Synergistic enzyme-nanozyme catalysis on a porous carbon electrode for sensitive L-phenylalanine sensing

Tandem catalysis that integrates enzymes with abiotic catalysts (nanozymes) offers a promising strategy for advanced biosensing applications. However, its analytical performance is highly dependent on the electrode architecture, which determines the efficiency of cascade catalysis and signal transduction. Here, we report a confined enzyme-nanozyme cascade system in which an L-Phe-selective amino acid oxidase and Prussian blue nanoparticles are co-localized within a macroporous carbon framework. The macroporous architecture promotes rapid mass transport while creating a confined microenvironment that enriches enzymatically generated H2O2 near the nanozyme active sites, thereby enhancing cascade efficiency. As a result, the platform exhibits substantially amplified electrochemical signals compared with micro-mesoporous carbon and planar electrode architectures. The enhancement is particularly pronounced at low L-Phe concentrations, where an approximately sixfold increase in current response is achieved. The resulting biosensor exhibits a clinically relevant linear detection range of L-Phe and a low limit of detection of 17.1 +/- 1.4 mu M. In addition, it demonstrates excellent selectivity against various amino acids and retains 72% of its initial sensitivity after 25 days of dry storage under repeated-use conditions. Furthermore, the sensor enables continuous monitoring of L-Phe, exhibiting no noticeable decay in current response during 3 h of continuous operation, highlighting its excellent operational stability and suitability for real-time sensing applications. These findings highlight the potential of confined enzyme-nanozyme cascade systems as a versatile platform for electrochemical biosensors.