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Multiplex Real-Time Loop-Mediated Isothermal Amplification Using an Electrochemical DNA Chip Consisting of a Single Liquid-Flow Channel.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-02-08 00:00:00 , DOI: 10.1021/acs.analchem.8b05284 Koji Hashimoto 1 , Mika Inada 1 , Keiko Ito 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2019-02-08 00:00:00 , DOI: 10.1021/acs.analchem.8b05284 Koji Hashimoto 1 , Mika Inada 1 , Keiko Ito 1
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We developed a multiplex system capable of simultaneously quantifying different target sequences by applying an electrochemical DNA chip that consists of single liquid-flow channel with primers designed for loop-mediated isothermal amplification (LAMP). We applied this system for detecting mature microRNAs (miRNAs). miRNAs extracted from serum were enzymatically lengthened to about 100 base pairs by reverse-transcription and elongation reactions. The LAMP primers for amplifying the lengthened miRNAs were adsorbed and immobilized on the surface of the liquid-flow channel at five different positions. A LAMP solution containing the lengthened miRNAs, Tin DNA polymerase, and ruthenium hexaamine (RuHex) as a redox compound was injected into the DNA chip. The electrochemical reaction of RuHex in the LAMP solution was then measured continuously via linear-sweep voltammetry at 65 °C. The LAMP reaction of the positive control revealed that the cathodic peak current of RuHex increased. Additionally, the initial number of miRNA copies was correlated with the time when the cathodic current began to increase. Five miRNAs were simultaneously detected at 103–106 copies per 50 μL within 2 h. We expect these results will be useful for developing a simple and stable electrochemical-based method for the real-time monitoring of miRNAs, while also facilitating the implementation of electrochemical DNA chips for molecular analyses.
中文翻译：
使用包含单个液流通道的电化学DNA芯片进行多重实时环介导的等温扩增。
我们开发了一种多重系统，该系统能够通过应用电化学DNA芯片来同时量化不同的靶序列，该DNA芯片由单个液流通道和设计用于环介导的等温扩增（LAMP）的引物组成。我们将该系统用于检测成熟的microRNA（miRNA）。通过逆转录和延伸反应，将从血清中提取的miRNA酶促延伸至约100个碱基对。用于扩增延长的miRNA的LAMP引物被吸附并固定在液流通道表面的五个不同位置。含有加长的miRNA，灯溶液锡将DNA聚合酶和六氧化钌（RuHex）作为氧化还原化合物注入DNA芯片中。然后通过线性扫描伏安法在65°C下连续测量RuHex在LAMP溶液中的电化学反应。阳性对照的LAMP反应表明RuHex的阴极峰值电流增加。另外，miRNA拷贝的初始数量与阴极电流开始增加的时间相关。在2 h内以每50μL10 3 –10 6拷贝的数量同时检测到5个miRNA 。我们希望这些结果将有助于开发一种简单且稳定的基于电化学的miRNA实时监测方法，同时也有助于实现用于分子分析的电化学DNA芯片。
更新日期：2019-02-08
中文翻译：
使用包含单个液流通道的电化学DNA芯片进行多重实时环介导的等温扩增。
我们开发了一种多重系统，该系统能够通过应用电化学DNA芯片来同时量化不同的靶序列，该DNA芯片由单个液流通道和设计用于环介导的等温扩增（LAMP）的引物组成。我们将该系统用于检测成熟的microRNA（miRNA）。通过逆转录和延伸反应，将从血清中提取的miRNA酶促延伸至约100个碱基对。用于扩增延长的miRNA的LAMP引物被吸附并固定在液流通道表面的五个不同位置。含有加长的miRNA，灯溶液锡将DNA聚合酶和六氧化钌（RuHex）作为氧化还原化合物注入DNA芯片中。然后通过线性扫描伏安法在65°C下连续测量RuHex在LAMP溶液中的电化学反应。阳性对照的LAMP反应表明RuHex的阴极峰值电流增加。另外，miRNA拷贝的初始数量与阴极电流开始增加的时间相关。在2 h内以每50μL10 3 –10 6拷贝的数量同时检测到5个miRNA 。我们希望这些结果将有助于开发一种简单且稳定的基于电化学的miRNA实时监测方法，同时也有助于实现用于分子分析的电化学DNA芯片。
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