Biosensors and Bioelectronics 21 (2005) 175181

Immunosensor for the diagnosis of Chagas disease

Antonio Aparecido Pupim Ferreiraa , Walter Collib , Paulo In acio da Costac , Hideko Yamanakaa,
c

Instituto de Qu mica, UNESP, R. Prof. Francisco Degni, s/n, 14800-900 Araraquara, SP, Brazil b Instituto de Qu mica, USP, Av. Prof. Lineu Prestes 748, 05508-900 S ao Paulo, SP, Brazil Faculdade de Ci encias Farmac euticas, UNESP, Rodovia Araraquara/Ja u Km 1, CP 502, 14801-902 Araraquara, SP, Brazil Received 16 June 2004; received in revised form 27 July 2004; accepted 4 August 2004 Available online 8 October 2004

Abstract Trypanosoma cruzi proteins from epimastigote membranes, herein referred as antigens, have been used for the construction of an amperometric immunosensor for serological diagnosis of Chagas disease. The proteins used had a molecular mass ranging from 30 to 100 kDa. The gold electrode was treated with cysteamine and glutaraldehyde prior to antigen immobilization. Antibodies present in the serum of patients with Chagas disease were captured by the immobilized antigens and the afnity interaction was monitored by chronoamperometry at a potential of 400 mV (versus Ag pseudo-reference electrode) using peroxidase-labeled IgG conjugate and hydrogen peroxide, iodide substrate. The incubation time to allow maximum antigenantibody and antibodyperoxidase-labeled IgG interactions was 20 min with a reactivity threshold at 0.104 A. 2004 Elsevier B.V. All rights reserved.
Keywords: T. cruzi; Immunosensor; Chagas disease

1. Introduction Chagas disease is an American trypanosomiasis caused by the hemoagellate Trypanosoma cruzi. According to the World Health Organization, there are between 16 and 18 million people infected from the south of the USA to the south of Argentina and Chile (WHO, 1991). Chagas disease vectors in Brazil are being controlled by insecticides, leading to a reduction of Triatoma infestans population (Silveira and Vinhaes, 1999). The control measures to eradicate the populations of native triatomine species have signicantly reduced the risk of T. cruzi transmission by contaminated hematophagous insects (Dias et al., 2002; Costa et al., 2003). Notwithstanding, contamination by blood transfusion is a serious problem in countries where screening of blood bank donors do not include serum monitoring for this particular infectious agent. The risk of receiving contami

Corresponding author. Tel.: +55 16 201 6622; fax: +55 16 222 7932. E-mail address: hidekoy@iq.unesp.br (H. Yamanaka).

nated blood increases in direct proportion to both the prevalence of the infection in the donor population and the number of transfusions received, with either infected whole blood or blood components. Diagnosis of infection by T. cruzi is based on epidemiology and clinics but must have its etiology conrmed by laboratory diagnosis. The latter may be performed through direct visualization of the parasite or through the immunological response of the infected host. The detection of antigen in the blood sera could be useful just for the acute phase of the Chagas disease. Detection of anti-T. cruzi antibodies in the serologic investigation is the method of choice for the etiological diagnosis of Chagas disease in the chronic phase, considering the specicity and sensitivity of the tests used in the clinical analysis routine. Prominent in clinical practice are the following serological tests using T. cruzi antigens: indirect hemaglutination, indirect immunouorescence and enzyme-linked immunosorbent assay (ELISA). Western blot has been used whenever sera titration gives uncertain results or in cases of divergence between results from

0956-5663/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.08.001

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the different conventional techniques. However, these traditional methods are time-consuming and may have sensitivity problems. The immobilization of antigens and antibodies on the surface of transducers led to the development of immunosensors for several substrates of interest in the biological, clinical and industrial areas (Mello and Kubota, 2002; Pravda et al., 2002; DOrazio, 2003; Dai et al., 2003; Shah and Wilkins, 2003). The methods that employ immunosensors are very rapid and have both high specicity and sensitivity (Riccardi et al., 2002). In addition, they have the advantage of requiring small sample volumes affording an increase in the number of analyzed samples, thus lowering costs when compared to the conventional analytical methods. The aim of this manuscript is to describe the antigen immobilization process in order to develop a biosensor for the detection of Chagas disease antibodies in serum samples using as comparison the spectrophotometric indirect immunoenzymatic assay, ELISA. Specicity, reproducibility, reaction simplicity, costbenet of the method, reaction time and precision to generate viable analytical determinations has been compared.

solutions (50 L) were put direct on the screen-printed electrodes and the enzymatic response was monitored by chronoamperometric measurement at 0.400 V at 25 C. 2.3. T. cruzi antigenic proteins Epimastigotes (2.5 109 ), Y strain, were washed three times with 0.9% NaCl and then resuspended in 6 mL of lysis buffer with the following composition: 0.5 103 mol L1 DTT, 1 103 mol L1 EGTA, 5 104 mol L1 sodium bicarbonate, 1 102 mol L1 HEPES, 1 104 mol L1 TLCK and 1 g mL1 leupeptin. Addition of protease inhibitors is necessary to avoid rapid hydrolysis of most proteins in the extract. The suspension was sonicated six times for 10 s in the cold with 1 min intervals, followed by centrifugation at 20,000 g for 45 min. The pellet was resuspended in 2 mL of lysis buffer and liophylized. The protein extract was dialyzed versus buffer solution, submitted to electrophoresis in 10% polyacrylamide gel with 0.1% sodium dodecyl sulphate (SDS-PAGE) and then transferred to a nitrocellulose membrane (0.8 mA cm2 current was applied). The immunoenzymatic assay was carried out using a pool of serum from positive Chagas patients, previously analyzed by the conventional techniques (ELISA, indirect hemaglutination, indirect immunouorescence and card tests). The total protein amount present in the extract 30 kDa < cut-off < 100 kDa was determined according to Bradford (1976) and used for further investigations. Dilution of the antigen was accomplished with 0.1 mol L1 (pH 6.9) sodium phosphate buffer solution. 2.4. Modication of the screen-printed electrode surface and immobilization of the T. cruzi antigens The screen-printed electrode was washed in ethanol and then incubated for 2 h in 2 102 mol L1 of cysteamine prepared in water. Washing in water and incubated for 1 h with 2.5% glutaraldehyde solution in 5 102 mol L1 phosphate (pH 7.5) and nally washed in water (Hor acek and Skl adal, 1997). Thirty-ve micrograms of antigenic protein was immobilized on the modied electrode, incubated overnight at 4 C, washed and stored in Petri dish paralm wrapped under refrigeration. 2.5. Interaction antigenantibody (AgAb) on the electrode surface (a) Application of 50 L of phosphate buffer solution containing 0.1369 mol L1 NaCl and 0.02% of Tween 20 (pH 7.5; PBST buffer solution) over the surface of the sensor and incubation for 10 min. (b) Washing. (c) Blocking of the sensor surface with 50 L of PBST buffer solution containing 0.1% bovine serum albumin and incubation for 20 min. (d) Washing.

2. Experimental 2.1. Reagents dl-Dithiothreitol (DTT), N-2-hydroxyethylpiperazineN -2-ethanesulfonic acid, sodium salt (HEPES), N--l-tosyll-lysine chloromethylketone hydrochoride (TLCK), ethyleneglycol-bis-(-aminoethyl)-N,N,N ,N -tetraacetic acid (EGTA) anti-human IgG-peroxidase (Ab*) and cysteamine were purchased from SigmaAldrich. Anti-T. cruzi positive and negative sera were from Diagnosis Laboratory of Faculty of Pharmaceutical Sciences. Sera from hosts of systemic infections (schistosomiasis, leprosy, paracoccidioidomicosis, rheumatic arthritis, leishmaniasis (sp), systemic lupus erythematosus and scleroderma), were donated by Instituto Adolf Lutz (S ao Paulo, SP) and Instituto Lauro de Souza Lima (Bauru, SP). Chagatek ELISA kit was purchased from Organon Teknika Argentina. All other chemicals were of reagent grade or better. H2 O2 (1 103 mol L1 ) and KI (3 103 mol L1 ) solutions were prepared in 0.1 mol L1 phosphate buffer solution (pH 7.0). The solutions were prepared with high-purity water ( = 18.2 M cm1 ). The screen-printed gold electrodes (Au/98% Pd, Ag/98% Pd and Au/98% Pd as working, reference and auxiliary electrodes, respectively), were supplied by BVT Technologies, Brno, Czech Republic. 2.2. Apparatus Electrochemical measurements were performed with a PGSTAT potentiostatgalvanostat from Eco-Chemie. The

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(e) Application of 50 L of T. cruzi positive serum (Ab) and incubation for 20 min. (f) Washing. (g) Application of 50 L of the anti-human IgG monoclonal antibody conjugated to peroxidase and incubation for 20 min. (h) Washing. The peroxidase was monitored by addition of 50 L of the solutions of hydrogen peroxide (substrate) and potassium iodide (mediator) on a rate of 1:3 prepared in a 0.1 mol L1 sodium phosphate buffer solution and incubation for 10 min. For the blank reaction, neither positive serum nor conjugate were applied on the electrode. The immunoenzymatic reaction was investigated by chronoamperometry at 400 mV. 2.6. Optimization of the amperometric immunosensor for Chagas disease The optimization of the immunosensor was carried out with the antibody anti-T. cruzi positive and negative sera from Chagatek ELISA kit. The incubation time for the primary (Ab) and secondary (Ab*) antibodies were investigated. Each modied printed electrode received 50 L of antigenic sample, partially puried, containing 0.142 g of proteins. The primary antibody dilution was 1:20 and the secondary antibody, a dilution of 1:1000 corresponding to the reactivity threshold for the research of antibodies IgG anti-T. cruzi in Western blot. Solutions of hydrogen peroxide and potassium iodide in concentrations of 1 103 and 3 103 mol L1 , respectively, were used. The concentration of Ag and Ab were titrated by diluting the antigen 1:5, 1:10, 1:20 and 1:40, 1:80 (stock solution: 0.142 mg mL1 ) and corresponding to decreasing dilutions of the primary antibody (1:160, 1:80, 1:40, 1:20 and 1:10) (see Table 1). The dilutions of 1:1000 for the secondary antibody and 1 103 and 3 103 mol L1 for the solutions of substrate and mediator, respectively, were xed. The effects of the Ab* and H2 O2 /KI concentration were also investigated. The concentration of Ag and Ab was xed and the secondary antibody (stock solution: 1.66 mg protein mL1 ) was diluted 1:1000, 1:2000, 1:4000, 1:8000 and 1:10,000. Simultaneously, concentrations of the substrate and the mediator (proportion 1:3) were varied from 1 106 to 1 102 mol L1 (see Table 2).
Table 1 Effect of antigen and antibody dilution on the immunosensor response Dilution Ag 1:5 1:10 1:20 1:40 1:80 Ab 1:160 1:80 1:40 1:20 1:10 0.242 0.013 0.276 0.021 0.320 0.022 0.390 0.021 0.441 0.022 I (A)

Table 2 Effect of the anti-human IgGHRP (Ab*) dilution and H2 O2 /KI concentration on the immunosensor response Ab* dilution 1:1000 1:2000 1:4000 1:8000 1:10000
a

H2 O2 /KI concentrationa (mol L1 ) 1 106 1 105 1 104 1 103 1 102

I (A) 0.459 0.010 0.365 0.053 0.227 0.022 0.166 0.021 0.083 0.042

H2 O2 /KI (1:3).

2.7. Determination of the reactivity threshold on the amperometric immunoassay The negative control (negative serum Chagatek ELISA kit) and sera samples from patients non-infected with Chagas disease, previously analyzed through conventional techniques (indirect immunouorescence and ELISA), were analyzed by the proposed methodology. The experimental conditions were xed as follows: dilutions of Ag (1:20), Ab (1:40), Ab* (1:4000), H2 O2 (1 104 mol L1 ) and KI (3 104 mol L1 ). The reactivity threshold was taken as the current intensity averages obtained with negative sera plus twice the standard deviation. 2.8. Analysis of sera using the immunosensor The measurements with the proposed methodology were done using anti-T. cruzi positive and negative sera and sera from hosts of systemic infections: schistosomiasis, leprosy, paracoccidioidomicosis, rheumatic arthritis, leishmaniasis (sp), systemic lupus erythematosus and scleroderma.

3. Results 3.1. Antigenic composition of the T. cruzi extract The electrophoretic separation of the protein extract of T. cruzi showed bands ranging from 18.4 to 200 kDa. The polypeptides were transferred to the nitrocellulose membrane and developed with sera from patients having Chagas disease. Stronger immunochemical reaction was obtained with bands between 35 and 39, 43 and 45, and 53 and 64 kDa. For further investigations, the protein extract of T. cruzi was ltered on polysulfone membranes and the proteins with molecular masses ranging from 30 to 100 kDa were used. The total protein of the extract was determined as 2.28 0.01 mg mL1 . 3.2. Amperometric monitoring of the HRP enzyme After modication of the gold surface and immobilization of Chagas disease antigens (Ag), the anti-T. cruzi (Ab) antibodies present in the serum sample are captured by Ag, remaining connected to the solid phase; the human antiIgG antibody conjugated to peroxidase (Ab*) reacts with

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Fig. 1. Scheme of the reactions involved in the determination of the antibodies using an immunosensor.

further experiments, the monitoring of the immunoreaction will be investigated at 400 mV. Riccardi (2001) previously studied the behavior of the system H2 O2 /KI in the presence of different peroxidase concentrations. The electrode surface modied with Ag and blocked with both Tween 20 and bovine serum albumin was incubated in presence or absence of Ab* and nally the substrates were added. The reduction current intensity at 400 mV obtained in both cases had no significant difference. It means the non-specic adsorption of the conjugate was avoided (Jenkins et al., 1990) and so the blank signals for immunosensor experiments were obtained without the Ab*. 3.3. The optimization of experimental conditions Effect of the incubation time for the AgAb and AbAb* interaction is presented in Fig. 3. The current intensity increased up to 20 min, and at 40 min values decreased. Decrease in current intensities may possibly be explained by enzyme partial denaturation in the complex system antigenantibody on the surface of the gold electrode. Another important parameter is the amount of Ag on the electrode surface and the dilution of the primary antibody. The results are displayed in Table 1 and the curve of Ag dilution versus I (Fig. 4a) and Ab dilution versus I (Fig. 4b) are represented. As shown, for lower dilutions of the antigen or the antibody the system becomes saturated, indicating that the reaction antigenantibody obeys the mass law. Small variations in the primary antibody concentration do not express signicant current values, thus impairing assay sensitivity. The dilutions of 1:20 and 1:40 for, respectively, the parasite antigen and the primary antibody correspond to points falling in the linear interval that contains the intersection of the curves. In that intersection, discrimination of current intensity is maximal for variations in antigen and antibody concentrations. If an important role on the establishment of the immunoassay sensitivity and precision is the adequate selection of the

immunocaptured anti-T. cruzi antibodies. The peroxidase catalyzes the reaction with H2 O2 , and the iodide reacts with the reduced peroxidase. Under these conditions, the current intensity of I2 reduction is proportional to the amount of anti-T. cruzi antibodies in the serum sample. A scheme of the immunosensor is presented in Fig. 1. A voltammetric investigation was carried out with the immunosensor and the results are shown in Fig. 2. Linear voltammograms of 0.1 mol L1 (pH 6.9) phosphate buffer solution without (Fig. 2a) and with (Fig. 2b) 1 103 mol L1 H2 O2 was performed over a potential interval ranging from +800 to 800 mV. A peak was observed around 660 mV, however, the H2 O2 /KI solution (Fig. 2c) showed peaks at 560 and 400 mV referring to the reduction of the product formed by the enzymatic catalysis (I2 ). For monitoring the anti-T. cruzi antibody reaction, the potential of 560 mV is too high, making it difcult the application of the system to samples from patients affected or not by Chagas disease, because it could allow the oxidation or reduction of compounds present in the complex matrix. The blank of the enzyme presented a peak at 400 mV in H2 O2 /KI solution (Fig. 2d) but the current intensity was low in comparison to Fig. 2c. In

Fig. 2. Linear sweep voltammogram of the 0.1 mol L1 (pH 6.9) sodium phosphate buffer solution (a), 1 103 mol L1 H2 O2 (b) and H2 O2 /KI (c) on printed electrode containing Ag, Ab and Ab*; and H2 O2 /KI (d) on printed electrode containing Ag and bovine serum albumin, = 100 mV s1 .

Fig. 3. Effect of the incubation time on the immunosensor response.

A.A.P. Ferreira et al. / Biosensors and Bioelectronics 21 (2005) 175181 Table 3 Values of current intensity for negative sera Negative serum Control 1 2 3 4 5 Table 4 Positive sera analyzed with the immunosensor Serum 1 2 3 4 5 6 7 8 I (A)

179

I (A) 0.054 0.082 0.089 0.075 0.091 0.065

Fig. 4. Effects of the dilution of antigen (a) and primary antibody (b) on the immunosensor response.

0.161 0.052 0.164 0.061 0.356 0.075 0.381 0.049 0.387 0.041 0.384 0.073 0.222 0.069 0.381 0.077

primary antibody dilution, equivalent attention must be assigned to the secondary antibody dilutions and H2 O2 /KI concentrations. The results are displayed in Table 2 and the current intensity is represented versus Ab* dilution (Fig. 5). In the AB interval, there is more discrimination in the variation of current intensity when compared to the other regions of the analytical curve. The point x of Fig. 5 corresponds to the dilution of 1:4000 for the secondary antibody and concentrations of 1 104 mol L1 for the substrate and 3 104 mol L1 for the mediator. 3.4. Determination of the cut-off on the amperometric immunoassay for Chagas disease As the conditions for the construction of the immunosensor have been dened, investigation on the current inten-

sity value that would indicate anti-T. cruzi antibodies in serum was undertaken. Due to sera complexity, an average of the current intensities of ve negative serum samples was analyzed (Table 3). The average of amperometric values was 0.076 0.013 A. Twice the standard deviation was added to that average establishing the reactivity threshold as 0.104 A. A sample is considered non-reactive when the current intensity is equal or lower than 0.104 A and reactive when values are above that current. 3.5. Analysis of sera using the immunosensor According to Table 4, the serum of all eight Chagas disease-infected patients samples presented the current intensity more than 0.104 A. The methodology herein described was also applied to sera of patients having different systemic diseases as schistosomiasis, leprosy, paracoccidioidomicosis, rheumatic arthritis, leishmaniasis (sp), systemic lupus erythematosus and scleroderma but cross-reactivity was observed with sera from patients having schistosomiasis (I = 0.242 0.048 A) and leishmaniasis (I = 0.296 0.024 A). The cross-reactivity for those diseases are as high as for the ELISA method.

4. Discussion Chagas disease is a chronic and incapacitating illness, caused by the protozoan parasite T. cruzi when trypomastigotes invade host cells (Colli, 1993). The protozoan is transmitted to humans by wound contamination with insect feces during blood sucking. Other forms of transmission like blood transfusion are becoming increasingly important, particularly in northern hemisphere regions that received intense migratory currents from Latin American countries. Thus, a method-

Fig. 5. Effects of the dilution of the Ab* and concentrations of the H2 O2 /KI (1:3) on the immunosensor response.

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ology with high sensitivity and rapid analysis is welcome for the screening of donors in blood banks. Immunosensing satises those requirements. Although immunosensors have been used for other conditions like allergies (Su et al., 2000), Plasmodium falciparum malaria (Mya et al., 2003) and Schistosoma japonicum (Wu et al., 2003), it is the rst time they are employed for the diagnosis of Chagas disease. The enzymatic reaction of peroxidase (HRP) occurs in three distinct steps, as shown in the reactions (1)(3) (Ghindilis et al., 1997; Lindgren et al., 1997; Furtmuller et al., 2001). The enzyme catalyzes the reduction of hydrogen peroxide oxidizing the heme group (1): HRP(Fe3+ ) + H2 O2 compound I + H2 O (1)

electrode and by the fact that the protein was not puried. It is important to stress that all samples that were positive using the ELISA method were also positive using the immunosensor, thus reinforcing the validity of the method herein proposed. For routine analysis, the methodology should be standardized and a detailed comparison of advantages or disadvantages of the electrode method over ELISA will be pursued in the near future.

5. Conclusions The method proposed is both simple and sensitive and quite easy to repeat whenever results difcult to interpret appear. The cross-reactivity with schistosomiasis and leishmaniasis will most probably be eliminated when reactive antigens are puried, including recombinant proteins specic for the trypomastigote stage (Umezawa et al., 2001). This method may prove important not only for the diagnosis of the presence of antibodies against T. cruzi in blood donors, but also to follow antibody decay during treatment of chagasic patients with the available drugs.

The oxidized enzyme (compound I) is reduced to compound II (2) that is further reduced to the HRP(Fe3+ ) active form (3): compound I + AH compound II + A compound II + AH HRP(Fe3+ ) + A + H2 O (2) (3)

Reducing agents (AH) like iodide are able to donate electrons to both compounds I and II. Consequently, the reduction of species A (iodine) on the electrode surface can be used to follow the peroxidase-catalyzed reaction (Ghindilis et al., 1997; Yamanaka and Skl adal, 2001). As shown, the negative control serum (dilution 1:40) presented an average current intensity value of 0.057 A. The dilution curve of positive control sera (Fig. 6) gave an interpolation of the cut-off value leading to amperometric reaction sensitivity up to the dilution of 1:560. This fact demonstrates the high sensitivity of the proposed methodology. The incubation time for AgAg and AbAb* using the ELISA method was 1 h for each interaction while for the immunosensor this time was reduced to 20 min. According to the results in Table 3, the variability is high. This could be explained by the low amount of protein onto the

Acknowledgements This work was supported by grants from FAPESP. The technical expertise of Marinei Ferreira Gonc alves in antigen preparation is greatly acknowledged. A patent is under requirement at INPI (PI0202214-1).

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