Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R. China.
Key Laboratory of Luminescent and Real-Time Analytical System (Southwest University), Chongqing Science and Technology Bureau, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China.
Anal Chem. 2021 Nov 2;93(43):14545-14551. doi: 10.1021/acs.analchem.1c03544. Epub 2021 Oct 24.
MicroRNAs (miRNAs) are found in extremely low concentrations in cells, so highly sensitive quantitation is a great challenge. Herein, a simple dual-amplification strategy involving target-activated catalytic hairpin assembly (CHA) coupled with multiple fluorophores concentrated on one X-shaped DNA is reported. In this strategy, four hairpin probes (H1, H2, H3, and H4) are modified with FAM and BHQ1 at both sticky ends, while a circulating hairpin probe (H0) is used to activate CHA circuits once it binds to complementary sequences in the target miR-21 (T). The powerful dual-amplification cascades in Förster resonance energy transfer (FRET)-based nonenzymatic nucleic acid circuits are triggered by T-H0-activated formation of the X-shaped DNA nanostructure, freeing T-H0 for the next CHA reaction cycle. CHA circuits increase the fluorescence due to the wide distance between FAM and BHQ1 in the formed X-shaped DNA nanostructure, resulting in signal amplification and highly sensitive detection of miR-21, with a limit of detection (LOD, 3σ) of 0.025 nM, which is 25.6 or 57.6 times lower than that obtained through a single-amplification strategy without multiple fluorophores on one X-shaped DNA or CHA circuit. Furthermore, this cascade reaction was completed in 45 min, effectively avoiding target degradation. This new enzyme-free signal amplification strategy holds promising potential for sensitively detecting different DNA or RNA sequences by simply adapting the fragment of the H0 sequence complementary to the target.
微小 RNA（miRNAs）在细胞中的浓度极低，因此高度敏感的定量分析是一个巨大的挑战。在此，我们报道了一种简单的双重扩增策略，涉及靶标激活的催化发夹组装（CHA）与集中在一个 X 形 DNA 上的多个荧光团的结合。在该策略中，四个发夹探针（H1、H2、H3 和 H4）在粘性末端修饰有 FAM 和 BHQ1，而循环发夹探针（H0）在与靶标 miR-21（T）的互补序列结合后被用于激活 CHA 电路。基于 FRET 的非酶核酸电路中的强大双重扩增级联反应是由 T-H0 激活形成 X 形 DNA 纳米结构触发的，释放 T-H0 以进行下一个 CHA 反应循环。CHA 电路由于在形成的 X 形 DNA 纳米结构中 FAM 和 BHQ1 之间的宽距离而增加了荧光，从而实现了 miR-21 的信号放大和高灵敏度检测，其检测限（LOD，3σ）为 0.025 nM，比没有多个荧光团的单一扩增策略低 25.6 或 57.6 倍在一个 X 形 DNA 或 CHA 电路上。此外，该级联反应在 45 分钟内完成，有效地避免了靶标降解。这种新的无酶信号放大策略通过简单地适应与靶标互补的 H0 序列片段，具有通过简单地适应与靶标互补的 H0 序列片段来灵敏地检测不同 DNA 或 RNA 序列的巨大潜力。
