基于链替代信号放大灵敏检测端粒酶活性的电化学方法

Sensitive and Electrochemical Detection of Telomerase Activity Based on the Signal Amplification of Strand Displacement Reaction

利用标记二茂铁基团的DNA(T-DNA)分子作为信号探针,基于端粒酶特异性延长其底物链(TS)所引发的链替代反应,建立了一种检测端粒酶活性的电化学信号放大法.将巯基化的发夹型DNA分子(H-DNA)通过金-硫键自组装于金电极表面,辅助DNA(A-DNA)与二茂铁修饰的T-DNA部分互补杂交形成双链AT-DNA;当端粒酶存在时,可在TS的3′末端合成TTAGGG的重复序列;A-DNA与TS延长链杂交置换出T-DNA;T-DNA与发夹H-DNA杂交使得二茂铁靠近电极表面;一条TS延长链可以释放出多条T-DNA,将二茂铁富集到金电极表面,从而实现信号放大检测端粒酶活性.HeLa细胞个数在5~100范围内与电流值成正比,最低可检测5个HeLa细胞中端粒酶的活性.因此,本文建立了一种简单灵敏检测端粒酶活性的电化学方法.

An electrochemical method was developed for sensitive detection of telomerase activity based on strand displacement reaction.A ferrocene(Fc)-labeled DNA probe(T-DNA)was designed as signal probe.The thiolated hairpin DNA(H-DNA)was used as capture probe and was self-assembled on the Au electrode surface via Au-S bond.Assisted DNA(A-DNA)partially hybridized with T-DNA to form AT-DNA duplex.In the presence of telomerase,telomerase substrate(TS)primer was elongated by adding multiple TTAGGG re⁃peats at its 3′terminal.The elongated DNA hybridized with A-DNA to displace T-DNA which can hybridize with H-DNA.As a result,the Fc tags are close to the Au electrode surface,producing an electrochemical sig⁃nal.One elongated DNA can displace multiple T-DNAs,leading to the enrichment of Fc onto the Au electrode for signal amplification.Based on this,the peak current is linearly related with the number of HeLa cells in the range of 5―100 cells,and detection limit is down to 5 HeLa cells.Therefore,it provides a simple and sensi⁃tive approach for detecting telomerase activity.