Vol. 7, Issue 10 (2018)

Author(s):
Pukhraj Meena

Abstract:
Amperometric biosensor was prepared by using of alkaline phosphatase (ALP) against caffeine by a chemical covalent immobilization of alkaline phosphatase (ALP) with a cross-linking agent, glutaraldehyde on a ceramic based gold screen printed electrode that was modified with cysteamine by forming a self-assembled monolayer. Caffeine (CAF) competitively inhibits ALP enzyme and the determination method of caffeine by the biosensor was based on this inhibition effect of caffeine. The principle of the measurement was based on the determination of the differentiation of biosensor responses in the enzymatic reaction catalyzed by ALP in the absence and the presence of caffeine. Differences between the biosensor responses were related to caffeine concentration which was added in to the reaction medium. Caffeine concentration can be determined accurately between 0.2 and 10 µM using the biosensor. Detection limit (LOD) of the biosensor is 0.1 µM. In the optimization studies of the biosensor, glycine buffer (pH 10.5; 50 mM) and 30 ºC were obtained as the optimum working conditions. The optimum pH value was obtained as 10.5 by using of glycine buffer more comparable with another buffer systems such as Tris/NaOH and borate etc. Below and above this pH value decreases in the biosensor response were observed.