Target-initiated triplex signal amplification cascades for non-label and sensitive fluorescence sensing of microRNA

Abstract

The aberrant expression of microRNAs (miRs) in cells is closely linked to the initiation and progression of various diseases. Sensitive monitoring of their level is hence vital for biomedical research and disease diagnosis. Herein, a highly sensitive and non-label fluorescence sensor based on multiple recycling signal amplification cascades is constructed for the detection of miR-21 in human sera. The presence of miR-21 initiates the primer-fueled target recycling process for the generation of many primer/hairpin templates for the subsequent auto-cycling primer extension (APE) amplification cycles, which result in the formation of lots of long-stem hairpins. The enzyme-based cleavage of such hairpins via polymerization/excision cycles further leads to the generation of abundant G-quadruplex strands, which associate with the thioflavin T (ThT) dye to emit remarkably magnified fluorescence for detecting miR-21 in the range of 1 pM–100 nM with a 0.32 pM detection limit without labeling the probes. Besides, the proposed assay can selectively discriminate miR-21 against other control molecules and realize the sensing of low levels of miR-21 in diluted sera. With features of high sensitivity via the triplex signal amplification cycles and simplicity in a non-label homogeneous manner, our miR sensing protocol can be a robust means for detecting various nucleic acids for the early diagnosis of diseases.