Open-tubular affinity capillary electrochromatography with MOF-caged thrombin: Effective mobility ratio strategy for anticoagulant screening in complex systems.

Affinity capillary electrophoresis (ACE) is a powerful tool for drug-protein interaction screening, but conventional ACE suffers from the narrow dissociation constant (Kd) detection range (10-3-10-5 mol L-1). To overcome this limitation, we developed an affinity capillary electrochromatography (ACEC) platform based on thrombin immobilized within meso-MOF-1 cages (Thr/meso-MOF-1@capillary). Via the effective mobility ratio strategy, the method accurately determined the Kd of benzamidine hydrochloride (19 μM) and successfully distinguished the interaction strengths of argatroban, benzamidine hydrochloride, and four flavonoids with thrombin. The experimental results were consistent with the conclusions of molecular docking analysis. Moreover, the reliability of this method was further verified by in vitro anticoagulant tests and actual sample screening of anticoagulant components in honeysuckle extracts. The Thr/meso-MOF-1@capillary column exhibited excellent reusability (≥46 runs) and good enzymatic activity (Km = 0.397 mM). This work provides an efficient tool for screening anticoagulant active components from complex systems.