Nanoamphizymes: Self‐Assembled Amphiphilic Enzymatic Nanomaterials for High‐Performance Biocatalysis and Biosensing

ABSTRACT Nanomaterials offer versatile platforms for biocatalysis by modulating enzyme stability and activity at the nano–bio interface. However, strong interfacial interactions often disrupt enzyme structure and cause substantial activity loss, while the largely empirical nature of enzyme–nanomaterial assemblies limits rational design. Here, we propose a rational strategy to regulate enzymatic activity by constructing amphiphilic enzymes that self‐assemble into stable nanoparticles (NPs) in aqueous environments. Using 4‐piperidino‐1,8‐naphthalimide‐functionalized glucose oxidase (PPNI–GOx) as a model system, we systematically varied glutaraldehyde crosslinking to control NP size and catalytic performance. The optimized PPNI–GOx NPs displayed a catalytic efficiency comparable to, and even surpassing that of native GOx in solution, while achieving a remarkable 66‐fold increase relative to previously reported GOx NPs. Integration of the optimized nanoamphizymes into an electrochemical glucose sensor afforded high sensitivity, broad linear range (up to 24 mM), low detection limit (53.3 µM), and excellent selectivity against common interferents. The sensor also demonstrated exceptional operational stability, retaining 92% of its initial response after 7 days of long‐term testing. Collectively, this work establishes nanoamphizymes as a powerful and tunable platform for enhancing enzyme activity, stability, and sensor performance, offering a transformative strategy for developing high‐performance biocatalysts and next‐generation biosensing systems.