State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China.
Anal Chim Acta. 2011 Mar 4;688(2):163-7. doi: 10.1016/j.aca.2011.01.004. Epub 2011 Jan 13.
An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.
基于表面连接反应和寡核苷酸（ODN）修饰金纳米粒子（AuNPs）的电化学点突变检测方法得到了证实。使用大肠杆菌 DNA 连接酶实现了点突变鉴定。该点突变检测系统依赖于夹心测定法，包括固定在 Au 电极上的捕获 ODN、靶 ODN 和连接 ODN。由于大肠杆菌 DNA 连接酶的序列特异性表面反应，只有在存在完全互补的靶 ODN 时，连接 ODN 才能与捕获 ODN 共价连接。通过与修饰有报道 ODN 的 AuNPs 杂交，使用计时库仑法检测 Au 电极上的连接产物。在这种方法中，AuNPs 的使用提高了计时库仑法的灵敏度，获得了 0.9 pM 互补 ODN 的检测限。对于单碱基错配 ODN（smODN），观察到可以忽略不计的信号。即使互补 ODN 与 smODN 的浓度比降低至 1:1000，也可以观察到可检测的信号。这项工作可能为点突变检测提供了一种特异性、敏感性和成本效益高的方法。
