Hierarchical enzyme immobilization: encapsulating mesoporous polymer nanospheres in covalent organic frameworks for enhanced reusability and stability.

Enzymes, as efficient and specific natural catalysts, are limited in industrial applications by their instability and non-recyclability under extreme conditions. Combining enzymes with solid supports can address separation issues and enhance stability. This study constructed an enzyme nanoreactor by co-loading glucose oxidase (GOx) and horseradish peroxidase (HRP) into mesoporous melamine-formaldehyde (MF) resin microspheres (denoted as Ps) and covalent organic frameworks (COFs). Specifically, enzymes were immobilized on mesoporous MF resin microspheres via electrostatic adsorption, followed by in-situ encapsulation of COF shells to form the core-shell immobilized enzyme system (GOx&HRP@TpBD/Ps). This system showed enhanced reusability (retaining ~ 80% initial activity after 7 catalytic cycles) due to COF shells’ smaller pore size than enzymes, reducing leaching. It also exhibited higher catalytic activity and stability than free enzymes, as the carrier’s porous structure provides a compartmentalized environment for catalysis, facilitating substrate transport, reducing diffusion limitations, and shielding enzymes from external interference to resist structural distortion. Its limit of detection for glucose determination using absorbance measurement was 2.32 µM.