A Millifluidic Device with Embedded Cross-Linked Enzyme Aggregates for Degradation of H₂O₂

Millifluidic devices decorated with enzymes have been used for enzymatic reactions in continuous processes, but low enzymatic activity and enzyme leaching remain as challenges. Herein, we develop a strategy to embed cross-linked enzyme aggregates (CLEAs) on the surfaces of millifluidic devices to achieve higher enzymatic activity and better stability. Catalase was chosen as a model enzyme to degrade H₂O₂ in wastewater samples. First, CLEA of catalase (153 ± 10 nm) was formed by simultaneous precipitation and cross-linking with 25.0 wt % acetonitrile containing 0.025 wt % glutaraldehyde in a millifluidic device. To immobilize CLEA, we first swell a piece of plastic tubing by using 5.0 wt % acetonitrile and then immerse it in an aqueous solution with 5.0 wt % (3-aminopropyl)triethoxysilane (APTES) and 5.0 wt % dextran polyaldehyde (DPA) subsequently. After CLEA is absorbed inside the expanded polymer network of the tubing, the tubing is tightened by using a vacuum to secure the immobilized CLEA. The millifluidic device decorated with CLEA of catalase has total activity of 660 U for degradation of H₂O₂, and it shows good stability under a flow rate of 200 μL/min. The tubing can be used to degrade 0.1 wt % H₂O₂ solution continuously for 3 h or remove 2 wt % residual H₂O₂ in wastewater for 2 h. The technique is general enough and can be applied to other types of enzymes for continuous enzymatic reactions.