The detection of miRNA shows great promise in disease diagnosis. In this work, a ratiometric fluorescent biosensor based on multi-walled carbon nanotubes@gold nanoclusters (MWCNTs@Au NCs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for miRNA detection. Colorectal cancer (CRC)-associated miR-92a-3p extracted from exosomes was selected as the target. MWCNTs@Au NCs performs the dual functions of fluorescence quencher and internal fluorescence reference. In the absence of miR-92a-3p, an Atto-425-modified single-stranded DNA probe is adsorbed on MWCNTs@Au NCs, resulting in the quenching of Atto-425. In the presence of miR-92a-3p, the duplex is formed by hybridization of the probe and miR-92a-3p and leaves the MWCNTs@Au NCs, resulting in the fluorescence recovery of Atto-425. DSN can cleave the probe and result in the release of miR-92a-3p. The released miR-92a-3p can hybridize with other probes to form a signal amplification cycle. The fluorescence of MWCNTs@Au NCs remains stable and constitutes a ratiometric fluorescence system with that of Atto-425. A detection concentration interval of 0.1-10 pM and a limit of detection of 31 fM was obtained under optimized measurement conditions. In addition, the accuracy of the biosensor was validated by detecting the concentration of miR-92a-3p extracted from clinical exosome samples.
Keywords: Au nanoclusters; biosensor; duplex-specific nuclease; exosomal miRNA; multi-walled carbon nanotubes; ratiometric fluorescence.