Title page for ETD etd-04202010-185750

Type of Document Dissertation
Author Lee, Wonbae
URN etd-04202010-185750
Title Cross-talk-free Dual-color Fluorescence Cross-correlation Spectroscopy For High-throughput Screening
Degree Doctor of Philosophy (Ph.D.)
Department Chemistry
Advisory Committee
Advisor Name Title
Soper, Steven A. Committee Chair
Murphy, Michael C. Committee Member
Murray, Kermit K. Committee Member
Russo, Paul Committee Member
Johnson, Sheldon A. Dean's Representative
  • Fluorescence Cross-correlation Spectroscopy
  • Single-molecule Detection
  • Microfluidic Devices
  • Aptamer
  • FRET
  • Enzyme Activity
Date of Defense 2010-03-30
Availability unrestricted
High throughput processing of chemical/biochemical information is critical in many areas, such as genome sequencing, drug discovery and clinical diagnostics. Integral to collecting information at high rates with the necessary throughput is the development of systems that can not only monitor the results with high precision and accuracy, but also prepare and process samples prior to the analytical measurement. To achieve the required throughput, we have conducted work directed toward developing a system that provides detection sensitivity at the single-molecule detection (SMD) level. The research was first focused on the development of a sensitive strategy for the detection of proteins (thrombin) at the SMD level. Nucleic acid-based fluorescence sensors were used as recognition elements for the detection of single protein molecules with single-pair fluorescence resonance energy transfer. The technique provided higher analytical sensitivity compared to bulk analog measurements due to the digital readout format (i.e., molecular counting) and also reduced assay turn-around-time. Research was then directed toward the design and construction of a two-color FCCS system, which employed two spectrally separate fluorophores, Cy3 (λabs = 532 nm, λem= 560 nm) and IRD800 (λabs = 780 nm, λem= 810 nm). The system provided negligible color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET). To provide evidence of cross-talk free operation, the system was evaluated using microspheres and quantum dots. Experimental results indicated no color leakage from the microspheres or quantum dots into inappropriate color channels. The enzymatic activity of APE1 was monitored by FCCS using a double-stranded DNA substrate that was dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid). To improve sample processing throughput, a multi-phase (water-in-oil) segmented flow microfluidic chip was studied using the FCCS system to monitor APE1 enzyme activity. Aqueous droplets were generated in a perfluorocarbon (FC-3283) carrier fluid with a nonionic surfactant (Perfluorooctanol, 10% v/v) in a polymer microchip. The optical system successfully monitored the controlled generation of highly regular droplets loaded with fluorescent beads at delivery rates ranging from 40 - 60 droplets per sec.
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