基于目标物引发的滚环扩增信号放大策略构建电致化学发光传感器用于microRNA检测

A novel electrochemiluminescenc biosensor was applied for micro RNA detection based on cyclic binding-induced rolling circle amplification(CI-RCA) strategy

该实验设计了基于目标物循环引发滚换扩增(CI-RCA)的电致化学发光(ECL)传感器。首先,设计了由结合区域与多鸟嘌呤区域两部分组成的哑铃型挂锁探针作为滚环扩增模板。该模板中的结合区域能够与目标miRNA以及固载电极表面的引物杂交形成三元"Y"形结构,进而引发滚环扩增,产生连环的多胞嘧啶序列的单链DNA。当滚环扩增完成对模板的一次复制时,目标miRNA被置换下来,进而协助下一条引物与模板的结合,引发下一个滚环扩增,生成大量的引物3端延伸出的多胞嘧啶DNA单链。基于C-Ag-C的配位作用,Ag+被固定在电极表面,进而增强过硫酸根体系的ECL信号。因此,随着目标miRNA浓度的增加,ECL信号强度越高。实验结果表明该传感器在1.0 fmol/L^1.0 nmol/L浓度范围内对miRNA有良好的线性响应。

In this work, an electrochemiluminescenc（ECL） biosensor based on cyclic binding-induced rolling circle amplification（CI-RCA） strategy was first proposed and applied in ECL biosensor for micro RNA（miRNA） detection.Herein, a dumbbell-like padlock probe was elaborated with the combination of guanine-rich sequence and binding region. The target miRNA acted as a binder which supported the hybridization of the primer and the padlock probe.Then the rolling circle amplification was triggered and the target miRNA would be released on account of the strand displacement polymerization and join into another reaction cycle. As a result, large amount of cytosine-riched DNA single strands were synthesized on the surface of the biosensor. As the specific affinity with cytosine, silver ions were assembled on the biosensor, which efficiently enhanced the ECL emission of the peroxydisulfate/oxygen（S2O82-/O2）system. Hence, the concentration of target miRNA could be read out through the ECL intensity. With the amplification of CI-RCA, the biosensor required very simple operations and demonstrated ultrasensitive response to target miRNA. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 1.0 fmol/L to 1.0 nmol/L and limit of detection down to 0.3 fmol/L.