State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College Hunan University, Changsha 410082, PR China.
Biosens Bioelectron. 2012 Jun-Jul;36(1):123-8. doi: 10.1016/j.bios.2012.04.012. Epub 2012 Apr 26.
This work develops a simple, sensitive and signal-on electrochemical sensor for methyltransferase (MTase) activity analysis. The sensor is composed of a methylene blue-modified "signaling DNA probe" and a "capture DNA probe" tethered methylation-responsive hairpin DNA (hairpin-capture DNA probe). The thiol- modified hairpin-capture DNA probe at 5' end was firstly self-assembled on gold electrode via Au-S bonding. Methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the methylene blue-modified "signaling DNA probe", mediating methylene blue onto the gold electrode surface to generate redox current. It was eT on state. The developed facile signal-on electrochemical sensing system showed a linear response to concentration of Dam MTase range from 0.1 to 1.0 U/mL. The detection limit of Dam MTase activity was determined to be 0.07 U/mL and the total detection time is 7h. The sensor also has the ability to provide information about the dynamics of methylation process. Furthermore, we demonstrated that this sensor could be utilized to screen inhibitors or drugs for Dam MTase.
这项工作开发了一种简单、灵敏且信号开启的电化学传感器，用于甲基转移酶（MTase）活性分析。该传感器由亚甲基蓝修饰的“信号 DNA 探针”和连接的甲基化反应性发夹 DNA（发夹捕获 DNA 探针）组成。5'端巯基修饰的发夹捕获 DNA 探针首先通过 Au-S 键自组装在金电极上。发夹捕获 DNA 探针的甲基化诱导断裂会导致发夹部分解离，并使“捕获 DNA 探针”部分留在金电极上。随后，留在金电极上的“捕获 DNA 探针”可以与亚甲基蓝修饰的“信号 DNA 探针”杂交，介导亚甲基蓝在金电极表面生成氧化还原电流。该系统表现出对 Dam MTase 浓度在 0.1 至 1.0 U/mL 范围内的线性响应。检测 Dam MTase 活性的检测限为 0.07 U/mL，总检测时间为 7 小时。该传感器还具有提供关于甲基化过程动力学信息的能力。此外，我们证明了该传感器可用于筛选 Dam MTase 的抑制剂或药物。
