Impedimetric evaluation for diagnosis of Chagas’ disease: antigen–antibody interactions on metallic eletrodes

https://doi.org/10.1016/S0956-5663(03)00213-6Get rights and content

Abstract

A polypeptide chain formed by recombinant antigens, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) (CF-Chimera) of Trypanosoma cruzi, was adsorbed on gold and platinum electrodes and investigated by electrochemical impedance spectroscopy on phosphate buffer saline solutions (PBS) containing a redox couple. It was found that the adsorption is strongly sensitive to the oxide layer on the electrode surface. In the majority of the experiments the antigens retained their activity as observed through their interaction with sera from chronic chagasic patients. The results expressed in terms of the charge transfer resistance across the interface, indicate the viability of using the impedance methodology for the development of a biosensor for serological diagnosis of Chagas’ disease.

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.

References (21)

There are more references available in the full text version of this article.

Cited by (38)

  • Recent advances on the piezoelectric, electrochemical, and optical biosensors for the detection of protozoan pathogens

    2022, TrAC - Trends in Analytical Chemistry
    Citation Excerpt :

    cruzi Ab up to a dilution of 1:10, 240 in canine samples. Before that in 2003, Diniz et al. [69] have prepared gold (Au) and platinum electrodes to adsorb flagellar repetitive Ag (FRA) and cytoplasmic repetitive Ag (CRA) of T. cruzi by EIS. Several drugs that have a nitro aromatic groups in their structures can be reduced in the nitro group [70].

  • Label-free electrochemical impedance immunosensor based on modified screen-printed gold electrodes for the diagnosis of canine visceral leishmaniasis

    2019, Talanta
    Citation Excerpt :

    With the recent development of miniaturized electrodes, such as screen-printed electrodes (SPEs), electrochemistry has become increasingly accessible for POCT applications [18]. In this context, the literature already presents some successful applications, such as: a label-free impedimetric immunosensor for detection of a biomarker of cancer [19]; EIS to detect Escherichia coli [20]; an electrochemical immunosensor for detection of avian leukosis virus subgroup J [21]; a platform for the impedimetric transduction for Chagas disease [22]; and a genosensor for Leishmania major [23]. Taking into account the need for advances in diagnosis of canine leishmaniasis, this work shows the development of a low-cost platform for the detection of anti-Leishmania infantum antibodies in canine clinical serum and real blood samples through a label-free electrochemical impedance method.

  • Method for equivalent circuit determination for electrochemical impedance spectroscopy data of protein adsorption on solid surfaces

    2014, Electrochimica Acta
    Citation Excerpt :

    Generally, with protein adsorption, EIS data are fit to an Equivalent Electrical Circuit (EEC) to extract quantitative information about the processes occurring at the electrode surface [1,2]. Typically, researchers examine the fit between the circuit and the Nyquist plot to determine whether or not the circuit is a good model for the system [2–4]. In this paper, we propose a more thorough method of circuit choice, based on relative residual errors between the experimental data and fit data, generated circuit parameter values relating to known physical parameters, and standard deviations based on replicate experiments.

View all citing articles on Scopus
View full text