Label-free photoelectrochemical detection of carboxylesterase activity based on carboxylesterase-induced amide bond hydrolysis

Carboxylesterase (CE), a promising biomarker for hepatocellular carcinoma (HCC), demands rapid and precise detection for clinical diagnosis. However, current detection methods are hampered by significant technical challenges. This study presents a novel label-free photoelectrochemical (PEC) biosensor based on amide-bond interactions between photoactive materials to detect CE activity. We constructed a Cd_0.6Zn_0.4S/In₂O₃ heterojunction with optimized band alignment, which enabled efficient separation of photogenerated electron-hole pairs (electron lifetime as long as 0.127 s) and significantly enhanced photocurrent generation efficiency (incident photon-to-electron conversion efficiency (IPCE) reaching 15.9 %). In this biosensing system, Cd_0.6Zn_0.4S nanoparticles were covalently linked to In₂O₃ nanospheres via amide bonds crosslinking. Upon the introduction of CE, enzymatic hydrolysis of the amide bond induced the detachment of Cd_0.6Zn_0.4S from the electrode surface, leading to a proportional decrease in the photoanodic signal. Experimental results showed that the photocurrent response exhibited a linear relationship with the logarithm of CE activity within the range of 10⁻⁵ to 0.4 U L⁻¹, with a detection limit of 3.2 × 10⁻⁶ U L⁻¹. Furthermore, the proposed PEC detection platform exhibits high selectivity and good stability for the detection of CE activity in human serum samples, demonstrating a spike recovery of 94 % to 102 % for CE determination in human serum. This label-free strategy enables sensitive detection of CE by modulating interfacial charge transport, providing a simplified yet effective approach for the early diagnosis of hepatocellular carcinoma.