Stepwise control of reduction of graphene oxide and quantitative real-time evaluation of residual oxygen content using EC-SPR for a label-free electrochemical immunosensor

The aim of this study was to develop an electrochemical surface plasmon resonance (EC-SPR) method for the stepwise reduction of graphene oxide (GO) by monitoring the real-time refractive index in an effort to control the residual oxygen functionality and conductivity in GO sheets, and then to enhance sensitivity to detect immunoaffinity. The EC-SPR technique acts as a real-time operating system to be used for the observation of oxygen chemical element on the surface of GO. Experimental results demonstrated that EC-SPR signals could quantitatively detect real-time changes in the refractive index of GO films during the stepwise removal of oxygen functional groups. Cyclic voltammetry (CV) in the initial cycles of the electrochemical reduction of GO showed that the oxygen content of the GO film declined by approximately 60%. The SPR angle shifts during the electrochemical reduction for 10, 50 and 100 CV cycles were 164, 218 and 223 mdeg, and the corresponding X-ray photoelectron spectroscopy (XPS) spectra carbon-to-oxygen (C/O) ratios were 17.35, 21.07 and 30.95, respectively. The obtained electrochemically-reduced graphene oxide (ERGO) film chip had a very high sensitivity to detect anti-BSA, and this electrochemical immunosensor was more sensitive than an SPR immunosensor. Our results confirm that this EC-SPR technique could be used to develop electrochemical biosensors with immediate modification of GO film surfaces.