State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, 410082, PR China.
Hunan Key Lab of Biomedical Materials and Devices, College of Life Sciences and Chemistry, Hunan University of Technology, Zhuzhou, 412008, PR China.
Talanta. 2022 Apr 1;240:123169. doi: 10.1016/j.talanta.2021.123169. Epub 2021 Dec 22.
Herein, a label free and sensitive miRNA detection method with enhanced practical applicability was developed based on the locked nucleic acid (LNA) assisted repeated fishing amplification strategy. The working mechanism of the proposed method is as follows: 1) a DNA probe (i.e, L-DNA) with LNA bases is immobilized onto the surface of a gold foil. The L-DNA hybridizes with the 3' terminus of the first strands of complementary deoxyribonucleic acid (cDNA) of the target miRNA in the test samples; 2) The protruding 5' terminus of the cDNA serves as a 'fishhook' to repeatedly fish the products of a hybridization chain reaction (HCR) out from a 'reaction tube'; 3) The HCR products can be unloaded from the gold foil into a 'product tube' through temperature-controlled dehybridization; 4) The concentration of the target miRNA is determined based on the fluorescence intensity generated by the addition of SYBR-Green I (SG) into the 'product tube'. The proposed platform was applied to the detection of miRNA-122 in cell lysate samples and obtained quantitative results with accuracy comparable to the quantitative reverse transcription PCR method (qRT-PCR). It is worth pointing out that the proposed platform achieved a limit of detection value of 2.9 fM for miRNA-122 by a simple but effective LNA-assisted repeated fishing amplification strategy instead of complicated enzyme-based amplification techniques. It is reasonable to expect that the proposed method provides a competitive alternative for designing practically applicable, cost-effective and label-free miRNA detection methods.
在此，我们基于锁核酸（LNA）辅助重复钓取扩增策略开发了一种无标记且灵敏的 miRNA 检测方法，该方法具有增强的实际适用性。该方法的工作原理如下：1）将带有 LNA 碱基的 DNA 探针（即 L-DNA）固定在金箔表面。L-DNA 与测试样品中目标 miRNA 的第一链互补脱氧核糖核酸（cDNA）的 3' 末端杂交；2）cDNA 的突出 5' 末端充当“鱼钩”，将杂交链式反应（HCR）的产物从“反应管”中反复钓出；3）通过控温解杂交，将 HCR 产物从金箔转移到“产物管”中；4）通过向“产物管”中加入 SYBR-Green I（SG）来测定目标 miRNA 的浓度，基于生成的荧光强度进行定量分析。该平台被应用于细胞裂解物样品中 miRNA-122 的检测，并获得了与定量逆转录 PCR 方法（qRT-PCR）相当的定量结果。值得指出的是，该平台通过简单而有效的 LNA 辅助重复钓取扩增策略，而非复杂的基于酶的扩增技术，实现了对 miRNA-122 的检测限达到 2.9 fM。可以合理地预期，该方法为设计实际可行、经济高效且无标记的 miRNA 检测方法提供了一种有竞争力的替代方案。
