Impedimetric evaluation for diagnosis of Chagas’ disease: antigen–antibody interactions on metallic eletrodes
Introduction
Chagas’ disease, an endemic chronic parasitic disease which occurs in Latin America, is caused by the protozoan Trypanosoma cruzi. According to the World Health Organization (WHO, 1996), it is estimated that 16 million people are infected and that about 100 million individuals, living in endemic areas, are at risk of contracting T. cruzi infection. Under natural conditions, infected reduviid bugs transmit the T. cruzi to humans. However, T. cruzi may bypass the vector bugs and be transmitted to man by a number of alternative mechanisms: blood transfusion, congenital transmission, accidental laboratory contamination, organ transplantation from infected donors and transmission by oral route (Umezawa et al., 1996, Gomes, 1997).
T. cruzi induces in humans an acute phase infection, with patent parasitemia, which is followed by a life-long chronic phase, characterized by subpatent parasitemia and scarce tissue parasitism. In the acute phase, diagnosis is based upon detection of these organisms by direct parasitological methods—thin or thick blood smears, Strout method, and buffy coat on slide (WHO, 1991). In the chronic phase, antibodies detection is the best way to perform etiological diagnosis and it is the tool for blood bank screening. These antibodies are detected by serological assays that, according to Brazilian Health Agency, requires at least two methods in parallel to confirm the infection in order to obtain a safe diagnosis. There are standard techniques for assays, as indirect haemagglutination (IHA), indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). However, cross-reaction to T. cruzi has been observed with related protozoan diseases, particularly leishmaniasis. This problem may be overcome by using recombinant polypeptides containing specific T. cruzi epitopes that elicit an immune response in the majority of chagasic patients.
Two recombinant antigens, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA), expressed in the bacterium Escherichia coli were analyzed in a diagnostic test for Chagas’ disease. The data indicated that recombinant antigens displayed better results when used in combination (CRA+FRA) than separately (Krieger et al., 1992). These antigens were characterized and shown to display a repetitive epitope structure (Lafaille et al., 1989, Krieger et al., 1990). FRA is located in the flagellum of the parasite and displays a 68-amino acid repeat, while CRA is distributed throughout the cytoplasm and has a 14-amino acid repeat (Lafaille et al., 1989). A kit for diagnosis of chronic Chagas’ disease, using CRA and FRA antigens, (EIE-Recombinant-Chagas-Biomanguinhos), was developed by Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil which showed high performance for serodiagnosis (Gomes et al., 2001) and were evaluated to monitoring the cure of chagasic infection (Silva et al., 2002). The molecular characteristics of CRA+FRA makes it an attractive antigen to use in other diagnosis methods such as immunosensors.
Impedance has been considered as a plausible alternative method for immunological assays (Newman et al., 1988). While some studies were carried out on silicon heterostructures (Souteyrand and Martelet, 1994, Maupas et al., 1996), other were performed directly on metallic electrodes (Ma Jie et al., 1999). Depending on configuration, information about the antigen–antibody interaction may be obtained from capacitance or resistance.
In order to develop a biosensor for Chagas’ disease, based on impedance methodology, the behavior of CRA+FRA antigens adsorbed on gold and platinum electrodes was investigated and the results are presented in this article.
Section snippets
Experimental
CRA and FRA recombinant proteins were obtained from Biomanguinhos/FIOCRUZ, that detain the patent (BR PI 1100552-1; US 5736348; Europe 919203356.2). Human sera blood were collected from two cardiac patients with confirmed clinical, epidemiological and serological diagnosis and one nonchagasic individual with negative serological evaluation. Blood samples were taken by venopuncture and the sera obtained were stored at −20 °C until use. The Au and Pt disc electrodes with diameters of 2 mm were
Results and discussion
As initial characterization of the electrode/solution interface, impedance measurements were carried out for the Au/PBS system at several electrode potentials. In general, for this system, the impedances are very large, as shown in Fig. 1. Nonetheless, it can be observed that at −100 and 0 mV the impedance is smaller than at other potentials. This fact was associated with the presence of oxygen dissolved in the solution. After repeating the measurement in nitrogen purged solution, the
Conclusions
The results shown above allow the following statements to be made, concerning the use of electrochemical impedance spectroscopy to investigate the eletrode/antigen interface aiming at the development of an affinity biosensor:
- i
It is possible to detect and perhaps quantify the adsorption of the antigens CRA+FRA in the electrode surface using electrochemical impedance spectroscopy.
- ii
The oxide layer on the electrode surface has strong effect on the adsorption of the antigens CRA+FRA on gold and
Acknowledgements
This work was partially financed by Instituto de Tecnologia em Imunobiológicos-Bio-Manguinhos/FIOCRUZ (grant no. CC 05/2000). Roseli R. Ueta and Alziana M. da C. Pedrosa, and Valéria R. A. Pereira gratefully acknowledge scholarships from CNPq. Thanks due to Dr. Marco Krieger for helpful discussions.
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