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QD FRET for Cellular Imaging and Sensing

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Title: QD FRET for Cellular Imaging and Sensing
Author(s): Samareh Afsari, Hamid
Advisor(s): Miller, Lawrence W.
Contributor(s): Snee, Preston T.; Min, Jung-Hyun; Mohr, Justin T.; Liu, Ying; Miller, Lawrence W.
Department / Program: Chemistry
Degree Granting Institution: University of Illinois at Chicago
Degree: PhD, Doctor of Philosophy
Genre: Doctoral
Subject(s): FRET, Quantum Dot, Microscopy,
Abstract: Fluorescence microscopy is an essential tool to study biological molecules in living cells or tissues. Forster resonance energy transfer (FRET) microscopy is commonly used to monitor the relative distance between two fluorescently labeled proteins or other biomolecules and can be leveraged to report on binding dynamics or conformational changes. FRET microscopy needs bright probes with good resistance to photobleaching and narrow emission spectra. Imaging probes may be based on fluorescence proteins (FPs), organic fluorophores, nanoparticles (Quantum Dots or QDs) or lanthanide complexes. There is a huge interest in applications of QDs in live cell microscopy due to their exceptional brightness and photo-stability. Using QDs as FRET donors, offers several practical advantages over conventional fluorophores (FPs or dyes). For example, broad absorption spectra allow for the excitation of QD far from the acceptor’s absorption region, which minimizes the direct excitation of the acceptor. QDs can also be FRET acceptors when paired with lanthanides as donors for use in time-gated microscopy. Time-Gated FRET microscopy is based on the difference in excited state lifetimes between lanthanides (0.1 – 2 ms) and QDs (<100 ns) with the correct spectral overlap. Time-gated FRET microscopy provides a better signal-to-noise ratio by using pulsed excitation and delayed detection to isolate ms-scale lanthanide and lanthanide-sensitized FRET signals from ns-scale cell autofluorescence and directly excited acceptor fluorescence. Successful use of QDs as microscopic imaging sensors poses several challenges including appropriate surface functionalization, delivery into living cells, and targeting to select biomolecules. This dissertation reports the results of efforts to engineer QDs for a variety of live-cell imaging and sensing applications, including ratiometric metabolite sensing, lanthanide-based FRET, and selective labeling of a protein inside a cell. Chapter one is comprised of relevant background information on QDs, lanthanides, FRET, and other materials and techniques explored herein. In chapter two, the design and characterization of QD-based ratiometric sensors is described. These sensors were developed to monitor oxygen, hydrogen sulfide, and endosomal escape in live cells. Chapter three explores terbium-to-quantum dot FRET microscopy. We developed several time-gated Tb-to-QD FRET systems that can be used for intracellular imaging with very efficient suppression of non-specific fluorescence background. With the removal of the acceptor fluorescence due to direct excitation, there is no need for control images such as donor only or acceptor only (as required for conventional FRET imaging experiments). For the demonstration of intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET relays, we used Tb-QD-dye nanoassemblies (Tb-pep and dye-peptides self-assembled via His6 to the QD surfaces) that were microinjected into live HeLa cells. In chapter 4, we show that His6 self-assembly, combined with amide coupling, increases the stability of QD complexes, and we used this system to conjugate methotrexate to the surface of QDs. Synthesis of methotrexate-peg-maleimide and the required peptides are described. The methotrexate-QD complex selectively targets a protein with an eDHFR fusion tag inside the cell and enables the real-time study of proteins.
Issue Date: 2017-12-08
Type: Thesis
Date Available in INDIGO: 2018-02-08
Date Deposited: December 2

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