Gold Nanoparticle-Driven Phenylketonuria Monitoring via Colorimetric Biosensing of Phenylalanine

Phenylalanine has long been recognized as a reliable biomarker for the diagnosis and monitoring of phenylketonuria (PKU), a rare genetic disease characterized by the impairment of phenylalanine hydroxylase. As part of the current efforts toward the design of point-of-care devices for the day-to-day management of PKU, we set up a coupled enzymo–plasmonic assay of phenylalanine based on the combination of the nicotinamide adenine dinucleotide (NAD + )-dependent enzyme phenylalanine dehydrogenase and gold nanoparticles to visually detect physiologically meaningful concentrations of phenylalanine and quantify them with a lightweight portable spectrophotometer compatible with smartphone readout. The principle of the assay relies on the dissolution of gold nanoparticles (AuNPs) induced by mixtures of HAuCl 4 and cetyltrimethylammonium bromide (CTAB) and its inhibition by (enzymatically produced) NADH, establishing a relationship between optical readout and phenylalanine concentration with a limit of detection of ca. 1 μM. The transposition of this assay to the analysis of serum samples containing phenylalanine concentrations spanning from 60 to 2000 μmol/L was achieved, validating the nanobiosensor as a promising tool for PKU management.