Application of an enzyme chip to the microquantification of l-phenylalanine

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase–resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 × 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)6- and (His)9-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The Vmax and kcat values of the (His)6-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)9-PheDH variant, and the (His)6-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-μl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.