Ultrasensitive supersandwich-type biosensor for enzyme-free amplified microRNA detection based on N-doped graphene/Au nanoparticles and hemin/G-quadruplexes

Abstract

A simple, enzyme-free supersandwich-type biosensor is fabricated for the ultrasensitive detection of microRNAs (miRNAs) using N-doped graphene/Au nanoparticles (NG–AuNPs) and hemin/G-quadruplexes. In the proposed strategy, AuNPs are deposited on the surface of a MoSe₂ modified electrode to immobilize the thiol-modified hairpin probe through the strong Au–S bond. When the target miRNA is added, capture DNA hybridizes with it and unfolds its stem-and-loop structure. The NG–AuNP hybrids are the main amplification element and are modified by hybridization with assistance DNA and the terminus of capture DNA, resulting in the formation of the supersandwich structure. The assistance DNA is embedded into the hemin/G-quadruplex complexes in the presence of hemin and K⁺ to provide an exceptional current signal for the detection of miRNAs. Under the optimized experimental conditions, a detection limit of 0.17 fM is obtained with a linear range of 10 fM–1 nM. In addition, the present biosensor shows outstanding selectivity towards mismatched miRNAs. This biosensor platform successfully realized the combination of the signal amplification technique with the supersandwich structure, providing a promising approach for the detection of miRNA-21 in practical applications.