Thionine/nanogold multilayer film for electrochemical immunoassay of alpha-fetoprotein in human serum using biofunctional double-codified gold nanoparticles

Abstract

A simple and sensitive electrochemical immunoassay method was developed for the detection of alpha-fetoprotein (AFP) in human serum. The immunoassay is based on the use of an AFP-functionalized thionine/nanogold multilayer film ({Thi/AuNP}_n) on a single-walled carbon nanotubes (CNT)-coated gold electrode and double-codified gold nanoparticles with horseradish peroxidase-labelled anti-AFP antibodies (HRP-anti-AFP-AuNPs). With a competitive immunoassay format, the assay was performed using HRP-anti-AFP-AuNP as trace and H₂O₂ as enzyme substrate. The electrochemical behavior of the multilayer {Thi/AuNP}_n films was studied. The maximal signal was obtained at n = 3 (i.e. {Thi/AuNP}₃). Under optimal conditions, the obtained electrochemical responses were proportional to the AFP levels in the sample. The dynamic range for AFP quantification was 0.25–45 ng mL⁻¹. Both the intra- and inter-assay coefficients of variation were less than 9.5%. The proposed assay had a detection limit of 0.05 ng mL⁻¹, which was 20-fold vs. 10-fold lower than that obtained using monolayer Thi/AuNP film vs. conventional HRP-labelled anti-AFP antibodies. Importantly, no significant differences at the 0.05 confidence level were encountered in the analysis of clinical serum samples between the proposed immunoassay and the commercially available Roche 2010 Electrochemiluminescent (ECL) Automatic Analyzer for AFP determination.