Microwell plates coated with graphene oxide enable advantageous real-time immunosensing platform

Abstract Immunoassays are fundamental analytical tools in molecular diagnostics, therapy monitoring and drug discovery. Nevertheless, they often take around 6 h and require cumbersome procedures. We introduce a breakthrough in immunosensing based on the photoluminescence quenching capabilities of graphene oxide (GO) and the versatile format offered by the famous microwell plates. Taking advantage of the highly efficient non-radiative energy transfer occurring between photoexcited fluorophores (donors) and GO (acceptor), we discovered that flurophore-labelled antibodies (Fl-Abs) are quickly and strongly quenched by the studied GO-coated microwell, whereas Fl-Abs complexed with the respective analyte are weakly quenched by the same surface due to the low affinity between the GO-coated surface and the relatively long distance between these photoluminescent complexes and the GO-coated surface. In doing so, we developed a conceptually innovative single-step immunosensing platform, avoiding blocking, separation and washing steps and exploiting a single antibody. Importantly, the biosensing response can be interrogated in real time. This leads to an advantageous immunodetection phenomenon which is observable in few minutes (e.g. 5 min). The resulting highly transformative biosensing platform operates with different photoluminescent agents and different analytes. Besides, this biosensing platform was demonstrated to operate with real samples of human urine doped with different concentrations of prostate specific antigen.