De novo synthesis of enzyme-embedded covalent organic frameworks (COFs) using deep eutectic solvent: Pushing the COF limits

Covalent organic frameworks (COFs), highly stable and porous crystalline materials, are an attractive host for embedding enzymes to protect them from harsh ex vivo conditions. However, because COF pore size must match enzyme size, larger enzyme molecules cannot be absorbed into COF matrices via COF pores. Furthermore, the COF synthesis using high temperatures, acid catalysts, and organic solvents is ineffective for the de novo construction of enzyme-embedded COFs. Herein, enzyme embedding within imine-based COFs regardless of pore dimensions is presented by pre-functionalizing the enzyme with COF aldehyde precursor and facilitating “armor-like” COF exoskeleton formation from the enzyme surface via condensation of COF aldehyde and amine precursors at ambient conditions using choline chloride/urea deep eutectic solvent as both solvent and catalyst. This de novo approach affords glucose oxidase (GOx) embedding within two kinds of COFs with pores smaller than GOx size and activity recovery of 55–62% is achieved, whereas GOx embedding into pre-synthesized COFs was not possible. According to time-dependent TEM analysis, GOx@COF morphology is dependent on enzyme and COF precursor geometry which was governed by reversible Schiff-base and Ostwald ripening. Enzyme confinement within the COF architecture, as well as chemical crosslinking of the enzyme surface with the COF exoskeleton, endows GOx@COF with excellent pH and thermo-stability, tolerance to denaturation by urea, trypsin, and organic solvent, and stable bactericidal activity over 5 recycles. The morphological structure of GOx@COF and extent of GOx cross-linking with COF precursor influence its kinetic performance. This work could open a new avenue for utilizing COFs to create a suite of stable enzyme-COF composites for various applications.