Pore-Size Confinement Strategy in Hierarchical Porous MOFs for Efficient Enzyme Immobilization and Thiol Identification

Metal-organic frameworks (MOFs) demonstrate considerable potential for enzyme immobilization, yet their applications are often limited by enzyme leakage or denaturation. Herein, we present a strategy for enzyme immobilization using hierarchically porous UiO-66 (a stable microporous MOF) frameworks. By partially replacing the conventional 1,4-benzenedicarboxylic acid (H₂BDC) linkers with monocarboxylic acids of controlled-chain lengths, we successfully engineered tailored mesopores within the stable microporous UiO-66 structure. Subsequently, a pore-size confinement strategy enables the selective adsorption of size-matched enzymes (horseradish peroxidase and glucose oxidase) into the tailored hierarchical mesopores. Impressively, the immobilized enzymes exhibited enhanced apparent catalytic activity compared to their free counterparts and outstanding stability against leaching tests. Benefiting from interactions between enzymes and thiol compounds, we developed a multichannel sensor array capable of identifying six thiols with a detection limit of 1 μM. This work offers a universal strategy for enzyme immobilization through rational MOF pore engineering, opening avenues for developing stable and high-performance enzyme-MOF hybrid systems in biosensing and biocatalytic applications.