Spheres-on-sphere silica microspheres as matrix for horseradish peroxidase immobilization and detection of hydrogen peroxide

Abstract

In this work, spheres-on-sphere (SOS) silica microspheres are prepared via a facile one-pot synthesis. After functionalization with carboxyl groups, the carboxylated SOS silica microspheres (SOS-COOH microspheres) can serve as a support for horseradish peroxidase (HRP) covalent immobilization. The obtained enzyme hybrid (SOS-COOH-HRP) is more stable under alkaline conditions than the free counterpart, and exhibits longer-term storage stability and higher resistance toward the denaturing agents such as guanidine hydrochloride (GdmCl) and urea. The Michaelis–Menten constant (K_m) of the immobilized enzyme is decreased slightly while the maximum rate of reaction (V_max) is very close to that of free HRP, resulting in the catalytic efficiency of SOS-COOH-HRP being enhanced significantly. For evaluating its utility, a SOS-COOH-HRP-based colorimetric method has been developed for selectively and sensitively detecting H₂O₂ both in buffer and 10% diluted human serum. Furthermore, the SOS-COOH-HRP displays excellent reusability and reproducibility in cycle analysis. The results demonstrate that the SOS-COOH-HRP has great potential for practical applications in biosensing and industrial fields.