Ratiometric Electrochemiluminescence/Electrochemistry Sensor for miRNA Based on Toehold-Mediated Strand Displacement Amplification

A simple and highly sensitive method for detection of miRNA was constructed by combining the high-efficiency thiolated-mediated strand displacement (TMSD) non-enzymatic amplification and the high -reproducibility electrochemiluminescence/electrochemistry (ECL/EC) ratio sensing technique. The EC signal generated by methylene blue (MB) modified on the electrode was used as the internal reference signal, and Ru(phen)(3)(2+) was embedded in DNA nanoparticles produced by the branched hybridization chain reaction as the ECL probe. The presence of target miRNA facilitated the detachment of MB from electrode surface and the modification of ECL beacon on the electrode, resulting in a decrease of the EC signal and an enhancement of the ECL signal. The method of using the ratio of ECL signal to EC signal reduced potential interference and improved reproducibility and sensitivity of the biosensor. The ratio of ECL/EC signals exhibited linear relationship with miRNA-21 concentration in the range from 10 fmol/L to 500 pmol/L, with a detection limit of 6.5 fmol/L, and the relative standard deviation of the assay was reduced from 8.2 % to 4.1 % when determining 1 pmol/L target miRNA-21 compared to that using single ECL signal. This method was preliminarily applied to the detection of miRNA-21 in complex cell samples.