Reiterative Signal Amplification by Gold Deposition

描述（由申请人提供）：我们提出了一种新的信号放大方法，用于细胞和组织中目标的超灵敏，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，每个循环引入受控数量的标签和一致数量的新结合位点，用于下一个循环。由于每个循环都是由添加试剂开始的，因此循环次数以及由此产生的放大程度和检测灵敏度可以在所研究的系统中进行编程，以获得最佳性能。与酶活性相比，化学反应受批次变化的影响要小得多，从而提高了可重复性，这将加速新的分子诊断方法（如原位杂交分析）的开发和临床验证。将使用三种相互作用：(i)用多组氨酸/硝基三乙酸(NTA) -镍螯合；（II）螯合试剂对；（II）互补寡核苷酸杂交；（iii）电离的正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签将是14nm纳米金颗粒，结合自动金属增强来产生可见光信号，用于光学显微镜或肉眼解释，同时在必要时启用电子显微镜定位。

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.