Enzyme‐Embedded Amorphous Fluorescent Covalent Organic Frameworks for the Construction of Pesticides Biosensor

Abstract Enzymes as signal amplifiers and highly specific recognition elements offer promising opportunities for enhancing the sensitivity and selectivity of fluorescent biosensors. However, the intrinsic fragility of enzymes presents significant challenges in achieving effective integration with fluorescent probes and ensuring reliable biorecognition‐to‐signal transduction functionality. Herein, a robust photo‐enzymatically integrated biosensor is prepared by in situ assembly of enzymes into amorphous fluorescent covalent organic frameworks (COFs). The fluorescence properties of amorphous COFs (aCOFs) are significantly enhanced by restricting the intramolecular rotation effect through hydrogen‐bond‐assisted design. Owing to the confinement protective effect of the framework, enzyme@aCOFs demonstrate superior stability against acidic conditions, organic solvents, and elevated temperatures compared to free enzymes, along with excellent recycling capabilities. Benefiting from the integration of fluorescent properties and the catalytic ability in enzyme@aCOFs, a highly sensitive and selective biosensor is constructed, where the highly synergistic action of the photo‐enzyme significantly enhances the biorecognition‐to‐signal transduction functionality to achieve the detection of chlorpyrifos pesticide at the nanogram per milliliter level. This study offers critical insights into the effective immobilization of enzymes and the rational design of the photo‐enzymatically integrated system, providing essential guidance for the development of advanced biosensors for practical applications.