Immobilisation of enzyme throughout a polytyramine matrix: a versatile procedure for fabricating biosensors Original Research Article

A simple method is described for the construction of biosensors based on the immobilisation of a variety of enzymes throughout a polytyramine membrane electrodeposited on a platinised glassy carbon electrode. Tyramine was polymerised from a mixed phosphate buffer/methanol solution at an `enzyme-friendlyʹ `pHʹ of 6.0. The free amine groups located on each tyramine molecule were exploited to covalently attach the enzyme to the polymer chains using carbodiimide coupling. The resultant enzyme electrodes are shown to have excellent reproducibility (<1% relative standard deviation between electrodes at low substrate concentrations), good stability and to be applicable to several enzymes (glucose oxidase, sulphite oxidase, l-amino acid oxidase and lactate oxidase). For example, the linear response range to glucose is 0.1–15 mM, with a limit of detection (S/N=3) of 0.03 mM. An experimentally designed interference study demonstrated that the presence of uric acid, acetaminophen and ascorbic acid, over a range of physiological concentrations, did not significantly interfere with the response of a glucose oxidase electrode to glucose. The ability of the glucose oxidase electrode to operate in natural samples was demonstrated with the analysis for glucose in human sera. The glucose concentration in the sera measured with the polytyramine enzyme electrode was more precise than the standard spectrophotometric method and the same as the assigned values within experimental error. The immobilisation of oxidase enzymes throughout the polytyramine layer provided considerable advantages over previously reported electrodes in which the enzyme was immobilised only on the surface of the polymer, with regard to tunability of the biosensor response, the screening out of interferants and reproducibility.