Date of Award
Master of Science
Civil, Construction, and Environmental Engineering
Kaoru . Ikuma
Carmen . Gomes
After the Flint Water Crisis, there has been heightened public awareness on threats to potable drinking water, especially those induced by existing infrastructure. One such threat has been identified as a group of waterborne pathogens known as opportunistic premise plumbing pathogens (OPPPs). OPPPs exhibit thermal-tolerance, disinfectant-resistance, and growth under oligotrophic conditions, which make water distribution systems favorable habitats for their survival. Legionella pneumophila is one of the most notorious OPPPs and is presently one of the most threatening waterborne pathogens, particularly in developed countries. Reported cases of Legionnaires’ disease have increased dramatically since the turn of the century. It is clear that this is a serious public health concern that could be better mitigated by improved forms of detection of the pathogen before human exposure. Biosensors have been identified as a potential source of on-site monitoring for pathogenic threats. This thesis focuses on optimizing the application of DNA as a bioreceptor for detection of L. pneumophila. The first chapter provides information on existing forms of detection for monitoring waterborne pathogens, OPPPs, DNA-based biosensors, as well as DNA quantification and hybridization. There is a discussion on current gaps and challenges with existing methods of waterborne pathogenic monitoring and the review of numerous electrochemical DNA-based biosensing schemes developed specifically for pathogen detection. The second chapter explains two different methodologies employing the Qubit fluorometer developed for selective quantification of DNA that isolates and measures single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The first method allows for accurate quantification of DNA probes that contain both ssDNA and dsDNA, such as hairpins. The next method provides a means to quantify dsDNA present post-hybridization, when in solution with excess unhybridized ssDNA. These methodologies prove important to measure the number of probe copies in solution and the hybridization efficiency of a DNA probe. The third chapter compares hybridization efficiencies capable for linear (LSP) and dangling-ended hairpin (Key) DNA probes, both in solution and immobilized on a magnetic microparticle surface. In the third chapter, data is provided that displays linear DNA probes that have higher affinity for the target DNA sequence than dangling-ended hairpin probes, when hybridized in a solution of ultrapure water. Ultimately, when hybridization efficiency is quantified again with the probes immobilized, there is not a significant difference in hybridization between the two different structures. The final chapter covers conclusions, engineering significance, and recommendations for future work. Through this research, more information about the application for DNA probes in biosensors was collected. The linear DNA probes observed higher sensitivity than the hairpin probes for detection in solutions absent of salt. However after immobilization, probe structure did not yield a significant change in sensitivity for detection, in a buffered saline solution. These results can be used to further optimize DNA-based biosensors.
Parker James Dulin
Dulin, Parker James, "Development of potential strategies for DNA quantification and DNA probes for detection of Legionella pneumophila" (2019). Graduate Theses and Dissertations. 17673.