Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062 China.
Biosens Bioelectron. 2017 Jan 15;87:1071-1076. doi: 10.1016/j.bios.2016.08.038. Epub 2016 Aug 13.
Human telomerase RNA (hTR), works as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes, is overexpressed in tumor cells and its concentration has a positive correlation with telomerase activity. The lack of facile and reliable method for detection of hTR in complex matric limited its application for clinical diagnosis. To address the limitation, herein, we proposed a facile and reliable flow cytometric assay for sensitive and specific detection of hTR by combing magnetic enrichment with signal amplification of DNA toehold strand displacement reaction (TSDR). Two hairpin DNA probes of TSDR are ingeniously designed, including biotinylated hairpin DNA1 (H1) and carboxyfluorescein (FAM)-labeled hairpin DNA2 (F-H2). Firstly, H1 was immobilized on streptavidin-functionalized magnetic beads (STV-MBs) through biotin-avidin interaction. In the presence of hTR DNA, TSDR between H1 and F-H2 was triggered to continuously form H1/H2 duplex, resulting in a "turn on" fluorescence on the surface of MBs. Due to fluorescence amplification of TSDR and magnetic enrichment, hTR-DNA can be sensitively, specifically and facile analyzed by flow cytometry and fluorescence microscopy imaging. The detection limit of flow cytometry is 0.3pM, which is superior to those of most existing approaches. Moreover, the proposed strategy can be successfully utilized to detect hTR in complex biological media as well. Therefore, an enzyme-free amplification approach is provided for robust and rapid detecting hTR DNA, which offers a facile, reliable and sensitive method for studying disease-related gene.
人端粒酶 RNA（hTR）作为线性真核染色体末端端粒 DNA 重复合成的模板，在肿瘤细胞中过度表达，其浓度与端粒酶活性呈正相关。由于缺乏检测复杂基质中 hTR 的简便可靠方法，其在临床诊断中的应用受到限制。为了解决这一限制，我们提出了一种简便可靠的流式细胞术检测方法，通过将磁富集与 DNA 触发链位移反应（TSDR）的信号放大相结合，来灵敏和特异地检测 hTR。巧妙地设计了两个 TSDR 的发夹 DNA 探针，包括生物素化发夹 DNA1（H1）和羧基荧光素（FAM）标记的发夹 DNA2（F-H2）。首先，H1 通过生物素-亲和素相互作用固定在链霉亲和素功能化的磁性珠（STV-MBs）上。在 hTR DNA 存在的情况下，H1 和 F-H2 之间的 TSDR 被触发，以连续形成 H1/H2 双链，导致 MBs 表面的荧光“开启”。由于 TSDR 的荧光放大和磁富集，hTR-DNA 可以通过流式细胞术和荧光显微镜成像进行灵敏、特异和简便的分析。流式细胞术的检测限为 0.3pM，优于大多数现有方法。此外，该策略还可成功用于检测复杂生物介质中的 hTR。因此，为 hTR DNA 的稳健和快速检测提供了一种无酶扩增方法，为研究疾病相关基因提供了一种简便、可靠和灵敏的方法。
