Point-of-care diagnostics for malaria currently rely on antibody-based rapid diagnostic tests (RDTs), but these tests are in danger of becoming obsolete as the world moves to eliminate malaria. More sensitive and robust techniques are needed to detect lower parasite loads using a more diverse array of biomarkers. This work encompasses several strategies to improve low-resource malaria diagnostics. First, a fluorescent on-bead sandwich detection method for PfHRP2 was developed that relies on a robust, stable transition metal compound rather than enzyme-tagged antibodies to produce signal. Then, circular dichroism was used to determine that heme binding causes the protein rcPfHRP2 to adopt a more helical structure than its typical unstructured form. This shift in structure also adversely affects the proteinâs ability to bind to antibodies on RDTs and in ELISAs. Work then transitioned to focus on DNA-based diagnostics due to the valuable information that DNA biomarkers can provide, and the large unmet need for point of care DNA diagnostic tests. Initial work focused on simplifying the DNA extraction process into an automated format, which was then expanded to include PCR in-line with the extraction to eliminate operator steps. Finally, a DNA-based RDT was developed to detect a âbarcodeâ DNA sequence, as part of the bio-barcode assay. This assay amplifies target DNA by hybridizing the captured target to a gold nanoparticle tagged with hundreds of barcode DNA sequences. When the barcode sequences are released, the signal from one target DNA strand is amplified to a level detectable by the RDT. This particular bio-barcode assay has issues with nonspecific binding, so unfortunately the two aspects of this assay were never combined with good results. However, the DNA-based RDT could be used as a platform for future work to bring DNA-based malaria diagnostics to low-resource settings.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03222017-144926 |
| Date | 31 March 2017 |
| Creators | Bitting, Anna Lynn |
| Contributors | Raymond L. Mernaugh, David W. Wright, David E. Cliffel, Janet E. Macdonald |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03222017-144926/ |
| Rights | restrictone, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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