Tuning Microenvironment over Metal–Organic Frameworks for Efficient Enzyme Immobilization and Sensitive Immunoassay

Immobilizing enzymes within metal-organic frameworks (MOFs) enables enzymes to act against extreme environments. However, immobilized enzymes usually face confined and inappropriate microenvironments, resulting in decreased bioactivity and significantly affecting their practical applications. Herein, we propose a functionalized hierarchically porous MOF (HP-MOFs) for efficient enzyme immobilization. The functionalized ligands were introduced to tune the microenvironments, followed by the utilization of the acid etching strategy to further enrich the mesoporous structure. The obtained HP-MOFs with a matching pore size with enzyme (cytochrome c, Cyt c) not only show high loading amounts (19.00%) but also facilitate the accessibility of enzymes. Importantly, the -OH functionalization enhanced the hydrophilicity of the carrier for maintaining the secondary structure of Cyt c and achieving a superior catalytic activity. Furthermore, the engineered HP-MOF-OH@Cyt c exhibits excellent recyclability and tolerance to inhospitable conditions. Capitalizing on the unique interaction of the Zr-O-P bond, the resultant HP-MOF-OH@Cyt c-based biosensor is constructed for a sensitive chlorpyrifos assay. The proposed biosensor has a good linear relationship with the concentration from 10 pg mL^-1 to 10 000 pg mL^-1, with a low detection limit of 4.63 pg mL^-1. This work represents a good advance in enzyme immobilization and is expected to show great prospects in practical enzyme-involved applications.