Using tungsten oxide quantum-dot enhanced electrochemiluminescence to measure thrombin activity and screen its inhibitors

A thrombin-activity-based electrochemiluminescence (ECL) biosensor was successfully constructed using tungsten oxide quantum dots (WO_3-x QD_S) as the co-reactant and thrombin-cleavable peptides as the recognizer. Specifically, Ru(bpy)₃ ²⁺ were doped on silica nanoparticles (Ru@SiO₂), which greatly enhanced the ECL potential. AuNPs@WO_3-x QDs composite was then prepared to accelerate electron transfer and improve the ECL signal by 219 times. Under ideal conditions, the limit of detection for thrombin in serum was determined to be 0.28 μU/mL with a linear range from 1 μU/mL to 1 U/mL. In addition, the developed ECL biosensor was used to screen for thrombin inhibitors from 12 compounds in Artemisiae Argyi Folium. Among the compounds tested, it was observed that 100 μmol/L luteolin exhibited a significantly higher inhibition rate (exceeding 80%) compared to apigenin, isorhamnetin, naringin, or eriodictyol. In an in-vitro anticoagulation experiment, luteolin (100 μmol/L) prolonged APTT by 49%, and the molecular docking assay indicated that luteolin had binding sites of Gly219 and Asp189 in the active pockets of thrombin. This may have been the main reason underpinning luteolin's anticoagulation effects. Overall, the Ru@WO_3-x QD_S ECL biosensor provided a reliable strategy for thrombin activity assay and screening of anticoagulant agents.