Microfluidic Fabrication Of Poly(ε-caprolactone)/Silica Hybrid Microspheres With Hierarchically Porous Architecture And Research Of Enzymatic Immobilization

In recent years, porous microspheres have received widespread attention in the enzymatic immobilization field due to their high surface area and ability to encapsulate enzymes within the pores. Specifically, co-immobilization of enzyme cascades have engendered tremendous interest. In this paper, based on the synergy effect between sol-gel process and solvent extraction in microdroplets, hierarchically porous poly（ε-caprolactone）/silica hybrid microspheres are fabricated in situ on a microfluidic flow-focusing device without the use of porogens and templates. Because of the competition between the hydrolysis/condensation rate and solvent extraction process when droplets are generated, hierarchically porous architecture with different surface porosity can be formed by precisely tuning the reaction rate with ammonia catalyst and the amount of TEOS.Alcohol dehydrogenase（ADH）, superoxide dismutase（SOD） and,catalase（CAT） are immobilized via either physical adsorption or covalent binding to evaluate the performance of hierarchically porous PCL/SiO₂ hybrid microspheres as enzyme carriers. The results of enzymatic activity, immobilization yield, and activity yield reveal that enzymes immobilized on hierarchically porous microspheres exhibit the optimal immobilization capacity, which can be attributed to the unique features of carriers providing a more stable microenvironment for enzymes. Notably, these enzymes immobilized on modified carriers via covalent binding both show excellent recycling performance with the remain activity of 90%（after more than 10 cycles）. More importantly, the structure and size distribution of these microspheres is highly stable after reusability assay.Compared to immobilization of single enzyme, cascaded enzymes exhibit enhancement of synergy between SOD and CAT and overall performance after co-immobilization. The superiority of double-enzymatic catalysis SOD/CAT lies in the improvement of enzymatic activity（35%, 23%） and the total antioxidant capacity（98.8%）. What’s more, the relative activities of SOD/CAT via covalently immobilized on PCL/SiO₂ hybrid microspheres with deep pores are still as high as 90.3% and 89.2%（after 16 cycles）, which contributes to expansion of promising application of enzyme cascades. Furthermore it focuses on SOD/CAT optimal proportion（1:2） and optimization co-immobilized condition for offering a reliable reference for realization double-enzymatic catalysis with excellent performance in immobilized forms.