Carbon quantum dot-based fluorescence quenching coupled with enzyme-assisted multiple cycle amplification for biosensing of miRNA

In this work, a new fluorescence biosensing platform based on carbon quantum dot (CQDs) quenching coupled with enzyme-assisted multiple cycle amplification was fabricated for miRNA detection. Single-stranded (ss) DNA labeled FAM was used as signal probe, and the small CQDs with good biocompatibility were used as both quenching agent and carrier for FAM, which resulted in fluorescence change. In the presence of target miRNA, the enzyme-assisted polymerization reaction was activated with the help of hairpin primer 1 (HP1), hairpin primer 2 (HP2) and driver primers (DP), forming two double-stranded DNA (dsDNA). Then double-cycle drivendsDNA nanostructure induced two successive amplification reactions, generating a large amount of ssDNA. When ssDNA hybridized with the FAM-probe and formed dsDNA, it went far away from the CQDs surface and led to the recovery of fluorescence for detection of miRNA. Using this enzyme-assisted multiple amplification strategy, the proposed fluorescence biosensor displays a wide linear range from 10 fM to 10 nM and the detection limit of 4.7 fM. This proposed biosensor shows great potential for early cancer diagnosis in clinic assay.