Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide
Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide
复制标题
基于等温指数扩增和氧化石墨烯的灵敏 microRNA 检测的无标记荧光策略
DOI：
10.1016/j.talanta.2015.10.078
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发表时间：
2016-02-01
期刊：
影响因子：
6.1
通讯作者：
Li, Feng
中科院分区：
文献类型：
--
作者：
Li, Wei;Hou, Ting;Li, Feng
MicroRNAs （miRNAs）在许多生物过程中发挥着重要作用，已被认为是癌症诊断和治疗的潜在靶点和生物标志物。此外，为了应对miRNA体积小、易被酶消化等特性带来的巨大挑战，开发准确、灵敏、简单的miRNA检测方法具有重要意义。在此，我们通过结合等温指数扩增和SYBR Green I （SG）和氧化石墨烯（GO）的独特特性，开发了一种无标记荧光策略，用于敏感的miRNA检测，其中SG在插入双链DNA （dsDNAs）后显着增强荧光，并且GO选择性地吸附miRNA，单链DNA和SG，以保护miRNA免受酶切，并熄灭被吸附的SG的荧光。在目标miRNA存在的情况下，巧妙设计的发夹探针（HP）展开，随后发生聚合和链位移反应，启动目标再循环过程。新形成的dsDNAs随后被切割酶识别和切割，产生与目标miRNA序列相同的新DNA触发器，其与完整的HPs杂交以启动新的延伸反应。结果实现了靶miRNA的环状指数扩增，并在插入SG后形成了大量的dsDNAs，产生了显著增强的荧光。从而实现了灵敏、选择性的荧光miRNA检测，并获得了3 fM的检测限。此外，该方法还具有简单和低成本的优点，因为它避免了昂贵和繁琐的标记过程。因此，所提出的无标记荧光策略在mirna相关的临床实践和生化研究中具有很大的应用潜力。(C) 2015 Elsevier B.V.版权所有
MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. (C) 2015 Elsevier B.V. All rights reserved.