Graduation Semester and Year

2019

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Aerospace Engineering

Department

Mechanical and Aerospace Engineering

First Advisor

Hyejin Moon

Abstract

In recent years there have been significant advances in micro/nanotechnologies employing miniaturized platforms to achieve efficient sample pre-processing, separation, and quantification processes. The idea of bringing several lab-scale protocols on to a single chip, i.e., a lab-on-a-chip (LOC) device along with automation, is a reality now. Miniaturization of biochemical operations usually handled in a laboratory has numerous advantages, such as cost effectiveness, multiplexing, assay speed, and sensitivity. Microfluidic technologies used in LOC devices can manipulate tiny volumes of samples and reagents to perform on-chip protocols with minimal human intervention. Electrowetting-on-dielectric (EWOD)-based digital microfluidics (DMF) is one of the LOC platforms that show the potential to achieve rapid processing through its capabilities, such as multiplexed operation, simple instrumentation, flexible device geometry, and easy coupling with other technologies. There has been much effort in integrating protocols of DNA analysis workflow into EWOD microfluidics recently. However, much of the sample preparation (mainly DNA extraction) is still done hands-on using standard lab protocols. In this work, an EWOD microfluidic platform has been designed to demonstrate and integrate the microscale drop-to-drop (DTD) DNA extraction via the liquid-liquid extraction (LLE) method. The first study is focused on the demonstration of LLE protocol in a DTD format on EWOD DMF devices. The study done was to show the separation of color dye analytes in a binary solution mixture using an EWOD platform. Absorbance-based concentration measurement was miniaturized and integrated with the platform to evaluate the performance of the on-chip LLE protocol. The study showed the capability of EWOD-based LLE to separate molecules in a sample. The second and the third studies are focused on extending the capability of EWOD DTD LLE for the separation of DNA molecules from other impurities in the sample. Plasmid DNA was used as the model DNA, and bovine serum albumin (BSA) protein molecules were used as impurities to show the capability of EWOD LLE to isolate the DNA molecules selectively. Different liquid-liquid systems were studied for the on-chip DNA extraction other than the traditional phenol extraction method. The second study is focused on the LLE of DNA using ionic liquid (IL) as an extractant. Hydrophobic IL, which forms a two-phase system with the aqueous sample, is an excellent solvent for DNA isolation. The capability of EWOD devices to isolate DNA molecules using aqueous/IL (Aq./IL) is highlighted in the second study. The third study is focused on the integration of aqueous two-phase systems (ATPS) for DNA extraction on the EWOD device. EWOD has already shown the capability to handle ATPSs, and this last study was done to demonstrate the integration of a aqueous two-phase extraction (ATPE) process for isolating DNA molecules on EWOD devices for the first time. It was observed that by changing the liquid-liquid systems, there were significant changes in the final extraction yield from the on-chip LLE process. This research validates the initial steps for the creation of an efficient on-chip system for DNA based sample preparation, recognized by the miniaturization of DNA LLE and novel DTD extraction techniques. This DNA sample preparation can eventually be integrated with post-extraction based manipulations of DNA utilized by genomic analysis modules.

Keywords

Microfluidics, EWOD, DNA extraction, Liquid-liquid extraction

Disciplines

Aerospace Engineering | Engineering | Mechanical Engineering

Comments

Degree granted by The University of Texas at Arlington

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