Electrochemical Sensor for Carboxypeptidase Y Using a 4‐Aminonaphthol‐Conjugated Peptide Substrate Lacking a Free Carboxyl Group at the C‐Terminus

Carboxypeptidase Y (CPY) is a serine carboxypeptidase crucial for understanding protein processing, degradation pathways, and intracellular transport, yet no electrochemical detection method has been reported. Here, we present the first electrochemical sensor for CPY, leveraging its ability to cleave 4‐aminonaphthol (AN)‐conjugated succinyl‐Leu–Leu‐Val‐Tyr (Suc‐LLVY‐AN) even in the absence of a free carboxyl group at the C‐terminus. Upon proteolysis, the electroactive species AN is released and detected using electrochemical–enzymatic redox cycling. We optimized pH, temperature, and incubation time to maximize the signal‐to‐background ratio. Under optimal conditions (pH 7.4, 37°C, 30 min), the sensor achieved a detection limit of 0.2 µg/mL in phosphate‐buffered saline, outperforming a comparable fluorescence‐based method (0.6 µg/mL). Even in artificial saliva, the sensor maintained favorable sensitivity (0.5 µg/mL), demonstrating its potential for complex sample analysis. Selectivity tests against other proteases confirmed high specificity, as only CPY effectively cleaved the Suc‐LLVY‐AN substrate. Overall, this novel electrochemical approach offers enhanced sensitivity and specificity for CPY detection, broadening the scope of electrochemical protease sensors and providing a valuable tool for diverse biochemical and diagnostic applications.