MXene-sensitized electrochemiluminescence sensor for thrombin activity detection and inhibitor screening

Thrombin, a crucial enzyme involved in blood coagulation and associated diseases, requires accurate detection of its activity and screening of inhibitors for clinical diagnosis and drug discovery. To address this, an electrochemiluminescence (ECL) method was developed to detect thrombin activity based on the sensitization of Ti₃C₂T_x MXene, which could sensitize the Ru(bpy)₃²⁺ ECL system greatly. The thrombin-cleavable substrate bio-S-G-R-P–V-L-G-C was used as recognizer to evaluate the activity of thrombin. Under the optimal conditions, the limit of detection for thrombin in serum was 83 pU/mL (S/N = 3) with a linear range from 0.1 nU/mL to 1 µU/mL. Moreover, the developed ECL biosensor was employed to screen for thrombin inhibitors from Artemisiae argyi Folium. Four potential thrombin inhibitors (isoquercitrin, nepetin, L-camphor, L-borneol) were screened out with inhibition rates beyond 50%, among which isoquercitrin had the best inhibition rate of 90.26%. Isoquercitrin and nepetin were found to be competitive inhibitors of thrombin, with Kiapp values of 0.91 μM and 2.18 μM, respectively. Molecular docking results showed that these compounds could interact with the active sites of thrombin through hydrogen bonds including ASP189, SER195, GLY216, and GLY219. The electrochemical biosensor constructed provides a new idea for the detection of thrombin activity and screening of its inhibitors. Graphical abstract: [Figure not available: see fulltext.].