Methylated phananthrene as petroleum marker: Toxicology assessment and engineering antibody reagents for environmental contamination detection.

摘要

Petroleum has long been a vital source of energy for the world, and will likely to remain so for decades to come. However, environmental contamination by petroleum is also a constant problem. Over the past decades, accidental oil spills have occurred all around the world and have released large amounts of crude oil into the aquatic ecosystem. These oil spill events pose risks to marine life, endanger the ecosystem, and ultimately jeopardize human health through bioaccumulation in seafood.;Polycyclic aromatic hydrocarbons (PAHs) in petroleum are of greatest concern due to their abundance, persistence, genotoxicity, carcinogenicity, and toxic effects on immune and reproductive systems and development. PAHs consist of multiple aromatic rings, with occasional substitution with alkyl groups around the benzene rings. Most previous studies have focused on unsubstituted PAHs, while methylated structures have not been specially distinguished and analyzed. However, the methylated derivatives account for a relatively large proportion of total PAH contaminants in cases such as oil spills (Blumer, 1976). Among the PAHs present in the environmental samples, phenanthrenes are often one of the most abundant families. At present, the toxic effects of petrogenic PAHs have not been fully characterized, and part of this thesis is to understand the significance of the alkyl substitutions on the toxic effects in a unified system that analyzes specific steps in the processes that lead to toxicity.;A better understanding of natural remediation processes and development of strategies to enhance contaminated site cleanup depends on accurate detection and measurement of contaminants prior to, during and following the remediation process. Immunosensors can be used to detect ligand at extremely low-levels; their sensitivity and potential for automation/high throughput provide new ways to solve problems in field and to improve the interface of environmental detection systems. However, the efficiency of the sensor systems depends highly on the quality of antibody employed. Antibody development against haptens has been a tough problem for decades. The possibility of raising monoclonal antibodies with a high affinity to the antigen is greatly reduced for haptens with a molecular weight of 300Da or less and monoclonal antibodies to such small-sized haptens can only be generated using the hapten conjugated to a carrier molecule, typically a large protein. Luckily, the technology of antibody production process has been revolutionized by the development of molecular biology methods for the expression of recombinant DNA. In this thesis, the essential variable domains of antibodies were cloned and restructured into recombinant antibodies, and valuable tools like phage and yeast display technologies were used to produce and select these recombinant antibodies with specific binding properties.