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Bacterial infections in marine fishes are linked to mass mortality issues; hence, rapid detection of an infection can contribute to achieving a faster diagnosis using point-of-care testing. There has been substantial interest in identifying diagnostic biomarkers that can be detected in major organs to predict bacterial infections. Aspartate was identified as an important biomarker for bacterial infection diagnosis in olive flounder (Paralichthys olivaceus) fish. To determine aspartate levels, an amperometric biosensor was designed based on bi-enzymes, namely, glutamate oxidase (GluOx) and aspartate transaminase (AST), which were physisorbed on copolymer reduced graphene oxide (P-rGO), referred to as enzyme nanosheets (GluOx-ASTENs). The GluOx-ASTENs were drop casted onto a Prussian blue electrodeposited screen-printed carbon electrode (PB/SPCE). The proposed biosensor was optimized by operating variables including the enzyme loading amount, coreactant (α-ketoglutarate) concentration, and pH. Under optimal conditions, the biosensor displayed the maximum current responses within 10 s at the low applied potential of −0.10 V vs. the internal Ag/AgCl reference. The biosensor exhibited a linear response from 1.0 to 2.0 mM of aspartate concentrations with a sensitivity of 0.8 µA mM−1 cm−2 and a lower detection limit of approximately 500 µM. Moreover, the biosensor possessed high reproducibility, good selectivity, and efficient storage stability.

A novel electrochemical sensor based on Cu-loaded carbon nanospheres (Cu–CNSs) was designed and fabricated. Initially, the CNSs were synthesized using a natural or inexpensive carbon source (dark brown sugar), and Cu was loaded to enhance the electrocatalytic properties of the material. Subsequently, the synthesized Cu–CNSs were modified onto a screen-printed carbon electrode (SPCE), termed Cu–CNS/SPCE, to simultaneously detect the biomarkers dopamine (DA) and melatonin (MT) through differential pulse voltammetry. The surface characterization of the Cu–CNSs confirmed the formation of carbon spheres and Cu nanoparticles covering the spheres. Electrochemical studies showed that the Cu–CNS/SPCE had a high selectivity and sensitivity toward DA and MT, with a significant peak separation of 0.502 V. The two linear ranges of DA were 0.125–20 μM and 20–100 μM and the linear range of MT was 1.0–100 μM, with corresponding detection limits of 0.34 μM and 0.33 μM (S/N = 3), respectively. The quantification limits for DA and MT were 2.19 and 1.09 μM (S/N = 10), respectively. The sensor performance is attributed to the high conductivity and large, electrochemically active surface area of the Cu–CNS. In human serum samples, the Cu–CNS/SPCE exhibited good selectivity and satisfactory reproducibility for the simultaneous determination of DA and MT.