CRISPR/Cas12a System coupling polyfluorene nanoreporter enriched by magnetic bead-based high-efficiency DNA nanocarrier for fluorescence analysis

Currently reported CRISPR/Cas12a-based fluorescence systems are usually constructed with fluorescent dyes as reporter probes, which are performed the detection in homogeneous solutions. The instability and non-repeatability of labeled fluorescent reporters and the interference of complex components in homogeneous detection systems couldn't be ignored. This work exploited a novel CRISPR/Cas12a-based fluorescence system using carboxyl-functionalized poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alt-2,7-(9,9-dioctylfluorene)] nanoparticles (c-PFN NPs) as fluorescence donor and Au-polydopamine-Au nanoparticles (Au-PDA-Au NPs) as fluorescent acceptor. Meanwhile, magnetic bead-assisted high-efficiency DNA nanocarrier (MHDN) was introduced to enrich and separate the reporter probes to effectively reduce the interference and background signal. Firstly, c-PFN NPs were covalently bonded with S2 and the resulting S2-PFN hybridized with MHDN to form S2-PFN-MHDN. The presence of miRNA-155 would trigger multi-sited rolling circle amplification (MRCA) reaction to output second targets (ST) in multiple directions, further activating CRISPR/Cas12a to cleave S3 in the quenching probe. The digested S3-Au-PDA-Au cannot hybridize with S2-PFN-MHDN, leading to an obviously increased fluorescence signal. The MRCA-mediated CRISPR/Cas12a system endowed the fluorescence system with high sensitivity and the limit of detection for miRNA-155 was 170 aM. More importantly, fluorescent reporter c-PFN NPs coupling MHDN overcame drawbacks of labeled fluorescence reporters and homogeneous detection, creating an attractive CRISPR/Cas12a-based fluorescence system for bioanalysis.