A label-free electrochemical assay for methyltransferase activity detection based on the controllable assembly of single wall carbon nanotubes.
A label-free electrochemical assay for methyltransferase activity detection based on the controllable assembly of single wall carbon nanotubes.
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DOI：
10.1016/j.bios.2012.08.034
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发表时间：
2013-03
影响因子：
12.6
通讯作者：
Yonghong Wang;Xiaoxiao He;Kemin Wang;Jing Su;Z. Chen;Genping Yan;Yudan Du
中科院分区：
文献类型：
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作者：
Yonghong Wang;Xiaoxiao He;Kemin Wang;Jing Su;Z. Chen;Genping Yan;Yudan Du
基于单壁碳纳米管（SWCNTs）的信号传导和放大作用，建立了一种灵敏的无标记“信号开启”电化学方法，用于甲基转移酶（MTase）活性的检测。在该方法中，寡核苷酸I首先通过Au-S键合自组装在电极上。在与其互补ssDNA（寡核苷酸II）杂交后，然后在电极上形成含有Dam MTase和甲基化敏感性限制性内切酶Dpn I的特异性识别序列的双链DNA（dsDNA）探针。在Dam MTase和Dpn I的存在下，dsDNA探针被甲基化，随后切割成两个dsDNA片段。加热后，电极上剩余的dsDNA片段熔化成ssDNA片段。然后，单壁碳纳米管可以可控地组装在保留在电极上的ssDNA片段上，介导电极和电活性物质之间的有效电子转移。它产生可测量的电流信号（eT ON），其与Dam MTase的浓度相关。在Dam MTase浓度低至0.04U/mL时，通过差示脉冲伏安法容易地测量电子转移效率的变化。该方法不需要在甲基化反应DNA探针上标记电活性分子。所建立的Dam MTase信号启动电化学传感系统的线性响应范围为0.1-1.0U/mL。此外，这种基于SWCNT的电化学测定也具有筛选Dam MTase抑制剂的能力。
A sensitive label-free “signal-on” electrochemical approach for detection of methyltransferases (MTase) activity is developed based on the signal transduction and amplification of single wall carbon nanotubes (SWCNTs). In this method, the oligonucleotide I is first self-assembled on the electrode via Au–S bonding. After hybridization with its complement ssDNA (oligonucleotide II), duplex strand DNA (dsDNA) probes containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I is then formed on the electrode. In the presence of Dam MTase and Dpn I, the dsDNA probes are methylated and subsequently cleaved into two dsDNA fragments. After heating, the remained dsDNA fragments on the electrode melted into ssDNA fragments. Then the SWCNTs can be controllably assembled on the ssDNA fragments remained on the electrode, mediating efficient electron transfer between the electrode and electroactive species. It generates measurable current signal (eT ON), which is related to the concentration of the Dam MTase. The resulting change in electron transfer efficiency is readily measured by differential pulse voltammetry at Dam MTase concentrations as low as 0.04U/mL. This method does not need electroactive molecules labeling on the methylation-responsive DNA probes. The linear response of the developed facile signal-on electrochemical sensing system for Dam MTase is in the range of 0.1–1.0U/mL. In addition, such a SWCNTs based electrochemical assay also has the ability to screen inhibitors for Dam MTase.