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

描述(由申请人提供)：要了解生命系统中固有的异质性，需要开发新的活体单分子(SM)光学方法，以跟踪蛋白质动力学，而不是整体平均的面纱。贵金属纳米团簇在可见光和近红外光谱中表现出非常强的尺寸依赖的发射，但比可比的半导体量子点的尺寸小得多(&lt；L nm)。金属纳米团簇的高极化率导致了极短的、高效的辐射寿命(约30ps，量子产率约为50%)，甚至增强了来自封装支架的拉曼信号，使其能够在单分子水平上观察到。我们将继续使用聚酰胺胺树枝状大分子(PAMAM)来增溶和稳定这些高发射性的纳米团簇。通过树枝状大分子合成，a)在无背景光谱区加入特定的拉曼活性标记，b)模块化树枝状大分子功能化，以加入广义树枝状大分子包裹的纳米簇(‘纳米点’)生化功能，我们将开发独特的能够在体内进行单分子成像的材料。独特的光物理(极快的辐射寿命、高量子产率，以及在没有大纳米颗粒的情况下产生拉曼信号的能力)使这些亚纳米纳米团簇与更大的(3-10 nm)半导体量子点一样强吸收，但由于它们不受大约10 ns的长量子点辐射寿命的限制，纳米点的发射率和亮度至少高出两个数量级。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。它们的优势特性使时间和光谱门控检测能够获得非常高的单分子信号，即使在活细胞特有的高自发荧光背景下也是如此。 我们已经组建了一个优秀的团队，对包裹和稳定高发射性Au和Ag纳米团簇的PAMAM支架进行化学功能化，并在体外和体内对它们进行光学和生物化学表征。通过三个具体的目标，我们将把这些坚固的超亮和超小的纳米点开发成无与伦比的、特定的、体内的生物标记。在AIM I中，我们将使用定向化学合成将模块化结合到PAMAM支架中，以便可以结合用于生化靶向和识别单元的模块化连接的任何官能团。在AIM II中，我们将合成纳米点并将膜传输功能附加到纳米点上，并表征它们的吸收和光学性质。这些研究导致了AIM III，在该目标III中，多功能纳米点被制成以与胞浆中的融合蛋白特异性结合，并将其输送到特定的细胞器。将开发单分子成像方法，以便通过在时间上和光谱上排除更长寿命(Ns)自体荧光物种的几乎所有背景，可以直接成像这些极其明亮的探针。这些组合方法应该能够将电流信噪比提高三个数量级以上，而不是基于纳米颗粒或有机荧光团的方法。这种模块化、超明亮、超小和短辐射寿命纳米点工具箱将普遍适用于广泛的系统，并将通过这个探索中心向社区提供。

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.