Ultrasensitive protein and exosome analysis based on a rolling circle amplification assisted-CRISPR/Cas12a strategy.

CRISPR/Cas12a system has attracted extensive concern in biosensing due to its high specificity and programmability. Nevertheless, existing Cas12a-based assays mainly focus on nucleic acid detection and have limitations in non-nucleic acid biomarker analysis. To broaden the application prospect of the CRISPR/Cas technology, a cascade Cas12a biosensing platform is reported by combining dual-functionalized gold nanoparticles (FGNPs)-assisted rolling circle amplification (RCA) and Cas12a trans-cleavage activity (GAR-Cas) for ultrasensitive protein and exosome analysis. FGNPs serve as a critical component in the transduction of protein or exosome recognition information into nucleic acid amplification events to produce Cas12a activators. In the GAR-Cas assay, by integrating the triple cascade amplification of FGNPs-assisted transduction, RCA, and Cas12a signal amplification, ultralow abundance of target molecules can arouse numerous concatemers to activate Cas12a trans-cleavage activity to release intense fluorescence, allowing the ultrasensitive detection of as low as 1 fg/mL (∼41 aM) cTnI and 5 exosomes per μL. Furthermore, the presented strategy can be applied to detect exosome levels from clinical samples, showing excellent performance in distinguishing cancer patients from healthy individuals. The GAR-Cas sensing platform exhibits great potential in clinical diagnosis and enlarges biosensing toolboxes based on CRISPR/Cas technology for non-nucleic acid target analysis.