Condensation of GO x by Restoring Global Protein Fold on Diazo-Enriched SG Surfaces: Reinstallation of FADs Restores Biocatalytic Sustainability in Quantifying Blood Glucose

Immobilization of glucose oxidase (GO x ) on silica gel (SG) via multipoint mode at high-level condensation, leveraging heterogeneous enzyme–product by restoring global protein fold and native enzyme’s activity, is a long-pending issue. Herein, the application of potent diazo-coupling at the O -carbon of tyrosine’s phenolic moiety was harnessed, which behaved as the inert center. The molecular dimensions of the product {SiO 2 }@GO x was assessed as {Si(OSi) 4 (H 2 O) x } n {−O-Si(CH 3 ) 2 –O–C 6 H 2 (N + ≡N) 3 (GO x )} 4 ·(H 2 O) y, where x = 0.35, n = 4309, y = 13112, and GO x = C 6994 H 10552 O 2627 N 1598 S 30 P 4 . The immobilized GO x (20.1 ± 0.2 μmol·g –1 ) protein groups remained in a state of the native enzyme’s natural interactions, wherein FAD remained at its normal position and restored the global protein-folded status quo. At freezing temperature, the FAD in 0.025 M (NH 4 ) 2 SO 4 at the respective low (1.4) and high (5.5) pH values moves out from and is reinstated at the enzyme’s sphere by the opening and enclosing of the protein fold, as evidenced by the CD spectroscopic signature and biocatalytic activity. The heterogeneous enzyme framework, {SiO 2 }@GO x -{SiO 2 }@HRP, in the column reactor exhibited biocatalytic performance within the native enzyme’s activity range at optimized conditions (pH 6.5 and 300 K) in a transient cascade response. The reactor was precise (SD: 0.003) in determining human blood glucose with an impressive LOD of 2.25 mg.dL –1 . The product(s) was stable under continuous spinning at 2500 rpm in a buffer and remained stable up to 360 K, and at the thrusts of mineral acids (0.05–0.2M). The dried product at 28-month storage exhibits appreciable stability, wherein the biocatalytic activity remained unchanged and appeared outstanding.