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 nonrepeatability 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.

Automated summary

This study presents a 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. Magnetic bead-assisted high-efficiency DNA nanocarrier (MHDN) is introduced to reduce interference and background signal. The system achieves high sensitivity for miRNA-155 detection through multi-sited rolling circle amplification and enzymatic cleavage of quenching probes.