A circular crRNA-triggered CRISPR/Cas12a fluorescent platform for detection of myeloperoxidase activity

Myeloperoxidase (MPO) is a key enzymatic biomarker for the diagnosis and therapeutic monitoring of acute leukemia. Here, we developed a CRISPR/Cas12a-based fluorescent sensing platform using a circular phosphorothioate-modified crRNA (crRNA-PS) for selective MPO activity detection. In this system, MPO catalyzes hypochlorous acid (HClO) generation, which oxidizes and linearizes the circular crRNA-PS, activating Cas12a-mediated trans-cleavage of a fluorescent reporter. The assay exhibited high sensitivity with detection limits of 0.79 ng/mL for MPO and 0.21 μM for HClO, along with excellent selectivity against reactive oxygen/nitrogen species and biomolecular interferents. Reliable performance was achieved in 1 % fetal bovine serum with recoveries of 92.87-112.54 %. The sensor also maintained stable responses over 8 weeks, indicating strong structural integrity of the circular crRNA-PS. Furthermore, the system was applied for inhibitor screening, yielding IC₅₀ values of 127.2 μM for SHA and 0.81 μM for 4-ABAH. This strategy provides a rapid, sensitive, and robust platform for MPO detection with promising clinical potential.