Antibody testing for the glutamic acid decarboxylase 65 antibody (GADA) is widely used as a golden standard for autoimmune diabetes diagnosis, while current methods for antibody testing are not sensitive enough for clinical usage. Here, a label-free electrochemiluminescent (ECL) immunosensor for detecting GADA in autoimmune diabetes is fabricated and investigated. In the designed immunosensor, a composite film including the multiwalled carbon nanotubes (MWCNTs), zinc oxide (ZnO), and Au nanoparticles (AuNPs) was prepared through nanofabrication processes to improve the performance of sensor. The MWCNTs, which can provide a larger specific surface area, ZnO as a good photocatalytic material, and AuNPs that can enhance the ECL signal of luminol and immobilize the GAD65 antigen were applied to prefunctionalize indium tin oxide (ITO) glass based on a nanofabrication process. The GADA concentration was detected using the ECL immunosensor after incubating with GAD65 antigen-coated prefunctionalized ITO glass. After a direct immunoreaction, it is found that the degree of decreased ECL intensity has a good linear regression toward the logarithm of the GADA concentration in the range of 0.01 to 50 ng mL-1 with a detection limit down to 10 pg mL-1. Human serum samples positive or negative for GADA all nicely fell in the expected area. The fabricated immunosensor with excellent sensitivity, specificity, and stability has potential capability for clinical usage in GADA detection.
Keywords: GAD65 antibody; composite electrode; electrochemiluminescence; label-free immunosensor; type-1 diabetes mellitus.