FLUORESCENT SENSORS FOR SUBCELLULAR COPPER IMAGING IN LIVE-CELLS

用于活细胞亚细胞铜成像的荧光传感器 项目摘要 背景和意义：铜是大多数生命形式所必需的微量金属营养素，是第三大 人体内丰富的过渡金属铜的氧化还原活性对几个关键生理过程至关重要; 然而，不受调节的铜水平可诱导细胞中的氧化应激和毒性。要么是缺铜要么是铜 体内的超负荷与各种疾病有关。铜和其他金属之间联系的生物化学基础 不平衡和人类疾病仍然不清楚。 由于缺乏分析手段，细胞或亚细胞区室中铜离子的测量是一项具有挑战性的任务 技术具有足够的空间和时间分辨率和灵敏度。为了填补这一空白，本研究项目的重点是 开发新的"开启"和比率荧光传感器，旨在跟踪积累，物种形成和 铜离子在活细胞中的运输通过光学成像，这将有助于详细研究这种离子的作用， 人类健康和疾病。配位诱导的荧光共振能量转移（CIFRET）机制将在 用于开发铜成像探针。探针将能够进入亚细胞区室，如 将在活细胞中测试线粒体在亚细胞水平对铜成像的能力以及它们对铜离子成像的能力。 荧光探针已经成为现代生物医学和生物技术中不可或缺的工具，因为它们提供了真实的 关于活细胞内感兴趣的离子或分子的量的时间信息。因此，铜特异性 在这个项目中开发的荧光传感器可能会刺激我们对细胞生物学的理解取得重大进展。大部分 所报道的探针在与铜离子结合时经历荧光猝灭，并且荧光变化仅能 这极大地限制了它们潜在的生物应用。因此开发 在生理条件下表现良好的铜离子的新的"开启"和比率荧光探针， 令人向往该项目的一个组成部分是使用密度泛函理论（DFT）和含时DFT， 理解铜离子结合、能量稳定和荧光成像的能力。该项目的目标将 通过以下具体目标实现。目标1：开发高效环保微波炉 辅助有机合成法制备检测人神经母细胞瘤SH-SY5Y细胞中铜离子的探针。 我们的工作将集中在开发具有良好水溶性的罗丹明-香豆素衍生物， 渗透性和光稳定性。在传感器的开发中，将注意利用和开发 环境友好的过程。目标2：开发在水介质中有效的传感器和化学工具，用于研究 生物铜的荧光技术。该策略涉及修改受体的结构， 水溶性基团，增强生物铜在水和活细胞中的传感活性。的作用 影响荧光传感器分析特性的各种因素，它们对荧光的选择性和灵敏度， 生物铜，以及其他重要特征将被研究。目的3：检查 罗丹明-香豆素探针和人神经母细胞瘤SH-SY5Y细胞中铜离子的荧光成像。 由于罗丹明-香豆素染料的生物相容性，探针检测和定位 将通过荧光显微镜成像检查神经母细胞瘤SH-SY5Y细胞中的铜。 此外，这项建议将加强摩根州立大学的研究和教育基础设施， 向代表性不足的少数民族和女学生介绍生物化学和生物医学研究经验， 否则就没有这样的机会。这将使他们能够体验广泛的技术， 获得现代科学调查中使用的数据分析等技能，同时开发一个庞大的 国家和国际研究所科学家之间的伙伴关系。

FLUORESCENT SENSORS FOR SUBCELLULAR COPPER IMAGING IN LIVE-CELLS Project Summary Background and significance: Copper is a trace metal nutrient essential for most forms of life and is the third most abundant transition metal in humans. The redox activity of copper is critical for several key physiological processes; however, unregulated levels of copper can induce oxidative stress and toxicity in cells. Either copper deficiency or copper over-load in the body is linked to various disease conditions. The biochemical basis for the connection between copper imbalance and human disease remains obscure. Measurement of copper ions in cellular or subcellular compartments is a challenging task due to the lack of analytical techniques with adequate spatial and temporal resolution and sensitivity. To fill this gap, this research project focuses on the development of new “turn-on” and ratiometric fluorescent sensors designed to track the accumulation, speciation and trafficking of copper ion in live cells by optical imaging, which will facilitate detailed studies of the role of this ion in human health and disease. Coordination-induced fluorescence resonance energy transfer (CIFRET) mechanisms will be applied to develop the copper-imaging probes. The probes will be capable of entering subcellular compartments such as mitochondria to image copper at subcellular level and their ability to image copper ions will be tested in live cells. Fluorescent probes have become indispensable tools in modern biomedicine and biotechnology because they provide real time information concerning the quantity of ions or molecules of interest within the living cell. Thus, the copper-specific fluorescent sensors developed in this project may spur significant advances in our understanding of cell biology. Most of the reported probes undergo fluorescence quenching upon binding with copper ion and the fluorescence changes can only be observed in non-aqueous solvent, which greatly limits their potential biological application. Therefore, developing new “turn-on” and ratiometric fluorescent probes for copper ion that behave well under physiological conditions is highly desirable. An integral part of this project is the use of density functional theory (DFT) and time-dependent DFT to understand the ability of copper ion binding, energy stabilization and fluorescence imaging. The goal of this project will be accomplished by the following specific aims. Aim 1: Develop an efficient and environmentally friendly microwave assisted organic synthesis for the development of probes to detect copper ion in human neuroblastoma SH-SY5Y cells. Our efforts will be focused on the development of rhodamine-coumarin derivatives with good water solubility, cell permeability and photostability. In the development of sensors, care will be taken to utilize and development in environmentally friendly process. Aim 2: Develop sensors effective in aqueous media and chemical tools for the study of biological copper by fluorescence-based techniques. The strategy involving modifies the structure of the receptor with water soluble groups that enhance the sensing activity of biological copper in aqueous and living cells. The role of various factors affecting the analytical characteristics of fluorescent sensors, their selectivity and sensitivity toward biological copper, as well as other important characteristics will be studied. Aim 3: Examine the cytotoxicity of rhodamine-coumarin probes and fluorescence imaging of copper ion in human neuroblastoma SH-SY5Y cells. Owing to biocompatibility nature of rhodamine-coumarin dyes, the ability of the probes for detection and localize of copper in neuroblastoma SH-SY5Y cells will be examined by fluorescence microscope imaging. Furthermore, this proposal will enhance the infrastructure of research and education at Morgan State University, introducing biochemical and biomedical research experience to underrepresented minority and female students, who would otherwise lack such opportunities. This would allow them to experience a broad spectrum of techniques, and acquire skills such as data analysis used in modern scientific investigations, while developing a vast network of partnership among scientists from national and international institutes.