Development of a novel potentiometric PHD/SPE biosensor for the determination of phenylalanine

In this paper, we report the first example of a novel potentiometric sensor for the real-time monitoring of phenylalanine. The biosensor consists of a commercial screen-printed electrode (SPE) platform, where the gold working electrode has been covalently functionalized with the enzyme phenylalanine dehydrogenase (PHD). The PHD/SPE membrane-free biosensor was fabricated by immobilizing the PHD enzyme to the gold electrode using 3-3'-dithiopropionic acid of N-succinimide ester (DSP) as a linker capable of creating a thiol bond with the gold electrode and an amide bond with the amine group present on the PHD enzyme. The ability of the enzyme to catalyze the transformation of phenylalanine to pyruvic acid, NADH, and ammonia, under the conditions used for analysis through the biosensor, was evaluated by UV-Vis analysis. Under the experimental conditions adopted, the enzymatic reaction is almost completed in 40-60 minutes. The biosensor was then tested for the Phe monitoring using the Open Circuit Potential (OCP) potentiometric technique, recording the potential of the biosensor as the reaction proceeded over time. The potentiometric response with respect to the Ag/AgCl reference electrode was found to be linear over a relatively wide concentration range (0-5000 mu mathrm{M}). The results demonstrated the feasibility of developing a novel membrane-free enzymatic biosensor for monitoring Phe in patients with phenylketonuria (PKU). © 2022 IEEE.