Simple and low-cost thrombin activity assay using screen-printed electrodes with portable electrochemiluminescence device

Real-time monitoring of thrombin activity is essential for preventing, diagnosing, and treating coagulation-related diseases. Herein, we present a simple, disposable electrochemiluminescence (ECL) biosensor for thrombin activity assay based on screen-printed electrodes (SPE). The thrombin-specific peptide substrate Mpr (4-Methoxybenzyl group)-GGRK-Fc (ferrocene) was identified from four candidates using molecular docking and specificity constant experiments. Mpr-GGRK-Fc exhibited the highest apparent specificity constant (k_cat/K_M) of 2.09 × 10¹² M⁻¹ s⁻¹, indicating superior affinity and specificity for thrombin. Thrombin catalyzes the cleavage of Mpr-GGRK-Fc, resulting in the detachment of Fc from the electrode surface, which leads to an increase in ECL intensity. Under optimal conditions, the biosensor exhibits a wide linear range from 1.0 × 10⁻⁴ U/mL to 1.0 U/mL, with a low detection limit of 4.02 × 10⁻⁵ U/mL. The biosensor shows high specificity for thrombin among a range of proteins. Compared to commercial fluorescence kits, our biosensor offers superior sensitivity, selectivity, and stability in detecting thrombin activity in whole blood, making it a promising tool for bedside clinical applications.