Spatially confined enzymatic tandem system with GOx and HRP compartmentalized in ultrafiltration membrane

The precise control of timely and spatially defined enzymatic reactions is of considerable interest in biology, biomedical engineering, and bio-catalysis. In this study, we report a robust multi-enzymatic cascade system with glucose oxidase (GOx) and horseradish peroxidase (HRP) spatially compartmentalized in a polyether sulfone ultrafiltration (UF) membrane by co-deposition of polydopamine (PDA) and polyethyleneimine (PEI). The PDA/PEI nanolayers reduce enzyme leakage, retaining ∼98 and ∼70 % of GOx and HRP, respectively, and facilitate substrates and intermediates passage and enrichment near the enzyme's active sites. The membrane serves as a porous scaffold facilitating enzyme immobilization and recyclability, improving stability and catalytic efficiency. The spatially defined cascade system exhibits superior catalytic activity, leading to a 1.48-fold and 3-fold increase compared to free GOx and HRP, respectively. This improvement is likely due to substrate channeling, substrate diffusion and product convection. Furthermore, the dynamics of the GOx and HRP tandem catalysis was investigated in a closed system which demonstrated the crucial roles of dissolved oxygen in both the oxidoreductase and peroxidase reactions. The concept of tandem enzyme reactions in UF membranes can be generalized to create efficient and stable enzyme systems for applications in biosensing and industrial processes.