Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Institute of Life Science and Green Development, Hebei University, Baoding, 071002, Hebei Province, PR China.
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Institute of Life Science and Green Development, Hebei University, Baoding, 071002, Hebei Province, PR China.
Talanta. 2020 Nov 1;219:121332. doi: 10.1016/j.talanta.2020.121332. Epub 2020 Jul 6.
A visualized assay for miRNAs detection has been developed in this work. The presented method is based on a combination of enzyme-free amplification cascades of catalyzed hairpin assembly (CHA) and hybridization chain reaction (HCR) and fluorescence quenching of dual-emission ratiometric fluorescent probes (RF probes). MiRNAs can efficiently initiate enzyme-free amplification reactions (CHA and HCR) and produce the long nicked dsDNAs with a lot of glucose oxidases (GOD) on the surface of dynabeads bridged by the GOD-labeled hairpin DNA probes. Hydrogen peroxide (HO) is generated by oxidation of glucose catalyzed by GOD, which can quench the outer green fluorescence without affecting the internal red fluorescence of RF probes. Therefore, increased miRNA amount can result in change of the two fluorescence intensity ratios of RF probes with continuous color changes from green to red under a UV lamp, which can be easily recognized by naked eye. The proposed assay exhibits high sensitivity toward let-7a with dynamic range from 10 M to 10 M, and which is applied successfully to detecting let-7a in the small RNA samples.
本工作开发了一种用于 miRNA 检测的可视化分析方法。所提出的方法基于无酶扩增级联反应（CHA）和杂交链式反应（HCR）的组合以及双发射比率荧光探针（RF 探针）的荧光猝灭。miRNA 可以有效地启动无酶扩增反应（CHA 和 HCR），并产生带有大量葡萄糖氧化酶（GOD）的长缺口双链 DNA，这些酶通过 GOD 标记的发夹 DNA 探针桥接在 dynabeads 上。由 GOD 催化的葡萄糖氧化产生过氧化氢（HO），可以猝灭外绿光而不影响 RF 探针的内红光。因此，随着 miRNA 数量的增加，RF 探针的两个荧光强度比会发生变化，在紫外灯下会发生连续的颜色从绿到红的变化，这可以通过肉眼轻松识别。该测定法对 let-7a 具有高灵敏度，动态范围为 10^-15 M 至 10^-10 M，并成功应用于检测小 RNA 样品中的 let-7a。
