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Bioconjugation of Semiconductor Nanocrystals and Characterization of Nanocrystal–Bioconjugates

Bioconjugation of Semiconductor Nanocrystals and Characterization of Nanocrystal–Bioconjugates
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Semiconductor nanocrystals are frequently used as fluorescent probes for biological imaging because they offer several advantages over organic dyes or fluorescent proteins. In comparison with conventional fluorophores, nanocrystals yield narrow, symmetric emission, which is size-tunable, and permit broad excitation wavelengths. They also offer high molar extinction coefficients and high photoluminescence quantum yields with long-term photostability. To utilize nanocrystals prepared by organic-based colloidal synthesis in biological applications, one has to make them water-soluble and biocompatible. In this regard, water-soluble nanocrystals ought to have reactive functional groups tethered on the surface to conjugate them with biomolecules, and hopefully nanocrystal–bioconjugates show small hydrodynamic size and little nonspecific interactions in biological setting. In this thesis, I discuss water-soluble surface passivation of fluorescent nanocrystals with several functional groups, bioconjugation of nanocrystals, and characterization of nanocrystals and nanocrystal–bioconjugates using various spectroscopic tools. Zinc-blende (ZB) CdSe/ZnS core/shell nanocrystals were synthesized by organic-based colloidal growths of the ZnS shell over CdSe core nanocrystals. The lattice structure and size distribution were characterized by powder X-ray diffraction and high-resolution transmission electron microscopy, respectively. Organic-soluble CdSe/ZnS nanocrystals were modified to water-soluble ones with carboxylic acid, hydroxyl, and amine functional groups tethered on the surface through metal–thiolate linkage. Bifunctional thiols used in this thesis work include 3-mercaptopropionic acid (MPA), 3-mercapto-1-propanol (MPO), and 2-aminoethanethiol (AET). All three thiols contain a short alkyl chain (either C2 or C3) between the two terminal functional groups. Effects of surface functional groups on optical properties of nanocrystals were examined by absorption and emission spectroscopy. The surface ligands of various nanocrystals were characterized by secondary ion mass spectrometry. The fluorescence intermittency (blinking) of single nanocrystal was also studied as a function of ligand. Water-soluble nanocrystals were conjugated with biomolecules, such as biotin, DNA, and peptides. Moreover, efficient bioconjugation strategies for hydroxylated and aminated nanocrystals were discovered. Nanocrystal–bioconjugates were applied to fluorescence imaging of biomolecules. Specific and nonspecific interactions between nanocrystal–bioconjugates and biomolecules were examined in biological setting. Further, hydroxylated nanocrystal–bioconjugates were applied to visualization of cancer cells as well as to Förster resonance energy transfer (FRET) detection of DNA hybridization. Chapter 1 introduces thesis works, Chapter 2 summarizes results from the study of ligand-dependent blinking of single nanocrystals, Chapter 3 presents an efficient bioconjugation strategy using hydroxylated nanocrystals for the background-free fluorescent detection of biomolecules, Chapter 4 describes Förster resonance energy transfer detection of DNA hybridization using hydroxylated nanocrystal–bioconjugates, and Chapter 5 presents a newer strategy for preparing aminated CdSe/ZnS nanocrystals and their bioconjugation. In conclusion, hydroxylated nanocrystals are outstanding fluorescent probes
they are bright, compact, photostable, and biocompatible in diverse physiological conditions. Most importantly, hydroxylated nanocrystal–bioconjugates enable highly sensitive, background-free detection biomolecules of without nonspecific interactions in solution as well as on the solid substrate.
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