Microfluidic Systems for Sample Preparation with a Focus on RNA Analysis

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.