MicroRNAs play a crucial role in regulating gene expression and cellular function. Reliable detection of miRNA is highly demanded in clinical diagnosis and therapy. Herein, we designed a structure-convertible DNA switch and constructed a novel switch-conversional ratiometric fluorescence biosensor (SCRF biosensor) for highly sensitive miRNA detection by the use of amplicon fragments to convert the structure of the switch. The DNA switch was a sophisticated designed single-strand DNA with a stem-loop structure and modified with two fluorophores (Cy3 and Cy5) and one quencher at specific sites of the switch. Amplicon fragments (c*) were produced by an exponential amplification reaction. When the c* hybridized to the loop of a DNA switch, the structure of the switch would convert, and fluorescence resonance energy transfer occurred between Cy5 and Cy3. Then two fluorescence signals with different trends would be observed. As a result, by the ratio of the two signals, we can quantitatively and quickly detect the target miRNA with the concentration range from 100 fM to 100 nM and the excellent detection limit down to 70.9 fM, providing this new SCRF biosensor broad application prospects.
Keywords: Exponential amplification reaction; Förster resonance energy transfer; Ratiometric fluorescence biosensor; Switch conversion.
Copyright © 2020 Elsevier B.V. All rights reserved.