Amino acid oxidases are selective for amino acids, but not for particular amino acids. That is, they act on a considerable number of amino acids. In addition, amino acid oxidases have an optical specificity for D-or L-isomers; L-amino acid oxidase (L-AAO) is specific for L-amino acids and D-amino acid oxidase (D-AAO) is specific for D-amino acids. The general enzyme reaction is Amino acids + O2 + H2O AAO → 2-oxo acids + NH3 + H2O2.Many research groups have proposed the enzyme sensors for the specific detection of L-1-7 or D-3,8,9 isomers of amino acids. Most of the enzyme sensors proposed are based on the amperometric 1,2,4-6,8 and potentiometric 7 detections or chemiluminometric 3,9 detection of hydrogen peroxide or ammonia produced by the amino acid oxidases. In addition, the development of new biosensors which may enable the simultaneous determination of L-and D-isomers of amino acids is also important, but it had not been achieved except for the successive detection 3,10 of both isomers. In this note, a dual enzyme electrode suitable for the simultaneous amperometric detection of D-and L-amino acids is described. The proposed dual electrode consists of two platinum disks modified by an L-AAO or D-AAO membrane cross-linked by glutaraldehyde.Furthermore, the electropolymerization to coat with a thin film of poly(1,2-diaminobenzene) was subsequently carried out to protect the electrode from direct (nonenzymatic) oxidation of electroactive amino acids such as methionine and tyrosine or electroactive interferents such as L-ascorbate, urate, and cysteine, which may otherwise cause electrode fouling. The analytical performance of the dual enzyme electrode is assessed under flow-injection conditions, particularly on substrate selectivity within a group of amino acids and on optical specificity for D-and L-isomers of amino acids.
ExperimentalReagents D-AAO (EC 1.4.3.3, 9.9 U mg -1 of solid from porcine kidney), L-AAO (EC 1.4.3.2, 4 U mg -1 of solid from Crotalus adamanteus), and the D-and L-forms of alanine, arginine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, tyrosine, and valine were obtained from Sigma. Bovine serum albumin (BSA, 96 -99 % albumin), glutaraldehyde (20% solution), ascorbic acid, uric acid, L-cysteine, and 1,2-diaminobenzene were obtained from Wako. They were used as received. The phosphate buffers were prepared from sodium dihydrogen phosphate. Distilled water purified with the use of a Millipore Milli-Q system was used throughout.
Construction of the dual enzyme electrodeA BAS cross-flow electrochemical flow-cell was used for the surface modification of the electrode. The electrode assembly consisted of a dual electrode with two platinum disks (3 mm in diameter) as a working electrode, a silver-silver chloride reference electrode, and a stainless-steel as an auxiliary electrode. Prior to the enzyme coating, the surface of the platinum disks was polished with 6 µm diamond particles (BAS) and then with 0.05 µm alumina particles (BAS), then rinsed with distilled water an...