基于金纳米颗粒信号放大效应电化学检测DNA聚合酶

基于金纳米颗粒(AuNPs)比表面积大、 尺寸小和能够承载大量DNA片段的特点, 建立了一种免标记、 简便、 快速检测DNA聚合酶Klenow fragment exo-(KF^-)的电化学方法. 首先将巯基化的DNA引物片段修饰在金电极上, 然后加入模板DNA链以及修饰有报告DNA链的金纳米颗粒(AuNPs-DNA), 模板DNA链能同时与DNA引物片段和修饰在AuNPs上的报告DNA链进行互补杂交形成"三明治"结构, 从而将AuNPs-DNA修饰在电极表面; 当加入电活性物质钌铵(RuHex)后, RuHex可通过静电吸附作用结合在DNA上. AuNPs上修饰的报告DNA链能够吸附大量RuHex, 导致电化学信号放大. 当加入脱氧核糖核苷三磷酸(dNTPs)以及KF^-聚合酶后, 引物片段发生延伸反应, 将与模板DNA链杂交的AuNPs-DNA竞争下来, 带走大量的RuHex, 使电信号降低, 从而实现对聚合酶的检测. 实验结果表明, 利用该方法可以检测到5 U/mL的KF^-.

Electrochemical Detection of Klenow Fragment Exo-(KF~-) Activity Based on Gold Nanoparticles Amplification

A label-free,simple and fast electrochemical method for Klenow fragment exo-(KF-) detection was developed based on the properties of gold nanoparticles(AuNPs) such as the large specific surface,small size and the ability of binding much DNA.In this work,thiolated primer DNA was first modified on the surface of electrode.In the presence of template DNA and reporter DNA tethered with AuNPs(AuNPs-DNA),primer DNA and report DNA were hybridized with template DNA to form a sandwich-like hybridization mode,which brings AuNPs-DNA to the electrode surface.When put the modified electrode into the electrolyte containing Ru(NH3)6]3+(RuHex),the RuHex quantitatively binds to the DNA via electrostatic interaction.Since AuNPs was loaded with much reporter DNA that can bind a large number of RuHex,this offers the electrochemical signals amplification for the detection.In the presence of dNTPs and KF-,the extension reaction was triggered,which results in the dissociation of the AuNPs-DNA and release of abundant RuHex molecules.As a result,the electrochemical signal was reduced and the KF-was detected.Using this method,a detection limit as low as 5 U/mL for KF-was achieved.