The use of nanomaterials for the construction of electrochemical immunosensors has provided greatly sensitive and selective detection devices, as a consequence of their bio-compatibility, adsorption capability and high specific surface area1. These affinity-based sensors are especially relevant for applications to the “in-situ” analysis of biological samples and for screening methods in the clinical field, in general, and in sport medicine, in particular. This is due to their low cost, robustness, low detection limits, small sample volumes required, easiness and rapidity for monitoring events of biological interaction. Testosterone, cortisol and related substances (other anabolic steroids or corticoids, respectively) are key molecules of high scientific-technological interest for which rapid and reliable determination methods are required to be applied as screening procedures in food and health areas.
Our research has developed some immunosensor configurations for the amperometric detection of these compounds. The common characteristic of all of them is they use composite electrodes prepared with carbon nanotubes. A first simple design involves a mixture of carbon nanotubes and colloidal gold using Teflon as the binder. This mixture has demonstrated to be a highly stable immobilization medium for anti-testosterone, besides exhibiting an intense electrocatalytic activity towards H2O2, thus allowing a competitive assay with HRP-labelled testosterone and the analyte to be establish. Using catechol as mediator, it is possible to detect the amperometric signals at a potential value of 0.0 V. Another configuration implied hybrids of PDDA-functionalyzed carbon nanotubes and gold nanoparticles. The resulting material, deposited on a glassy carbon electrode, is used as an immunosensing surface for testosterone in a sandwich-type assay with a HRP-labelled secondary antibody. Moreover, an immunosensor for cortisol has been designed by using a carbon nanotubes-colloidal gold- 1-n-octylpyridinium hexafluorophosphate composite electrode. After immobilization of anticortisol on the electrode surface, the antigen determination is carried out by means of a sandwich-type scheme using an alkaline phosphatase-labelled secondary antibody. The electrode surface exhibits very low background currents due to the presence of the ionic liquid. The detection of the enzyme reaction product, 1-naftylphenol, was accomplished at a potential value of + 0.3 V.
The optimization of the sensing surfaces bio-functionalization conditions was carried out in all cases. The influence of the experimental variables affecting the immunosensor performance: type of mediator, detection potential value, pH, reagents concentrations and incubation times, were evaluated.
