Graphene-based multiplexed disposable electrochemical biosensor for rapid on-farm monitoring of NEFA and βhBA dairy biomarkers

Elevated circulating concentrations of non-esterified fatty acids (NEFA) and beta hydroxy butyrate (βHBA) in biological fluids are recognized as critical biomarkers for early diagnosis of negative energy balance (NEB) in dairy cows. Herein, we report the development of a cost-effective, bio-friendly and electrochemically active dual screen printed electrode (SPE) sensor platform composed of electro-reduced graphene oxide nanosheets (E-rGO) modified with specific antibodies against NEFA and βHBA. The chemically synthesized graphene oxide (GO) was reduced directly on the screen printed electrode (SPE) surface via a green electrochemical approach without using toxic chemicals. The E-rGO was characterized using various analytical techniques, like XPS, SEM, TEM, AFM, UV-Vis, Raman spectroscopy, FTIR, and XRD, to get insight into its properties. Electrochemical analysis demonstrates that the E-rGO-modified SPE electrodes exhibit enhanced and durable redox properties as compared to the pristine graphite and GO electrodes. Target specificity is accomplished through immobilization of specific antibodies against NEFA and βHBA over the nanostructure-modified surface of the SPE, which only interacts with its counterpart NEFA and βHBA only. The antibodies retain their characteristic immuno-complex formation property upon immobilization and exhibit changes to amperometric signals upon interaction with various concentrations of NEFA and βHBA in standard, spiked blood and real clinical samples. The DPV signals resulted from the developed immunosensor platform exhibited a good correlation (R² ∼ 0.99 for both NEFA and βHBA) for a wide range of target concentrations from 0.1 mM to 10 mM. The proposed immunosensor design not only provides a rapid analytical response time (≥1 min), but also simplicity in fabrication and instrumentation, which may provide a promising approach for on-farm diagnostics of ketosis and metabolic disorders associated with NEB.