Sample Preparation and Nucleic Acid Extraction for Molecular Diagnostics: Innovations for Resource Limited Settings

Deraney, Rachel Nouha

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Year:: 2019
Contributor:: Deraney, Rachel Nouha (creator); Tripathi, Anubhav (Advisor); Cu-Uvin, Susan (Reader); Shukla, Anita (Reader); Kofron, Celinda (Reader); Brown University. Biology and Medicine: Biomedical Engineering (sponsor)
Genre:: theses
Subject:: Biomedical engineering; Microfluidics; Point-of-care testing; Electroosmotic flow; Nucleic Acid Extraction; HIV Viral Load Monitoring
Extent:: 16, 112 p.

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Abstract:: Many diagnostic tests are dependent on the effective capture and amplification of nucleic acids. However, the biological samples which contain the target molecules (blood, urine, saliva, etc.) have an abundance of contaminants and inhibitors. Sample preparation is the crucial first step to accurate and reliable molecular diagnostics. Although technological advances have been made to remove nucleic acids from these undesirable sample conditions, these approaches are often inappropriate in resource constrained areas. This thesis will focus on my research efforts in 1) increased understanding of fundamentals of microfluidic chip operation and design in relation to sample preparation and 2) appropriate application of molecular diagnostics in a resource limited setting. First, I describe a PDMS based microchip and how paramagnetic particles were used to extract nucleic acids. We minimize our carryover volume to 0.2ng/uL.This is followed by a discussion on the effects of surfactants on sample inhibitor carryover. To accommodate a larger, clinically relevant sample volume, a new chip was designed. I report results on nucleic acid extraction and the effect of carrier RNA extraction rate. The model I derived using the Arrhenius equation to explore the relationship between temperature and unbinding of nucleic acids from paramagnetic particles is explained. Once a protocol was established which minimized carryover volume, optimized nucleic acid yield, and required no costly lab equipment, I traveled to Kenya and used HIV viral load as a measure of our microchip nucleic acid extraction efficiency from patient plasma samples. In HIV spiked serum samples, we report 100% nucleic acid yield and approximately 60% nucleic acid yield on clinical patient plasma samples. The results of these clinical applications are discussed as well as suggestions for future work. Finally, I introduce the concept of electroosmotic flow within a microfluidic chip and discuss results on carryover volume and nucleic acid extraction in this system. In this system, we minimize carryover volume to below 0.22 0.03% and show a statistically significant difference in both carryover volume and spiked DNA extraction yield at higher applied voltages.
Notes:: Thesis (Ph. D.)--Brown University, 2019

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Deraney, Rachel Nouha, "Sample Preparation and Nucleic Acid Extraction for Molecular Diagnostics: Innovations for Resource Limited Settings" (2019). Biomedical Engineering Theses and Dissertations. Brown Digital Repository. Brown University Library. https://repository.library.brown.edu/studio/item/bdr:1129476/

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Biomedical Engineering Theses and Dissertations

Theses and Dissertations for the Biomedical Engineering department.
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