Wang Qian, Li Ru-Dong, Yin Bin-Cheng, Ye Bang-Ce
Lab of Biosystem and Microanalysis, Biomedical Nanotechnology Center, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China.
Analyst. 2015 Sep 21;140(18):6306-12. doi: 10.1039/c5an01350j.
Developing simple and rapid methods for sequence-specific microRNA (miRNA) analysis is imperative to the miRNA study and use in clinical diagnosis. We have developed a colorimetric method for miRNA detection based on duplex-specific nuclease (DSN)-assisted signal amplification coupled to the aggregation of gold nanoparticles (AuNPs). The proposed method involves two processes: target-mediated probe digestion by a DSN enzyme and probe-triggered AuNP aggregation as a switch for signal output. The reaction system consists of a rationally designed probe complex formed by two partly complementary DNA probes, and two sets of different oligonucleotide-modified AuNPs with sequences complementary to a DNA probe in the probe complex. In the presence of target miRNA, the probe complex is invaded, resulting in the formation of a miRNA-probe heteroduplex as the substrate of the DSN enzyme, and releasing the other probe to link to the AuNPs. The proposed method allows quantitative detection of miR-122 in the range of 20 pM to 1 nM with a detection limit of ∼16 pM, and shows an excellent ability to discriminate single-base differences. Moreover, the detection assay can be applied to accurately quantify miR-122 in cancerous cell lysates which is in excellent agreement with the results from a commercial miRNA detection kit. This method is simple, cost-effective, highly selective, and free of dye label and separation procedures.
开发用于序列特异性微小RNA（miRNA）分析的简单快速方法对于miRNA研究及其在临床诊断中的应用至关重要。我们基于双链特异性核酸酶（DSN）辅助的信号放大与金纳米颗粒（AuNP）的聚集相结合，开发了一种用于miRNA检测的比色法。所提出的方法涉及两个过程：由DSN酶介导的靶标介导的探针消化以及作为信号输出开关的探针触发的AuNP聚集。反应体系由两个部分互补的DNA探针形成的合理设计的探针复合物以及两组与探针复合物中的DNA探针序列互补的不同寡核苷酸修饰的AuNP组成。在存在靶标miRNA的情况下，探针复合物被侵入，导致形成作为DSN酶底物的miRNA-探针异源双链体，并释放另一个探针以连接到AuNP。所提出的方法能够在20 pM至1 nM范围内对miR-122进行定量检测，检测限约为16 pM，并且显示出出色的区分单碱基差异的能力。此外，该检测方法可用于准确量化癌细胞裂解物中的miR-122，这与商业miRNA检测试剂盒的结果高度一致。该方法简单、经济高效、具有高度选择性，并且无需染料标记和分离程序。
