Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams
Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams
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DOI：
10.1021/ac990644t
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发表时间：
1999-11-15
影响因子：
7.4
通讯作者：
Mathies, RA
中科院分区：
文献类型：
--
作者：
Haab, BB;Mathies, RA
DNA分离已使用微加工毛细管电泳芯片和检测使用单分子荧光猝发计数技术。我们通过1 μ m直径的聚焦激光束聚焦样品，提高了检测到的电迁移DNA分子的百分比。通过在分离通道中引入锥体并通过现在从交叉通道递送的鞘管来聚焦样品。样品流宽度、单分子速度和单分子计数率与预期的电流密度比呈线性变化。使用pBluescriptDNA的稀溶液研究每个聚焦条件的最佳激光功率。虽然荧光猝发高度和背景随激光功率而变化，但使用我们的单分子计数技术，信噪比仅微弱地依赖于此参数。聚焦单分子计数用于检测100-1000-bp DNA大小梯的分离。通过在梯状分离的中途施加聚焦电流并将观察到的计数率与条带中的已知分子浓度进行比较来量化分子检测效率的增加。分子检测效率随所施加的电流密度比线性变化，并且实现了检测效率的大于3倍的增强。
DNA separations have been performed using microfabricated capillary electrophoresis chips and detected using a single-molecule fluorescence burst counting technique. We enhanced the percentage of electromigrating DNA molecules that were detected by focusing the sample through the 1-mu m-diameter focused laser beam. The sample was focused by introducing a taper in the separation channel and by a sheath now delivered from cross channels. The sample stream width, single-molecule velocities, and single-molecule count rates varied linearly with the current density ratio as expected. The optimal laser power for each focusing condition was investigated using dilute solutions of pBluescript DNA. Although fluorescence burst heights and background varied with laser power, the signal-to-noise ratio was only weakly dependent on this parameter using our single-molecule counting technique. Focused single-molecule counting was used to detect separations of a 100-1000-bp DNA sizing ladder. The increase in molecular detection efficiency was quantified by applying a focusing current midway through the ladder separation and comparing observed count rates to the known molecular concentration in the bands. The molecular detection efficiency varied linearly with the applied current density ratio, and greater than 3-fold enhancements in detection efficiency were achieved.