| Summary: | Thermally reduced graphene oxide (TRGO) is a graphene-based nanomaterial that has been identified as promising for the development of amperometric biosensors. Urease, in combination with TRGO, allowed us to create a mediator-free amperometric biosensor with the intention of precise detection of urea in clinical trials. Beyond simplicity of the technology, the biosensor exhibited high sensitivity (2.3 ± 0.1 µA cm<sup>−2</sup> mM<sup>−1</sup>), great operational and storage stabilities (up to seven months), and appropriate reproducibility (relative standard deviation (RSD) about 2%). The analytical recovery of the TRGO-based biosensor in urine of 101 ÷ 104% with RSD of 1.2 ÷ 1.7% and in blood of 92.7 ÷ 96.4%, RSD of 1.0 ÷ 2.5%, confirmed that the biosensor is acceptable and reliable. These properties allowed us to apply the biosensor in the monitoring of urea levels in samples of urine, blood, and spent dialysate collected during hemodialysis. Accuracy of the biosensor was validated by good correlation (R = 0.9898 and R = 0.9982) for dialysate and blood, utilizing approved methods. The advantages of the proposed biosensing technology could benefit the development of point-of-care and non-invasive medical instruments.
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