Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
Fujian Research Institute of Metric Science, Fuzhou 350003, China.
Biosens Bioelectron. 2014 Sep 15;59:365-9. doi: 10.1016/j.bios.2014.03.053. Epub 2014 Apr 13.
In this work, using methylene blue (MB) as a redox marker and exonuclease III (Exo III) as an amplificatory enzyme, we developed a facile and a label-free electrochemical method for sensitive DNA detection. A double-stranded DNA (dsDNA) probe was prepared by hybridizing two single-stranded DNA (ssDNA) probes. In the ssDNA probes, one ssDNA was guanine bases free and the other one consisted of many unbound guanine bases. MB could be absorbed on the unbound guanine bases owing to the specific interaction between MB and the guanine bases. When the dsDNA probe was challenged with target DNA, it induced a simple Exo III assisted cleavage process, accompanied by the release of the unbound guanine bases. Thus, the amount of MB absorbed on the electrode was much less compared to the initial signal. The detection limit for DNA was found to be as low as 20 fM. Moreover, it could discriminate mismatched DNA from perfectly matched target DNA. This detection method is simple in design, fast in operation and can be applied to detect different DNA sequences.
在这项工作中，我们使用亚甲蓝 (MB) 作为氧化还原标记物和核酸外切酶 III (Exo III) 作为扩增酶，开发了一种简单且无标记的电化学方法，用于灵敏的 DNA 检测。双链 DNA (dsDNA) 探针通过杂交两条单链 DNA (ssDNA) 探针制备。在 ssDNA 探针中，一条 ssDNA 无鸟嘌呤碱基，另一条 ssDNA 包含许多未结合的鸟嘌呤碱基。由于 MB 与鸟嘌呤碱基之间的特异性相互作用，MB 可以被吸附在未结合的鸟嘌呤碱基上。当 dsDNA 探针受到目标 DNA 的挑战时，它会诱导简单的 Exo III 辅助切割过程，同时释放未结合的鸟嘌呤碱基。因此，与初始信号相比，吸附在电极上的 MB 量要少得多。发现 DNA 的检测限低至 20 fM。此外，它可以区分错配 DNA 与完全匹配的靶 DNA。这种检测方法设计简单，操作快速，可用于检测不同的 DNA 序列。
