We report herein the development of a novel glucose chemiluminescence (CL) biosensor based on covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell and direct coupling of chitosan-induced Au/Ag alloy nanoparticles on it, and how it may be useful for determination of glucose due to CL detection of enzymatically generated H2O2. In addition, the nanoalloy offers excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose and increases stability of covalent-linked enzyme. Chitosan molecules act as both the reducing and stabilizing agents for the preparation of nanoparticles (NPs) and also as a coupling agent between GOD and Au/Ag alloy NPs, which made possible the fabrication of a sensitive, accurate and stable biosensor for glucose. Under the optimum conditions, the biosensor can be used for the determination of glucose in the range of 1.2 × 10–6 to 6.25 × 10–3 M with a detection limit of 5.0 × 10–7 M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2 months storage at pH 7.0. The present CL biosensor has been used to determine glucose in real serum and urine samples and validated against colorimetric spectrophotometry method.
