Magnetic bead-amplified voltammetric detection for carbohydrate antigen 125 with enzyme labels using aptamer-antigen-antibody sandwiched assay

Conventional affinity assays used for carbohydrate antigen (CA125) detection are still problematic due to the heterogeneity in the structure and molecular nature of CA125. This makes the conventional antibody based immunoassay difficult to detect CA125 levels accurately in clinical samples which demands the need for the development of new affinity-based detection without compromising specificity and sensitivity. In this study, for the specific detection of CA125, a unique hybrid affinity biosensor involving DNA aptamers and monoclonal antibodies in a sandwich assay format was developed. To suppress the electrode biofouling from interfering blood proteins, CA125 in human blood/serum samples were specifically captured and transferred to the electrode using monoclonal antibodies conjugated magnetic beads (MBs). The affinity binding between CA125 and the protein binding aptamer was investigated using electrochemical methods and computational modelling. Two different electrochemical detection modalities, viz., electrocatalytic activity of signaling enzymes conjugated to MBs and utilizing external redox probe in the solution, have been evaluated for monitoring the immuno-complex formation CA125 detection. In human serum samples, a sensitivity of 0.017 mA U mL(-1) and the detection limit of 0.08 U mL(-1) for CA125 is demonstrated. The immunosensor was able to detect CA125 levels with a broad clinical range of 2 U/mL to 100 U/mL.