An electrochemical biosensor for highly sensitive detection of microRNA-377 based on strand displacement amplification coupled with three-way junction

Abstract In this research, a novel electrochemical biosensing strategy has been developed for detection of microRNA (miRNA) by integrating strand displacement amplification (SDA) with three-way junction (TWJ). The target miRNA triggers the SDA cycle of polymerization, nicking and displacement reaction in the presence of the template hairpin structure containing the recognition sites for the nicking restriction enzyme and its downstream linker sequence. This reaction cycle is amplified by polymerization and cleavage reactions, producing large quantities of linkers. The linkers are employed in hybridization reaction between the TWJs and capture probes, which can transform the single signal readout into multiple signal output. Thus, the electrochemical signal is obtained by using streptavidin linked to alkaline phosphatase (ST-AP) toward the synthetic enzyme substrate α-naphthyl phosphate (α-NP). The proposed biosensing strategy exhibits a high sensitivity and specificity in the dynamic range of 1 fM to 1 nM with a low detection limit down to 0.68 fM and also achieves acceptable reproducibility for the determination of miRNA-377. This strategy presents a promising platform toward highly sensitive miRNAs detection in biomedicine and early clinical diagnostic application.