DETERMINATION OF L-PHENYLALANINE BASED ON AN NADH-DETECTING BIOSENSOR

An enzyme carbon paste electrode containing three different enzymes wa s developed for the determination of L-phenylalanine, This sensor is b ased on the enzymatic/electrochemical recycling of tyrosinase in combi nation with salicylate hydroxylase and L-phenylalanine dehydrogenase ( PADH). The enzymes salicylate hydroxylase and tyrosinase were coimmobi lized first in a carbon paste electrode for the sensitive detection of NADH. The principle of the bienzyme scheme is as follows: the first e nzyme, salicylate hydroxylase, converts salicylate to catechol in the presence of oxygen and NADH. The second-enzyme, tyrosinase, then oxidi zes the catechol to o-quinone, which is electrochemically detected and reduced back to catechol at the electrode at an E-appl = -50 mV vs Ag /AgCl, This results in an amplified signal due to the recycling of the catechol and o-quinone between tyrosinase and the surface of the elec trode, Prior to adding PADH, the salicylate hydroxylase-tyrosinase car bon paste electrode was characterized in terms of its sensitivity to N ADH, pH dependence, buffer composition, interferences, and stability, Interference from ascorbic acid and uric acid was found to be minimal, Human serum was used to investigate whether this bienzyme system was suitable for the detection of NADH in serum and blood samples, The sen sitivity for NADH was increased by a factor of 33 times using the bien zyme amplification scheme (electroreduction of o-quinone at E-appl = - 50 mV) as opposed to the salicylate hydroxylase single-enzyme system ( at which catechol would have been oxidized at E-appl = +150 mV vs Ag/A gCl), The detection limit for NADH achieved by the bienzyme carbon pas te electrode was 1 vs 100 mu M for the single-enzyme carbon paste elec trode, The salicylate hydroxylase-tyrosinase system was then coupled w ith phenylalanine dehydrogenase for L-phenylalanine determination, Thi s multienzyme sensor was able to achieve a linear range of 20-150 mu M and a detection limit of 5 mu M for L-phenylalanine. The sensitivity is sufficient since the reference clinical range for L-phenylalanine i s 78-206 mu M.