Facile electrochemical assay of thrombin activity in biological products

Thrombin, a critical protein-based therapeutic agent, is widely used in modern medicine. Precise quantification of thrombin enzymatic activity represents a fundamental requirement for both manufacturing quality control and clinical therapeutic monitoring. Here, we developed a “signal-off” electrochemical method for the rapid detection of thrombin activity within 15  min. A thrombin-specific substrate peptide was anchored to a gold electrode via Au S bonds at its C-terminal, with an N-terminal ferrocene (Fc) tag as a redox probe. A cellulose acetate (CA) membrane was added to reduce contamination and signal loss. The detection mechanism relies on thrombin-mediated peptide cleavage, detaching the Fc-tagged fragment and generates a decreasing current proportionally to enzyme activity. The detection limit of the described method is 81.1 U/mL, with a linear response over the range of 200–1500 U/mL (R 2  = 0.9941), satisfying the quality control requirements for commercial thrombin drugs. This biosensing strategy combines operational simplicity with analytical reliability, showing significant potential for rapid and efficient assessment of thrombin biological products activity.