Dual signal amplification strategy for enzyme-free electrochemical detection of microRNAs

The combination of catalyzed hairpin assembly reaction (CHA) and hybridization chain reaction (HCR) is introduced to develop an electrochemical biosensor without the assistance of enzyme for highly sensitive microRNA (miRNA) detection. Firstly, the hairpin-shaped capture probe H1 immobilized on the electrode surface was opened by target. In the presence of another hairpin probe H2, hybridization of H1 to H2 resulted in the release of target from H1-target complex by strand-displacement reaction. The released target further hybridized with the remaining capture probe H1. After the target recycling process, H1-H2 complex was achieved with an exposed stem of H2. Then, the exposed stem of H2 served as initiator to trigger HCR event, yielding long double strands (dsDNA) molecule. Ultimately, numerous methylene blue (MB) as redox probes intercalated into the minor groove of the long dsDNA polymers to achieve amplified electrochemical signal. The proposed miRNA biosensor achieved a linear range from 10 fM to 1 nM with a wide dynamic range of six orders of magnitude.