Indirect ELISA with amperometric detection as a diagnostic tool: Characterization of a self-assembled electrode for Trypanosoma cruzi antibodies quantification

RIBONE, M. E.; BELLUZO, M. S.; MARCIPAR, I. S.; LAGIER, C. M.

Coimbra, Portugal

Simposio; XVIII International Symposium on Bioelectrochemistry and Bioenergetics and 3rd Spring Meeting Bioelectrochemistry; 2005

A selfassembled electrode was designed for an indirect immunoassay to detect Chagas disease antibodies. The modifying layer consists of Trypanosoma cruzi (T-cruzi) proteins obtained from a homogenate of parasite culture as antigen, attached either covalently or by electrostatic forces to a gold electrode, previously modified using 3-mercaptopropane sulfonic acid or  3-mercaptopropanoic acid as thiols. The immunoelectrode assemblage is completed with two subsequent steps consisting of adsorption of anti-T-cruzi antibodies (from human sera blood ofconfirmed infected patients) and anti-human IgG conjugate with horseradish peroxidase. The assay is performed using ferrocenemethanol (FcMe) as a soluble enzyme co-substrate mediating between the electrode surface and the redox site of the enzyme. The charge transport between FcMe and the electrode surface was confirmed by cyclic voltammetry, whereas the FcMe redox mediation between the enzyme and the modified electrode was verified by measuring the current occurring after addition of the enzyme natural substrate, hydrogen peroxide, into the solution containing the mediator. The immunoassay proposed therefore relays on the amperometric  measurements of the catalytic current occurring at 80 mV (vs. a Ag/AgCl reference electrode), when a human serum positive to Chagas disease is used to build-up the immunosensor. The measured signal increased with the concentration of the specific T-cruzi antibody used to assemble the electrode. The sensibility of the assay was, at least, equivalent to that obtained with the currently used commercial ELISA kit with spectrophotometric detection for Chagas disease  diagnosis.