Electrochemical Determination of Phenylalanine Using A Phenylalanine Hydroxylase–Modified Graphite Pencil Lead Electrode

Objective: Phenylalanine (L-Phe) is an essential aromatic amino acid whose abnormal accumulation is associated with serious metabolic disorders, particularly phenylketonuria (PKU). Therefore, the development of rapid, reliable, and cost-effective analytical methods for L-Phe determination is of significant clinical and biochemical importance. In this study, a novel and low-cost electrochemical biosensor platform was developed for the determination of L-Phe based on phenylalanine hydroxylase (PAH) immobilized on a graphite pencil lead electrode. Methods: The electrode surface was first activated by nitric acid treatment and electrochemical etching to introduce oxygen-containing functional groups, thereby increasing surface roughness and effective surface area. PAH enzyme immobilization was achieved via carbodiimide chemistry using an EDC/sNHS coupling strategy, enabling stable covalent attachment of the enzyme to the graphite surface. The electrochemical performance of the biosensor was evaluated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in a PAH–BH₄–O₂ reaction system. Results: The biosensor exhibited a distinct anodic signal in the potential range of 0.65–0.75 V, corresponding to the electrochemical oxidation of L-tyrosine generated by enzymatic conversion of L-Phe. DPV measurements enabled improved selectivity and resolution of the anodic response compared to CV, confirming that the electrochemical signal originated from the enzymatic product rather than direct oxidation of L-Phe. Conclusion: Overall, the proposed biosensor offers a simple, accessible, and cost-effective approach for L-Phe determination and provides a promising foundation for the further development of enzyme-based electrochemical sensing platforms.