A label-free microRNA biosensor based on DNAzyme-catalyzed and microRNA-guided formation of a thin insulating polymer film

Herein we report a label-free microRNA (miRNA) biosensor in which the formation of a thin insulating film is used to amplify the analytical signal. Briefly, the biosensor is made of an oligonucleotide-coated gold electrode. After hybridizing with a target miRNA, free capture probe (CP) strands on the biosensor are removed by a nuclease digestion. A second hybridization with an oligonucleotide-tailed DNAzyme is performed to introduce the DNAzyme to the biosensor. The DNAzyme triggers the polymerization of 3,3′-dimethoxybenzidine (DB) in the presence of H2O2 and the hybridized miRNA-CP duplexes serve as templates to guide the deposition of poly (3,3′-dimethoxybenzidine) (PDB). The formation of the insulating PDB film alters the impedance of the biosensor, rendering it readily distinguishable by electrochemical impedance measurements. The accumulative nature of the PDB deposition drastically improves the detectability of the biosensor. A proof-of-concept study is conducted on the detection of miRNAs in total RNA extracted from cultured cells.

► A PCR-free and label-free microRNA biosensor is developed.
► The accumulative nature of the deposition of a thin insulating film drastically improves the detectability of the biosensor.
► The amplifier—DNAzyme was found to be much more tolerant than natural enzymes in terms of reaction conditions.
► Attempts were made in detecting miRNAs in total RNA extracted from cancer cells.