical

Degree

Ph.D.

Abstract

Electrochemical detection of chemical DNA damage is of high biomedical importance. This thesis has addressed development rapid, inexpensive electrochemical sensors for chemical toxicity and oxidative stress. Sensors for screening chemicals for potential toxicity at early stages in their commercial development is very important as it can be used to reduce the number of candidates for standard bioassays. In chapter 2 of this thesis, metallopolymer films adsorbed on pyrolytic graphite electrode detected DNA by oxidation of guanines in DNA. ss-DNA gave twice the square-wave voltammetric catalytic current compared to an equivalent amount of ds-DNA suggesting that these metallopolymer films may be useful for detection of DNA hybridization and damage. ^ In chapter 3, layer by layer films constructed on [PVP-ClRu(bpy) 2]2+ metallopolymer detected damage of both calf thymus and salmon sperm ds-DNA in both solution and on films after DNA induced damage by incubation with styrene oxide a known carcinogenic molecule. Detection of DNA damage was achieved after 2-min reaction, showing high sensitivity of this method. Detection of pro-carcinogenic molecules like styrene was achieved by incorporating bio-activating enzymes and DNA in a single film built one layer at a time with an inner layer of metallopolymer film. Myoglobin and hemoglobin were used as model enzymes in the sensor construction. Styrene oxide generation from styrene was used as the test metabolic reaction. These reactions mimic a very important process in human liver where relatively non-harmful organic molecules are converted to carcinogenic metabolites by enzymes such as cytochrome P450s. ^ Chapter five describes novel sensors for detection of DNA oxidation by detecting 8-oxoguanine as one of the products of in situ reaction of DNA with reactive oxygen species (ROS) and a clinical biomarker for oxidative stress. Amount of 8-oxoguanine in DNA can serve as an index of cancer risk. [Os(bpy) 2(PVP)10Cl]+ catalyst with a lower oxidation potential was synthesized and used to make other type of films. These films selectively oxidized 8-oxoguanine without requiring DNA hydrolysis hence fast and sensitive (could detect 1 oxidized base in 6000 bases). ^ Sensors developed in chapter 2–5 were used to investigate DNA damage inhibition by dietary antioxidants. These sensors were used to assess the rates of DNA damage from incubations with styrene oxide, and hydroxyl radicals. These sensors were also used to assess DNA damage by pro-carcinogens in films made using bio-activating enzymes. A clearer picture of effectiveness of these antioxidants was achieved and would in future help unravel the mechanisms involved in inhibition. ^

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