Determination of Phenylalanine by a Novel Enzymatic PHD/SPE Biosensor

In this article, we report the first example of a novel potentiometric sensor for the real-time monitoring of phenylalanine (Phe). The developed enzymatic biosensor consists of a commercial screen-printed electrode (SPE) platform with the gold working electrode covalently functionalized with the enzyme Phe 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 Phe to pyruvic acid, NADH, and ammonia, under the conditions used for the enzymatic biosensor, was first assessed by UV-Vis analysis under batch conditions. Under the experimental conditions adopted, the enzymatic reaction was almost completed in 40–60 min. 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–
$5000 \mu \text{M}$
). The reported results demonstrated the feasibility of developing a novel membrane-free enzymatic biosensor for Phe monitoring in patients affected with phenylketonuria (PKU).