Sensitive detection of microRNAs using hemin/G-quadruplex concatamers as trace labels and RNA endonuclease-aided target recycling for amplification

In this work, a novel electrochemical biosensor based on endonuclease-aided target recycling and hybridization chain reaction (HCR) was constructed for the sensitive detection of microRNAs (miRNAs). To construct the biosensor, the hairpin-like capture probe (CP) which contained the complementary RNA sequence of target miRNA was immobilized on gold nanoparticles (nano-Au) modified electrode through AuS bond for the following hybridization. In the presence of target miRNA, a linear RNA-RNA hybrid duplex was formed instead of the hairpin structure, and ribonuclease A (RNase A) selectively cuts the RNA fragment of CP in hybrid duplex, leaving the miRNA strand intact for analyte recycling. Subsequently, the cleaved single-strand fragment on electrode surface could initiate HCR, which resulted in the hybridization reaction to form double-strand DNA concatamers on the electrode surface. Consequently, hemin could stack into the G-quadruplex-forming region and the hemin/G-quadruplex can be formed to give an amplified electrochemical signal by differential pulse voltammetry (DPV). Using microRNA-101 as a model, the resulting biosensor presented high sensitivity and a broad linear response from 1.0 × 10−13 to 1.0 × 10−8 M with the detection limit of 1.0 × 10−13 M under optimal assay conditions.