An enzyme-free sensitive electrochemical microRNA-16 biosensor by applying a multiple signal amplification strategy based on Au/PPy–rGO nanocomposite as a substrate

In present study, a sensitive and effective electrochemical microRNA (miRNA) sensing platform is successfully developed by integrating gold nanoparticles/polypyrrole-reduced graphene oxide (Au/PPy-rGO), catalyzed hairpin assembly (CHA) and hybridization chain reaction (HCR) multiple signal amplification strategy. Firstly, Au/PPy-rGO was employed onto a bare GCE by electrodeposition that can greatly enhanced conductivity and effectively immobilize probes. Then, the thiolated capture probes (SH-CP) were self-assembled on the Au/PPy-rGO modified GCE via Au-S bond. The target miRNA triggered the dynamic assembly of the two hairpin substrates (H1 and H2), leading to the cyclicality of the target miRNA and the formation of H1-H2 complexes without the assistance of enzyme. Subsequently, the newly emerging DNA fragment of H2 triggered the HCR when a mixture solution (hairpins H3 and H4) and produced dsDNA polymers. Finally, a substantial amount of methylene blue (MB) as signal indicator was intercalated into the minor groove of the long dsDNA polymers to achieve detected electrochemical signal. The fabricated sensor is able to detect miRNA-16 (model target) with concentration range from 10 fM to 5 nM with a low detection limit (LOD) of 1.57 fM (S/N = 3). Current research suggests that the developed multiple signal amplification platform has a great potential for the applications in the field of biomedical research and clinical analysis.