Dual signal amplification strategy for specific detection of Circulating microRNAs based on Thioflavin T

Abstract

Circulating microRNAs (c-miRNAs) are emerging as new non-invasive biomarkers for human cancers diagnosis and prognosis. Herein, in this study, an ideal biosensing system with highly sensitivity and excellent selectivity for rapid and facile detection of c-miRNAs has been developed. The sensing system mainly consists of two unlabeled hairpin probes (HP1 and HP2) and a circular probe. Upon binding with the target miRNA, HP1 is opened, which serves as a toehold to hybridize with HP2. Since HP1-HP2 duplex is more stable than HP1-miRNA duplex, the target miRNA can be displaced from HP1, and again binds with a new HP1 to initiate another reaction cycle. Moreover, the newly formed HP1-HP2 duplex can be further used as a primer to initiate rolling circle amplification (RCA) with the circular probe, producing extremely long single-stranded DNA molecules with repetitive sequence units which can form into a large number of G-quadruplexes. These G-quadruplexes can bind with ThT, resulting in a significantly enhanced fluorescent signal. This newly developed sensing system has great potential to be applied in biochemical research and clinical diagnosis based on the high-performance of circulating miR-21 detection in this study.