Preparation and characterization of alkaline phosphatase, horseradish peroxidase, and glucose oxidase conjugates with carboxylated carbon nano-onions.

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO-enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ∼0.5 mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.