Bioinformatic Insights into AuNP-Directed Enzyme Orientation for Enhanced Phenylalanine Electrochemical Biosensing

Phenylalanine (Phe) accumulation in blood is a hallmark of phenylketonuria (PKU), a metabolic disorder that requires early diagnosis and lifelong monitoring to prevent irreversible neurological damage. Conventional analytical methods rely on centralized laboratory infrastructure, limiting their use in decentralized and point-of-care (POC) settings. Here, we report an enzymatic electrochemical biosensor based on screen-printed carbon-graphene electrodes modified with gold nanoparticles (AuNPs) and functionalized with phenylalanine dehydrogenase (PheDH). The biosensor exhibited a linear response across physiological and pathological Phe concentrations, with a sensitivity of 13.6 μC (mg dL-1)-1 and a limit of detection of 0.12 mg dL-1. Michaelis-Menten analysis yielded an apparent Km of 1.31 mg dL-1 and a maximum charge (Qmax) of 316 μC, indicating high affinity between the immobilized enzyme and Phe. The platform also enabled quantitative detection in spiked biological matrices, demonstrating its applicability to clinically relevant samples. Bioinformatic and structural analyses revealed that AuNPs promote a favorable enzyme orientation that enhances substrate accessibility and electron transfer. By integrating electrochemical measurements with molecular-level modeling, this work establishes a rational strategy for designing high-performance enzymatic biosensors. The compatibility of the platform with portable electrochemical instrumentation highlights its translational potential for decentralized and self-monitoring applications in PKU management, although further validation with larger clinical sample sets and long-term stability studies is required.