Gold Nanoparticle-Based Enzyme-Assisted Cyclic Amplification for the Highly-Sensitive Detection of miRNA-21
Yang Qing, Yuxing Yang, Ping Ouyang, Chenxin Fang, Haobin Fang, Yazhen Liao, Haiyu Li, Zhencui Wang, Jie Du
Abstract
Because microRNAs (miRNAs) are biological indicators for the diagnosis, treatment, and monitoring of tumors, cancers, and other diseases, it is significant to develop a rapid, sensitive, and reliable miRNA detection platform. In this study, based on miRNA-21 detection, DNA-a with a 3′ end overhang and Texas Red fluorophore-labeled 5′ end was designed, which reacts with miRNA-21 and hybridizes with exonuclease III (Exo III), where the part connected to miRNA-21 is hydrolyzed, leaving a-DNA. At the same time, miRNA-21 is released to participate in the following reaction, to achieve cyclic amplification. a-DNA reacts with DNA-b conjugated to gold nanoparticles to achieve fluorescence quenching, with the quenching value denoted as F; additionally, after adding DNA-d and linked streptavidin immunomagnetic beads (SIBs), fluorescence recovery was achieved using DNA-c, with the recovered fluorescence recorded as F0. By comparing the difference in the fluorescence (F0 − F) between the two experiments, the amount of DNA-a hydrolyzed to produce a-DNA was established to determine the target miRNA-21 content. Under optimized conditions, by comparing the changes in the fluorescence signal, the developed strategy shows good sensitivity and repeatability, with a detection limit of 18 pM, good discriminative ability and selectivity, and promise for the early diagnosis of breast and intestinal cancers.