Phenylalanine-responsive fluorescent biosensor based on graphene oxide-chitosan nanocomposites catalytic film for non-destructive fish freshness grading

An optical enzymatic phenylalanine biosensor for non-invasive evaluation of fish freshness using biofluid from the fish body surface has been developed based on phenylalanine dehydrogenase (PDH) and nicotinamide adenine dinucleotide (NAD+) chemisorbed on the graphene oxide-chitosan (GO/CT) nanocomposites film. The immobilized PDH on the biosensor surface via EDC/NHS coupling strategy catalyzed the oxidative deamination of phenylalanine to phenylpyruvate, and concomitantly converted the cofactor NAD+ (oxidized form) to fluorophore NADH (reduced form), which can be quantified by using fluorescence spectrophotometer. The phenylalanine-responsive fluorescent biosensor showed a dynamic linear concentration range between 0.03 mM and 0.24 mM phenylalanine at 644 nm emission wavelength with an excitation wavelength of 320 nm. The limit of detection (LOD) of the fluorometric phenylalanine biosensor was estimated at 0.02 mM with a fast response time of 1 min. No significant interference by amines-synthesized amino acids, such as histamine, creatine, sarcosine, creatinine, tyrosine and uric acid at typical physiological levels commonly co-exist with phenylalanine in the biofluid metabolite of fish towards fresh fish quality grading. Validation study of the GO/CT catalytic film revealed excellent practicality of the biosensor in biological fluid sample of tuna fish (Euthynnus affinis) for detection of phenylalanine. The proposed GO/CT nanocomposites biofilm offers rapid test that can be assessed with an easy manner to rapidly analyze large number of samples in a short period of time.