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Dissertation/Thesis Abstract

Beyond Equilibrium: Using Ion Concentration Polarization to Enhance the Detection and Selectivity of Nucleic Acids and the Isolation of Exosomes
by Marczak, Steven, Ph.D., University of Notre Dame, 2017, 190; 13836453
Abstract (Summary)

Nucleic acids such as DNA and RNA represent a growing class of potential biomarkers for disease diagnosis and prognosis. Early diagnosis could potentially save lives through early treatment while continual prognosis may help to identify the most beneficial types of treatment. To avail the medical community of nucleic acid biomarkers, microfluidics has arisen to supplant traditional instrumentation by offering portable, affordable, and simple microfluidic chips which match the performance of conventional laboratory techniques. However, while substantial progress has been achieved, there remain significant obstacles to full implementation in the clinical setting. Two of the most significant impediments are the ability to detect very low levels of nucleic acids and to differentiate between nearly identical nucleic acid sequences. To address these challenges, we designed a new nucleic acid biosensor based on the principles of preconcentration by ion concentration polarization, gel depletion isotachophoresis, and nanoparticle aggregation. In our proof-of-concept study, we detected a 69 base long model DNA target down to a concentration of 10 pM using only 2 μL of sample. In further studies, we demonstrated our assay could selectively identify perfectly complementary DNA targets by 100-fold more than sequences with only two base mismatches. In the same study, we illustrated the importance of designing selective DNA sensors based on nonequilibrium, or kinetic, properties rather than thermodynamic equilibrium properties. It provided a justification of the framework for the design of new DNA sensors based on kinetic considerations. In addition to designing a nucleic acid sensor, we adapted our microfluidic chip to the isolation of exosomes which are subcellular species responsible for transporting biomarkers such as microRNA in extracellular fluid. Because exosomes are common in extracellular fluid and carry notable biomarkers, they make excellent targets for noninvasive detection. Therefore, their efficient isolation is of critical importance. Conventional techniques are too time consuming, too costly, and yield low recovery rates. Using gel filtration and ion-selective membrane, we simultaneously isolated and preconcentrated exosomes for subsequent analysis. Our microfluidic chip consistently recovered approximately 70% of exosomes in various media such as buffer and cell media culture. With the development of a new nucleic acid sensor and exosome isolation chip, we move closer to achieving a fully integrated microfluidic chip.

Indexing (document details)
School: University of Notre Dame
Department: Chemical and Biomolecular Engineering
School Location: United States -- Indiana
Source: DAI-B 80/06(E), Dissertation Abstracts International
Subjects: Analytical chemistry, Biomedical engineering, Chemical engineering
Keywords: DNA selectivity, Exosome isolation, Gold nonparticles, Ion concentration polarization, Microfluidics, Nucleic acid hyvridization
Publication Number: 13836453
ISBN: 978-0-438-83639-6
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