Cell-Mimetic Compartmentalization of Natural Enzymes within MOF-Core/COF-Shell Heterostructures as a Versatile Cascade Biosensing Platform

The burgeoning demand for efficient multienzyme cascades necessitates advanced scaffolds capable of precise spatial organization; however, constructing heterostructured multilayer frameworks (HMFs) remains a formidable challenge due to harsh solvothermal conditions and imprecise enzyme localization. Herein, a biocompatible, interface-directed strategy is reported to engineer a cell-mimetic metal–organic framework@covalent organic framework (MOF@COF) heterostructure as a versatile colorimetric biosensing platform. Specifically, glucose oxidase immobilization within defect-rich (COOH)₂-UiO-66 via competitive coordination generated robust sensing elements. Subsequently, ionic liquid-catalyzed interfacial nucleation orchestrated the in situ growth of a horseradish peroxidase-encapsulated COF shell, yielding a compartmentalized architecture with hierarchical channels. Mechanistic investigations and density functional theory (DFT) calculations reveal that the support’s defect density and interfacial adsorption energy, rather than long-range crystallinity, dictate the directional assembly of the COF layer. This heterostructure exhibits enhanced cascade activity and analytical reliability through spatial proximity and reduced mass transfer resistance over conventional one-pot or stepwise assemblies, while control studies with zeolitic imidazolate framework-8 and hydrogen-bonded organic framework counterparts underscore its superiority in proximity-induced intermediate transfer. Integrating monomer reactivity with topological symmetry offers a predictive blueprint for customizing structurally diverse HMF libraries. Ultimately, flexible replacement of the core enzyme enables the functional validation of this platform through the colorimetric quantification of glucose and hypoxanthine, offering a rational blueprint for engineering biointerfaced heterostructures in cascade biocatalysis.