基于杂交链式反应辅助多重信号放大的端粒酶灵敏检测

Sensitive Detection of Telomerase Based on Hybridization Chain Reaction-assisted Multiple Signal Amplification

端粒酶是真核细胞维持端粒长度的关键逆转录酶,其生物活性的高低可以为多种癌症的临床诊断和预后治疗提供有价值的信息。本研究以人宫颈癌细胞(He La细胞)裂解液中的端粒酶为研究对象,通过借助杂交链式反应辅助多重信号放大策略,提出了一种新颖、灵敏的检测端粒酶电化学方法。首先将端粒酶的延伸引物自组装在金电极表面,当端粒酶存在时,端粒酶能够催化引物的延伸,产生与发卡环探针H1部分互补的序列,进而引发杂交链式反应,形成由两个发卡环探针(H1和H2)交替杂交而形成的DNA长链。由于H1和H2末端均修饰有生物素,加入链霉亲和素修饰辣根过氧化物酶后,辣根过氧化物酶被被连接到电极表面,催化邻苯二胺氧化生成2,3-二氨基吩嗪,产生显著的电化学信号。实验结果表明,本研究建立的端粒酶电化学检测方法高效、可行,线性范围宽,灵敏度高,可以检测每毫升10个He La细胞裂解液中的端粒酶。本方法具有较好的选择性,能有效区分端粒酶和对照蛋白。

A new electrochemical method for telomerase activity assay was developed on the basis of hybridization chain reaction( HCR)-assisted multiple signal amplification,aiming at improving the sensitivity and specificity of telomerase assay. The experiments utilized He La cells as original source of the telomerase in the electrochemical studies. The telomerase primer was firstly self-assembled on the surface of gold electrode.The telomerase catalyzed the elongation of the primer,producing the complementary sequences of hairpin probe H1. In this case,HCR was then initiated by interacting with two hairpin probes H1 and H2. Because both H1 and H2 were modified by biotin,horseradish peroxidase could be captured on the electrode surface through the high-affinity interaction between biotin and streptavidin, catalyzing the oxidation of o-phenylenediamine to produce 2,3-diaminophenazine. Therefore,the telomerase assay was realized by tracing the electrochemical signals with differential pulse voltammetry. This electrochemical method was of high efficiency and feasibility for detecting telomerase activity,and could trace the telomerase activity down to 10 cells/m L He La cells with a wide linear range. Besides,it could also easily distinguish the target enzyme from the control proteins with high specificity.