Converting Nonspecific Adsorption into Bioaffinity Recognition: Defect-Engineered MOF-808 Creates a Synergistic Enzyme Interface for Selective Ligand Fishing and Closed-Loop Target Confirmation

Abstract Nonspecific adsorption of metal −organic frameworks (MOFs) limits the analytical reliability of immobilized-enzyme ligand fishing in complex natural systems. In this work, an activity-guided defect-engineering strategy was developed to convert MOF-808 into a synergistic enzyme-MOF biointerface for selective molecular screening. Mild NaOH etching generated OH-MOF-808 with hierarchical porosity and exposed Zr4+ sites, which facilitated neuraminidase (NA) immobilization while preserving its native-like secondary structure (circular dichroism (CD) spectroscopy) and operational stability (64.47% activity after 28 days; >80% after 5 cycles). X-ray photoelectron spectroscopy (XPS) confirmed that the exposed Zr4+ sites anchored NA through coordination interactions. Active/inactive enzyme controls and pristine/defect MOF comparisons demonstrated that the conformationally preserved NA layer contributed to target discrimination: the recovery of the NA ligand Scutellarein increased from 52.65% to 71.49%, while nonspecific interferents were suppressed. Applied to a traditional Chinese medicine formula, the platform enabled selective capture of 25 candidate constituents with enhanced antiviral activity. A reverse fishing strategy, in which the captured ligands were re-immobilized to selectively retrieve NA from infected-cell lysates (2.5 mg·g−1, 72% of total captured protein), closed the validation loop and confirmed the target specificity of the enriched constituents. This work establishes a transferable strategyconverting nonspecific adsorption toward enzyme-programmed recognition through defect chemistryfor constructing high-specificity biointerfaces in complex biological systems.