Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures
Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures
复制标题
DOI：
10.1073/pnas.0710982105
复制
发表时间：
2008-01-29
影响因子：
11.1
通讯作者：
Turner, Stephen W.
中科院分区：
文献类型：
--
作者：
Korlach, Jonas;Marks, Patrick J.;Turner, Stephen W.
光学纳米结构使单分子荧光显微镜亚衍射检测体积的创建成为可能。它们的适用性通过将分子放置在受限的观察体积中而不干扰其生物功能的能力而扩展。在这里，我们证明了进行性DNA合成数千个碱基的长度进行了固定在零模波导（ZMW）的观察体积在高密度阵列中的单个DNA聚合酶分子。聚合酶选择性固定到ZMW的熔融石英底板通过使用聚膦酸酯化学钝化金属包层表面来实现，产生超过400：1的玻璃与铝的酶密度对比。对于直径范围（70-100 nm）的ZMW，获得了约30%的单分子占有率。本文的结果支持了固定化单DNA聚合酶在ZMW阵列中用于长读长DNA测序的应用。
Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximate to 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.