One-Step, Ultrasensitive, and Electrochemical Assay of microRNAs Based on T7 Exonuclease Assisted Cyclic Enzymatic Amplification

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• 作者：Mo Wang ; Zhengliang Fu ; Bingchen Li ; Yunlei Zhou ; Huanshun Yin ; Shiyun Ai
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：June 17, 2014
• 年：2014
• 卷：86
• 期：12
• 页码：5606-5610
• 全文大小：278K
• 年卷期：v.86,no.12(June 17, 2014)
• ISSN：1520-6882

文摘

Taking advantage of the special exodeoxyribonuclease activity of T7 exonuclease, a simple, sensitive, selective, and label-free microRNA biosensor based on the cyclic enzymatic amplification method (CEAM) has been proposed. First, thiol functionalized DNA probes were assembled onto a gold nanoparticles modified gold electrode surface through a Au鈥揝 bond, followed by hybridizing with target miRNA. Subsequently, DNA in RNA/DNA duplexes was digested by T7 exonuclease, which can release the microRNA molecules from the electrode surface and return into the buffer solution. Meanwhile, the released microRNA can further hybridize with the unhybridized DNA probes on the modified electrode surface. On the basis of it, an isothermal amplification cycle is realized. The T7 exonuclease-assisted CEAM achieved a low detection limit of 0.17 fM. Moreover, this assay presents excellent specificity with discriminating only a single-base mismatched microRNA sequence. Furthermore, this work can also be applied to detect avian leukemia based on the decreased expression level of microRNA-21.