Photoinduced Electron Transfer‐Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA
Photoinduced Electron Transfer‐Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA
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DOI：
10.1002/slct.201601485
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发表时间：
2016-12
期刊：
影响因子：
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通讯作者：
Fulin Zhou;R. Meng;Qiang Liu;Yan Jin;Baoxin Li
中科院分区：
文献类型：
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作者：
Fulin Zhou;R. Meng;Qiang Liu;Yan Jin;Baoxin Li
MicroRNAs(MiRNAs)的检测对于进一步了解miRNAs的生物学功能以及癌症的早期诊断和治疗是至关重要的，但现在仍然是一个巨大的挑战。在这里，我们提出了一种新的均相生物传感策略来检测miRNA的荧光。该策略以初步合成的环状DNA为识别探针，与miRNA靶标杂交，然后进行滚环扩增(RCA)。RCA产物可与数千个羧基荧光素(FAM)标记的线性DNA探针杂交，使FAM接近RCA产物的-GGG碱基，并伴随着FAM与鸟氨酸之间的光诱导电子转移(PET)而产生的显著荧光猝灭。这是首次将RCA和PET整合到一个检测过程中。由于RCA的高效扩增和PET信号的良好读出，该方法对靶miRNA具有很高的灵敏度，检测下限为6am靶标依赖的挂锁探针环化和连接反应可以有效地提高特异性，从而导致对miRNA家族成员的区分。该方法为miRNA的灵敏检测提供了一种简单、恒温、低成本的方法，为基因相关疾病的早期诊断提供了巨大的潜力。
MicroRNAs (miRNAs) detection is crucial for further understanding the biological functions of miRNAs and early cancer diagnosis and therapeutics, but now remains a great challenge. Here, we present a novel homogenous biosensing strategy for fluorescence detection of miRNA. In this strategy, a preliminary synthesized circular DNA was used as recognition probe for hybridization with miRNA target, and then miRNA target primed a rolling circle amplification (RCA) reaction. The RCA product could be hybridized with thousands of carboxyfluorescein (FAM)-labeled linear DNA probes, which led FAM to be close to -GGG- base of RCA product, accompanying with the significant fluorescence quenching due to photoinduced electron transfer (PET) between FAM and guanine. This is for the first time to integrate RCA and PET into one detection process. With highly efficient amplification of RCA and excellent signal readout of PET, this method exhibited a high sensitivity toward target miRNA with a detection limit of 6 aM. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to discrimination between miRNA family members. This method provides a simple, isothermal, and low-cost approach for sensitive detection of miRNA and holds great potential for early diagnosis in gene-related diseases.