Simultaneous electrochemical determination of two hepatitis B antigens using graphene-SnO2 hybridized with sea urchin-like bimetallic nanoparticles.

The hepatitis B virus (HBV) can cause chronic hepatitis and hepatocellular carcinoma. Hepatitis B surface antigen (HBs-Ag) and Hepatitis B e-antigen (HBe-Ag) are key markers for the diagnosis of HBV. In this study, electrodeposited gold was used as a sensing platform. Three-dimensional (3D) SnO2-loaded graphene sheets functionalized by Thionine (Thi) and ferrocene (Fc) and hybridized by sea urchin-like bimetallic nanoparticles (GS-SnO2-BMNPs) were used as redox probes for labeling antibodies to fabricate sandwich-type immunosensors for the simultaneous determination of HBs-Ag and HBe-Ag. The bimetallic nanoparticles, gold hybrid platinum nanoparticles (Au@Pt) and L-cysteine-connected gold-silver nanoparticles (Ag-cys-Au), have large electroactive surface areas. They were prepared by an efficient and economical method. Additionally, the sea urchin morphology accelerates spatial utilization, thus increasing the number of combination sites. Therefore, the immune probe can load a mass of signal source molecules (Thi and Fc). Furthermore, GS-SnO2-BMNPs (GS-SnO2-Au@Pt and GS-SnO2-Ag-cys-Au) with excellent electrical conductivity and bimetallic synergy can enhance the square wave voltammetry (SWV) signal. SWV was used to record the electrochemical signal by scanning the potential from - 0.6 to 0.6 V (vs. SCE). The signal peaks resulted from the reduction reaction of Thi and Fc, and two signal peaks were completely separate. The peak position and current intensity reflect the identity and level of the corresponding antigens. Therefore, the simultaneous detection of two viral biomarkers was achieved by the proposed immunosensor. The fabricated immunosensor showed a linear concentration range for HBs-Ag (0.01-100 ng·mL-1) and HBe-Ag (0.01-100 ng·mL-1), with detection limits for HBs-Ag and HBe-Ag of  4.67 pg·mL-1 and 4.68 pg·mL-1, respectively.  The RSD of HBs-Ag ranged between 2.0 and 4.4%and the recovery was in the range 98.7 to 99.4%. For  HBe-Ag the RSD  was between 2.6 and 3.3% andrecoveries  in the range 99.2 to 100.5% were obtained.