Gas-Shearing Microfluidic Fabrication of Alginate/Chitosan Hydrogel Microreactors for Genipin-Mediated Dual-Mode Lipase Immobilization.

Enzyme immobilization is an effective strategy to improve catalyst recovery and operational stability, but conventional approaches are often limited by diffusion resistance, steric hindrance, and insufficient control over the enzyme microenvironment. Hydrogel-based materials are attractive supports because their hydrated, tunable, and porous networks help preserve enzyme conformation while improving substrate diffusion and mass transfer. Here, a gas-shearing microfluidic strategy was developed to fabricate alginate/chitosan hydrogel microreactors for dual-mode lipase immobilization through entrapment and genipin-mediated covalent binding. By tuning gas-liquid shear, microspheres with controllable and narrow size distribution were generated without surfactants or UV-initiated polymerization. Importantly, genipin was introduced as a mild and biocompatible cross-linker to reinforce the alginate/chitosan network and create a stable interfacial microenvironment for enzyme immobilization. Compared with conventional cross-linkers, the milder and more controllable reactivity of genipin is advantageous in preserving enzyme conformation and catalytic performance. The resulting microreactors exhibited enhanced catalytic activity, improved storage stability, and good reusability. After five consecutive cycles, the entrapped and covalently immobilized systems retained 70% and 81% of their initial activity, respectively. These results demonstrate that gas-shearing microfluidics combined with genipin-mediated network reinforcement provides a robust and biocompatible platform for constructing hydrogel-based enzymatic microreactors with improved catalytic performance and operational durability.