Wang Mo, Fu Zhengliang, Li Bingchen, Zhou Yunlei, Yin Huanshun, Ai Shiyun
College of Chemistry and Material Science, Shandong Agricultural University , Tai'an, Shandong 271018, PR China.
Anal Chem. 2014 Jun 17;86(12):5606-10. doi: 10.1021/ac5010376. Epub 2014 Jun 6.
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-S 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.
利用T7核酸外切酶的特殊核酸外切酶活性，提出了一种基于循环酶扩增法（CEAM）的简单、灵敏、选择性好且无标记的微小RNA生物传感器。首先，通过Au-S键将硫醇功能化的DNA探针组装到金纳米颗粒修饰的金电极表面，随后与靶标微小RNA杂交。接着，RNA/DNA双链中的DNA被T7核酸外切酶消化，这能使微小RNA分子从电极表面释放并回到缓冲溶液中。同时，释放出的微小RNA可进一步与修饰电极表面未杂交的DNA探针杂交。在此基础上，实现了等温扩增循环。T7核酸外切酶辅助的CEAM实现了0.17 fM的低检测限。此外，该检测方法具有出色的特异性，仅能区分单碱基错配的微小RNA序列。此外，基于微小RNA-21表达水平的降低，这项工作还可应用于检测禽白血病。
