The development of a voltammetric immunosensor for determination of alpha-fetoprotein (AFP) in serum is presented. ELISA assays with voltammetric reading were carried out exploiting the peculiar properties of nanobiocomposite materials based on gold nanoparticles for the immobilization of Antibody (Ab)/Antigen/Antibody-HRP (Horseradish Peroxidase) sandwich on the glassy carbon (GC) electrode surface. The electrochemical transduction was mediated by thionin, which was used in its monomeric form dissolved in the reading solution, so avoiding critical immobilization procedures. The study was aimed at the development and validation of an immunosensor able to provide results in short time, simple to use, rugged and cost-effective for AFP monitoring purposes. A crucial aspect of the study was the development of an experimental protocol leading to highly standardized and consequently reproducible sensors. Two-way analysis of variance (ANOVA) was applied to study the effect of the concentration of the solutions used for the incubation of the antibodies. The sensor was validated in serum assessing stability of the immunocomplex, linearity of response, limit of detection (3.7 ng/ml) and limit of quantitation (11 ng/ml), precision (intra- and inter-sensor repeatability) and recovery rate (103%). The stability of the GC/Ab functionalized substrate was demonstrated over one month, showing variation coefficients below 5%. Experiments carried out with real samples of clinical interest evidenced that the developed immunosensor can be considered as powerful tool in cancer screening programmes.
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