Electrochemical protease detection strategies based on proteolytic cleavage

Abstract Proteases play essential roles in both physiological and pathological processes, driving the need for sensitive and specific detection methods. Among these, electrochemical detection strategies based on proteolytic cleavage have emerged as powerful tools thanks to their high sensitivity, rapid response, and suitability for point‐of‐care applications. In this review, we classify electrochemical protease detection strategies into heterogeneous and homogeneous formats, and further organize them by signal‐transduction modes such as signal‐on, signal‐off, and impedance‐based assays. Heterogeneous formats utilize proteolytic cleavage at electrode surfaces, using protein, hydrogel, or peptide layers, which are particularly effective for endopeptidases. Homogeneous formats, by contrast, rely on proteolytic cleavage in solution and are suitable for detecting both endopeptidases and exopeptidases. We also examine substrate‐design principles tailored to each protease class, and compare the strengths and limitations of each detection format. Despite significant progress, electrochemical detection of low‐abundance protease biomarkers with high specificity remains a challenge. Future efforts should focus on improving sensitivity and selectivity via advanced electrode architectures, redox‐cycling amplification, and rational substrate design. The continued development and integration of these technologies promise impactful applications in point‐of‐care diagnostics, environmental monitoring, and biomedical research.