Reiterative Signal Amplification by Gold Deposition

通过金沉积重复信号放大

• 批准号: 6647381
• 负责人: RICHARD DENIS POWELL
• 金额: $ 17.37万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6647381
• 关键词: analytical method; chelating agents; chemical reaction; fluorescent in situ hybridization; gold; histidine; human tissue; immunocytochemistry; method development; oligonucleotides; reagent /indicator

DESCRIPTION (provided by applicant): We propose a new approach to signal amplification for ultrasensitive, high-resolution localization of targets in cells and tissues. Amplification will be conducted using reiterative cycles of discrete, highly specific chemical reactions, each of which introduces a controlled number of labels and a consistent number of new binding sites for the next cycle. Because each cycle is initiated by the addition of reagent, the number of cycles, and therefore the resulting degree of amplification and detection sensitivity, can be programmed for optimum performance in the system under study. The chemical reactions will be much less subject to batch variations than enzymatic activity, yielding improvements in reproducibility which will accelerate the development and clinical validation of new molecular diagnostics such as in situ hybridization assays. Three interactions will be used: (i) chelation using polyhisitidine / nitrilotriacetic acid (NTA) - nickel (II) chelate reagent pairs, (ii) complementary oligonucleotide hybridization, and (iii) noncovalent ion pairing of ionized cationic and anionic reagent pairs. The detection label of choice for the new system will be the 1 4 nm Nanogold gold particle, combined with autometallic enhancement to generate a visible signal for interpretation by light microscopy or by eye, while enabling electron microscope localization if necessary.

描述（由申请人提供）：我们提出了一种新的信号放大方法，用于细胞和组织中靶点的超灵敏、高分辨率定位。扩增将使用离散的、高度特异性的化学反应的重复循环进行，每个循环为下一个循环引入受控数量的标记和一致数量的新结合位点。由于每个循环都是通过添加试剂来启动的，因此可以对循环次数以及由此产生的扩增程度和检测灵敏度进行编程，以使所研究的系统具有最佳性能。与酶活性相比，化学反应受批次变化的影响要小得多，从而提高了重现性，这将加速新分子诊断学（如原位杂交测定）的开发和临床验证。将使用三种相互作用：（i）使用多组氨酸/次氮基三乙酸（NTA）-镍（II）螯合试剂对进行螯合，（ii）互补寡核苷酸杂交，以及（iii）离子化阳离子和阴离子试剂对的非共价离子配对。新系统选择的检测标记将是14 nm的纳米金颗粒，结合自动增强以产生可见信号，用于通过光学显微镜或眼睛进行解释，同时在必要时实现电子显微镜定位。