Abstract
Detection and analysis of nucleic acids (NAs) is essential across numerous disciplines, including clinical diagnostics, forensic science, and molecular biology. Unfortunately, ubiquitously-implemented laboratory-based instrumentation for amplification, quantitation, and electrophoretic separation of NAs are costly, time-consuming, and prone to contamination, ultimately hindering successful analysis and subsequent action. There is a growing demand for alternative technologies that are not only faster and more cost-effective relative to benchtop instrumentation but are also amenable to point of need implementation. These demands are being addressed through advancements in microfluidic technologies, which offer many advantages, including rapid analysis times, low reagent consumption, and reduced contamination risks. Among microfluidics, centrifugal microdevices present promising opportunities for powerful, portable alternatives to traditional instrumentation, particularly as they can be developed into micro total analysis systems (µTAS) that facilitate sample-to-answer analysis or simply automate a portion of the NA analysis workflow with minimal user intervention.
The chapters that follow describe the development and application of rotationally-driven microdevices that adapt existing laboratory processes to a cost-effective platform for rapid NA analysis. For context, Chapter 1 provides an introduction to conventional NA analysis with a focus on amplification, quantitation, and downstream detection methods, including high resolution melting (HRM) and electrophoresis. Discussion regarding considerations for adapting each assay to the microscale is given along with relevant recent advances in the field. Chapter 2 purports an expansion of the centrifugal microfluidic toolbox in the form of a two-stage metering process to meter and aliquot nanoliter volumes on an inexpensive device for automated assay optimization, a portion of the NA analysis process particularly relevant to successful, efficient amplification. The work in Chapter 3 highlights development and optimization of an ultra-rapid microfluidic instrument for real-time RT-PCR of polynucleic acids with fluorescence detection. The capacity of the system was expanded to perform high resolution melting for discrimination of specific from non-specific amplification as well as differentiation of amplicons with varied sequences. Chapter 4 details progress towards a portable, sample-to-answer microfluidic system that automates messenger RNA (mRNA) extraction, amplification, electrophoretic separation, and fluorescent detection for rapid body fluid identification. Specifically, research focused on optimizing the amplification workflow, designing the corresponding centrifugal microdevice, and engineering an existing instrument for rapid, automated amplification. The final chapter highlights the potential implications for the techniques described in Chapters 2-4 with a section dedicated to future research needed, and persistent challenges to overcome, to transform these microdevices into µTAS.
