Development of gradient guidance assays using nanogold surface patterning

描述（由申请人提供）：大多数大脑的感觉输入被映射成地形图，在目标区域内，投射神经元的最近邻关系保持在它们的连接中。例如，视网膜神经节细胞（RGC）轴突以地形图方式投射到大脑中的视网膜目标，从而允许视觉图像以空间完整的形式传输。地形图的形成是由轴突引导分子在投射神经元和目标神经元中表达的梯度决定的。虽然在地形测绘中使用梯度是一个共识，但关于轴突如何读取分子梯度并在适当的位置分支的许多问题和假设仍然未知。本文提出了一种在固体基质上制备生物分子密度梯度的新方法。该方法采用分层策略，首先将蛋白质附着在纳米大小的金属颗粒上，然后将所得的蛋白质-纳米颗粒生物偶联物从溶液组装到底物表面。通过原子力显微镜对金属纳米颗粒进行成像，监测金属纳米颗粒的大而独特的可见光吸收，从而表征其表面形貌。这种纳米级表面工程方法能够在一维和二维上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，由于金纳米粒子和银纳米粒子吸收不同能量的可见光，可以在单个底物上组装和表征含有两种不同蛋白质的表面密度梯度。二维蛋白质图谱将显著增加体外细胞检测的复杂性。包含蛋白质密度梯度的表面的效用将通过研究视觉系统中的地形映射来说明。EphA受体和ephrin-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过程中的梯度表达的方式构建。表面结合神经元生长试验将通过更紧密地模拟生命系统中遇到的蛋白质模式，加深我们对轴突引导的分子机制的理解。这种理解不仅能让我们了解大脑中的神经连接是如何形成的，还能指导我们在受伤后重新连接神经。

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.