A Label-Free Biosensor for Electrochemical Detection of Femtomolar MicroRNAs

A simple and ultrasensitive label-free microRNA (miRNA) biosensor, based on hybridized miRNA-templated deposition of an insulating polymer film and electrochemical impedance spectroscopic detection, is described in this report. The biosensor is made of a monolayer of charge-neutral morpholino capture probes on an indium tin oxide (ITO)-coated glass slide. Upon hybridization, the neutral surface of the biosensor is converted to anionic by the hybridized miRNA strands. The deposition of the insulating polymer film, poly(3,3'-dimethoxybenzidine) (PDB), is then carried out by the horseradish peroxidase-catalyzed polymerization of 3,3'-dimethoxybenzidine in the presence of H2O2. The cumulative nature of the PDB deposition process significantly enhances the sensitivity of the biosensor. Under optimized conditions, miRNA expression profiling can be performed label-freely from 5.0 fM to 2.0 pM with a detection limit of 2.0 fM. The biosensor is applied to the detection of circulating miRNAs in blood and miRNAs in total RNA extracted from cultured cells.