An Improved Amperometric Acetylcholine Biosensor by Integrating Enzyme Nanoparticles with Graphene Oxide Nanoparticles

Abstract

An improved amperometric acetylcholine (ACh) biosensor was developed by co-immobilizing enzyme nanoparticles (ENPs) i.e. acetylcholine esterase (AChENPs) and choline oxidase (ChONPs) onto graphene oxide nanoparticles (GrONPs) electrodeposited onto pencil graphite electrode (PGE). The GrONPs were prepared from graphite rod and characterized by cyclic voltammetry (CV) transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), UV and visible spectroscopy. ENPs/GrONPs/PGE was studied by scanning electron microscopy (SEM) and FTIR at different stages of its construction. The biosensor showed optimum current response at an applied potential of 0.5V within 2s at pH 7.0 and 30˚C in acetylcholine concentration range, 0.001μM to 1000μM, with a detection limit (LOD) of 0.001 μM. The analytical recovery of added acetylcholine in serum, as measured by this bio sensor was 98%. Within and between batch coefficient of variations were 0.04% and 0.06%, respectively. A good correlation (R2 = 0.989) was found between sera ACh level measured by standard enzymic colorimetric method and the present biosensor. The biosensor measured ACh level in sera of apparently healthy persons and patients suffering from Alzheimer’s. The working electrode lost 25% of its initial activity, over 240 days, when stored dry at 4˚C.