基于DNA杂交链式反应和杂交空位的无标记荧光检测DNA研究

摘要

基于DNA杂交链式反应(Hybridization chain reaction, HCR)的信号放大策略,通过在参与HCR反应的发夹型DNA中设计一个特殊碱基,HCR反应后,该碱基对位出现杂交空位,利用杂交空位与荧光小分子(2-Amino-5,6,7-trimethyl-1,8-naphthyridine, ATMND)特异性结合产生的荧光淬灭效应,构建了一种无标记、无酶、灵敏的DNA检测体系.利用凝胶电泳和原子力显微镜等对目标DNA 引发两个发夹型DNA交替自组装形成的超级长链进行了表征.通过对杂交盐浓度和ATMND浓度等条件的优化,获得了满意结果.相比于未利用此放大信号策略的分析方法,灵敏度提高了两个数量级,目标DNA浓度在5. 0~72. 7 nmol/L 浓度范围内与荧光比值(F/F0)呈现良好的线性关系,检出限为2. 0 nmol/L.%A label-free and enzyme-free fluorescent DNA sensor based on hybridization chain reaction ( HCR) and vacant site-binding molecule was developed. By rationally incorporating a cytosine in hairpin DNAs for HCR and utilizing vacant site-binding fluorophore ATMND, sensitive DNA detection was achieved, resulting from the large amount of vacant sites presented on the long DNA nanostructures upon HCR. Gel electrophoresis and atomic force microscopy were performed to characterize the hybridization chain reaction. Under the optimized conditions, the method could sensitively detect the target DNA with the low detection limit of 2. 0 nmol/L. The method shows great potential in signal amplification for DNA detection with advantages such as simple, label-free and low-cost.