A simple and sensitive turn-off biosensor for miRNA-21 detection based on tyramine signal amplification

Abstract

The expression level of microRNA (miRNA) is strongly associated with a variety of diseases, and the development of new approaches for miRNA assay with high sensitivity will facilitate a deeper understanding of miRNA biological functions. Herein, a simple and sensitive turn-off biosensor for miRNA-21 detection based on tyramine signal amplification (TSA) was developed. In the absence of the target miRNA, the DNA capture probe on the surface of magnetic bead (MB) complementarily hybridizes with the DNA reporter probe; subsequently, a large amount of horseradish peroxidase (HRP) is bound by the biotin-streptavidin reaction; later, in the presence of hydrogen peroxide (H2O2), HRP catalyzes the deposition of biotinylated tyramine, and then the fluorescent phycoerythrin (PE) is successfully introduced via a biotin-streptavidin reaction, producing a robust fluorescence (FL) signal upon excitation. When the target miRNA is present, it competitively occupies the binding site of the DNA capture probes, leading to a decrease in the amount of the DNA reporter probes hybridized to the MB surface; subsequently, the effect of TSA is diminished, which ultimately leads to a weakened FL and generates the “turn-off” signal. This method utilizes the negative correlation between target miRNA concentration and FL intensity, which can achieve high sensitivity detection of miRNA with low detection limit. This method can be successfully applied to the determination of miRNA-21 in human lung cancer cells, which has great application potential for disease diagnosis.