Biosensor for L-phenylalanine based on the optical detection of NADH using a UV light emitting diode

Abstract

A biosensor was developed for the detection of L-phenylalanine (Phe) and demonstrated for use in the diagnosis of phenylketonuria (PKU). It consists of L-phenylalanine dehydrogenase (L-PheDH) immobilized on a membrane, an ultraviolet light-emitting diode excitation system, and a photomultiplier tube. The L-PheDH was immobilized on a teflon membrane modified with 2-methacryloyloxyethyl phosphorylcholine and placed at the distal and of an optical fiber. The concentration of Phe was determined by immersing the sensor into a sample solution that also contained NAD+ and measurement of the fluorescence of the NADH produced by enzymatic reaction. Two L-PheDHs (from Thermoactinomyces intermedius and Sporosarcina sp.) were studied and compared. The fluorescence intensities of the biosensor are linearly related to the L-Phe concentrations in the range from 10 μmol L−1 to 10 mmol L−1. The sensor also was operated in the kinetic mode by differential determination of the slope of the signal within 2 min. The analytical range of the sensor is adequate for application in the genotypic diagnosis of PKU (diagnostic value >600 μmol L−1). High sensitivity, good cost-benefit ratio, and low power consumption are typical features of this biosensing system that can can be applied to routine screening of newborn.