Herein, using a novel bifunctional DNA-hybridization-DNA (DHD) probe, a symmetric DNA polymerase/NEase-assisted target signal amplification reaction into a single probe coupled with dual “light-up” colored silver nanoclusters (AgNCs) was introduced for label-free simultaneous detection of multiple microRNAs (miRNAs) for the first time. The DHD probe contained two single-stranded loops complementary to two different miRNA targets. Upon the recognition of the specific target miRNAs (miR-21 or miR-141), a symmetric polymerase/NEase-assisted signal amplification reaction was initiated, which generated massive triggers (trigger 1 and trigger 2). Subsequently, the released triggers hybridized with two different dark AgNCs and formed a partial double-stranded structure; thus, “light-up” red or yellow fluorescence readout was observed. The established biosensing approach was capable of simultaneously detecting multiple miRNAs with a limit of detection (LOD) down to 9.8/6.1 pM for miR-21/miR-141, respectively. This innovative approach displayed acceptable reproducibility and high specificity. Furthermore, the “AND” logic gate was activated by combining the dual-functional DHD probe with the dual “light-up” colored AgNCs, with/without miRNAs as inputs.

在本文中，使用新型的双功能DNA杂交DNA（DHD）探针，将对称DNA聚合酶/ NEase辅助的目标信号扩增反应引入与双“亮”色的银纳米簇（AgNC）耦合的单个探针中首次免费同时检测多个microRNA（miRNA）。DHD探针包含两个与两个不同miRNA靶标互补的单链环。识别出特定的靶标miRNA（miR-21或miR-141）后，就启动了对称聚合酶/ NEase辅助信号放大反应，产生了大量的触发信号（触发信号1和触发信号2）。随后，释放的触发器与两种不同的深色AgNC杂交并形成部分双链结构。因此，观察到“点亮”的红色或黄色荧光读数。建立的生物传感方法能够同时检测多种miRNA，而miR-21 / miR-141的检测限（LOD）分别为9.8 / 6.1 pM。这种创新方法显示出可接受的重现性和高特异性。此外，通过将双功能DHD探针与双色“点亮”彩色AgNC结合使用，可以将“ AND”逻辑门激活，无论是否输入miRNA。