▎ 摘　　要

This work demonstrated for the first time a simple and rapid approach to endow the electrode with the excellent discrimination ability over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) through the robust electrochemical grafting of in situ generated 1-naphthalenesulfonate (NS-) diazonium salt onto the surface of indium tin oxide (ITO) electrode. On the basis of understanding the influence of sequence and length on the binding affinity of ssDNA and dsDNA toward NS- grafted ITO (NS--ITO) electrode, these interesting findings were successfully employed to rationally develop a ratiometric homogeneous electrochemical biosensing platform for micro-RNA based on the affinity-mediated signal transduction. The achievement of ultrasensitive detection of microRNA lies in a compatibly designed T7 exonuclease-assisted isothermal amplification strategy, in which the presence of target microRNA initiated the continual and opposite affinity inversion of two rationally engineered electrochemical signal reporters, methylene blue (MB) labeled hairpin reporter and ferrocene (Fc) labeled dsDNA reporter, toward NS--ITO electrode, thereby providing the ratiometric transduction and amplification of the homogeneous electrochemical output signal. By measuring the distinct variation in the peak current intensity ratios of Fc and MB tags, this ratiometric homogeneous electrochemical microRNA biosensing platform showed a detection limit of 25 aM, which is much lower than that of the reported homogeneous electrochemical biosensors. Therefore, we envision that the proposed approach will find useful applications in disease molecular diagnoses and biomedicine.