Proximity binding-initiated DNA walker and CRISPR/Cas12a reaction for dual signal amplification detection of thrombin

We report here a highly sensitive fluorescent thrombin biomarker sensing method by integrating the DNA walker and CRISPR/Cas12a system. The presence of thrombin causes the localization of DNA moving arms on AuNP tracks via their proximity bindings with the dye-labeled probes immobilized on AuNPs. With the assistance of the primer and DNA polymerase, the arm sequences move continuously on the AuNP tracks to generate many CRISPR/Cas12a-responsive dsDNAs, which push the dye to move away from AuNPs to restore its fluorescence. Moreover, the dsDNAs can be recognized and cut by the CRISPR/Cas12a to trigger its trans-cleavage activity for cyclically cleaving the fluorescently quenched signal probes on the AuNP tracks, which liberates the dye from AuNPs to further enhance the fluorescence restoration to achieve highly sensitive thrombin assay with detection limit of 29.5 fM. Selectively detecting thrombin against other interference proteins and in diluted serums by such sensing method has also been verified, making it an attractive approach for monitoring other protein biomarkers at low levels for the diagnosis of diseases.