Urea contamination in milk poses significant health risks, including kidney failure, urinary tract obstruction, fluid loss, shock, and gastrointestinal bleeding. This highlights the need for sensitive, rapid, and reliable methods to detect traces amount of urea in milk. In this study, we designed an electrochemical transducer for urea detection by utilizing purified watermelon urease (Urs), gold nanoparticles (AuNPs), and graphene oxide (GO). The nanomaterials and biosensor probe were characterized using UV-vis spectroscopy, XPS, TEM, XRD, FTIR, AFM, CV, EIS, and DPV. The engineered probe (GCE/AuNPs/GO/Urs) demonstrated a broad linear detection range of 5 to 90 mg/dL and a low limit of detection (LOD) of 0.037 (±0.012) mg/dL (RSD < 3.7%). The biosensor was tested for potential interferents that may be present in adulterated milk and an exceptionally low coefficient of selectivity (ksel <0.1) was obtained. Evaluation of milk samples from a local dairy farm showed good recovery rates from 93.13% to. 98.79% (RSD < 4.28%, = 3), indicating reliable detection capabilities. Stability tests confirmed the sensor's reproducibility and consistent performance. Additionally, a comparison study of the system was carried out using the purified watermelon urease and the commercially available urease. Herein, the results obtained using the sensor probe was finally validated with the gold standard method.
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