Quantification of low concentrations of DNA using single molecule detection and velocity measurement in a microchannel
Quantification of low concentrations of DNA using single molecule detection and velocity measurement in a microchannel
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DOI：
10.1007/s10895-007-0194-0
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发表时间：
2007-11-01
影响因子：
2.7
通讯作者：
Wang, Tza-Huei
中科院分区：
文献类型：
--
作者：
Chao, Shu-Yi;Ho, Yi-Ping;Wang, Tza-Huei
我们提出了一种基于单分子检测（SMD）的微通道内分子计数和流量测量的低浓度DNA定量的新方法。实现定制的共聚焦荧光光谱系统以检测从染色的DNA分子发射的荧光爆发。测量是一次一个分子，因为它们流过一个飞秒大小的激光聚焦探针。单分子荧光猝发的持续时间，这被发现是强烈相关的分子通过检测区域的渡越时间，进行统计分析，以确定在原位的流速和随后的样品体积流过焦点探针。因此，DNA样品的绝对浓度可以基于来自DNA分子的单分子荧光计数和测量的时间过程的相关探针体积来定量。为了验证这种用于定量低浓度生物分子的方法，我们测试了范围为1 pM至10 fM（类似于3 ng/ml至30 pg/ml）的pBR 322 DNA样品。除了分子定量，我们还证明了这种方法是一种精确和非侵入性的方式在微通道内的流动分析。
We present a novel method for quantifying low concentrations of DNA based on single molecule detection (SMD) for molecular counting and flow measurements inside a microchannel. A custom confocal fluorescence spectroscopic system is implemented to detect fluorescent bursts emitted from stained DNA molecules. Measurements are made one molecule at a time as they flow through a femtoliter-sized laser focal probe. Durations of single molecule fluorescent bursts, which are found to be strongly related to the molecular transit times through the detection region, are statistically analyzed to determine the in situ flow speed and subsequently the sample volume flowing through the focal probe. Therefore, the absolute concentration of a DNA sample can be quantified based on the single molecule fluorescent counts from the DNA molecules and the associated probe volume for a measured time course. To validate this method for quantifying low concentrations of biomolecules, we tested samples of pBR322 DNA ranging from 1 pM to 10 fM (similar to 3 ng/ml to 30 pg/ml). Besides molecular quantification, we also demonstrate this method to be a precise and non-invasive way for flow profiling within a microchannel.