Co-Immobilized Multi-Enzyme Systems with Spatial Arrangement for Glucose Detection

Multi-enzyme immobilization offers promise for sensitive glucose detection, but traditional random co-immobilization hampers spatial control. Here, a compartmentalized system (HRP.PDA.GOx@NPC-10) was constructed by localizing GOx and HRP in the inner and outer polydopamine layers on hierarchical porous carbon spheres. This method can precisely control the spatial arrangement of enzyme molecules, effectively reduce the inhibitory effects of H₂O₂, and enhance the catalytic efficiency of multi-enzyme catalysis. The immobilization yields of GOx and HRP reached 54.6% and 65.0%, respectively. The resultant HRP.PDA.GOx@NPC-10 exhibited 1.65-folds higher activity than free enzymes and maintained favorable stability under extreme pH and temperature conditions. After 7 cycles, HRP.PDA.GOx@NPC-10 retained 75.66% of its initial activity, demonstrating reusability. Especially, HRP.PDA.GOx@NPC-10 has been demonstrated to possess a glucose detection limit as low as 0.28 μM and a reaction time of only 3 min, indicating its potential for practical glucose detection.