Date Degree Awarded

Spring 5-18-2013

Degree Type

Open Access Dissertation

Degree Name

PHD in Applied Life Sciences

First Thesis/Dissertation Advisor

Angelika Niemz

Second Thesis/Dissertation Advisor

Jim Sterling

Terms of Use & License Information

Terms of Use for work posted in Scholarship@Claremont.

Abstract

Technologies that enable infectious diseases diagnosis in low-resource settings could greatly facilitate effective treatment and containment of such diseases. Nucleic acid amplification testing can be used to identify pathogens, but typically requires highly-trained personnel and large, expensive lab equipment, neither of which is available in low-resource settings. Our overall goal is to develop a portable diagnostic system that utilizes a low-cost, disposable, mesofluidic cartridge and a handheld electronics unit to perform fully-integrated nucleic acid testing at the point of care in low-resource settings. As a first step toward this goal, we developed a subunit to execute isothermal nucleic acid amplification coupled with lateral flow detection, in parallel with the development of a sample preparation subunit by our collaborators at Claremont BioSolutions. Fluid handling inside the amplification and detection cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard, scalable manufacturing techniques, such as injection molding. Using an initial prototype system, we demonstrated detection of purified Mycobacterium tuberculosis genomic DNA. We then developed a refined amplification and detection cartridge in conjunction with an improved portable instrument, which automates pumping, heating, and timing, using a design format compatible with eventual integration with the sample preparation subunit. This refined cartridge incorporates a novel, inexpensive, stand-alone, passive valve, smaller, integrated pump components, a more complex injection molded polycarbonate cartridge core piece, and enhanced lateral flow chambers to improve visual detection. The independent valve component can be tailored for a variety of fluidic systems. We demonstrated appropriate fluidic and thermal control, and successful isothermal nucleic acid amplification within this refined amplification and detection subunit. We have developed a separate fluidic module for master-mix reagent storage and reconstitution that is designed to act as the interface between the amplification and detection subunit and the upstream sample preparation subunit. We envision that the merger of these two subunits into a fully-integrated cartridge will enable user-friendly, automated sample-in to answer-out diagnosis of infectious diseases in primary care settings of low-resource countries with high disease burden.

Rights Information

© 2013 Kristina Roskos

DOI

10.5642/kgitd/2

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