Development of gradient guidance assays using nanogold surface patterning

描述（由申请人提供）：对大脑的大多数感觉输入被地形映射，投射神经元的最近邻关系在它们的连接中保持在目标区域内。 例如，视网膜神经节细胞（RGC）轴突以拓扑学方式投射到大脑中的视网膜靶，从而允许视觉图像以空间完整的形式转移。 据信，地形图使用在投射神经元和靶神经元中表达的轴突引导分子的梯度形成。 虽然有一个共识，梯度用于地形图，许多问题和假设轴突如何读取分子梯度和分支在其适当的网站仍然未知。 该方案描述了一种在固体基底上制备生物分子密度梯度的新方法。 该方法采用分层策略，其中蛋白质首先附着到纳米尺寸的金属颗粒上，随后将所得的蛋白质-纳米颗粒生物缀合物从溶液组装到基底表面上。 所得到的表面形貌可以通过用原子力显微镜对纳米颗粒成像来监测金属纳米颗粒的大的且独特的可见光反射来表征。 这种纳米级表面工程方法能够在一维和二维中形成不同浓度和斜率的分子梯度，并且不需要昂贵或复杂的设备。 此外，由于金和银纳米颗粒吸收不同能量的可见光，因此可以组装和表征在单个基底上含有两种不同蛋白质的表面密度梯度。 二维蛋白质模式化将显著增加体外基于细胞的测定的复杂性。 包含蛋白质密度梯度的表面的效用将通过视觉系统中地形图的研究来说明。 EphA受体和肝配蛋白-A配体的一维和二维表面梯度将以再现其在视觉系统发育期间的分级表达的方式构建。 表面结合的神经元生长分析将加深我们的理解轴突的分子机制的指导，更密切地模仿蛋白质图案中遇到的生活系统。 这种理解不仅可以让我们了解大脑中的神经连接是如何形成的，还可以指导我们在受伤后重新连接。

DESCRIPTION (provided by applicant): Most sensory input to the brain is mapped topographically, with nearest neighbor relationships of the projecting neurons maintained in their connections within target areas. For example, retinal ganglion cell (RGC) axons project topographically to retinal targets in the brain, allowing visual images to be transferred in a spatially intact form. It is believed that topographic maps form using gradients of axon guidance molecules expressed in both projection and target neurons. Although there is a consensus that gradients are used in topographic mapping, many questions and hypotheses about how axons read molecular gradients and branch at their appropriate sites remain unknown. This proposal describes a new method for fabricating density gradients of biomolecules on solid substrates. The approach employs a hierarchical strategy in which proteins are first attached to nanometer-sized metal particles, and the resulting protein-nanoparticle bioconjugates are subsequently assembled from solution onto a substrate surface. The resulting surface topography can be characterized by monitoring the large and distinctive visible light absorptions of metal nanoparticles by imaging the nanoparticles with atomic force microscopy. This method of nanoscale surface engineering is capable of forming molecular gradients of varying concentration and slope, in one and two- dimensions, and does not require expensive or sophisticated equipment. Moreover, because gold and silver nanoparticles absorb visible light of different energies, surface density gradients containing two different proteins on a single substrate may be assembled and characterized. Two dimensional protein patterning will increase the complexity of in vitro cell-based assays significantly. The utility of surfaces containing protein density gradients will be illustrated through studies of topographic mapping in the visual system. One and two-dimensional surface gradients of EphA receptors and ephrin-A ligands will be constructed in a manner that recapitulates their graded expression during visual system development. Surface-bound neuronal growth assays will deepen our understanding of the molecular mechanisms of axon guidance by more closely mimicking protein patterning encountered in living systems. This understanding will not only allow us to understand how neural connections are made in the brain but will also guide us to be able to rewire connections after injury.