School of Chemical Engineering, Shandong Institute of Petroleum and Chemical Technology, Dongying 257061, P. R. China.
School of Nursing, Shandong Shengli Vocational College, Dongying 257061, P. R. China.
Analyst. 2024 May 13;149(10):2925-2931. doi: 10.1039/d4an00277f.
Sensitive detection of microRNA (miRNA), one of the most promising biomarkers, plays crucial roles in cancer diagnosis. However, the low expression level of miRNA makes it extremely urgent to develop ultrasensitive and highly selective strategies for quantification of miRNA. Herein, a DNA machine is rationally constructed for amplified detection and imaging of low-abundance miRNA in living cells based on the toehold-mediated strand displacement reaction (TMSDR). The isothermal and enzyme-free DNA machine with low background leakage is fabricated by integrating two DNA circuits into a cascade system, in which the output of one circuit serves as the input of the other one. Once the DNA machine is transfected into breast cancer cells, the overexpressed miRNA-203 initiates the first-layer circuit through TMSDR, leading to the concentration variation of fuel strands, which further influences the assembly of hairpin DNA in the second-layer circuit and the occurrence of fluorescence resonance energy transfer (FRET) for fluorescence imaging. Benefiting from the cascade of the two-layer amplification reaction, the proposed DNA machine acquires a detection limit down to 4 fM for quantification of miR-203 and a 10 000-fold improvement in amplification efficiency over the single circuit. Therefore, the two-layer circuit cascade-based DNA machine provides an effective platform for amplified analysis of low-abundance miRNA with high sensitivity, which holds great promise in biomedical and clinical research.
灵敏检测 microRNA（miRNA）作为最有前途的生物标志物之一，在癌症诊断中起着至关重要的作用。然而，miRNA 的表达水平较低，因此迫切需要开发超灵敏和高选择性的策略来定量检测 miRNA。本文基于引发链置换反应（TMSDR），合理构建了一种 DNA 机器，用于在活细胞中对低丰度 miRNA 进行放大检测和成像。该 DNA 机器具有等温、无酶和低背景泄漏的特点，是通过将两个 DNA 电路集成到级联系统中而构建的，其中一个电路的输出作为另一个电路的输入。一旦将 DNA 机器转染到乳腺癌细胞中，过表达的 miRNA-203 通过 TMSDR 启动第一层电路，导致燃料链的浓度发生变化，这进一步影响第二层电路中发夹 DNA 的组装以及荧光共振能量转移（FRET）的发生，从而进行荧光成像。得益于两层放大反应的级联，所提出的 DNA 机器的检测限低至 4 fM，用于定量检测 miR-203，并且与单个电路相比，放大效率提高了 10000 倍。因此，基于两层电路级联的 DNA 机器为高灵敏度的低丰度 miRNA 的放大分析提供了一个有效的平台，在生物医学和临床研究中具有广阔的应用前景。
