Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA.
Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA.
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DOI：
10.1021/ja200279y
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发表时间：
2011-05-11
影响因子：
15
通讯作者：
Wang, Tza-Huei
中科院分区：
文献类型：
--
作者：
Liu, Kelvin J.;Rane, Tushar D.;Zhang, Yi;Wang, Tza-Huei
单分子自由溶液流体动力学分离 (SML-FSHS) 将圆柱照明共焦光谱与自由溶液流体动力学分离结合在一起。该技术能够以 100% 的质量检测效率、高尺寸分辨率和宽动态范围对尺寸分离的 DNA 进行单分子分析，超越了单分子毛细管电泳的性能。此外，SML-FSHS 仅需要裸露的熔融石英微毛细管和简单的压力控制，而不需要复杂的高压电源、筛分矩阵和墙壁涂层。通过在相同条件下分离大 DNA（23 与 27 kbp）和小 DNA（100 与 200 bp），证明了 SML-FSHS 的宽动态范围和高尺寸分辨率。使用少至 5 pL 的样品和 240 yocmolole（约 150 个分子）的 DNA 成功进行分离，样品消耗量几乎为零。定量精度主要受到分子散粒噪声的限制。此外，还研究了该方法分析单分子纳米传感器的能力。 SML-FSHS 用于检查大肠杆菌 16s rRNA 靶标检测中随机开放分子信标和靶标结合分子信标之间的热力学平衡。
Single molecule free solution hydrodynamic separation (SML-FSHS) cohesively integrates cylindrical illumination confocal spectroscopy with free solution hydrodynamic separation. This technique enables single molecule analysis of size separated DNA with 100% mass detection efficiency, high sizing resolution and wide dynamic range, surpassing the performance of single molecule capillary electrophoresis. Furthermore, SML-FSHS required only a bare fused silica microcapillary and simple pressure control rather than complex high voltage power supplies, sieving matrices, and wall coatings. The wide dynamic range and high sizing resolution of SML-FSHS was demonstrated by separating both large DNA (23 vs. 27 kbp) and small DNA (100 vs. 200 bp) under identical conditions. Separations were successfully performed with near zero sample consumption using as little as 5 pL of sample and 240 yoctomoles (~150 molecules) of DNA. Quantitative accuracy was predominantly limited by molecular shot noise. Furthermore, the ability of this method to analyze of single molecule nanosensors was investigated. SML-FSHS was used to examine the thermodynamic equilibrium between stochastically open molecular beacon and target-bound molecular beacon in the detection of E. coli 16s rRNA targets.
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