Electrochemical\nBiosensing Platform Based on Toehold-Mediated\nStrand Displacement Reaction and DSN Enzyme-Assisted Amplification\nfor Two-Target Detection

Simultaneous\ndetection of multiple targets is of great significance\nfor accurate disease diagnosis. Herein, based on duplex-specific nuclease\n(DSN) assisted signal amplification and the toehold-mediated strand\ndisplacement reaction (TSDR), we constructed an electrochemical biosensor\nwith high sensitivity and high specificity for dual-target detection.\nMiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc)\nand methylene blue (MB) with significant peak potential differentiation\nwere used as the electrochemical signal probes. The elaborately designed\nhairpin probe H1, which was fixed on the electrode surface, could\nbe hybridized with the target miRNA-141 to perform signal amplification\nby the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be\ndetected by Fc signal changes at 0.41 V. The hairpin H1 can also combine\nwith the MB-labeled signal probe (SP) output from miRNA-133a-induced\nTSDR, and the detection of miRNA-133a can be realized according to\nthe response signal generated by MB at −0.26 V. The two sensing\nlines are independent of each other, and there is no mutual interference\nin the detection process. Therefore, two independent detection lines\ncould be connected in series, and the simultaneous detection of two\ntargets can be achieved on a single electrode. This novel detection\nstrategy provides a new way to simultaneously detect different biomarkers.