Electrochemiluminescence biosensor for miRNA-21 based on toehold-mediated strand displacement amplification with Ru(phen)32+ loaded DNA nanoclews as signal tags

A novel electrochemiluminescence (ECL) biosensor was developed for high sensitive and selective detection of miRNA-21 based on the efficient and specific toehold-mediated strand displacement (TMSD) amplification with Ru(phen)(3)(2+) loaded DNA nanoclews (NCs-Ru(phen)(3)(2+)) as signal tags. The stable DNA nanoclews, synthesized by a simple rolling circle amplification reaction, were employed to load with Ru(phen)(3)(2+) efficiently as ECL signal tags to amplify the signals. As for TMSD, the substrate strand (Sub) was initially hybridized with P1 and P2 to form DNA duplex structures with a toehold 1. miRNA-21 could hybridize with the toehold 1 and trigger the TMSD amplification with the help of assist strand, releasing lots of P1 stands from DNA duplex structures. The TMSD technique realized the converting and amplification of the single miRNA-21 input to lots of output DNA (namely P1) with good selectivity, simultaneously. Output P1 were designed to expand the stem-locked region of HP, which were immobilized on the Au electrodes firstly. Subsequently, the opened HP could hybridize with the Ru(phen)(3)(2+), capturing the ECL signal tags closed to the sensing surface. The ECL intensity of the system had a linear relationship with the logarithm of the miRNA-21 concentration in the range of 1.0 fM to 100 pM with a limit of detection of 0.65 fM. The strategy was further applied to detect miRNA-21 in complex samples, and the result was consistent with the qRT-PCR.