Development of electrochemical HE4 immunosensor

In this study, a new immunosensor was developed for the electrochemical determination of human epididymis protein 4 (HE4) that is a cancer biomarker and its application was performed on human blood serum samples. In this work, firstly the screen printed carbon electrodes (SPCE) were modified with reduced graphene oxide (RGO), then the electrode surface was coated with poly(thionine) by applying the electropolymerization technique, and finally, gold nanoparticles (AuNP) were created by electrodeposition on the surface. Electrochemical characterizations of the SPCE/RGO/PTH/AuNP electrode were performed using cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) methods. Immobilization of Anti-HE4 to the surface of SPCE/RGO/PTH/AuNP electrodes were carried out by covalent binding method using 3-mercaptopropionic acid (3-MPA) and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide/N-hydroxy succinimide (EDC/NHS). The optimum working parameters of the prepared HE4 immunosensor were determined by DPV and EIS methods. The optimum Anti-HE4 concentration, incubation time of Anti-HE4 and incubation time of HE4 were found to be 33.33 µg/mL, 45 min and 30 min, respectively. The analytical characterization of electrochemical HE4 immunosensors were performed under the optimum working parameters. It was calculated that the linear ranges were 1-100 pg.mL-1 by EIS, DPV and SWV methods, limit of detections (LOD) were 2.16 pg.mL-1, 1.04 pg.mL-1 and 1.16 pg.mL-1, respectively. The repeatability of the HE4 immunosensor was tested for 50 ng.mL-1 HE4 concentration and relative standard deviation (R.S.D.) values were calculated as 4.09% (n = 10), 4.29% (n = 10) and 1.95% (n = 10) for EIS, DPV and SWV methods, respectively. The operation stability of the developed HE4 immunosensor was examined by voltammetric methods for 60 days, and the storage stability for 12 weeks. The selectivity of the HE4 immunosensor was tested by performing HE4 analysis in the presence of 9 different antigen mixes and it was found that the antigen mixture made a 3.67% interference. The prepared HE4 immunosensors were used for the analysis of different concentrations of HE4 added to the human blood serum samples in a known amount and over 95% recoveries were obtained.