State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China; Yunnan Academy of Tobacco Science, Kunming 650106, PR China.
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Biosens Bioelectron. 2014 May 15;55:187-94. doi: 10.1016/j.bios.2013.12.019. Epub 2013 Dec 16.
A novel homogenous fluorescent sensor for signal-on detection of Cu(2+) has been developed based on intra-molecular G-quadruplex formed by DNA-templated click reaction and crystal violet (CV) as label-free signal reporter. The clickable DNA probe consists of two G-rich strands (A and B) bearing azide and alkyne group, respectively, and a template strand (C) locating two proximate reactants by pairing with A and B. The sequences of A and B are derived from asymmetric split of the G-quadruplex sequence (TTAGGG)4. In the presence of Cu(2+), the whole G-quadruplex sequence A-B is generated by chemical ligation of A and B via copper ion-catalyzed alkyne-azide cycloaddition, then released from template by toehold strand displacement, and consequently forming a stable intra-molecular G-quadruplex, which binds with CV to generate a strong fluorescent signal. Oppositely, weak fluorescence was obtained without Cu(2+) because of unstable intermolecular G-quadruplex formed by A and B and lack of lateral loop connection. Therefore, the Cu(2+) can be sensitively and specifically detected by the fluorescence of the CV-stained G-quadruplex with a low detection limit of 65nM and a linear range of 0.1-3µM. This method rationally integrated the DNA-templated synthesis and G-quadruplex structure-switch, presenting a simple and promising approach for biosensor development.
基于 DNA 模板点击反应形成的分子内 G-四链体和结晶紫（CV）作为无标记信号报告分子，开发了一种用于 Cu(2+)信号开启检测的新型均相荧光传感器。可点击的 DNA 探针由两条富含 G 的链（A 和 B）组成，分别带有叠氮化物和炔基基团，以及一条模板链（C）通过与 A 和 B 配对定位两个相邻的反应物。A 和 B 的序列来源于 G-四链体序列（TTAGGG）4 的不对称分裂。在 Cu(2+)存在下，通过铜离子催化的炔烃-叠氮化物环加成反应，A 和 B 化学连接生成完整的 G-四链体序列 A-B，然后通过碱基对置换从模板中释放，从而形成稳定的分子内 G-四链体，与 CV 结合产生强荧光信号。相反，由于 A 和 B 形成的不稳定的分子间 G-四链体和缺乏侧环连接，没有 Cu(2+)时则获得较弱的荧光。因此，通过 CV 染色的 G-四链体的荧光可以灵敏和特异性地检测 Cu(2+)，检测限低至 65nM，线性范围为 0.1-3µM。该方法合理地集成了 DNA 模板合成和 G-四链体结构切换，为生物传感器的发展提供了一种简单而有前途的方法。
