CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin

Background: Thrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited. Results: Here we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media. Significance: This assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.