Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, People's Republic of China.
Mikrochim Acta. 2019 Sep 5;186(10):669. doi: 10.1007/s00604-019-3789-7.
A highly sensitive fluorometric method is described for the determination of let-7a microRNA. It is based on the use of target-triggered cascade signal amplification that involves the use of (a) catalytic hairpin assembly (CHA), (b) exonuclease-assisted signal amplification (EASA), and (c) DNA-templated fluorescent silver nanoclusters (DNA-AgNCs) having excitation/emission maxima at 535/616 nm. The CHA reaction is initiated by the hybridization of the microRNA with hairpin probe HP1. The opening of HP1 results in the assembly of HP1 and another hairpin probe (HP2) to form a duplex. This releases the microRNA that which initiates another CHA reaction. The HP1-HP2 duplex binds to hairpin probe HP3 to form a stable Y-shaped junction structure HP2-HP1-HP3. The Y-shaped junction structure is cleaved by λ exonuclease to recycle the HP1-HP2 duplex to initiate the EASA reaction. This generates a large number of single-stranded reporter sequences. These act as scaffolds for the synthesis of fluorescent AgNCs by reduction of Ag (I) ions. A remarkably amplified fluorescent signal is observed whose intensity increases linearly in the 1 fM to 10 nM let-7a microRNA concentration range. The detection limit is 0.89 fM. The method can well discriminate let-7a microRNA from other microRNAs of the same family. Graphical Abstract Schematic representation of a fluorometric method based on target-triggered cascade signal amplification and DNA-templated silver nanoclusters (DNA-AgNCs) for sensitive detection of microRNA (miRNA).
一种高灵敏度的荧光测定法被用来测定 let-7a 微 RNA。该方法基于目标触发级联信号放大的原理，涉及到（a）催化发夹组装（CHA），（b）外切酶辅助信号放大（EASA）和（c）基于 DNA 的荧光银纳米簇（DNA-AgNCs）的使用，其激发/发射最大值为 535/616nm。CHA 反应是由微 RNA 与发夹探针 HP1 的杂交引发的。HP1 的打开导致 HP1 和另一个发夹探针（HP2）组装成双链。这释放了微 RNA，它引发了另一个 CHA 反应。HP1-HP2 双链与发夹探针 HP3 结合形成稳定的 Y 型连接结构 HP2-HP1-HP3。Y 型连接结构被 λ 外切酶切割，以回收 HP1-HP2 双链，引发 EASA 反应。这产生了大量的单链报告序列。这些序列作为银纳米簇（AgNCs）的合成模板，通过还原 Ag（I）离子。观察到一个显著放大的荧光信号，其强度在 1 fM 到 10 nM let-7a 微 RNA 浓度范围内呈线性增加。检测限为 0.89 fM。该方法可以很好地区分 let-7a 微 RNA 与同一家族的其他微 RNA。
