Cascade assembly of metal-organic Framework–Immobilized enzyme complexes for enhanced catalytic activity and sensing applications

Immobilized enzymes retain the high catalytic efficiency of natural enzymes while exhibiting improved stability under extreme conditions and reusability, which has led to their widespread application in sensing. This study presents the strategic immobilization of horseradish peroxidase (HRP) on magnetic UiO-66-NH 2 @PDA@Fe 3 O 4 frameworks, resulting in magnetic Metal-Organic Framework (MOF)-enzyme complexes (HRP@UiO-66-NH 2 @PDA@Fe 3 O 4 , named HUNPF). The immobilization of HRP led to the complexes exhibiting exclusively stable peroxidase activity. The Michaelis constant (Km) of immobilized HRP decreased to one-third of that of free HRP, indicating enhanced substrate affinity, while the ordered structure and large surface area of UiO-66-NH 2 @PDA@Fe 3 O 4 enhanced enzyme-substrate interactions. Furthermore, the creation of Zr-Fe active center synergistically enhances substrate catalysis alongside HRP, helping to counterbalance the activity loss caused by immobilization. Remarkably, the composite retained 90 % catalytic activity after 10 reuse cycles under magnetic recovery. A dual-mode colorimetric/fluorometric sensor was developed for nitrite detection using HUNPF, achieving detection limits of 0.20 μM and 0.13 μM for the colorimetric and fluorometric modes, respectively. This sensor offers low detection limits, excellent reliability, and high sensitivity, making it a valuable tool for environmental monitoring and food safety applications.