A miniaturized nanobiosensor for choline analysis.

A novel reusable chemiluminescence choline nanobiosensor has been developed using aligned zinc oxide nanorod-films (ZnONR). The chemically fashioned ZnONR were synthesized by hybrid wet chemical route onto glass substrates and used to fabricate a stable chemiluminescent choline biosensor. The biosensor was constructed by co-immobilization of the enzymes choline oxidase and peroxidase. The covalent immobilization of the enzymes on the ZnONR was achieved using 16-phosphonohexadecanoic acid as a cross-linker. The phosphonation of the ZnONR imparted significant stability to the immobilized enzyme as against physisorbed enzyme. A lower value of Michaelis-Menten constant (Km), of 0.062 mM for the covalently coupled enzyme over the physisorbed enzymes facilitated enhanced stability of ZnONR nanobiosensor. The ZnONR-choline biosensor has been investigated over a wide range of choline from 0.0005 mM to 2 mM. Importantly, the recovery of choline in milk samples was close to 99%. Using the developed biosensor, choline was measurable even after 30 days with 60 repeated measurements proving the stability of the sensor (Intraday RSD%=2.83 and Interday RSD%=3.51).