Dendrimer & peptide-encapsulated fluorescent Ag nanodots

描述（由申请人提供）：这项研究的长期目标是创造小的，高荧光的纳米材料，使生物系统中的单分子光学显微镜实验的全新阵列。单分子实验非常擅长探索机制异质性，它具有很大的潜力来解开导致生物活性的所有复杂步骤。不幸的是，所有的单分子实验都在一定程度上受到可用荧光标记的性质的限制。因此，单分子方法的任何重大进展将仅可能通过开发新的、极其光稳定的、高荧光的标记物来实现，所述标记物具有易于体外和体内标记的潜力。通过三个提出的具体目标，我们将调整我们最近发现的单个Ag纳米点荧光，以创建非常明亮的通用荧光标记。由于这些材料具有很强的吸收性，它们可以很容易地用弱汞灯激发，从而大大降低了与体内成像相关的自发荧光背景和与基于激光的方法相关的实验复杂性。在具体目标I中，我们将在单分子和散装水平上对不同的生物相容性树枝状聚合物封装的Ag纳米点进行全面的化学和生物学表征。由于成功的可能性很高，我们将大大扩展我们的初步结果，以创建和充分表征高荧光，水溶性银纳米点，从而为后续目标提供基础。在Specific Aim II中，我们将联合收割机生物多样性与材料科学相结合，以确定结合和稳定Ag纳米点的特定肽序列。使用多个肽文库，鉴定的Ag纳米点结合肽将产生与目前稳定Ag纳米点荧光的树枝状聚合物的荧光互补的荧光。因此，我们将通过质谱和单分子/本体荧光实验来研究肽-Ag纳米点相互作用的性质和特异性。将研究目标I中表征的树枝状聚合物包封的纳米点的Ag纳米簇转移到通过文库筛选鉴定的优化的Ag结合肽。特异性目的III探针将这些鉴定的肽连接到两个独立可定量的蛋白质上，以测定标记效率和蛋白质功能的保留。这些实验对于将Ag纳米点结合肽遗传地连接到感兴趣的蛋白质并将Ag转移到其上以用作高荧光、稳健的体内单分子标记的潜在用途是至关重要的。

DESCRIPTION (provided by applicant): The long-term goal of this research is to create small, highly fluorescent nanomaterials to enable a whole new array of single molecule optical microscopy experiments in biological systems. Beautifully adept at probing mechanistic heterogeneity, single molecule experiments hold great potential to unravel the all of the complex steps leading to biological activity. Unfortunately, all single molecule experiments are at some level limited by the properties of available fluorescent labels. Thus, any significant advances in single molecule methods will only be possible through the development of new, extremely photostable, highly fluorescent labels with the potential for both facile in vitro and in vivo labeling. Through the three proposed Specific Aims, we will adapt our recently discovered single Ag nanodot fluorescence to create extremely bright, general use fluorescent labels. As these materials are very strongly absorbing, they can be easily excited with weak Hg lamps, thereby greatly reducing both the autofluorescent background associated with in vivo imaging and the experimental complexity associated with laser-based methods. In Specific Aim I, we will fully chemically and photophysically characterize the different biocompatibie dendrimer-encapsulated Ag nanodots on both the single molecule and bulk levels. With a high probability of success, we will greatly expand our preliminary results to create and fully characterize highly fluorescent, water-soluble Ag nanodots, thereby providing the basis for the subsequent Aims. In Specific Aim II, we will combine biological diversity with materials science to identify specific peptide sequences that bind and stabilize Ag nanodots. Using multiple peptide libraries, identified Ag nanodot-binding peptides will yield fluorescence complementary to that of the dendrimers currently stabilizing the Ag nanodot fluorescence. Thus, we will study the properties and specificity of peptide-Ag nanodot interaction through mass spectrometry and single molecule / bulk fluorescence experiments. The dendrimer - encapsulated nanodots characterized in Aim I will be investigated for Ag nanocluster transfer to the optimized Ag-binding peptides identified by library screening. Specific Aim III probes attaching these identified peptides to two independently quantifiable proteins to assay labeling efficiency and retention of protein function. These experiments are crucial to the potential uses of genetically attaching the Ag nanodot binding peptide to the protein of interest and transferring Ag to it for use as a highly fluorescent, robust in vivo single molecule label.