Ultra-sensitive detection of microRNA-21 based on duplex-specific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification
Ultra-sensitive detection of microRNA-21 based on duplex-specific nuclease-assisted target recycling and horseradish peroxidase cascading signal amplification
复制标题
基于双链体特异性核酸酶辅助靶标回收和辣根过氧化物酶级联信号放大的microRNA-21超灵敏检测
DOI：
10.1016/j.snb.2018.02.143
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发表时间：
2018-06-15
影响因子：
8.4
通讯作者：
Chang, Jin
中科院分区：
文献类型：
--
作者：
Peng, Weipan;Zhao, Qian;Chang, Jin
microRNA （miRNA）的检测和分析可以为生物医学研究和临床诊断提供重要信息。本文构建了一种基于双特异性核酸酶（DSN）辅助靶标恢复和辣根过氧化物酶（HRP）级联信号放大的简便方法，实现对miRNA的超灵敏快速检测。在靶microRNA-21 （miR-21）存在的情况下，DSN酶特异性剪切了与靶miRNA杂交的单链DNA (ssDNA)，但仍保持了靶miRNA的完整，因此聚苯乙烯微粒中由于靶循环和信号放大，释放了更多的游离HRP。收集上清中的游离HRP与底物3,3',5,5'-四甲基联苯胺（TMB）反应。HRP-TMB体系的颜色和吸光度变化进一步导致肉眼定性筛选和酶标仪定量检测，检出限低至3.1 fM。该检测方案不仅具有较高的灵敏度和选择性，而且能够很好地区分miRNA家族成员之间的差异，且操作简单、检测快速、肉眼定性和微孔板定量。在生物医学研究和临床诊断中具有重要的应用价值。(C) 2018 Elsevier B.V.版权所有
The detection and analysis of microRNA (miRNA) can provide critical information for biomedical research and clinical diagnosis. In this paper, we constructed a simple method to achieve ultra-sensitive and rapid detection of miRNA based on the double-specific nuclease (DSN) assisting target recovery and horseradish peroxidase (HRP) cascading signal amplification. In the existence of target microRNA-21 (miR-21), the DSN enzyme specifically cut the single-stranded DNA (ssDNA) hybridized with the target miRNA but still kept the target miRNA intact, so there were more free HRP released from the polystyrene microparticlesssDNA-HRP conjugates (PS-ssDNA-HRP) because of the target recycle and signal amplification. The free HRP in the supernatant were collected to react with the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The color and absorbance changes of HRP-TMB system further resulted in qualitative screening by naked eyes and quantitative detection by the microplate reader with the detection limit as low as 3.1 fM. This assay protocol not only showed high sensitivity and selectivity, but also well distinguished the difference between the miRNA family members, as well as the simple operation, fast detection, naked eye qualitative and microplate reader quantification. It has great application value in the biomedical research and clinical diagnosis. (C) 2018 Elsevier B.V. All rights reserved.