Fabrication of Compartmentalized Multienzyme Reactor for Colorimetric Biosensing of Glucose and Phenol with High Sensitivity

Enzymatic cascade reactions are widely utilized in food security, environmental monitoring, and disease diagnostics, whereas their practical application was hindered due to their limited catalytic efficiency and intrinsic fragility to environmental influences. Herein, a compartmentalized dual-enzyme cascade nanoreactor was constructed in metal–organic frameworks (ZIF-8) by a shell-by-shell growth method. ZIF-8 provided a good microenvironment to maintain the activity of enzymes and protected them against harsh conditions. Importantly, experimental results revealed that the encapsulation order and enzyme ratio affected the cascade catalytic activity. When the cascade enzyme ratio was 1:1 and horseradish peroxidase (HRP) was encapsulated in the inner layer with glucose oxidase (GOx) in the outer layer (H@ZIF-8@G@ZIF-8), the nanoreactor facilitated the mass transfer process of substrates and showed the highest cascade catalytic efficiency. The maximum reaction rate (V_max) of H@ZIF-8@G@ZIF-8 was 294.96 nM s^–1, which was 1.6 times greater than G@ZIF-8@H@ZIF-8 (182.84 nM s^–1). Therefore, H@ZIF-8@G@ZIF-8 was effectively applied in glucose monitoring and phenol sensing. The glucose biosensor showed a low detection limit of 0.76 μM and a broad linear range of 5–300 μM. The phenol biosensor demonstrated a wide linear range (20–300 μM) with a detection limit of 0.60 μM. In addition, the spiked recovery experiments for glucose and phenol were carried out in serum (recovery: 95.26–100.04%) and tap water (recovery: 97.05–106.50%), respectively. The high accuracy demonstrated potential applications of the cascade system in biosensing and environmental detection.