Correlative Enzymatic and Gold Probes

相关酶和金探针

• 批准号: 6993472
• 负责人: RICHARD DENIS POWELL
• 金额: $ 16.94万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-19 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6993472
• 关键词: Microsporidia; RNA splicing; bioimaging /biomedical imaging; biotechnology; cell nucleus; crosslink; electron microscopy; enzyme substrate; fluorescent dye /probe; fungal proteins; genetic transcription; gold; immunofluorescence technique; light microscopy; molecular /cellular imaging; molecular probes; nanotechnology; particle; protein localization; technology /technique development

DESCRIPTION (provided by applicant): A new type of immunoprobe will be developed, containing both an enzyme and a covalently linked 1.4 nm, 3 nm, 5 nm or 10 nm gold label. These will be used for correlative microscopic staining of biologically significant targets at the cell and tissue level by fluorescence or brightfield light microscopy using either a fluorescent or chromogenic substrate, and at the macromolecular level by electron microscopy, using a single labeling procedure. By enzymatic deposition of a fluorescent substrate, the new probes will enable combined immunofluorescent labeling and 3, 5 or 10 nm gold labeling, a procedure previously prohibited by the strong fluorescence quenching properties of the gold particles. Correlation of the two complementary data sets will be improved by the elimination of the effects on specimen morphology of processing between labeling experiments. In addition, these probes will facilitate multiple labeling in which two or more targets are distinguished by different colors at the light or fluorescence level, and different sized gold particles at the electron microscopic level. A variety of synthetic cross-linking rationales will be used to prepare probes, and labeling performance of each component of the resulting probe will be carefully compared with the equivalent singly labeled conjugate using blots, light microscopy on cell and tissue control slides, and electron microscopy. Probes meeting targets for sensitivity, specificity and specimen penetration will be evaluated in two research applications: (i) localization of polar tube proteins in microsporida, and (ii) structural and localization studies on pre-mRNA transciption and splicing factors in the mammalian cell nucleus. This research will provide researchers with new tools for combining data on normal and disease processes in biological systems at the cellular and molecular level, enabling the acquisition of fundamental new knowledge of how these processes work that will support the development of improved dignostics and therapeutics. In addition, it is anticipated that the new probes will be capable of higher detection sensitivities than either detection component used alone, and this may be used for the earlier detection of cancer or other genetic abberations through improved molecular diagnostic procedures such as DNA and RNA in situ hybridization and immunohistochemistry of low abundance targets.

描述（由申请人提供）：将开发一种新型免疫探针，含有酶和共价连接的1.4 nm、3 nm、5 nm或10 nm金标记。这些将用于通过使用荧光或显色底物的荧光或明视野光学显微镜在细胞和组织水平上对生物学重要目标进行相关显微染色，并通过电子显微镜在大分子水平上使用单一标记程序进行染色。通过荧光底物的酶促沉积，新的探针将使联合免疫荧光标记和3，5或10 nm的金标记，一个程序以前禁止的强荧光猝灭性质的金颗粒。两个互补数据集的相关性将通过消除标记实验之间的处理对样本形态的影响来改善。此外，这些探针将促进多重标记，其中两个或更多个目标在光或荧光水平上通过不同的颜色区分，并且在电子显微镜水平上通过不同尺寸的金颗粒区分。将使用各种合成交联原理制备探针，并使用印迹法、细胞和组织对照载玻片上的光学显微镜检查和电子显微镜检查，将所得探针的各组分的标记性能与等同的单标记结合物进行仔细比较。将在两个研究应用中评估满足灵敏度、特异性和样本穿透目标的探针：（i）小孢子虫中极管蛋白的定位，以及（ii）哺乳动物细胞核中前mRNA转录和剪接因子的结构和定位研究。 这项研究将为研究人员提供新的工具，用于在细胞和分子水平上结合生物系统中正常和疾病过程的数据，从而获得有关这些过程如何工作的基本新知识，这将支持改进诊断学和治疗学的发展。此外，预期新探针将能够比单独使用的任一检测组分具有更高的检测灵敏度，并且这可用于通过改进的分子诊断程序（例如DNA和RNA原位杂交和低丰度靶标的免疫组织化学）来更早地检测癌症或其它遗传异常。