In situ growth of enzyme-inorganic hybrid nanoflowers on paper strips for the visual detection of saliva-level glucose

Enzyme-inorganic hybrid nanoflowers (HNFs) are crucial for non-invasive salivary glucose monitoring in diabetic patients due to their exceptional activity and stability. However, the size mismatch results in the low immobilization efficiency of HNFs through traditional polymeric entrapment, limiting their effectiveness in low salivary glucose level monitoring. Herein, a cellulose paper is immersed in an inorganic solution of CuSO₄ and PBS containing glucose oxidase (GOx) and horseradish peroxidase (HRP) for the in situ growth of GOx&HRP@Cu₃(PO₄)₂ HNFs on a paper matrix (HNF-based paper strip). This new approach for the in situ growth of multi-enzyme-inorganic HNFs on a paper matrix significantly improves the immobilization efficiency owing to close contact interaction and enough contact points between the small-sized nucleation sites of the primary crystal and wet cellulose paper. The results show that the sensing time of the HNF-based paper strip (60 s) is eight times faster than that of the traditional enzyme@gel@paper strip (480 s). Moreover, the HNF-based paper strip retains about 80% of its catalytic activity at 70 °C. Density functional theory (DFT) calculations reveal that the metal sites of Cu₃(PO₄)₂ enhance the binding of both H₂O₂ and the enzyme, thus enhancing the activity and stability of the HNF-based paper strip. Based on these results, the proposed HNF-based paper strip could be a powerful alternative tool for non-invasive glucose monitoring.