A Photostable Fluorescent Probe For Imaging Live Cells Alternative To Quantum Dot

用于活细胞成像的光稳定荧光探针替代量子点

• 批准号: 7276802
• 负责人: Zhenjun Diwu
• 金额: $ 10万
• 依托单位: ABD BIOQUEST, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7276802
• 关键词: Address; Affect; Alexa fluor 546; Amyloid beta-Protein Precursor; Antibodies; Apoptosis; Applications Grants; Biological; Cells; Cellular Structures; Complex; Disease; Dyes; Event; Fluorescence; Fluorescent Dyes; Fluorescent Probes; Image; Immunofluorescence Immunologic; Label; Lead; Life; Light Microscope; Location; Molecular; Molecular Weight; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Oranges; Perylene; Pharmaceutical Preparations; Phase; Process; Property; Protein translocation; Proteins; Quantum Dots; Reproducibility; Research Institute; Resolution; Science; Screening procedure; Semiconductors; Small Business Technology Transfer Research; System; Techniques; Technology; Toxic effect; base; cell fixing; cell injury; cellular imaging; design; fascinate; fluorescence imaging; functional group; imaging probe; light microscopy; novel; prevent; quantum; size; water solubility

DESCRIPTION (provided by applicant): Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. The precise understanding of the molecular mechanisms of neurodegenerative processes could lead to new biological targets for developing leading new drugs that can be effectively used for the ultimate cure of these diseases. In neurological sciences, for example, understanding the translocation of proteins, or protein complexes, from one subcellular location to another is an important aspect for effectively developing novel drugs for various neurodegenerative diseases. Live-cell fluorescence imaging (in particular, high content analysis), however, is seriously limited by the availability of photostable fluorescent probes. The existing fluorescent probes are not appropriate for long- term live-cell imaging and tracing due to their poor photostability. Additionally, these probes are quite toxic to cells if used at high concentrations. Recently, quantum dots (Qdots) that are made from fluorescent semiconductor materials have been proposed for live-cell imaging due to their extremely high photostability. However, their huge size seriously affects the activities of the biological target molecules that are tagged. Uniformity and reproducibility of Qdot labeling have been very challenging. Some of these fluorescent probes require excitation near UV wavelength that cause cell damage and early apoptosis and prevent these probes from being useful for long-term live-cell imaging. ABD Bioquest will collaborate with the Southern Research Institute to develop novel perylene-based fluorescent probes, which have the following properties, for live cell imaging. 1) Small molecular weight. 2) Minimal toxic to cells. 3) High intensity emission with narrow bandwidth. 4) High photostability. 5) Visible wavelength (prefer near to red) excitation. 6) Easy conjugation with antibodies. Two specific aims will be addressed in this Phase I application. Specific Aim 1: Design, synthesize and characterize novel perylene-based fluorescent dyes Specific Aim 2: Evaluate the perylene dye's feasibility for live cell immunofluorescence imaging. The successful completion of these aims will produce a powerful fluorescent probe that can be used for exploring novel applications of high content analysis and screening in live cells.

描述（由申请人提供）：由于最早检查了细胞结构，因此使用光学显微镜观察细胞的生物学家着迷。现在可用的荧光标签技术以及大量复杂的光学显微镜技术的出现使研究活细胞中的动态过程几乎平常。可视化，跟踪和量化具有高空间和时间分辨率的活细胞中的分子和事件的能力对于理解生物系统至关重要。对神经退行性过程的分子机制的精确理解可能会导致新的生物学靶标，用于开发主要的新药，这些药物可有效地用于这些疾病的最终治愈。在神经科学中，例如，了解蛋白质或蛋白质复合物的易位，从一个亚细胞位置到另一个亚细胞位置是有效开发各种神经退行性疾病的新型药物的重要方面。但是，活细胞荧光成像（尤其是高含量分析）受到光增生荧光探针的可用性受到严重限制。现有的荧光探针由于其光稳定性差而不适用于长期活细胞成像和跟踪。另外，如果在高浓度下使用，这些探针对细胞具有很大的毒性。最近，由于其极高的光稳定性，已经提出了由荧光半导体材料制成的量子点（QDOT）。但是，它们的巨大规模严重影响了标记的生物靶标分子的活动。 QDOT标签的均匀性和可重复性非常具有挑战性。这些荧光探针中的一些需要在UV波长附近激发，这会导致细胞损伤和早期凋亡，并防止这些探针可用于长期活细胞成像。 ABD BioQuest将与南方研究所合作开发具有以下特性的新型基于Perylene的荧光探针，用于实时细胞成像。 1）小分子量。 2）对细胞的最小有毒。 3）具有狭窄带宽的高强度发射。 4）高光稳定性。 5）可见波长（更喜欢接近红色）激发。 6）易于与抗体结合。在此I阶段应用程序中将解决两个具体目标。特定目标1：设计，合成和表征新型基于pery素的荧光染料特异性目标2：评估pery素染料对活细胞免疫荧光成像的可行性。这些目标的成功完成将产生强大的荧光探针，可用于探索高素质分析和筛选活细胞中的新应用。