生物成像用新型发光探针的研究

【作者】 余梦晓；

【导师】 李富友； 黄春辉；

【作者基本信息】 复旦大学 ， 无机化学， 2009， 博士

【摘要】 活细胞内单分子行为研究和活体动物成像已经成为荧光生物成像的前沿领域。然而在单分子和活体动物这两种极端的情况下,生物样品中大量存在的内源性荧光物质产生的自发荧光会对标记分子的荧光信号产生非常严重的干扰。为了解决生物成像中自发荧光干扰的问题,我们必须开发一些能完全消除自发荧光干扰的“新型发光探针”。本论文针对这一目标开展研究,主要包括四个部分。1.红色荧光化学计量器用于活细胞内Cu²⁺的荧光成像从发光波长方面考虑,利用生物组织对红光和近红外光的吸收非常弱的特点,并考虑到明确生理过程中铜的浓度和亚细胞分布的变化情况对于研究铜的复杂生理功能和致病机理具有重要的意义。我们设计合成了一个含有高电负性S原子的罗丹明B衍生物,实验表明这个化合物是一个Cu²⁺化学计量器,对Cu²⁺的响应表现为红光区的发射增强。利用一个Cu²⁺促进的罗丹明开环、氧化还原和水解反应的识别机理,加入Cu²⁺引起的荧光强度的增加和吸收强度的增加相当,说明以此化学计量器为探针可以有效地避免Cu²⁺顺磁性造成的荧光淬灭。更重要的是,这个探针能快速（响应时间≤1 min）、高灵敏（检出限≤10 ppb）、高选择地识别Cu²⁺。单光子、双光子荧光成像及台盼蓝细胞活性实验证实该探针可以用于检测活细胞中的Cu²⁺,显示Cu²⁺的亚细胞分布。2.磷光重金属配合物用于活细胞磷光成像利用磷光重金属配合物为探针,结合时间分辨成像技术,可以区分来自标记物的寿命较长的发光（～μs）和寿命较短的自发荧光（～ns）,有望完全消除自发荧光的干扰。铱配合物是一类性质优异的磷光重金属配合物,但其在生物成像中的应用尚未见文献报道。首先,我们合成了两个具有明亮的绿光和红光发射的阳离子型铱配合物,并且证明了它们是特异性染色活细胞胞浆的磷光染料。这两个铱配合物在缓冲溶液中发光效率较高,很容易穿过细胞膜进入细胞,专一性地染色细胞的胞浆区域,且细胞毒性低,光稳定性相较于有机染料DAPI有所提高,是一类性能优良的小分子磷光探针。其次,我们将一个对组氨酸有发光响应的铱配合物用于细胞成像,发现它能快速且特异性地染色活细胞和固定细胞的细胞核,并仅在细胞核区域表现出明亮的发光。3.稀土上转换发光纳米材料用于激光扫描上转换发光显微成像（LSUCLM）稀土上转换发光纳米材料（UCNPs）在980 nm连续光激发下具有独特的上转换发光过程,是一类非常有潜力的生物标记材料。我们发现UCNPs上转换发光的成像形式非常特殊,非焦面的上转换发光信号会使焦面细节完全模糊,导致图像分辨率很差。通过引入反式的激发二色分镜和共聚焦针孔技术,我们成功地消除了非焦面的上转换发光的干扰,并且发展了一种新的三维成像方法,即激光扫描上转换发光显微成像（LSUCLM）技术。随后,通过对UCNPs掺杂的薄膜、有机荧光染料和UCNPs同时标记的细胞进行成像实验,我们发现以UCNPs为发光探针的LSUCLM拥有很多独特的优势,如对有机染料和UCNPs的光漂白均非常低,完全消除了来自内源性荧光物质和同时标记的荧光染料的背景干扰,还可以与普通共聚焦荧光成像系统联用等。4.放射性核素标记的稀土纳米晶用于正电子发射断层扫描（PET）和上转换发光双模式成像PET技术具有最高的活体成像灵敏度;而对细胞和组织显像一般采用荧光成像方式。因此,开发同时具有放射性和荧光的探针可以构造出用于多尺度成像的显像剂。我们利用¹⁸F^-和稀土离子发生特异性结合反应的原理,发展了一个简单、快速、高效的方法制备了¹⁸F标记的稀土上转换发光纳米晶(即¹⁸F-UCNPs),放射性标记率＞60%。通过在体（in vivo）microPET及LSUCLM成像实验,我们研究了¹⁸F-UCNPs在小鼠体内的生物分布,证实了¹⁸F-UCNPs可以作为双模显像剂同时用于PET和上转换发光成像。在体PET成像和LSUCLM成像的联用,为实现¹⁸F-UCNPs标记物从细胞到活体多尺度上的高灵敏度的可视化提供了一种独特的方法。更多还原

【Abstract】 One of the current research interests in bioimaging is the ultra-sensitive detection of the targets in vivo or at the single-molecule scale.However,the signal from the targets labeled with fluorescent probes would probably be masked by the high autofluorescence from the endogenous components in biological samples.To solve this problem,it is necessary to develop some new luminescent labels to eliminate autofluorescence background in bioimaging.This thesis is focused on this project and composed of four parts.1.Organic Chemodosimeter for Fluorescence Imaging of Cu²⁺ in Live CellsBiological tissues have weak absorption of light in the red and near-infrared region. Therefore,a red fluorescence probe would greatly reduce the background fluorescence.In addition,visualizing the concentration and subcellular distribution of copper in physiological processes may greatly contribute to understanding its complex physiological functions and nosogenesis.A Rhodamine B derivative containing a highly electron-rich S atom has been synthesized as a red fluorescence turn-on chemodosimeter for Cu²⁺.As a result of Cu²⁺-promoted ring-opening,redox and hydrolysis reactions,comparable amplifications of absorption and fluorescence signals were observed upon addition of Cu²⁺,suggesting that the chemodosimeter effectively avoided the fluorescence quenching caused by the paramagnetic nature of Cu²⁺.Importantly,this compound can selectively recognize Cu²⁺ in aqueous media in the presence of other metal ions with high sensitivity（detection limit≤10 ppb） and a rapid response time（≤1 min）. Moreover,by virtue of the chemodosimeter as fluorescent probe for Cu²⁺,confocal and two-photon microscopy experiments revealed a significant increase of intracellular Cu²⁺ concentration and the subcellular distribution of Cu²⁺,which was internalized into the living HeLa cells upon incubation in growth medium supplemented with 50μM CuCl₂ for 20 hours.2.Heavy-metal Complexes for Phosphorescence Imaging of Live CellsPhosphorescent heavy-metal complexes have relatively longer luminescence lifetimes（～μs） than those of endogenous fluorescent substances（～ns）,and thus are appealing probes for completely avoiding background fluorescence in bioimaging through a time-gated technique.Being among the best class of phosphorescent heavy-metal complexes,iridium（Ⅲ） complexes exhibit many advantageous photophysical properties.To date,no luminescent staining of live cells using iridium（Ⅲ） complexes has been reported.Two cationic iridium（Ⅲ） complexes with bright green and red emissions were demonstrated as phosphorescent dyes for live cell imaging.In particular,their exclusive staining in cytoplasm,low cytotoxicity and reduced photobleaching,as well as cell membrane permeability,make the two complexes promising candidates for the design of specific bioimaging agents.Additionaly,an iridium（Ⅲ） complex with histidine-induced luminescence enhancement was demonstrated as a phosphorescent dye for exclusively staining the nuclei of cells.The advantage of phosphorescent complexes for bioimaging,the capability of visualizing nuclei of both live and fixed cells,as well as the short period time（～10 min） for staining promise wide applications in biological and medical studies.3.Rare-earth Nanophosphors for Laser Scanning Up-conversion Luminescence Microscopy（LSUCLM）Rare-earth up-conversion nanophosphors（UCNPs） as alternatives of conventional biological luminescent labels have attracted a tremendous amount of attention.We found that rare-earth nanophosphors exhibit a unique up-conversion luminescence mechanism and imaging modality and developed a new three-dimensional visualization method of laser scanning up-conversion luminescence microscopy（LSUCLM） with little photobleaching and no background fluorescence, by introducing a reverse excitation dichroic mirror and the confocal pinhole technique. Moreover,we demonstrated the up-conversion emission imaging of thin films containing embedded rare-earth nanophosphors and cells multilabeled with the nanophosphors and organic dyes.These data show that LSUCLM offers some distinct advantages,such as little photobleaching of both organic dyes and rare-earth nanophosphors,no background fluorescence from either endogenous fluorophores or colabeled fluorescent probes,and excellent compatibility with conventional confocal microscopy.4.¹⁸F-Labeled Rare-earth Nanophosphors for Dual-modality Positron Emission Tomography（PET） and Up-conversion Luminescence ImagingPET is a whole-body imaging technique with the highest sensitivity,while fluorescence imaging is widely used for cell and tissue imaging.Therefore,both radioactivity and fluorescence should ideally be combined into one probe for multi-level imaging. Radionuclide ¹⁸F^-has been incorporated into up-converting nanophosphors （UCNPs） in＞60%labeling yield by virtue of a simple,rapid and efficient strategy based on reaction between ¹⁸F^-and rare-earth elements.The effectiveness of ¹⁸F-labeled UCNPs for both PET and UCL imaging was futher evaluated by investigating the biodistribution of these nanoparticles using in vivo PET imaging and LSUCLM imaging experiments.The combination of in vivo PET and LSUCLM imaging indicates the potential of the ¹⁸F-labeled UCNPs for ultra-sensitive molecular imaging from the cellular scale to whole-body evaluation.更多还原