Electrochemical detection of the activities of thrombin and its inhibitor

An electrochemical approach is described to assess the activities of thrombin and one of its inhibitors. Thrombin is a vital proteinase, which is one kind of serine protease, and plays an important role in coagulation cascade, thrombosis, and hemostasis. A gold electrode immobilized with p-aminodiphenylamine (pADA) modified peptide (H-d-Pro-Phe-Arg-p-aminodiphenylamine) was prepared for the analyses of thrombin and its inhibitor. The peptide was employed as the recognition and cleavage site for thrombin while pADA was used as an electroactive reporter. Experimental results showed that this method is sensitive as a low concentration of 5 fM thrombin can be detected. The inhibition of thrombin activity by argatroban was monitored and IC50 was found to be about 10 μM. The protocol was further applied for the measurement of a healthy human serum sample.

► The protocol provides a facile readout of thrombin activity and its potential for application of human serum sample. ► The method is sensitive as a low concentration of 5 fM thrombin can be detected. ► The strategy offers a possible way to screen the throughput of thrombin inhibitors.