Antibodies directed to RNA/DNA hybrids: an electrochemical immunosensor for microRNAs detection using graphene-composite electrodes.

We report a simple and sensitive label-free immunosensor for detection of microRNAs (miRNA) based on a conducting polymer/reduced graphene oxide-modified electrode to detect miR-29b-1 and miR-141. Square wave voltammetry is used to record the redox signal. Current increases upon hybridization (signal on) from 1 fM to 1 nM of target miRNA. The limit of quantification is ca. 5 fM. The sensor exhibits high selectivity as it distinguishes mismatch. To double-check its selectivity, two specific RNA-DNA antibodies recognizing miRNA-DNA heteroduplexes, antipoly(A)-poly(dT) and anti-S9.6, were used. The antibody complexation with the hybrid leads to a current decrease that confirms the presence of miRNA, down to a concentration of 8 fM. The antibody-hybrid complex can be then dissociated by adding miRNA-DNA hybrids in solution, causing a shift-back on the signal, i.e., an increase in the current density (signal-on). This On-Off-On detection sequence was used as a triple verification to increase the reliability of the results.