Sensor Technology for Water Quality Monitoring - A. C. Cannons

Two methods for the detection of important human pathogens, Cryptosporidium parvum and Helicobacter pylori, were investigated: a fiber optic biosensor, and real time PCR. The mechanism for specific detection in both methods is recognition of specific DNA sequences in the target organisms. The biosensor that was used, the Analyte 2000, was originally developed for the detection of chemicals. It utilizes a fiber optic wave guide that propagates an evanescent light wave of very specific wavelength. The light excites fluorescent molecules bound to the waveguide, but not in the bulk solution, which theoretically enhances signal while reducing background interference. Attempts to develop this system for the detection of DNA were not successful due to poor detection of the target molecules. An assay analogous to a sandwich immunoassay was designed for use on the Analyte 2000. Specific oligonucleotide probes were designed to bind to the waveguides via biotin-streptavidin interaction, and were used to capture the target DNA. Pure target DNA representing unique genes in the organisms were synthesized by PCR. Detection of captured DNA was then attempted using an oligonucleotide detection probe designed to bind to the target. Two detection systems were employed: an indirect signal amplification system based on biotin-tyramide deposition, or direct detection of fluorescent signal from Cy-5 molecules. In all experiments performed there was very little difference between the signal gene
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