A dual-electrode label-free immunosensor based on in situ prepared Au-MoO3-Chi/porous graphene nanoparticles for point-of-care detection of cholangiocarcinoma.

A novel label-free electrochemical immunosensor was prepared for the detection of carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) as biomarkers of cholangiocarcinoma (CCA). A nanocomposite of gold nanoparticles, molybdenum trioxide, and chitosan (Au-MoO3-Chi) was layer-by-layer assembled on the porous graphene (PG) modified a dual screen-printed electrode using a self-assembling technique, which increased surface area and conductivity and enhanced the adsorption of immobilized antibodies. The stepwise self-assembling procedure of the modified electrode was further characterized morphologically and functionally. The electroanalytical detection of biomarkers was based on the interaction between the antibody and antigen of each marker via linear sweep voltammetry using ferrocyanide/ferricyanide as an electrochemical redox indicator. Under optimized conditions, the fabricated immunosensor showed linear relationships between current change (ΔI) and antigen concentrations in two ranges: 0.0025-0.1 U mL-1 and 0.1-1.0 U mL-1 for CA19-9, and 0.001-0.01 ng mL-1 and 0.01-1.0 ng mL-1 for CEA. The limits of detection (LOD) were 1.0 mU mL-1 for CA19-9 and 0.5 pg mL-1 for CEA. Limits of quantitation (LOQ) were 3.3 mU mL-1 for CA19-9 and 1.6 pg mL-1 for CEA. The selectivity of the developed immunosensor was tested on mixtures of antigens and was then successfully applied to determine CA19-9 and CEA in human serum samples, producing satisfactory results consistent with the clinical method.