Amperometric biosensor for rapid determination of alanine Original Research Article

An enzyme Clark electrode containing three different enzymes was developed for the determination of alanine. This sensor is based on specific dehydrogenation of l-alanine dehydrogenase (AlaDH) in combination with salicylate hydroxylase (SHL) and pyruvate oxidase (PyOD). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, AlaDH, catalyses the specific dehydrogenation of alanine consuming NAD+. The products, NADH and pyruvate, initiate two following reactions: SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH and PyOD decarboxylates pyruvate in the presence of oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of alanine by AlaDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. This results in an amplified signal due to the two-enzymatic consumptions of dissolved oxygen in the measurement of alanine. Interferences from different amino acids and electroactive substance were found to be minimal due to the specificity of AlaDH and the application of a Teflon membrane. The sensor has a fast response (2 s) and short recovery times (2 min) with a linear range between 10 and 800 μM alanine and a detection limit of 7.2 μM. A good agreement (R2 = 0.9902) with spectrophotometric method was obtained in beverage sample measurements.