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Title page for ETD etd-03222017-144926


Type of Document Dissertation
Author Bitting, Anna Lynn
URN etd-03222017-144926
Title Strategies to Improve Malaria Diagnostics at the Point of Care by Understanding Protein Behavior and Developing Low-Resource DNA Detection Methods
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
David W. Wright Committee Chair
David E. Cliffel Committee Member
Janet E. Macdonald Committee Member
Raymond L. Mernaugh Committee Member
Keywords
  • malaria
  • low-resource diagnostics
  • PfHRP2
  • DNA
  • PCR
  • circular dichroism
Date of Defense 2017-03-13
Availability restrictone
Abstract
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 protein’s 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 “barcode” 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.
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