State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, PR China.
Talanta. 2011 Jan 15;83(3):937-42. doi: 10.1016/j.talanta.2010.10.051. Epub 2010 Nov 5.
An electrochemical method for nicotinamide adenine dinucleotide (NAD(+)) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5'-SH and 3'-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD(+), E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD(+)-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD(+) range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD(+) from its analogues.
电化学方法检测烟酰胺腺嘌呤二核苷酸（NAD(+)）具有高灵敏度和选择性，该方法通过使用分子信标（MB）样 DNA 和大肠杆菌 DNA 连接酶实现。在该方法中，通过简单的金硫醇化学将 5'-SH 和 3'-生物素标记的 MB 样 DNA 自组装成金电极的双链体形式，从而阻断电子传递。此时处于关闭状态。在 NAD(+)存在的情况下，大肠杆菌 DNA 连接酶被激活，并且可以通过 NAD(+)-依赖性大肠杆菌 DNA 连接酶将与 MB 样 DNA 环互补的两个核苷酸片段连接起来。与 MB 样 DNA 杂交的连接 DNA 会引起这种表面受限 DNA 结构的大构象变化，从而将生物素从电极表面推开，使电子与电极自由交换。然后可以通过差分脉冲伏安法（DPV）测量产生的电化学信号。在优化条件下，得到了从 3 nM 到 5 μM 的对数浓度 NAD(+)的线性响应和 1.8 nM 的检测限。此外，该策略具有足够的选择性，可以区分 NAD(+)与其类似物。
