Hierarchical MOFs with spatially ordered cascade enzymes for sensitive nitrite immunosensing

Enhancing the bioactivity and utilization efficiency of immobilized enzymes is essential for the construction of sensitive immunosensors. However, conventional approaches are often limited by random enzyme spatial distribution, which compromises cascade efficiency. Here, we constructed a multilayer metal-organic framework (MOF) through a spatially ordered hierarchical assembly. This design enables ordered spatial compartmentalization of glucose oxidase (GOx) and horseradish peroxidase (HRP), thereby promoting intermediate transfer and significantly enhancing cascade catalytic activity. Further refinements included microenvironment modification and surface etching. Specifically, microenvironment regulation using polyvinylpyrrolidone (PVP) and histidine (His) resulted in a high GOx encapsulation efficiency of 74.43%. Additionally, Ni-triggered etching enlarged the MOF pores from 2.21 nm to 5.68 nm, reducing the mass transfer resistance of substrate and doubling the catalytic activity of cascade enzyme. As a proof-of-concept, when applied as a signal amplifier in an immunosensor, this composite exhibited ultrahigh sensitivity for nitrite detection with a detection limit of 0.05 μg mL^-1, achieving a 1000-fold improvement of sensitivity in comparison to the conventional enzyme-linked immunosorbent assay. This study provides a valuable strategy for efficient immobilization and activity enhancement of cascade enzymes, offering guidance for the employment of controllable nanostructures in constructing highly sensitive immunosensors.