Fluorescent Zinc Sensors for Cellular Imaging

用于细胞成像的荧光锌传感器

• 批准号: 6931997
• 负责人: CHRISTOPH J FAHRNI
• 金额: $ 27.84万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6931997
• 关键词: X ray crystallography; cell line; chemical synthesis; chromophore; cytotoxicity; fluorescence microscopy; fluorescence spectrometry; fluorescent dye /probe; intracellular transport; ionophores; molecular /cellular imaging; quantum chemistry; technology /technique development; thermodynamics; vesicle /vacuole; zinc

DESCRIPTION (provided by applicant): Zinc serves as a cofactor in various metallo enzymes and is involved in a broad range of biological processes, such as neurotransmission, gene expression, apoptosis, and immune system functions. Much of the intracellular zinc is bound to proteins and the concentration of free zinc in the cytoplasm is extremely low; however, a fraction of the total zinc is localized in cytoplasmic vesicles and freely exchangeable. There is strong evidence, that these vesicles play a central role in the intracellular regulation of Zn(ll) and possibly other vital biological processes. However, the nature of these processes and the biological function of the vesicles are elusive. The investigation of the dynamics of vesicular zinc has been hampered in particular by the lack of Zn(ll)-specific fluorescent probes, that are suitable for live cell studies. This proposal describes the design, synthesis and characterization of Zn(ll)-specific fluorescent probes, that should overcome problems of existing probes and enable researchers to elucidate the mechanisms of cellular zinc homeostasis. The proposed sensors are specifically designed for two-photon excitation microscopy, a noninvasive live cell imaging technique that drastically reduces photoxicity. The sensing mechanism is based on zinc-induced inhibition of excited state proton transfer (ESIPT), a new design concept that provides large spectral shifts suitable for quantitative emission ratiometric measurements. The proposed research fills a critical niche in the biological chemistry of metal ion speciation. The lack of a non-invasive zinc specific fluorescent sensor is currently a limiting factor in the study of intracellular zinc regulation and trafficking.

描述（由申请人提供）：锌在各种金属酶中充当辅因子，并参与了广泛的生物学过程，例如神经传递，基因表达，凋亡和免疫系统功能。大部分细胞内锌与蛋白质结合，细胞质中游离锌的浓度极低。但是，总锌的一部分位于细胞质囊泡中，并且可以自由交换。有强有力的证据表明，这些囊泡在Zn（LL）的细胞内调节以及可能的其他重要生物学过程中起着核心作用。但是，这些过程的性质和囊泡的生物学功能是难以捉摸的。尤其是由于缺乏适合活细胞研究的Zn（LL）特异性荧光探针，对囊泡锌动力学的研究受到了阻碍。该建议描述了Zn（LL）特异性荧光探针的设计，合成和表征，该探针应克服现有探针的问题，并使研究人员能够阐明细胞锌稳态的机制。所提出的传感器是专门针对两光子激发显微镜设计的，这是一种无创的活细胞成像技术，可大大降低光毒性。感应机制基于锌诱导的激发态质子转移（ESIPT）的抑制，这是一种新的设计概念，可提供适用于定量发射比率测量值的大光谱移动。拟议的研究填补了金属离子物种形成生物化学的关键利基市场。目前，缺乏非侵入性锌特异性荧光传感器是研究细胞内锌调节和运输的限制因素。