| The sensitive and selective detection of biomolecules is essential to many research areas including: neurochemistry, proteomics, forensics, and genomics. The recent completion of the first working draft sequence of the human genome has sparked interest in DNA and protein analysis technologies. The information in this initial sequence is a remarkable tool, which is currently being used to identify the mutations responsible for disease, track migratory patterns, and study the evolution of our species. The initial sequencing, however, is only the beginning. There is still a need to re-sequence the human genome, identify single nucleotide polymorphisms (SNPs), and sequence the genome of other organisms. Therefore, the improvement in detection techniques is necessary to increase the throughput, cost-effectiveness and simplicity of biomolecule analysis.; Described in this work is an electrochemical method, Sinusoidal Voltammetry (SV), which can selectively detect different electroactive molecules based on their redox properties at an electrode surface. SV utilizes data analysis in the frequency domain, which allows a unique "fingerprint" spectrum for a particular molecule to be visualized. The use of frequency-based analysis has many advantages when compared to the more commonly used time domain. To introduce the SV technique to the detection of biomolecules, it was used to selectively detect the neurotransmitter dopamine at a carbon microelectrode. Additionally, SV analysis was applied to the detection of native amino acids and peptides at a copper microelectrode achieving greater sensitivity and selectivity compared to DC amperometric techniques.; The main application discussed in this work involves the development of an electrochemical technique for DNA analysis utilizing SV, specifically for DNA sequencing and mutation detection. The development of a strategy analogous to four "color" DNA sequencing, which utilized electrochemical detection and four unique ferrocene tags was demonstrated. SV was coupled to capillary gel electrophoresis (CGE) and used to sensitively and selectively detect the electrochemically labeled DNA fragments. Additionally, the redox-active labels were used to demonstrate a single nucleotide polymorphism screening assay, utilizing CGE coupled to end-column SV detection. The SV detection methodology developed in this work has the potential to provide an inexpensive, simple, and easily miniaturized assay for biologically significant molecules. |
