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Title:Advances in molecular-based diagnostics and biomolecular interaction screening using silicon photonic microring resonators
Author(s):Kindt, Jared
Director of Research:Bailey, Ryan C.
Doctoral Committee Chair(s):Bailey, Ryan C.
Doctoral Committee Member(s):Hergenrother, Paul J.; Murphy, Catherine J.; Sweedler, Jonathan V.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biosensor
Micro Ribonucleic Acid (miRNA)
silicon photonics
microring resonator
protein detection
biomarkers
Abstract:Personalized medicine promises that improved information leads to improved treatments, that each individual has unique genetic, clinical, and environmental factors which are ultimately responsible for the distinct molecular mutations which drive disease as well as variation in treatment efficacy. The field of biomarker-based diagnostics attempts to characterize this variability by both identifying which biomolecules are indicative of disease condition as well as quantitate their abundance, thus enabling personalized and improved treatment. The challenge is thus to accurately quantify the abundance of relevant biomarkers at a sufficiently large scale and in a sufficiently inexpensive format. While numerous biomarker quantitation methodologies exist, they frequently are specialized, providing either high throughput analysis in a qualitative fashion, or highly quantitative analysis of a single biomarker. These extremes are ill-suited to biomarker-based diagnostics, which require quantitative rigor to discern the often subtle differences in biomarker abundance, as well as the multiparameter detection capabilities that lend biomarker panels meaningful predictive power. Silicon photonic microring resonators are a promising class of sensor well suited to biomarker quantitation due to their sensitivity, scalability and flexibility, enabling quantitative multiplexed analysis. Their sensing modality arises from interactions between light and matter—light circumnavigating the microring structure interacts via the accompanying evanescent field with the local environment of the ring, enabling binding events to be readily transduced as a change in the resonant wavelength circulating in the cavity. The resonating nature of the structure results in multi-pass interactions between surface bound analytes and the evanescent field, enabling the high sensitivity found in much larger devices, but in a small footprint amenable to high density sensor arrays. This architecture lends itself to scalability—the current chip contains 128 active sensors, with the potential for even higher plexity in the future. Additionally, the nearly planar sensor geometry enables facile and reproducible fabrication capitalizing on well-developed commercial semiconductor processes, resulting in very low costs per assay. The intrinsic flexibility of this sensing modality has enabled the development of a wide variety of assays quantifying DNA, RNA, and proteins, as well as the screening of more complex and subtle biomolecular interactions. Herein I describe assay developments using novel reagents and methodologies to specifically address the quantitation challenges associated with various analyte classes, with emphases on subsequent applications in complex matrix environments to demonstrate clinical utility. Chapter 1 contains a more thorough introduction to multiparameter biomolecular analysis using microring resonators, with particular emphasis on recent developments in the field and an eye towards likely future directions. Chapter 2 gives a near-exhaustive review of current microRNA (miRNA) analysis methodologies, emphasizing both advancements in classical molecular biology techniques as well as more recent in vitro diagnostic devices. Many of the techniques discussed, while in the context of miRNA, are used in slightly modified forms for general RNA or even DNA analysis. Chapter 3 describes the use of a novel anti-DNA:RNA antibody as a secondary label to improve the miRNA detection limit 400-fold relative to direct detection, enabling 4-plexed miRNA quantitation in total RNA extracts from mouse and soybean samples. Chapter 4 demonstrates the detection of bacterial transfer-messenger RNA in a multiplexed assay, employing a unique RNA fragmentation protocol to optimize signal response. Chapter 5 describes the quantitation of full length messenger RNA using a novel chaperone-assisted detection strategy, coupled with nanoparticle tertiary labels for signal enhancement. These developments enabled mRNA expression profiling in total RNA extracts from a differentiating cell line. Chapter 6 describes the recapitulation of the RNA Induced Silencing Complex (RISC) on the ring resonator surface, and demonstrates modulation of Argonaute 2 catalytic activity in response to varied guide strand miRNA. Chapter 7 explores an enzymatic enhancement strategy to achieve the highest signal response recorded on the ring resonator platform to date, achieving an ultrasensitive sub-pg/mL detection limit. Chapter 8 outlines future work, describing the importance of preconcentration, the enormous potential of enzymatic signal enhancement, and the capabilities of on-chip characterization of the biomolecular interactions of RISC.
Issue Date:2014-01-16
URI:http://hdl.handle.net/2142/46874
Rights Information:Copyright 2013 Jared Kindt
Date Available in IDEALS:2014-01-16
2016-01-16
Date Deposited:2013-12


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