Spin Sensor-Integrated Covalent Organic Frameworks Reveal the Effect of Host–Guest Interactions on the Catalytic Activity of Immobilized Enzymes

Encapsulating enzymes within covalent organic frameworks (COFs) offers a promising strategy to enhance enzyme stability and facilitate their separation and recycling. However, the complex host-guest interactions in such hybrid systems substantially affect enzyme conformation flexibility, which can dominate key catalytic processes such as substrate binding and catalytic conversion. Understanding these molecular interplays is crucial but remains challenging. In this work, we present a spin sensor-integrated COF (SSICOF) strategy to probe host-guest interactions across diverse enzyme@COF systems. This approach leverages the highly programmable reticular framework of structurally stable COFs, wherein pore channels are atomically engineered to integrate spin probes via a click chemistry reaction. The spin-lattice interactions between the spin probes and the encapsulated enzyme generate interpretable variations in electron paramagnetic resonance (EPR) signals, delivering the host-guest interactions that cannot be probed by traditional spectroscopic techniques. Using the SSICOF approach combined with computational simulations, we demonstrate that the catalytic activity of immobilized enzymes is closely linked to their degree of freedom and structural flexibility under COF pore confinement. This strategy offers a versatile and powerful tool for probing underlying interactions in complex host-guest systems and predicting the catalytic activity of enzyme@COF hybrid biocatalysts.