Thiol Specific Fluorogenic Agents for Laser Scanning Confocal Microscopy

用于激光扫描共焦显微镜的硫醇特异性荧光剂

• 批准号: 7924328
• 负责人: XIANGMING GUAN
• 金额: $ 43.03万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924328
• 关键词: Acetylcysteine; Affect; Biological; Biological Assay; Cell physiology; Cell surface; Cells; Chinese Hamster; Chinese Hamster Ovary Cell; Coupled; Detection; Disease; Disulfides; Dithionitrobenzoic Acid; Fluorescence; Fluorescent Dyes; Health; High Pressure Liquid Chromatography; Image; Laser Scanning Confocal Microscopy; Location; Maps; Measurement; Methods; Modeling; Monitor; Morphologic artifacts; Nature; Nitrogen; Ovary; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Play; Predisposition; Property; Reaction; Reagent; Role; Stimulus; Structure; Subcellular structure; Sulfhydryl Compounds; Techniques; Tissues; Validation; Work; adduct; analytical tool; base; density; design; disulfide bond; extracellular; interest; liquid chromatography mass spectrometry; oxidation; public health relevance; quantum; reaction rate; therapy development; tool

DESCRIPTION (provided by applicant): Oxidative stress has been found to be associated with various disease states. An analytical tool which can be used to monitor cellular and subcellular oxidative stress in intact cells will be a valuable tool in understanding how cells respond to oxidative stress stimuli and help develop treatments to oxidative stress- associated diseases. A combination of a proper fluorogenic or fluorescent agent with laser scanning confocal microscopy (LSCM) has been widely used to image the distribution of the molecule of interest in different subcellular structures in intact cells and will be a method of choice in monitoring oxidative stress at different subcellular locations. Thiol (-SH) status plays a significant role in determining cellular oxidative stress status due to its high susceptibility to oxidation and high abundance. Thiol density decreases as oxidative stress increases and its status has been used as a parameter to reflect cellular oxidative stress. A proper thiol specific fluorogenic or fluorescent agent in combination with LSCM can be used to image thiol density and distribution in intact cells, which in turns reflects redox state at subcellular levels. Numerous fluorogenic and fluorescent agents have been developed for thiol assays. However, most of these agents are not thiol specific. Their use for thiol assays needs to be coupled with a separation technique, such as high performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS), and is not suitable for LSCM. Consequently, their use is limited to thiol assays of tissue homogenates. Only a few fluorogenic agents are available for thiol assays in intact cells by LSCM with various drawbacks. In view of the significant role oxidative stress plays in various cellular functions, disease states, and valuable information which can be gained from an intact cell study of thiol distribution, it is highly desirable to develop thiol specific fluorogenic agents which can be used for LSCM. The aim of this proposed application is to develop thiol specific fluorogenic agents which can be used to image thiol density intracellularly and on the cell surface in intact cells with LSCM. The design of these agents is based on a thiol specific reaction: thiol-disulfide (-S-S-) exchange reaction. This reaction is the base of the most widely used thiol specific reagent, Ellman's reagent which enables measurement of thiols through a visible wavelength. This proposed project is to take advantage of the thiol specific reaction and integrate the disulfide bond (-S-S-) into a well studied and widely used benzofurazan fluorogenic structure. Specifically, rationally designed thiol specific fluorogenic agents and their thiol adducts will be synthesized. Their fluorescence properties will be fully characterized. Their application in determining intracellular thiol distribution and thiol density on the cell surface in intact cells will be validated by LSCM with Chinese hamster ovary (CHO) cells under different extents of thiol oxidative stress. PUBLIC HEALTH RELEVANCE: Oxidative stress has been associated with various health issues. A method which can detect the status of oxidative stress in intact cells will help understand the role of oxidative stress in various diseases and facilitate development of treatments to oxidative stress-associated diseases. This proposed application is aimed to develop a method which can be used to detect the status of oxidative stress in intact cells.

描述（由申请人提供）：已发现氧化应激与多种疾病状态相关。可用于监测完整细胞中的细胞和亚细胞氧化应激的分析工具将成为了解细胞如何响应氧化应激刺激并帮助开发氧化应激相关疾病的治疗方法的宝贵工具。适当的荧光剂或荧光剂与激光扫描共聚焦显微镜 (LSCM) 的组合已被广泛用于对完整细胞中不同亚细胞结构中目标分子的分布进行成像，并将成为监测不同亚细胞位置氧化应激的一种选择方法。 硫醇 (-SH) 状态由于其对氧化的高敏感性和高丰度，在确定细胞氧化应激状态中起着重要作用。硫醇密度随着氧化应激的增加而降低，其状态已被用作反映细胞氧化应激的参数。适当的硫醇特异性荧光剂或荧光剂与 LSCM 结合可用于对完整细胞中的硫醇密度和分布进行成像，这反过来又反映了亚细胞水平的氧化还原状态。 已开发出许多用于硫醇测定的荧光剂和荧光剂。然而，大多数这些试剂都不是硫醇特异性的。它们用于硫醇测定需要与分离技术结合使用，例如高效液相色谱 (HPLC) 或液相色谱/质谱 (LC/MS)，并且不适合 LSCM。因此，它们的用途仅限于组织匀浆的硫醇测定。只有少数荧光剂可用于通过 LSCM 对完整细胞进行硫醇测定，但存在各种缺点。鉴于氧化应激在各种细胞功能、疾病状态中发挥重要作用，并且可以从硫醇分布的完整细胞研究中获得有价值的信息，因此非常需要开发可用于 LSCM 的硫醇特异性荧光剂。 本申请的目的是开发硫醇特异性荧光剂，可用于通过 LSCM 对完整细胞的细胞内和细胞表面上的硫醇密度进行成像。这些试剂的设计基于硫醇特异性反应：硫醇-二硫化物（-S-S-）交换反应。该反应是最广泛使用的硫醇特异性试剂埃尔曼试剂的基础，该试剂能够通过可见波长测量硫醇。该项目旨在利用硫醇特异性反应，将二硫键（-S-S-）整合到经过充分研究和广泛使用的苯并呋喃荧光结构中。 具体而言，将合成合理设计的硫醇特异性荧光剂及其硫醇加合物。它们的荧光特性将被充分表征。它们在确定完整细胞的细胞内硫醇分布和细胞表面硫醇密度方面的应用将通过 LSCM 与中国仓鼠卵巢 (CHO) 细胞在不同程度的硫醇氧化应激下进行验证。 公共卫生相关性：氧化应激与各种健康问题有关。一种可以检测完整细胞中氧化应激状态的方法将有助于了解氧化应激在各种疾病中的作用，并促进氧化应激相关疾病的治疗方法的开发。本申请旨在开发一种可用于检测完整细胞氧化应激状态的方法。

项目成果

Benzofurazan sulfides for thiol imaging and quantification in live cells through fluorescence microscopy.

• DOI: 10.1021/ac301306s
• 发表时间: 2012-08-07
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Li, Yinghong;Yang, Yang;Guan, Xiangming
• 通讯作者: Guan, Xiangming