Development of Sensitive Fluorescent Probes for Physiological Zinc Over Large Con

描述（由申请人提供）：项目总结项目名称：开发用于大浓度范围生理Zn 2+的灵敏荧光探针主要研究者：Lei Zhu联合主要研究者：Michael W. Davidson联合主要研究者：Cathy W. Levenson我们的目标是开发用于在生理环境中成像游离锌离子（Zn 2+）的荧光探针，具有高灵敏度和大浓度覆盖范围。已知Zn 2+在人体生理学中起结构、催化和其他作用。Zn 2+稳态的破坏是病理性的。Zn 2+在不同生化途径中的确切功能尚未完全阐明，部分原因是缺乏准确确定Zn 2+在异质和动态生物介质中的分布和运动的工具。将Zn 2+与其他生理学上重要的物质区分开来的一个特殊挑战是Zn 2+在生物系统中的浓度范围超过六个数量级。哺乳动物细胞中游离Zn 2+（Zn 2+不与蛋白质紧密结合）的基础水平被认为在皮摩尔和纳摩尔之间。然而，在某些特定的细胞中，如脑神经元和某些亚细胞器，以及当细胞处于压力下时，经常发现接近毫摩尔的Zn 2+浓度升高。这种大的浓度范围尚未见过的其他生理物质提出了一个巨大的挑战，光学生物成像社区在智力和实际意义上。 这项研究的意义在于，如果成功，它将为在所有细胞类型中的完整生理浓度范围内定量分析Zn 2+提供有价值的工具。因此，所提出的研究将有助于阐明锌2+稳态途径，并确定治疗目标，以开发诊断和治疗的疾病，其病理学与锌2+稳态的偏差。所提出的研究的创新体现在我们合理设计的异配位平台上，该平台将三种配位状态（非配位、单配位和双配位）转化为三种荧光状态（非荧光、一种颜色的荧光和不同颜色的荧光），从而为在大浓度范围内进行定量Zn 2+分析提供了方便的分析方案。 在此授权申请中，我们提出了开发用于生理Zn 2+的荧光探针的策略，其具有大的有效浓度范围，将用于游离Zn 2+的定量活细胞成像。本论文以本实验室开发的异位芳基乙烯基-bipy体系为基础，具体研究了以下几个方面的问题：（1）设计和制备新的探针分子，以提高Zn ~（2+）定量的灵敏度（同时保持较大的有效浓度范围）;（2）将已知的激光染料结构结合到我们独特的异质结构框架中以产生用于活细胞荧光显微应用的探针;（3）开发了一种新的异质结构框架，其发射谱对溶剂极性不敏感;（4）我们的探针在生物成像中的应用，特别是在海马神经元中，使用荧光显微镜。

DESCRIPTION (provided by applicant): Project Summary Project Title: Development of sensitive fluorescent probes for physiological Zn2+ over large concentration ranges Principal Investigator: Lei Zhu Co-Principal Investigator: Michael W. Davidson Co-Principal Investigator: Cathy W. Levenson We aim to develop fluorescent probes for imaging free zinc ions (Zn2+) in physiological settings with both high sensitivity and large concentration coverage. Zn2+ is known to play structural, catalytic, and other roles in human physiology. The disruption of Zn2+ homeostasis is pathological. The exact functions of Zn2+ in different biochemical pathways are not completely elucidated, partly due to the lack of tools to accurately determine the distribution and movement of Zn2+ in heterogeneous and dynamic biological media. One particular challenge that distinguishes Zn2+ from other physiologically significant substances is the more than six orders of magnitude concentration range of Zn2+ in biological systems. The basal level of free Zn2+ (Zn2+ not tightly bound with proteins) in mammalian cells is believed to be between picomolar and nanomolar. However, elevated Zn2+ concentrations approaching millimolar have been often found in certain specialized cells such as brain neurons and certain subcellular organelles, as well as when cells are under stress. This large concentration range yet unseen for other physiological substances raise a great challenge to the optical bioimaging community both intellectually and in practical sense. The significance of the proposed research lies in the fact that if successful, it will provide valuable tools for quantitative analysis of Zn2+ over its complete physiological concentration range in all cell types. Therefore, the proposed studies will facilitate the elucidation of Zn2+ homeostatic pathways and to identify therapeutic targets for developing diagnosis and treatment of diseases whose pathology is related to deviation of Zn2+ homeostasis. The innovation of proposed research is reflected in our rationally designed heteroditopic platform that translates three coordination states (non-, mono-, and di-coordinated) to three fluorescence states (non- fluorescent, fluorescent at one color, and fluorescent at a different color), thus providing a convenient analytical protocol for quantitative Zn2+ analysis over a large concentration range. In this grant application, we propose strategies for developing fluorescent probes for physiological Zn2+ with large effective concentration ranges that will be used in quantitative live-cell imaging of free Zn2+. Built upon a heteroditopic arylvinyl-bipy system developed in our laboratory, we will specifically address the issues stipulated in the Specific Aims: (1) design and preparation of new probe molecules to increase the sensitivity of Zn2+ quantification (while maintaining a large effective concentration range); (2) incorporation of known laser dye structures into our unique heteroditopic framework to produce probes for live-cell fluorescence microscopic applications; (3) development of a new heteroditopic framework whose emission profile is insensitive to solvent polarity; and (4) applications of our probes in biological imaging, in particular in hippocampal neurons, using fluorescence microscopy.