Microfluidic Integration of Multi-Step Bioanalytical Assays
Dignan, Leah, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Landers, James, AS-Chemistry, University of Virginia
Molecular diagnostics are powerful analytical tools that permit acquisition of useful biochemical information across numerous sectors, including forensic human identification, toxicology, ancestral genotyping, and disease diagnosis. Despite significant recent advances, the vast majority of these bioanalytical techniques remain tethered to benchtop laboratory instrumentation operated by trained analysts via time-consuming, labor-intensive workflows. The growing demand for alternative technologies, especially those amenable for use at the point-of-need, is evidenced by an increase in the translation of microfluidic academic research efforts into commercially-available products. Among these, centrifugal microfluidic ‘lab-on-a-disc’ (LoaD) platforms are especially promising as powerful, portable alternatives for rapid, cost-efficient, and convenient biomolecular processing by nontechnical personnel on-site, eliminating laboratory dependence entirely.
Molecular diagnostics involving nucleic acids are among the most rapidly growing areas for the development of fieldable analytical tools. However, successful analysis of genetic material requires effective purification from biological samples, a step that has garnered much less attention than downstream amplification and detection techniques. Consequently, nucleic acid preparation is often overlooked and omitted from LoaD strategies entirely, despite the aim for comprehensive translation of the benchtop workflow. This body of work describes research efforts to develop highly integrated, rotationally-driven microfluidic tools to be employed at the point-of-need. For reference, chapter one describes existing centrifugal microfluidic approaches to nucleic acid sample preparation and describes their application in the few sample-to-answer systems that autonomously perform all processes required for genetic analysis. Much of the work highlighted in this dissertation is focused on efforts to expand the repertoire of such microfluidic methods, including the preconcentration of biological targets, cellular/virion lysis, and extraction or purification methods. Chapter two describes the development of a LoaD for dynamic solid phase extraction (SPE) of high-purity polynucleic acids; the preparation mode demonstrated compatibility with a panel of ubiquitous nucleic acid amplification tests. Similarly, chapter three is centered on the characterization, optimization, and microfluidic adaptation of viral preconcentration and subsequent RNA extraction, specifically intended for rapid pathogen detection. Both of these automatable, portable techniques stand to considerably advance capabilities for centrifugal microfluidic genetic analysis outside of traditional laboratory settings.
The comprehensive reliance of these LoaDs on rotational forces to direct flow confers a high degree of portability; yet, this fluidic control modality has also been cited as a key reason that very few sample-to-answer systems exist. Explicitly, leveraging centrifugal force provides unidirectional flow, whereby fluid moves radially outward towards the disc periphery and no further on-board processing is possible. Novel methods for on-disc liquid transport towards the center of rotation would enable sequential integration of nucleic acid preparation, amplification, and detection. Chapter 4 describes a biocompatible method for radially inward fluid displacement that relies on on-board gas generation and allows further rotationally-controlled processing, ultimately permitting increased assay complexity. Chapter 5 explores a second novel method for microfluidic flow control for LoaDs based on embedded cellulosic membranes to enable immunodetection of opioids. These novel flow control methods increase the capabilities of LoaD systems by facilitating the performance of increasingly complex bioassays without compromising portability.
Chapter 6 highlights the potential applications and implications of the techniques described in chapters two through five, future avenues for research exploration, and persistent challenges to be overcome.
PHD (Doctor of Philosophy)
microfluidics, bioanalytical chemistry
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