Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen.

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL for CEA and up to 30 ng mL for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented.