Xu Huo, Lin Yongju, Sun Lijun, Fang Xiaojun, Jia Lee
Institute of Oceanography, Minjiang University, Fuzhou, Fujian, 350108, China.
Institute of Oceanography, Minjiang University, Fuzhou, Fujian, 350108, China.
Talanta. 2020 Nov 1;219:121302. doi: 10.1016/j.talanta.2020.121302. Epub 2020 Jun 26.
Extremely sensitive and visual measurements of microRNA (miRNA) in situ for early detection and monitoring of diseases remains a major challenge. To address this issue, this work reports a rapid, highly sensitive and selective microRNA (miRNA) biosensing strategy based on isothermal circular strand-displacement polymerization (ICSDP), and miRNA imaging was performed inside cells. In this work, a double hairpin DNA probe (HP1/HP2 complex) embedded with a sensing region and polymerase primer region was designed. Briefly, after the specific binding of target miRNA with the HP1/HP2 probe, HP1/HP2 itself can function as a primer to initiate the ICSDP with the help of Klenow Fragment (KF), yielding target miRNA for new rounds of ICSDP. In this process, one target can produce multiple signal outputs (1: n), achieving low abundance of miRNA detection. Under optimized conditions, the proposed strategy showed high sensitivity with a detection limit of 5 pM within 15 min and can also easily distinguish the control miRNA from the target miRNA. This method can be further applied to image the intracellular miRNA of interest in situ inside the cancer cells.
对微小RNA（miRNA）进行极其灵敏且可视化的原位测量以实现疾病的早期检测和监测仍然是一项重大挑战。为解决这一问题，本研究报道了一种基于等温环状链置换聚合（ICSDP）的快速、高灵敏且选择性的微小RNA（miRNA）生物传感策略，并在细胞内进行了miRNA成像。在本研究中，设计了一种嵌入传感区域和聚合酶引物区域的双发夹DNA探针（HP1/HP2复合物）。简要来说，目标miRNA与HP1/HP2探针特异性结合后，HP1/HP2自身可作为引物，在klenow片段（KF）的帮助下启动ICSDP，产生用于新一轮ICSDP的目标miRNA。在此过程中，一个目标可产生多个信号输出（1:n），实现对低丰度miRNA的检测。在优化条件下，所提出的策略显示出高灵敏度，15分钟内检测限为5 pM，并且还能轻松区分对照miRNA和目标miRNA。该方法可进一步应用于原位成像癌细胞内感兴趣的细胞内miRNA。
