Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-Assisted Catalyzed Hairpin Assembly and "DD-A" FRET
Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-Assisted Catalyzed Hairpin Assembly and "DD-A" FRET
复制标题
通过磁性微珠辅助催化发夹组装和“DD-A”FRET 检测复杂样品中的核酸
DOI：
10.1021/acs.analchem.8b01330
复制
发表时间：
2018-06-19
影响因子：
7.4
通讯作者：
Wang, Kemin
中科院分区：
文献类型：
--
作者：
Fang, Hongmei;Xie, Nuli;Wang, Kemin
核酸作为一种重要的生物标志物，在基础研究和临床应用中显示出巨大的价值，引起了人们的广泛关注。在这项工作中，我们开发了一种基于磁性微珠（MMB）辅助催化发夹组装（CHA）和供体供体-受体荧光共振能量转移（“DD-A”FRET）信号传导机制的用于检测复杂样品中核酸的荧光分析。在该系统中使用了三种类型的DNA发夹探针，包括Capture、H1（作为FRET供体的双FAM标记探针）和H2（作为FRET受体的TAMRA标记探针）。首先，将固定在MMB上的捕获物与复杂样品中的靶标结合，并暴露捕获物中可以触发催化发夹组装（CHA）的序列。然后，分离靶富集的MMB复合物并重悬于含有Hl和H2的反应缓冲液中。结果，在CHA过程中形成了大量的H1-H2双链体，诱导了明显的FRET信号。相反，CHA不能被触发，FRET信号弱，而靶不存在。借助磁分离和“DD-A”FRET，有效消除了背景干扰的误差。重要的是，该策略实现了缓冲液中的扩增检测，microRNA的检测限低至34 pM。该方法已成功应用于血清和细胞培养液中microRNA-21的检测。结果表明，我们的方法具有潜在的生物医学研究和临床应用。
Nucleic acids, as one kind of significant biomarker, have attracted tremendous attention and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and a donor donor-acceptor fluorescence resonance energy transfer ("DD-A" FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labeled probe as FRET donor), and H2 (TAMRA-labeled probe as FRET acceptor). First, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMB complexes were separated and resuspended in the reaction buffer containing HI and H2. As a result, numerous H1-H2 duplexes were formed during the CHA process, inducing an obvious FRET signal. In contrast, CHA could not be triggered, and the FRET signal was weak, while target was absent. With the aid of magnetic separation and "DD-A" FRET, errors from background interference were effectively eliminated. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application.