Development of selective NBD-based fluorescent probes for in vitro detection of factor XIa and Thrombus imaging

Factor XIa (FXIa) is a promising target for safer anticoagulants. Herein, a series of NBD-based fluorescent probes (FNC2-FNC6) were developed by linking an FXIa inhibitor scaffold to an NBD fluorophore via alkyl chains of varying lengths. Molecular dynamics simulations revealed that shorter linkers, especially a two‑carbon chain, stabilize NBD binding in the FXIa pocket, enhancing fluorescence. Among these probes, FNC2 displayed potent FXIa inhibition (Ki = 14.2 nM) and selectivity over related proteases. It exhibited linear fluorescence increase upon FXIa binding (R² = 0.9952; LOD = 1.19 μg/mL), enabling accurate quantification in immunoglobulin products (recovery: 94.41%-103.76%; RSD < 2%). FNC2 visualized FXIa in human plasma clots with 2.7-fold higher signal than free NBD, suppressed dose-dependently by asundexian. This structure-guided approach yields selective probes for FXIa detection and thrombus imaging.