Monitoring blood coagulation is critical for assessing bleeding and cardiac disorders. A variety of bioanalytical techniques for coagulation analysis are based on measuring the activity of a key enzyme, thrombin. Recent advances in electrochemical analyzers have enabled miniaturized systems for rapid thrombin activity analysis, but a key challenge is the lack of universally available electrogenic substrates. This work presents a proof‐of‐concept electrochemical biosensor for continuous thrombin monitoring using a widely available peptide‐based substrate. The measuring system consists of a screen‐printed gold electrode and the substrate, Phe‐Pip‐Arg‐para‐nitroanilide, which is cleaved by thrombin to liberate para‐nitroaniline (pNA). We developed a square wave voltammetry technique to measure the oxidation current of the liberated pNA in just seconds, with a total sample volume of only 100 µL and a detection limit of 0.24 nM. The developed assay is compatible with self‐assembled monolayer (SAM)‐functionalized electrodes, which are highly demanded in biosensor research, such as aptamer‐based sensors. We found that thrombin retains its activity on gold and even on antibiofouled electrodes treated with mercaptohexanol. Our enzymatic amplification method can be integrated into coagulation analyzers and thrombin generation assays, while providing a better understanding of protein activity and biofouling on SAMs.
Ardalan et al. (Fri,) studied this question.