DNA strand displacement-based sensor for the detection of miRNA-141 in serum samples

Abstract

MicroRNA (miRNA) expression is commonly dysregulated in various diseases. Our aim was to develop a simple and sensitive fluorescence-based assay for detecting microRNA-141 (miR-141) activity using DNA strand displacement. In this approach, one strand of the DNA probe (P1) is labeled with a BHQ molecule at its 3’-end, while the complementary strand (P2) is labeled with a FAM molecule at its 5’-end. When P1 and P2 hybridize to form a duplex DNA probe (P1/P2), no fluorescence signal is observed (“off” state). Upon introduction of miR-141 target, the P1/P2 probe undergoes displacement based on the toehold-mediated strand displacement reaction (TMSD), resulting in the formation of a new double-stranded complex between miRNA and P1. This displacement releases a significant amount of single-stranded FAM-labeled nucleotides (P2), resulting in a robust fluorescence signal (“on” state) that enabling target detection. The assay achieved highly detection of miRNA with a detection limit (LOD) of 0.05 nM. Furthermore, in human serum samples, the sensor exhibited a satisfactory recovery rate ranging from 91.73% to 107.00%, with relative standard deviations (RSD) between 0.24% and 0.69%. These findings underscore its potential as a promising diagnostic tool for cancer.