Improving Both Stability and Sensitivity of Immunosensor by Spatially Controlled Enzyme‐Engineered Covalent Organic Frameworks

Abstract Immobilization of fragile enzymes in a suitable support material provides new opportunities to improve the performance of immunosensor, but construction remains challenging due to the dimensional limitation or denaturation during the process. Herein, an enzyme‐engineered assembly strategy that enables the in situ fusion of enzymes into the framework structure during the crystal formation process via the covalent‐polymerization‐permeation mechanism is demonstrated, utilizing the dynamic covalent chemistry property of covalent organic frameworks (COFs‐PB). By finely regulating the assembling order of building blocks, enzyme‐COFs composite with the spatial distribution structure of the enzyme (such as uniform, sub‐surfaces, or interface) can be evolved, featuring controllable activity and structural stability. Impressively, the apparent enzymatic kinetics of the enzyme can be well maintained after encapsulation in COFs‐PB, and enzyme@COFs‐PB composite has shown outstanding stability in strongly acidic environments. Benefiting from structural integration, a robust enzyme@COFs‐PB‐based immunosensor is built for the sensitive detection of isocarbophos pesticide, which performed a 56‐fold enhancement in sensitivity compared with that of the standard immunosensor. This study gives valuable insight into a comprehensive understanding of the interfacial interactions between enzymes and COFs in designing ideal biocatalytic nanosystems, providing important guidance for the development of advanced immunosensors in on‐site application.