Development of Sensitive Fluorescent Probes for Physiological Zinc Over Large Con

描述(申请人提供)：项目概要项目名称：开发大浓度范围内生理锌离子的灵敏荧光探针首席研究员：朱磊共同首席研究员：Michael W.Davidson共同首席研究员：Cathy W.Levenson我们的目标是开发用于在生理环境中对游离锌离子(Zn2)成像的高灵敏度和大浓度覆盖范围的荧光探针。众所周知，锌在人体生理学中起着结构、催化和其他作用。锌稳态的破坏是病理性的。锌在不同生化途径中的确切功能尚未完全阐明，部分原因是缺乏工具来准确确定锌在非均质和动态生物介质中的分布和运动。将锌与其他具有重要生理意义的物质区分开来的一个特殊挑战是，生物系统中锌的浓度范围超过六个数量级。哺乳动物细胞中游离锌(与蛋白质没有紧密结合的锌)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，经常发现升高的接近毫摩尔的锌浓度。这种对其他生理物质来说还未见过的大浓度范围对光学生物成像界提出了巨大的挑战，无论是在智力上还是在实践意义上都是如此。这项研究的意义在于，如果成功，它将为定量分析所有细胞类型中锌的完整生理浓度范围提供有价值的工具。因此，这些研究将有助于阐明锌的稳态途径，并为诊断和治疗与锌稳态失调相关的疾病提供治疗靶点。研究的创新体现在我们合理设计的异位平台，将三种配位状态(非、单、双配位)转换为三种荧光状态(非荧光、一种颜色的荧光和不同颜色的荧光)，从而为大浓度范围内的锌的定量分析提供了一个方便的分析方法。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锌的荧光探针的策略，这些荧光探针将用于游离锌的定量活细胞成像。在我们实验室开发的异位芳基乙烯基-bipy系统的基础上，我们将具体解决具体目标中规定的问题：(1)设计和制备新的探针分子，以提高锌离子定量的灵敏度(同时保持较大的有效浓度范围)；(2)将已知的激光染料结构纳入我们独特的异位框架，以生产用于活细胞荧光显微镜应用的探针；(3)开发一种新的异位框架，其发射轮廓不受溶剂极性的影响；以及(4)将我们的探针应用于生物成像，尤其是海马神经元的荧光显微镜成像。 公共卫生相关性：项目标题：为大浓度范围的生理锌开发灵敏的荧光探针首席研究员：朱磊联席首席研究员：Michael W.Davidson联席首席研究员：Cathy W.Levenson锌的健康相关性源于锌离子在许多生理过程中的关键参与。缺乏对锌离子作用的了解，部分原因是缺乏确定锌离子在细胞内的时空分布的手段。这些活动的成功完成将有助于确定锌离子在生物系统中的动态分布，从而有助于阐明锌稳态途径，并为锌稳态紊乱相关疾病的诊断和治疗寻找治疗靶点。

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. PUBLIC HEALTH RELEVANCE: 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 The health relevance of zinc is resulted from the critical involvement of zinc ion in many physiological processes. The lack of understanding of the roles of zinc ion is partly due to the lack of means to determine the spatiotemporal intracellular distribution of zinc ion. The successful completion of the proposed activities will aid the determination of dynamic zinc ion distribution in biological systems therefore to facilitate the elucidation of zinc homeostatic pathway and to identify therapeutic targets for diagnosis and treatment of diseases related to the disruption of zinc homeostasis.