Mechanochemical Encapsulation of Enzymes in a Highly Hydrophilic HOF: Harnessing Pore Microenvironment for Enhanced Biocatalysis

ABSTRACT Enzymes immobilized within hydrogen‐bonded organic frameworks (HOFs) often suffer from reduced catalytic activity due to mass transfer limitations and pore channel blocking by residual organic solvents from conventional solution‐phase synthesis. In addition, the correlation of HOF pore hydrophilicity‐hydrophobicity with enzymatic reactivity has rarely been underscored. Here, we report a mechanochemistry‐mediated HOF assembly strategy that enables non‐destructive, in situ enzyme encapsulation while generating an open porous network free from solvent‐induced pore blockage. Using a series of isostructural HOFs assembled from [1,1′:4′,1″‐terphenyl]‐3,3″,5,5″‐tetracarboxylic acid (H 4 TPTCA) bearing different substituents (‐NH 2 , ‐H, ‐CH 3 ), we demonstrate that mechanochemical synthesis affords enzyme@HOF biocomposites with up to a 449‐fold enhancement in catalytic rate compared to the conventional solution‐phase method. Notably, the introduction of ‐NH 2 groups substantially improves pore‐wall water adsorption capability, leading to 1.45–2.78 times and 116.1–330.3 times higher activity than the ‐H and ‐CH 3 analogues, respectively. Leveraging the high activity and stability of mechanochemical enzyme@HOF biocatalysts, we construct a sandwich‐type electrochemical immunosensor for carcinoembryonic antigen (CEA) that achieves a wide linear range and a detection limit as low as 15.6 fg mL −1 . This work establishes a green mechanochemical route to highly active enzyme@HOF biocatalysts and highlights the critical role of hydrophilic pore environments in preserving enzymatic function.