Graphene nanosheets as an electric mediator for ultrafast sensing of urokinase plasminogen activator receptor-A biomarker of cancer

Fluorine doped tin oxide (FTO) electrochemical immunosensor has been developed for rapid detection of urokinase type plasminogen activator receptor (uPAR) - a biomarker for cancer. uPAR is a GPI-anchored cell membrane receptor that shows increased expression in many types of human cancers which include breast, prostate, colorectal, and non-small cell lung cancer. In this study, a novel ultrasensitive FTO graphene nanosheets based electrode was used as a working probe to analyze the interaction between urokinase plasminogen activator (uPA) and monoclonal uPAR antibody (Ab). Graphene nanosheets (GNS) exhibited high conductivity, thereby increasing the sensitivity of the immunochemical assay. GNS were coupled with uPAR-Ab via carbodiimide activation chemistry with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as a heterobifunctional crosslinker. The confirmation of immobilization events was done by biophysical methods such as UV-Vis spectroscopy, fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), atomic force microscopy (AFM), differential pulse (DPV), and cyclic voltammetry (CV). The immobilization conditions were optimized in accordance with the best sensor response. Under optimum conditions, the proposed sensor displayed wide linear detection range (1 fM to 1 mu M) with a detection limit of 4.8 fM in standard. The developed sensor was profitably engaged to detect uPA in spiked serum samples up to 9.2 pM. Furthermore, the developed uPAR immunosensor showed good reproducibility, repeatability, and storage stability (75% of initial activity observed up to 4 weeks). FTO/GNS/uPAR-Ab/uPA-Ag immunosensor displayed acceptable performance for detection of uPA and exhibited low detection limit with high reproducibility. The proposed immunosensor is 'easy to use', highly specific, and can be used as a quantitative tool making it a tenable alternate for the detection of uPAR in cancer patients.