Development of Sensitive Fluorescent Probes for Physiological Zinc Over Large Con

项目名称：大浓度范围内生理锌离子（Zn2+）敏感荧光探针的开发项目负责人：朱雷，联合负责人：Michael W. Davidson，联合负责人：Cathy W. Levenson我们的目标是开发具有高灵敏度和大浓度覆盖范围的生理环境下游离锌离子（Zn2+）成像荧光探针。众所周知，Zn2+在人体生理中起着结构、催化和其他作用。Zn2+稳态的破坏是病理性的。Zn2+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Zn2+在异质和动态生物介质中的分布和运动的工具。将Zn2+与其他生理上重要的物质区分开来的一个特殊挑战是，生物系统中Zn2+的浓度范围超过6个数量级。哺乳动物细胞中游离Zn2+（不与蛋白质紧密结合的Zn2+）的基础水平被认为介于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细胞器，以及当细胞处于应激状态时，经常发现Zn2+浓度升高接近毫摩尔。如此大的浓度范围是其他生理物质所看不到的，这对光学生物成像界提出了巨大的挑战，无论是在知识上还是在实践上。本研究的意义在于，如果成功，将为定量分析所有细胞类型中完整生理浓度范围的Zn2+提供有价值的工具。因此，本研究将有助于阐明Zn2+体内平衡途径，并为诊断和治疗与Zn2+体内平衡偏离有关的疾病找到治疗靶点。本研究的创新之处在于，我们合理设计了异位平台，将三种配位态（非、单、非配位）转化为三种荧光态（非荧光、单色荧光、异色荧光），为大浓度范围内Zn2+的定量分析提供了一种便捷的分析方案。在这项拨款申请中，我们提出了开发具有大有效浓度范围的生理Zn2+荧光探针的策略，该探针将用于游离Zn2+的定量活细胞成像。基于我们实验室开发的异位芳基乙烯基-bipy体系，我们将专门解决具体目标中规定的问题：(1)设计和制备新的探针分子，以提高Zn2+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整合到我们独特的异位框架中，生产用于活细胞荧光显微应用的探针；(3)开发了一种新的异位骨架，其发射谱线对溶剂极性不敏感；(4)我们的探针在生物成像中的应用，特别是在海马神经元，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.