Microfluidic means of achieving attomolar detection limits with molecular beacon probes.
Microfluidic means of achieving attomolar detection limits with molecular beacon probes.
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DOI：
10.1039/b819605b
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发表时间：
2009-04-21
期刊：
影响因子：
6.1
通讯作者：
Wang TH
中科院分区：
文献类型：
--
作者：
Puleo CM;Wang TH
我们使用内联，微蒸发器浓缩和运输DNA目标的纳升单分子荧光检测室，随后的分子信标探针杂交和分析。在微量分析平台中使用溶剂去除作为靶向转运的独特手段，导致浓度增强超过5，000倍，并且检测限低于通常使用共聚焦荧光检测报告的飞摩尔屏障。这种用于单分子计数分析的简单微升至纳升互连解决了几个常见的限制，包括在低目标水平下需要过量的荧光探针浓度以及直接处理高度稀释的生物样品的效率低下。在这份报告中，通过微蒸发将约25微升50 aM样品中含有的数百种细菌特异性DNA分子运送到4纳升检测室。在这里，以前无法检测的目标被增强到pM制度，并通过微流体再循环通过共聚焦检测体积进行探针杂交和高效的荧光事件分析。在单分子检测（SMD）平台中使用微流体技术提供了无与伦比的灵敏度，并引入了互补技术，这些技术可能有助于使SMD在取代研究和临床实验室中检测稀有目标生物分子的传统方法方面得到更广泛的应用。
We used inline, micro-evaporators to concentrate and transport DNA targets to a nanoliter single molecule fluorescence detection chamber for subsequent molecular beacon probe hybridization and analysis. This use of solvent removal as a unique means of target transport in a microanalytical platform led to a greater than 5,000-fold concentration enhancement and detection limits that pushed below the femtomolar barrier commonly reported using confocal fluorescence detection. This simple microliter-to-nanoliter interconnect for single molecule counting analysis resolved several common limitations, including the need for excessive fluorescent probe concentrations at low target levels and inefficiencies in direct handling of highly dilute biological samples. In this report, the hundreds of bacteria-specific DNA molecules contained in ~25 microliters of a 50 aM sample were shuttled to a four nanoliter detection chamber through micro-evaporation. Here, the previously undetectable targets were enhanced to the pM regime and underwent probe hybridization and highly-efficient fluorescent event analysis via microfluidic recirculation through the confocal detection volume. This use of microfluidics in a single molecule detection (SMD) platform delivered unmatched sensitivity and introduced compliment technologies that may serve to bring SMD to more widespread use in replacing conventional methodologies for detecting rare target biomolecules in both research and clinical labs.
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影响因子：
7.4
作者：
Heo, Yun Seok;Cabrera, Lourdes M.;Takayama, Shuichi
通讯作者：
Takayama, Shuichi
影响因子：
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影响因子：
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作者：
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通讯作者：
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