Leishmaniasis is a complex of diseases caused by a protozoan of the genus Leishmania
] that affects millions of people worldwide. Visceral leishmaniasis, caused by Leishmania donovani and Leishmania infantum
, represents the most severe form and can lead to death if not treated [3
]. The onset of the infection and clinical manifestations are dependent on many factors including environmental and host immunologic status, especially in the early stages of infection [5
]. Visceral leishmaniasis represents a major health problem in some tropical areas of the world. The currently available serum diagnosis does not fit the proper criteria of sensitivity and specificity, especially for identification of asymptomatic and or low symptomatic patients due to the low concentration of antibodies in the serum, particularly in the case of asymptomatic patients, which results in high cross-reactions [6
]. Furthermore, due to its epidemiological characteristics, a diagnostic test that is accessible in remote areas is a desired tool for precise diagnosis and early therapeutic intervention.
The diagnosis of visceral leishmaniasis is made by combining clinical signs with parasitological or serological tests; however, they depend on extremely equipped laboratories, qualified labor, and a long period of time to carry out the tests [7
]. The parasitological diagnosis is the reference choice for exams for detecting the disease, which shows the parasite directly in tissues or in culture. Aspirates from the spleen, bone marrow, and lymph nodes are used, and liver biopsy can also be performed. These techniques have high specificity and variable sensitivity. Until 2014, the Ministry of Health used two tests: the fluorescence indirect antibody test (IFAT) for human leishmaniasis, developed by the Institute of Immunobiological Technology (Biomanguinhos), Fundação Oswaldo Cruz, Brazil, and the Kalazar Detect rapid test (InBios International, Seattle, WA, USA).
Several studies show a comparison between the techniques, such as rapid test rK39, direct agglutination test (DAT), and ELISA, with some even showing good sensitivities and specificities in these patients [9
]. However, the identification of asymptomatic infection remains challenging, since it depends on the sensitivity and specificity of the employed technique [10
]. Furthermore, there is no agreement among the available techniques, and thus it is necessary to search for the best method to measure visceral leishmaniasis positives not only in symptomatic patients [11
]. Hence, the average linear range of asymptomatic patients is a concern.
In this context, the presentation of new methodologies that show good performance, easy handling, speed, and detection of asymptomatic patients is essential to the control and early treatment of leishmaniasis. In other words, the development of tools that contribute to the optimization of a portable platform of leishmaniasis is a priority. Thus, the development of electrochemical biosensors shows advantages in comparison to traditional techniques, such as fast execution, a small amount of sample utilization, portability, selectivity, and specificity to obtain diagnoses.
The physical principle of the biosensor is to turn the biological sign into an electrical sign, making it possible in this way to monitor and quantification of signals. The immobilization of antigens, which specifically recognize the antibodies, can be provided on the surface of the sensor. The biorecognition, in the case of the evaluated pathologies, involves the antibodies as the target of detection, and they can be also quantified [9
Electrochemical biosensors were developed by some research groups seeking to solve the problem of diagnosis of visceral leishmaniasis, investigating novel platforms and modification procedures for the diagnostic needs of Leishmania
]. Amongst the platforms used in sensors, the carbon-based electrodes present conditions for immobilization (through adsorption) once they enable a random anchorage and orientation of the biomolecules on its surface. Meanwhile, gold electrodes enable oriented couplings that minimize the distance between the active biomolecule sites and the electrode surface, facilitating the electron transfer, and a greater number of antibodies can be immobilized on the electrode surface [24
]. Considering the use of portable platforms for point-of-care diagnosis, screen-printed electrodes play a key role in the development of electrochemical biosensors for several applications, as reported in the literature [27
In order to an immunosensor functions properly for the diagnosis of leishmaniasis, it should be highly sensitive, specific, fast, and simple, with potential application for the serological diagnosis of leishmaniasis, since the disease is directly related to cross-reactivity with Chagas disease and other diseases. There are some studies reporting sensors for the diagnostic of Leishmania
sp. Infection. Mohan et. al. [36
] developed a genosensor modified with NiO nanostructured on an indium-tin oxide conductive glass plate to distinguish DNA from parasites extracted from human DNA extracts; however, these tests have not yet been performed in clinical samples. Moradi et al. [13
] also developed a genosensor based on gold nanoparticles immobilized on polycrystalline gold discs and found high sensitivity in cutaneous leishmaniasis. However, the DNA extraction process has a higher cost compared to the use of protein antigens. Facing the diagnostic difficulties of cross-reactions, in this study, we describe how the electrode allows for differentiated interactions of the same biomolecule using a gold nanoparticle-modified surface, starting from the principle of this being biologically compatible and non-toxic. Such properties have attracted attention in diagnostic application because the molecules should not have their biological properties altered [26
In this context, this work shows the development of an immunosensor for the specific diagnosis of visceral leishmaniasis without the cross-reactivity with Chagas disease (CD). Linear response, sensitivity, selectivity, specificity, repeatability, reproducibility, and stability were researched. To our knowledge, the concentration of Leishmania antigens considered as potential risk to develop the disease is not accurately known, and for this reason the proposed biosensor provides a detection limit in very concentrations (ng mL−1) to detect antigens before the appearance of disease symptoms.
2. Materials and Methods
2.1. Reagents and Biomolecules
All used reagents were of analytical grade and were used without further purification. Ultrapure water (MilliQ, Resistivity value greater than 18.2 MΩ, Millipore Corporation, Burlington, MA, USA) was used in the preparation of all solutions. The aqueous solution of the mixture of potassium ferricyanide/ferrocyanide ([Fe(CN)6]3−/[Fe(CN)6]4−) in KCl (5 mmol L−1, 0.1 mol L−1, pH 7.4, LabSynth, Brazil) used for the electrochemical characterization of the immunosensor was prepared immediately before the use. Gold (III) chloride was dissolved in sulfuric acid medium (1 g L−1 AuCl3 in 10 mL of 0.5 mol L−1 sulfuric acid). All experiments were carried out at controlled room temperature (25 ± 1 °C).
Preparation of the leishmania infantum crude antigen: The PP75 strain of Leishmania infantum
cultured in the Schneider medium, supplemented with 20% fetal bovine serum, in the exponential phase was centrifuged 2000× g
at 25 °C for 20 min and then washed three times with phosphate buffer (PB) solution and discarded supernatant. The pellet was resuspended in PB containing 0.05% NP40 (Nonidet P-40 Substitute, Roche) with the COMPLETE protease inhibitor (ROCHE, SWI). The antigen was obtained by the method of freezing in liquid nitrogen and thawing in a 37 °C water bath and then centrifuging it at 10,000× g
for 30 min, and then the supernatant containing the soluble crude antigen was stored at −80 °C until the moment of use. The protein concentration of the antigen was determined by the Lowry method [38
]. Aliquots of the extract were stored with the total soluble antigens at −80 °C until further use. The preparation of crude antigens specific to Leishmania infantum
was performed. This species is characteristic of visceral leishmaniasis [39
]. The technique was performed and adapted on the basis of [40
]. Moreover, in our experiments, the efficiency of soluble and membrane-free extract of the parasite (data not shown) was proven, since these Leishmania infantum
antigens are able to specifically be recognized by the serum antibodies of patients with visceral leishmaniasis antibodies.
The real samples used in the experiments appeared favorable and were substantiated from CEP (Comitê de Ética em Pesquisa/Research Ethics Committee) by Plataforma Brasil. Leishmania sera have the CAAE 58301516.8.0000.5154 and were 1,846,584 in number. Chagas sera have the CAAE 64048117.3.0000.5154 and were 2,163,043 in number. The stock solutions of total antigens (0.01 μg mL−1) and serum (visceral leishmaniasis = 1:100–0.202 mg mL−1; CD = 1:100–0.146 mg mL−1) were diluted in deionized water and frozen until the electrochemical experiment.
Screen-printed carbon electrodes (DPR-110) and screen-printed gold electrodes (DPR-220 BT) were purchased from DropSens (Oviedo, Asturias, Spain), which consist of a ceramic strip containing a three-electrode system (working, counter, and reference electrodes) for a single-drop analysis. The reference was made of a silver ink (known as silver pseudo-reference electrode) and the counter and working electrodes were made of carbon ink (in DPR-110) or of made of a gold ink (in DPR-220 BT). The working electrode of the screen-printed carbon electrode was modified with gold by electrodeposition (next described), which is the third electrochemical device evaluated in this work. Electrochemical analyses were performed by cyclic voltammetry using Em Stat 1 equipment (PalmSens BV, The Netherlands) connected to a notebook. The changes in the electrochemical signals of [Fe(CN)6]4−/[Fe(CN)6]3− (5 mM) were evaluated (scan rate of 100 mV s−1). For ELISA tests (EnSpire/PerkinElmer), optical density (OD) values were determined on a microtiter plate reader at 490 nm.
2.3. Indirect ELISA
The indirect ELISA for the detection of immunoglobulin G (IgG) antibodies against leishmania used high affinity plaques (Thermo Scientific Tm Nunc Tm, Waltham, MA, USA), which were sensitized with the antigens (1 μg mL−1), diluted in 0.06 mol L−1 carbonate-bicarbonate buffer (pH 9.6), and incubated for 18 h at 4 °C. After this period, all plates were washed six times with PB containing 0.05% Tween 20 (PB-T) and blocked with PB containing 5% skimmed milk powder (Molico, Nestle, São Paulo, Brazil—PB-M5%) for 4 h at room temperature. After further washing, the serum samples were 1:40 diluted in 5% PB-M and incubated for 2 hours at room temperature. After six washes, the anti-human IgG antibody (1:2000) conjugated to peroxidase (IgG/horseradish peroxidase(HRP), Dako) was added and incubated for 2 h at room temperature. After further washing, the reaction was developed by addition of the enzymatic substrate 1,2-orthophenylenediamine (OPD, Dako) with 0.05% H2O2 and stopped with H3PO4. Positive and negative controls were included on the plate. The levels of antibodies were expressed in ELISA, according to the following formula: EI = Abs sample/cut-off, where cut-off is calculated as the mean of the Abs of negative control serum plus three standard deviations. EI values > 1.4 were considered positive.
2.4. Electrodeposition of Gold Nanoparticles on Carbon Electrodes
The electrodes were submitted to a 30-cycle cyclic voltammetry (CV) pre-treatment in 1 mol L−1
solution in the potential range between −0.3 and +1.2 V at 100 mV s−1
for surface cleaning and activation. After that, the electrodes were submitted to the deposition of gold nanoparticles by 15 voltammetric cycles in a gold chloride (HauCl4
, 1 g L−1
) solution prepared in 1 mol L−1
in the potential range between 0.3 and +1.0 V at scan rate of 0.1 V s−1
]. After the electrodeposition, it is possible to verify the color change of the working electrode evidencing the formation of gold nanoparticles.
The activation of the modified electrode was performed by 10 cycles in 1 mol L−1
solution to eliminate impurities that can hinder the adsorption of molecules, diminishing the reproducibility and stability of the modified surface [45
The first step involved the immobilization of the total soluble antigens on the surface of the working electrode (carbon, gold, and carbon modified with an electrodeposited gold nanoparticles) by drop-casting. The dissolution method of 4 μL lasted until the solution dried (15 min). In order to prevent nonspecific binding, we added 4 μL of 1% bovine serum albumin (BSA) as a blocking solution (15 min) after the first step. At the end, the serum was made available until it dried. After each step, the electrodes were washed and dried in a desiccator. For the interaction investigation between total antigens immobilized on each working electrode and total soluble antigens recognition, we used the solution of [Fe(CN)6
(5 mM) as a redox probe indicator. Thus, 80 μL of this solution was dropped over the three electrodes, closing the working electrode circuit between the other two electrodes (counter electrode and reference). Reactions occurred at room temperature (25 ± 1 °C). Using the cyclic voltammetry (CV) technique, we evaluated the behavior of the electrochemical signal of the supporting electrolyte (indirect detection) on the sensor, as shown in Figure 1
An aliquot of 4 μL of positive serum for Chagas disease (1:100 diluted) was pipetted on the immunosensor and kept for 15 min at room temperature. Thereafter, a final wash occurred with MiliQ water (50 μL), and the electrode was dried. Using the other electrode, the same protocol with positive serum for leishmaniasis (1:100 diluted) was performed, and on a third electrode, it was performed with serum negative (diluted 1:100, protein concentration in 5 μg mL−1). In all tests, including triplicates, the changes in the electrochemical signals of [Fe (CN)6]4−/[Fe (CN)6]3− (5 mM) were evaluated (scan rate: 100 mV s−1).
2.7. Sensor Stability
To evaluate the stability of the immunosensor, we stored modified electrodes containing the total soluble antigen at 4 °C for 5 days, protecting them from light and oxygen.
2.8. Calibration Curve
To validate the immunosensor sensitivity analyses, we added 4 μL of different serum dilutions (1:25, 1:50, 1:100, 1:250, 1:100,000) to the immunosensor. Incubation was for 15 min at 25 ± 1 °C.
2.9. Statistical Analysis
The analyses are descriptive and are based on the comparative study of the voltammograms and their reinterpretations in bar charts and linear graphs (calibration).