基于链置换和催化发夹组装策略用于miRNA高灵敏生物传感检测

Electrochemical biosensor based on strand displacement amplification and catalytic hairpin assembly strategy for highly sensitive detection of miRNA

摘要:

目的：基于链置换扩增（strand displacement amplification，SDA）和催化发夹组装（catalytic hairpin assembly，CHA）级联放大策略构建电化学生物传感器用于microRNA（miRNA）高灵敏检测。方法：靶标miR-21引发SDA获得大量与靶标miR-21相关的触发DNA，继而引发CHA，得到大量生物素标记的双链DNA。然后，所获得的双链DNA与修饰在电极表面的辅助探针杂交，再利用生物素与链霉亲和素的特异性识别作用，将含链霉亲和素的碱性磷酸酶（streptavidin-alkaline phosphatase，ST-AP）修饰在电极表面上，在底物α-萘酚磷酸酯（α-naphthyl phosphate，α-NPP）存在下，ST-AP催化α-NPP水解，在电极表面原位产生具有电化学活性的α-萘酚（α-naphthol，α-NP），获得电化学信号，从而实现对靶标miR-21的高灵敏检测。结果：在最优实验条件下，所制备的miR-21生物传感器的线性范围为0.1 pmol/L至10 nmol/L，最低检测限为60 fmol/L。结论：所设计的miR-21生物传感器展现出高的灵敏度和强的特异性，将为临床上miR-21的检测提供新思路和新平台。

Abstract:

Objective：To construct a high sensitive electrochemical biosensor for the detection of microRNA（miRNA） based on the strand displacement amplification（SDA） and catalytic hairpin assembly（CHA） cascading amplification strategy. Methods：Target miR-21 initiated SDA to obtain mass target-related DNA which could further trigger CHA to obtain mass biotin-labeled dsDNA. And then，the obtained biotin-labeled dsDNA was immobilized onto the surface of the electrode modified with auxiliary probe. Later，ST-AP was modified onto the electrode surface by the specific recognition of biotin and streptavidin. When the α-NPP was in the test buffer，α-NPP was catalyzed by ST-AP to in situ produce α-NP on the surface of the electrode. Finally，the electrochemical signal of α-NP was correlated with the concentration of miR-21. Results：Under the optimized experimental conditions，the prepared biosensor showed a linear response to miR-21 range from 0.1 pmol/L to 10 nmol/L with the limit of detection low to 60 fmol/L. Conclusion：The designed biosensor has been exhibited high sensitivity and excellent specificity，which may provide a new idea and new platform for clinical determination of miR-21.