Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Department of Physics, Brown University, Providence, Rhode Island 02912, USA.
Biomicrofluidics. 2013 Jul 29;7(4):44107. doi: 10.1063/1.4816943. eCollection 2013.
The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein.
从生物样本中分离目标核酸序列已成为当今诊断和检测策略中的重要过程。除了可能的临床应用外，对表面修饰的磁性珠上目标和序列特异性杂交的基本理解具有很高的价值。在本文中，我们描述了一种新颖的微流控平台，该平台利用静态微流道中的移动磁场，通过核酸杂交分离单链 DNA（ssDNA）分子。我们首先使用链霉亲和素包被的磁性珠建立了生物素化捕获探针（BP）的有效分离。随后，我们研究了与结合到珠上的 BP 的目标 ssDNA 的杂交，并使用双物种动力学模型解释了这些杂交动力学。由于空间位阻效应，结合到珠上的目标 ssDNA 分子的数量比 BP 上的数量少约 6.5 倍。还检查了与结合到珠上的非互补 BP 的目标 ssDNA 的杂交，发现非特异性杂交可以忽略不计。最后，我们在存在非目标 ssDNA 的情况下证明了目标 ssDNA 的高效捕获和分离，其中可以从混合物中检测到低至 1%的目标 ssDNA。本文描述的微流控方法具有重要的相关性，具有广泛的适用性，特别是对于需要结合和分离已知靶生物分子（如 RNA、ssDNA 或蛋白质）的即时生物诊断平台。
