Multilayered graphene (MLG) is an interesting material for electrochemical sensing and biosensing because of its very large 2D electrical conductivity and large surface area. We propose a less toxic, reproducible, and easy method for producing functionalized multilayer graphene from multiwalled carbon nanotubes (MWCNTs) in mass scale using only concentrated H2SO4/HNO3. Electron microscopy results show the MLG formation, whereas FTIR and XPS data suggest its carboxylic and hydroxyl-functionalized nature. We utilize this functionalized MLG for the fabrication of a novel amperometric urea biosensor. This biosensor shows linearity of 10–100 mg dL–1, sensitivity of 5.43 μA mg–1 dL cm–2, lower detection limit of 3.9 mg dL–1, and response time of 10 s. Our results suggest that MLG is a promising material for electrochemical biosensing applications.
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