Development of Molecular Diagnostic and Microfluidic Methods Based on Molecular Transport and Reactions.

다국어 초록 (Multilingual Abstract)

Molecular diagnostic testing provides critical information to patients that can lead to earlier detection and disease prevention, specific treatment options, and personalized medicine. The workflow for this type of testing begins with initial sample collection followed by target analyte extraction and purification from the original sample, then amplifying the analyte to a level where detection can be performed. This complex workflow can be laborious, time consuming, and expensive which is why many molecular diagnostic tests have turned to microfluidic technologies to improve all steps. In this thesis, we utilize experimental, computational, and theoretical approaches to characterize molecular transport and reactions. Moreover, we will use microfluidic geometries to develop and improve molecular diagnostic tests. In particular, we will present the design and use of microfluidic devices for nucleic acid extraction and purification using a combination of solid phase extraction on magnetic beads and electrokinetic purification within a microfluidic channel. We show the effect of magnetic beads, free DNA, and sample contaminant transport within this system and how that can change amplification, detection, and even DNA sequencing results. Additionally, this thesis further investigates methods for improved amplification and detection through exploring fundamental components of these steps. We show the development of a new method using targeted nucleic acid sequence to size-based separation analysis as well as the process for creating a model to be used for predicting false-positive results when using loop mediated isothermal amplification through understanding molecular interactions. Overall, the work described in this thesis aims to reduce false-positive diagnosis errors, increase sample purity, and reduce human errors all within the context of molecular diagnostics. The impact of this thesis work includes reducing costs, reagent volumes, and hands on work time using microfluidics, all while improving or maintaining the sensitivity and specificity of these tests particularly through the use of method designs and translatability for automation. Ultimately, through investigating the fundamentals underlying molecular diagnostics we have developed translatable technology useful to a variety of applications.