Crosslinking-regulated nanoconfined biointerface enables highly stable and sensitive cooperative cascade biocatalysis...

Engineering catalytic microenvironments within hybrid materials is a critical strategy for enhancing enzyme stability and catalytic efficiency; however, precise regulation of intermediate transfer and interfacial catalytic coupling remains challenging. Here, we report a crosslinking-regulated, double-crystallized hierarchical hybrid nanoflowers system (D-Cu@c-GOx NFs) that establishes a nanoconfined biointerface for cooperative cascade biocatalysis. The system is constructed via in situ enzyme incorporation, followed by glutaraldehyde-mediated crosslinking and a second crystallization step, generating a densely interconnected Cu-based framework that spatially organizes catalytic domains. This nanoconfined architecture minimizes intermediate diffusion loss and promotes proximity-driven catalytic interactions, resulting in enhanced catalytic efficiency and operational stability. The hybrid nanoflowers exhibit strong tolerance to pH and temperature variations and retain 83% of their initial activity after 30 days of storage. Kinetic analysis confirms that the hierarchical structure significantly improves catalytic turnover compared with physically mixed systems, highlighting the importance of spatial proximity and confined reaction pathways. Furthermore, electrochemical analysis demonstrates that the nanoconfined biointerface modulates interfacial redox behavior and electron-transfer kinetics, providing mechanistic insight into structure-function relationships in hybrid catalytic systems. Notably, the same D-Cu@c-GOx NFs architecture supports both enzyme-nanozyme cascade colorimetry and electrochemical signal transduction, enabling dual-mode biosensing from a common hybrid biointerface. While glucose oxidation is employed as a model reaction, the primary contribution of this work lies in demonstrating how crosslinking-regulated nanoconfinement governs enzyme-associated catalytic behavior. This study establishes a general strategy for designing hybrid nanostructures with controlled catalytic microenvironments, offering a versatile platform for developing stable and efficient enzyme-based systems.