Mechanochemistry has emerged as a sustainable alternative to solvent-intensive synthetic strategies, enabling rapid and environmentally benign material fabrication.1,2 Here, we report a one-step mechanochemical approach for the in situ immobilization of enzymes within hydrogen-bonded organic frameworks (HOF-1), producing robust and highly active biocatalytic nanoarchitectures. Using ball milling under near solvent-free conditions, glucose oxidase (GOx) and a cascade system of GOx/horseradish peroxidase (HRP) were co-assembled with HOF-1 during framework formation.
Structural characterization (PXRD, BET, FTIR, UV–Vis, confocal microscopy) confirmed successful enzyme encapsulation while preserving framework crystallinity and enzyme conformation.3 The GOx@HOF-1 composite exhibited remarkable resistance to extreme pH (4–12), elevated temperatures (up to 85 °C), chaotropic agents, and organic solvents, retaining >70% activity under conditions where free enzyme rapidly deactivated. Long-term stability and recyclability were maintained over multiple cycles.
Importantly, co-immobilization of GOx and HRP within the same HOF-1 scaffold enabled efficient substrate channeling.4 Transient-time analysis revealed near-zero lag time for the cascade system, resulting in a 2.4-fold enhancement in catalytic efficiency compared to free enzymes and a 12-fold increase relative to separately immobilized enzymes. The bienzyme construct demonstrated ultrasensitive glucose detection with a limit of detection of 0.1 µM, attributed to confined cascade amplification within the porous hydrogen-bonded network.
This work establishes mechanochemical nanoarchitectonics as a scalable and green platform for constructing enzyme@HOF biocomposites, offering new opportunities for sustainable catalysis, biosensing, and industrial biotransformations.