Spatially ordered immobilization of cascade enzymes for the construction of a robust colorimetric hydrogel sensor

The development of a suitable mimetic scaffolds for maintaining high activity and stability of co-immobilized multi-enzymes is a key challenge in biotechnology. Herein, we achieved the regular distribution of cascade enzymes through spatially controlled hierarchical loading into protein-inorganic hybrid nanoflowers using a mild biomineralization technique. The comprehensive understanding of sequential regulation in constructing controlled nanoarchitecture enables to combine a continuous reaction and achieve tailoring catalysis for biomimetic application. The ordered-assembled cascade enzymes showed stronger bioactivity in comparison with the disordered format or inappropriate loading format. The stability of the enzyme is incrementally improved by an efficient dual-enhanced mode of immobilizing the free enzyme into hybrid nanoflowers and encapsulating it in a hydrogel system, addressing the inherent fragility of natural enzymes. Benefiting from the structural integration, a protein-inorganic hybrid nanoflowers-embedded hydrogel sensor is constructed for on-site detecting NO₂^- with a detection limit of 5.08 μM. This work showcases a convenient approach for the efficient design of the ideal cascade biocatalysts, and supports the development of portable devices for practical application.