Synthesis with Glutaraldehyde and Characterization of Uniformly Coated Alkaline Phosphatase-Nanoparticle Bioconjugates

Alkaline phosphatase from raw milk was immobilized on cysteine-functionalized silver nanoparticles with high efficiency. The nanoparticles were characterized by ultraviolet–visible spectroscopy, X-ray diffraction, and transmission electron microscopy. The surface functionalization was confirmed by infrared spectroscopy. The spherical nanoparticles were from 40 to 60 nm in size and used for the covalent immobilization of alkaline phosphatase on the surface with glutaraldehyde treatment. As compared to soluble enzyme, an enhanced enzymatic activity of 79.87% was obtained with a percentage immobilization of 75.41%. The immobilization process did not significantly affect the structure and size of the nanoparticles, while providing a uniform coating of the enzyme on the nanoparticle as characterized by electron microscopy. The bioconjugates were reusable for up to eight times with 85% retention of the initial enzymatic activity. The synthesis of these enzyme–nanoparticle bioconjugates with high activity and stability suggests their use in biological applications.