Microfluidic Systems for Sample Preparation with a Focus on RNA Analysis

Hagan, Kristin Alice, Department of Chemistry, University of Virginia
Landers, James, Department of Chemistry, University of Virginia
Venton, Barbara Jill, Department of Chemistry, University of Virginia
Allen, Ralph, Department of Chemistry, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia

The development and characterization of microfluidic systems for sample
preparation with a focus on RNA analysis from a variety of biological samples stands to revolutionize the use of microfluidic technology for genetic analysis. Microfluidic solid phase extraction (SPE) of RNA from both forensically- and clinically-relevant samples is demonstrated, as well as microchip-based reverse transcription-polymerase chain reaction (RT-PCR). The characterization of two different solid phases and their respective chemistries is detailed, focusing on the advantages of each for the purification of RNA from a variety of commonly-encountered forensic sample sources that included semen and blood, as well as from clinical samples represented here by a common pediatric cancer cell line. Next, the development of a three-chip modular system for forensic human identification through STR (short tandem repeat) analysis of DNA is
described, utilizing separate microdevices for SPE, PCR, and microchip electrophoresis (ME). In addition, a single microdevice for integrated SPE-PCR was developed as a step towards a portable, valveless system for complete STR analysis on a single device. Finally, the culmination of this dissertation is the development of a valveless integrated system for the microfluidic integration of SPE-RT-PCR for rapid detection of biowarfare agents and pathogens. Influenza A was detected in nasal swab samples after sample preparation using the SPE-RT-PCR device in under one hour – the first demonstration of rapid virus identification via microfluidic sample processing harnessing the advantages of an infrared (IR)-mediated heating system for microchip-based RT-PCR.

PHD (Doctor of Philosophy)
RNA, DNA, forensic, microfluids
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