Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlOx membrane
Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlOx membrane
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DOI：
10.1039/b804624g
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发表时间：
2008-09-01
期刊：
影响因子：
6.1
通讯作者：
Gale, Bruce K.
中科院分区：
文献类型：
--
作者：
Kim, Jungkyu;Gale, Bruce K.
将纳米多孔氧化铝膜整合到设计用于从裂解的全血中提取hgDNA（人类基因组DNA）的微流体系统中。该提取系统的有效性通过使已知浓度的纯化hgDNA通过具有不同孔径的纳米多孔膜并测量沉积在膜上的hgDNA的量同时还改变溶液中的盐浓度来确定。DNA提取效率随着盐浓度的增加和纳米孔径的减小而增加。基于这些结果，从全血中提取hgDNA，同时改变盐浓度、纳米孔径和洗脱缓冲液，以找到产生最大浓度的hgDNA的条件。发现最佳条件是使用低盐裂解溶液、100 nm孔和阳离子洗脱缓冲液。在这些条件下，流动和离子破坏的组合足以将hgDNA从膜上除去。使用PCR（聚合酶链反应）分析和评价提取的hgDNA样品，以确定洗脱样品是否含有PCR抑制因子。从微流控系统洗脱的样品被扩增而没有任何抑制作用。使用提取的样品的PCR证明了几个感兴趣的基因。这种基于嵌入式膜的微流控DNA提取系统将减少微流控系统中DNA分析所需的时间、空间和试剂，并将证明对芯片实验室应用中的样品制备有价值。
A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.