Sub-nm dendrimer-metal nanoclusters as ultrabright, mod*

描述（由申请人提供）：了解生命系统内的固有异质性需要开发新的体内单分子（SM）光学方法，以跟踪蛋白质动力学，而无需系综平均的面纱。 贵金属纳米团簇在整个可见光和近红外光谱中表现出非常强的尺寸依赖性发射，但尺寸比可比的半导体量子点小得多（<l-nm）。 金属纳米团簇的高极化率导致极短的高效率辐射寿命（约30-ps，以及约50%的量子产率），并且甚至增强来自封装支架的拉曼信号，以使其在单分子水平上可观察。 我们将继续使用聚（酰胺胺）树枝状聚合物（PAMAM）来溶解和稳定这些高发射性纳米簇。通过树枝状聚合物合成，a）在无背景光谱区域中结合特定的拉曼活性标记，和B）模块化树枝状聚合物官能化，用于结合通用树枝状聚合物封装的纳米簇（“纳米点”）生物化学功能，我们将开发独特地实现体内单分子成像的材料。 独特的物理学（极快的辐射寿命、高量子产率和在没有大纳米颗粒的情况下产生拉曼信号的能力）使这些亚nm纳米团簇与大得多的（3-10 nm）半导体量子点一样强烈地吸收，但是，因为它们不受约10 ns的长量子点辐射寿命的限制，所以纳米点发射速率，因此亮度至少高两个数量级。 我们将充分表征这类新的重要纳米材料的光学响应，因为我们将它们用作生物标记。 它们的有利特性使得时间和光谱门控检测能够获得非常高的单分子信号，即使在存在活细胞特有的高自发荧光背景的情况下。 我们组建了一支优秀的团队，对PAMAM支架进行化学功能化，封装和稳定高发射性的Au和Ag纳米团簇，并在体外和体内对其进行光学和生物化学表征。 通过三个特定的目标，我们将把这些强大的超亮和超小纳米点开发成无与伦比的，特异性的体内生物标记。 在目标I中，我们将使用靶向化学合成来将模块性并入PAMAM支架中，使得可以并入用于生物化学靶向和识别单元的模块化连接的任何官能团。 在目标II中，我们将合成并将膜传输功能连接到纳米点上，并表征其吸收和光学特性。 这些研究导致了目标III，其中多功能化纳米点被制成特异性结合胞质溶胶内的融合蛋白，并且我们将其转运到特定的细胞器中。 将开发单分子成像方法，使得这些非常明亮的探针可以通过时间和光谱上拒绝来自更长寿命（ns）的自发荧光物质的基本上所有背景来直接成像。 这些组合的方法应该能够将当前的信号/噪声比增加超过当前的基于纳米颗粒或有机荧光团的方法的三个数量级。 这个模块化，超亮，超小和短辐射寿命纳米点的工具箱将普遍适用于各种系统，并将通过这个探索中心提供给社区。

DESCRIPTION (provided by applicant): Understanding the inherent heterogeneity within living systems demands the development of new in vivo single molecule (SM) optical methods to follow protein dynamics without the veil of ensemble averaging. Noble metal nanoclusters exhibit exceedingly strong, size dependent emission throughout the visible and near IR spectrum, but at much smaller sizes (<l-nm) than comparable semiconductor quantum dots. The high polarizability of metal nanoclusters leads to extremely short, high efficiency radiative lifetimes (approximately 30-ps, and quantum yield of approximately 50%), and even enhances the Raman signal from the encapsulating scaffold to make it observable on the single molecule level. We will continue using poly(amidoamine) dendrimers (PAMAM) to solubilize and stabilize these highly emissive nanoclusters. Through dendrimer synthesis to a) incorporate specific Raman active labels in background-free spectral regions and b) modular dendrimer functionalization for incorporating generalized dendrimer encapsulated nanocluster ('nanodot') biochemical functionality, we will develop materials that uniquely enable in vivo single molecule imaging. The unique photophysics (extremely fast radiative lifetime, high quantum yield, and ability to produce Raman signals without a large nanoparticle) make these sub-nm nanoclusters as strongly absorbing as much larger (3-10 nm) semiconductor quantum dots, but, because they are not limited by the long quantum dot radiative lifetime of approximately 10 ns, the nanodot emission rates, and therefore brightness are at least two orders of magnitude higher. We will fully characterize the optical response of this new class of important nanomaterials as we employ them as biological labels. Their advantageous properties enable time and spectrally gated detection to obtain very high single molecule signals even in the presence of high autofluorescent backgrounds characteristic of living cells. We have assembled an outstanding team to chemically functionalize the PAMAM scaffold encapsulating and stabilizing the highly emissive Au and Ag nanoclusters and optically and biochemically characterize them in vitro and in vivo. Through three specific Aims, we will develop these robust ultrabright and ultrasmall nanodots into unparalled, specific, in vivo biological labels. In Aim I we will use targeted chemical synthesis to incorporate modularity in the PAMAM scaffold such that any functional group for modular attachment of biochemical targeting and recognition units can be incorporated. In Aim II we will synthesize and attach membrane transport functionalities to the nanodots and characterize their uptake and optical properties. These studies lead to Aim III in which multifunctionalized nanodots are made to specifically bind fusion proteins within the cytosol and we gate their transport into specific organelles. The single molecule imaging methods will be developed such that these extremely bright probes can be directly imaged by temporally and spectrally rejecting essentially all background from the more long-lived (ns) autofluorescent species. These combined methods should be capable of increasing current signal/noise ratios by more than three orders of magnitude over current nanoparticle or organic fluorophore based methods. This toolbox of modular, ultrabright, ultrasmall, and short-radiative lifetime nanodots will be generally applicable to a wide range of systems and will be made available to the community through this Exploratory Center.