Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
Biosens Bioelectron. 2016 Mar 15;77:995-1000. doi: 10.1016/j.bios.2015.10.082. Epub 2015 Oct 29.
Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA-Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe-miRNA-CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu(2+)). Cu(2+) could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA-Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu(2+), which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5 pM to 1000 pM with a detection limit of ~0.73 pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit.
开发直接且方便的 microRNAs（miRNAs）分析方法对于理解 miRNAs 的生物学功能和癌症的早期诊断具有重要意义。我们开发了一种基于纳米粒子辅助信号放大结合荧光金属纳米团簇作为信号输出的 miRNA 快速无酶检测方法。该方法包括两个过程：目标 miRNA 介导的纳米粒子捕获，由磁性微球（MMP）探针和 CuO 纳米粒子（NP）探针组成，以及纳米粒子介导的信号产生放大，由荧光 DNA-Cu/Ag 纳米团簇（NC）和 3-巯基丙酸（MPA）组成。在存在靶 miRNA 的情况下，MMP 探针和 NP 探针通过各自的互补序列夹心捕获靶 miRNA。所得的夹心复合物（MMP 探针-miRNA-CuO NP 探针）通过磁场分离，然后通过酸解进一步溶解，将 CuO NP 转化为大量的铜（II）离子（Cu2+）。Cu2+通过优先与 MPA 反应形成二硫化合物作为中间产物，破坏 MPA 的硫醇部分与荧光 Cu/Ag NCs 之间的相互作用。通过这种方式，在存在 MPA 的情况下，DNA-Cu/Ag NCs 的荧光发射随着 Cu2+浓度的增加而增加，这与靶 miRNA 的量成正比。该方法可在 5 pM 至 1000 pM 的范围内对肝特异性 miR-221-5p 进行定量检测，检测限约为 0.73 pM，并且具有很好的单碱基差异分辨能力。此外，该检测方法可用于检测癌细胞裂解物中的 miRNA，与商用 miRNA 检测试剂盒的检测结果非常吻合。
