Improved sensitivity in graphene-mediated surface-enhanced Raman scattering via H2/Ar annealing

Graphene is well suited to biological applications, including biosensing and drug delivery, through uses such as field-effect transistors, surface-enhanced Raman scattering (SERS), fluorescence imaging, and photothermal therapy. For SERS-based biochemical sensing, the chemical stability of graphene against oxidation and other reactions is particularly advantageous. However, its low SERS enhancement factor hinders practical application. To address this limitation, we demonstrate that H2/Ar annealing at temperatures above 350 °C significantly improves sensitivity to a level comparable to that of metal nanostructured SERS substrates. We developed a sandwich enzyme-linked immunosorbent assay on graphene substrates as a biosensor, enabling the detection of interleukin-6, a cytokine associated with mastitis, at concentrations as low as 0.5 pg/ml. We also investigated the mechanism underlying the SERS enhancement observed in H2/Ar-annealed graphene. This enhancement can be attributed to the removal of polymethyl methacrylate residues and oxygen-containing groups from the graphene surface, as well as the adsorption of hydrogen and hydrocarbons, which facilitates the binding of negatively charged antibodies. Additionally, increased surface hydrophobicity enhances hydrophobic interactions, promoting the adsorption of antibodies.