螢光奈米鑽石及近紅外光螢光染料之螢光能量共振轉移

Abstract

具有NV0及NV-缺陷中心（defect center）的螢光奈米鑽石（fluorescent nanodiamond），是一種擁有許多獨特特性的新穎奈米材料，例如絕佳的生物相容性、容易進行表面修飾、具有較高組織穿透力的紅色螢光以及優異的光穩定性，很適合用來做為近紅外光螢光生物標記（biolabel）。近來，螢光能量共振轉移廣泛地被應用在研究生物分子結構、酵素動力學及蛋白質交互作用上，因此利用螢光奈米鑽石傑出的光穩定性，做為能量轉移之施子（donor），可提供一個以螢光奈米鑽石為主的生物螢光感測器應用。在本篇論文中，我們藉由測量19奈米直徑螢光奈米鑽石與近紅外光染料（IRDye 800 CW）在不同比例下螢光光譜，以及觀察在聚賴胺酸（poly-lysine）基質（matrix）上，23奈米直徑螢光奈米鑽石之螢光強度及螢光生命期（lifetime）在光漂白近紅外光染料前後之變化，可發現螢光能量轉移效率可達30％。同時，藉由蒙地卡羅模擬（Monte Carlo simulation）可估算平均每個螢光奈米鑽石微粒可與鄰近10個染料分子發生螢光能量共振轉移。這對於利用螢光奈米鑽石做為單分子螢光標記或具有奈米級解析度之分子尺（molecular ruler）提供了新的應用平台。 Fluorescent nanodiamond (FND) is a potent fluorescent probe possessing several unique properties such as excellent biocompatibility, facile surface modification, high tissue-penetrable red fluorescence, and outstanding photostability. The neutral and negatively charged nitrogen-vacancy (NV0 and NV-) defect centers embedded in the diamond lattice is responsible for the far red photon emission from this novel nanomaterial. FNDs are excellent biolabels and could be used to probe intricate biochemical processes. Fluorescence resonance energy transfer (FRET) has recently been widely introduced to study biomolecular configuration, enzyme kinetics and protein-protein interactions. Therefore, as a donor, with its perfect photostability, FND paves the way for a FND-based biosensor. In this thesis, we investigate the fluorescence spectra of 19-nm-sized FND conjugated with near-infrared dye (IRDye 800 CW) and demonstrate that it is possible to approach the FRET efficiency up to 30% between 23-nm-sized FND and dye molecules co-embedded in a poly-lysine matrix. We measured the changes in both fluorescence intensity and lifetime of FNDs before and after photobleaching of near-infrared dye. Moreover, according to Monte Carlo simulations, on an average, each FND transfers its energy to 10 dye molecules attached to its periphery. These results set the stage for FND-based single particle bio-labeling or molecular ruler with nanometric resolution.