Oriented protein attachment onto gold electrodes to perform a highly sensitive and selective immunoassay with amperometric detection

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

Bordeaux, Francia

Congreso; 11th International Conference on Electroanalysis; 2006

In a previous work, we have reported the feasibility of performing an immunoassay with electrochemical detection to sense anti-Trypanosoma cruzi (T-cruzi) IgG antibodies. The method consists of an indirect ELISA, using thiolmodified gold electrodes as the support to which proteins from a T-cruzi lysate are adsorbed electrostatically to capture the analyte from human serum samples. Then, a secondary antibody conjugated with the redox enzyme, HRP, is used to develop the analyte reaction. By adding the enzyme substrate in a three-electrode cell containinga redox mediator, which is oxidised when acting as enzyme cosubstrate, it is possible to subsequently reduce the mediator at the electrode surface. The occurrence of a catalytic reduction current evidences the analyte presence in the sample, thus allowing for the diagnosis of T-cruzi infection. Even when the method showed an appropriate performance in discriminating between positive and negative samples, false positive results arose when testing serum samples from patients with related parasitosis, such as leishmaniasis. To improve the specificity of themethod, we examined the possibility of using as capturing antigen one recombinant chimera protein, Q1, which we had already demonstrated to be suitable for leishmaniasic serum samples. Although it was possible to avoid false positive results, the signal measured diminished from 30 to 50% when using Q1, as compared to that obtained when using total parasite lysate as a source of antigen, thus revealing sensitivity loss. Consequently, we assayed to enhance the signal-to-noise relationship by attaching covalently the recombinant protein to the electrode surface previously activated with carbodiimide. However, the currents registered when using this strategy were in the same order to those obtained when adsorbing Q1 electrostatically. Therefore, we designed a new chimera recombinant protein, Q2, with a three-lysine chain at the end of the protein zone involved in the antigen-antibody reaction, to facilitate a conveniently directed binding of the antigen to the carbodiimide-activated electrode surface. This approach recovered the signal between 30 and 80%, as compared with the currents registered when attaching Q1 covalently. These results suggest that this antigen linking strategy results in an appropriately oriented proteinattachment that improves epitopes exposure to the analyte