Electrochemical Biosensor for Sensitive Detection of Hepatitis B in Human Plasma

In this work, we report the construction of a novel electrochemical device for molecular diagnosis of hepatitis B virus in the blood plasma of infected patients, using graphite electrodes functionalized with poly(4-aminophenol) and sensitized with a specific DNA probe. The recognition of genomic DNA was evaluated by electrochemical techniques (DPV and EIS) and scanning electron microscopy. The genosensor was efficient in detecting genomic DNA with a linear range from 1.176 to 4.825 μg mL−1 and detection limit of 35.69 ng mL−1 (4.63 IU ml−1 or 25.93 copies.ml−1), which is better than the 10.00 IU ml−1 limit of reference method, real-time PCR, used in point of care. EIS analysis shows that the genosensor resistance increased exponentially with the concentration of the genomic DNA target. This novel platform has advantages to its applicability in real samples, such as good sensitivity, selectivity, low sample volume, and fast assay time (36 min), thus interesting for application in the diagnosis of hepatitis B virus in blood plasma. Also, the ease of synthesis of the low-cost polymer by electrosynthesis directly on the electrode surface allows the translation of the platform to portable devices.


Introduction
Hepatitis B is a globally distributed, highly infectious, and difficult to treat viral infection caused by hepatitis B virus (HBV), the double-strand DNA virus. The infection causes irritation and inflammation of the liver, and it is estimated that more than 296 million people suffer from the chronic form of the disease [1]. The chronic form of hepatitis B is a serious and silent disease; once some patients are asymptomatic during an acute phase, the liver is gradually destroyed. In more severe cases, the disease may progress to hepatocellular carcinoma and liver cirrhosis and poses great risks to human health, resulting in 820,000 deaths in 2019 [2].
HBV can be found circulating in the bloodstream and other body fluids during infection, and therefore, the methods currently used for diagnosis of hepatitis B are performed through serological tests such as the enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and polymerase chain reaction (PCR) [3]. Nevertheless, these assays require the use of reagents in high quantity, skilled labor, and expensive equipment that prevents the implementation of these methodologies outside the laboratory environment [4]. When the diagnosis is not performed in the early stages, the patient has a serious medical condition and treatment and cure become more difficult [5]. It is reported that 95% of patients with acute hepatitis B who had early diagnosis showed a good recovery [6]. On the other hand, monitoring of patients in the chronic phase of hepatitis B must be constant to prevent disease progression. Therefore, it is of the utmost importance for diagnostic methods to be rapid, highly sensitive, and selective, and then allow to detect and/or monitor the disease in real time, and at the same time being accessible to the entire population [6,7].
The electrochemical biosensor technologies have such advantages as a fast procedure, low cost, high sensitivity and selectivity and are suitable for diagnostic use [8]. These can be modified with nanotechnological materials, such as polymeric films [9], carbon nanotubes [10], quantum dots [11], and metallic nanoparticles (gold, platinum, palladium, and other metals) [12][13][14][15]. Electrochemical genosensors are constructed using immobilized DNA sequences, functioning as a biological recognition element of the complementary sequence present in the sample to be analyzed, and hybridization can be monitored and analyzed with high sensitivity from a small amount of sample and reduced dimensions of the working electrode, which can be manufactured at a low cost [16,17].
The association of conducting polymers has been widely used in the development of biosensors to increase the sensitivity for detecting the target analyte. The preparation of polymers by electrochemical methods is relatively simple and highly reproducible [18]. There is a great variety of materials that can be used in the construction of polymer films, as they allow the immobilization of active species, increase the transfer of electrons, and improve the selectivity through the blocking of interferents [9]. Several electrochemical biosensors that use the genetic material for the detection of diseases can be found in the literature, for example, for meningococcal meningitis [19], Epstein-Barr virus [20], the oncogenic marker MYCN [21], hepatitis C virus [22,23], Zika virus [24], and Mycobacterium leprae [25]. Srisomwat and collaborators [2] described a device based on 3D microfluidic paper (μPAD) for detecting HBV DNA using hexacyanoferrate (III)/(II) as an electrochemical indicator, and a linear range of 50 pM-100 nM was obtained with a detection limit of 1.45 pM. Zhao and collaborators [26] built an electrochemical sensor for the analysis of HBV DNA using nanoflowers, gold nanoparticles, and two aptamers immobilized on the surface to amplify the signal, resulting in a detection limit of 1100 copies. ml −1 and a linear range from 1.10 × 10 3 to 1.21 × 10 5 copies.ml −1 .
This work describes the construction of a DNA biosensor based on a graphite electrode coated with poly(4-AMP) and functionalized with a specific oligonucleotide probe for HBV diagnosis. The representative scheme is demonstrated below (Fig. 1). The viral DNA detection was performed indirectly by monitoring the oxidation peaks of the ethidium bromide (EB) intercalator using electrochemical techniques. Furthermore, the electrode's surface modification was confirmed with surface scanning electron microscopy (SEM), and its functionality was successfully tested in plasma samples collected from HBV-positive patients.
Stock solutions of DNA probe and target were prepared in SSC buffer (sodium chloride 0.3 mol L −1 , sodium citrate 0.03 mol L −1 ; Sigma-Aldrich) at pH 7.0 and stored at − 12℃. The choice of HBV-specific probe oligonucleotide was based in the literature [27]

Apparatus
Abbott m2000sp extractor (Abbott Laboratories, Chicago, USA) was used for the purification of genomic DNA. The electrochemical studies were performed in a potentiostat from CH Instruments model 760C (CH Instruments, Austin, USA) using three-compartment and one-compartment cells for electrodeposition and detection of biomolecules, respectively. A graphite disk, 6 mm diameter, was used as the working electrode. Platinum plate and silver/ silver chloride (Ag/AgCl, KCl 3.0 mol L −1 ) electrodes were used as counter electrode and reference electrode, respectively. Electrochemical impedance spectroscopy was performed in an Autolab Electrochemical System (PGSTAT302 N and NOVA 1.10; Metrohm, Herisau, Switzerland).

Preparation and Functionalization of Graphite Surface
Working electrodes were fabricated using a graphite disk (6 mm in diameter and ∼2 mm in height), bonded with silver epoxy to the brass base in a teflon cylinder to establish electrical contact. The space between graphite and teflon was filled with epoxy. After drying, the surface was previously polished with coarse and fine sandpaper. The final polishing was done in alumina suspension (Buehler 0.3 µm in diameter). The polished electrodes were submitted to a sonication process, immersed in deionized water to remove the remaining alumina. This procedure is important to guarantee the homogeneity of the surface of the working electrodes, thus guaranteeing a better reproducibility of the system. A solution of 4-AMP (2.5 mmol L −1 ) was deoxygenated by bubbling with ultra-pure nitrogen gas for 45 min. The electropolymerization of the monomer on the surface of the graphite electrode was performed according to the literature [20 29]. In this step, cyclic voltammetry of graphite electrode was carried out in HClO 4 solution (0.5 mol L −1 ) containing 4-aminophenol (2.5 mmol L −1 ), from 0.0 to + 1.0 V, 50 mV.s −1 , for electrodeposition of poly(4-AMP). The ionic characteristics of the electrodes before and after modification with poly (4-AMP) were evaluated using the HClO 4 in the same potential range.

Hep1 Probe Immobilization on Modified Graphite Electrode and Hybridization Detection
For the probe immobilization, 18 uL of Hep1 (63 µmol L −1 ) was added onto the modified electrodes and the solution was dried at room temperature (25 ± 1℃) for 5 min, 10 min, 20 min, and 30 min for the probe adsorption. The electrode was immersed for 6 s in phosphate buffer under agitation to remove biomolecules which have not been adsorbed and then maintained in a solution of 0.5% w/v BSA (bovine serum albumin) by 1 h at 37℃. After this step, the electrode was washed in phosphate buffer and dried with ultra-pure nitrogen.
The complementary target detection (Hep2) was performed indirectly by monitoring the oxidation peaks of ethidium bromide (EB), which amplifies the response signal. For detection, 18 µL of Hep2 solution (189 µmol L −1 ) was added onto the modified electrode and maintained at 55℃ in an oven (Laboven, Brazil) for 5 min, 10 min, 20 min, and 30 min to promote hybridization. The hybridization temperature was calculated using Gene Runner v3.05 software. Thereafter, the electrode was rinsed in phosphate buffer, and 18 µL of EB solution (1 µmol L −1 ) was added and incubated for 5 min at room temperature, followed by the same washing and drying procedure.

Calibration Curve
The calibration curve was constructed based on the analysis of the oxidation voltammograms of EB. The concentration of the Hep1 probe (63 µmol L −1 in SSC buffer) was maintained and varying the concentrations of the purified genomic HBV DNA: 1.18 µg mL −1 , 2.17 µg mL −1 , 3.27 µg mL −1 , 3.95 µg mL −1 , 4.21 µg mL −1 , and 4.82 µg mL −1 . For the statistical analysis, each concentration was averaged and the standard deviation was based on the experimental triplicate.

Selectivity Study Using Blood Plasma
Plasma from five patients infected with HBV (plasma DNA positive) and one healthy patient (negative for HBV DNA) were collected, treated, and analyzed by Adolfo Lutz Institute and were used for testing the detection capacity of the genosensor. After extracting the genomic DNA, the samples were diluted at 1:100 (v/v) or 1:200 (v/v) in SSC buffer, maintaining the concentrations used for the construction of the analytical curve. Genomic DNAs extracted from plasma patients were subjected to a denaturing step prior to the hybridization process.
The solutions were prepared and incubated at 98℃ for 3 min. To avoid double-strand renaturation and to allow complementary and non-complementary DNAs to access the probe in the same condition, the modified electrodes, poly (4-AMP/Hep1), were also incubated at the same temperature. Immediately thereafter, 18 μL of the genomic DNA solutions was dripped into the modified electrodes. They were dried in an oven for 10 min at 55℃ to promote hybridization. After this process, washing with phosphate buffer was carried out for the removal of the unbound molecules.
The detection of genomic DNA was performed indirectly using the EB mediator, which amplifies the response signal. To this end, 18 μL of a solution of EB (1 µmol L −1 ) was added to the electrodes for 5 min at room temperature, followed by washing with the phosphate buffer solution. All experiments were performed in triplicate.

Electrochemical Impedance Spectroscopy
The fundamental steps for the construction of the genosensor were accompanied by the electrochemical impedance spectroscopy technique. The measurements of the electrochemical impedance spectroscopy (EIS) was performed at perturbation amplitude of 10 mV and frequency range of 10 kHz to 0.1 Hz in 5.00 mmol L −1 K 4 Fe(CN) 6 /K 3 Fe(CN) 6 containing 0.10 mol L −1 KCl. All experiments were performed in triplicate.

Data Presentation
The peak currents obtained by DPV were corrected with the Peak Analyzer tool in Origin 8, subtracting a straight line tangent to the base. The values of peak current variation (Δip) were measured as the peak height, whereas charge (Q) was measured as the area under the peak.

Immobilization and Oligonucleotide Hybridization
Graphite electrodes have chemical inertness, miniaturization potential, relative roughness, and low cost. Modification with polymeric films containing π electrons allows the immobilization of biological species by electrostatic interactions and/or covalent bonds. The most common technique to obtain it is electrochemical polymerization by cyclic voltammetry, which consists of delimiting a range of scanning potential containing the oxidation potential of the monomer at a given scanning speed [28]. 4-Aminophenol is a promising compound for electrochemical studies since this compound have two distinct substituent groups on the aromatic ring, which are chemically and electrochemically active (NH 2 and -OH). Castro and collaborators demonstrated that the polymer formed in an acidic medium is efficient for immobilization and detection of genetic material, suggesting a platform for the construction of DNA biosensors [26].
The sensitivity of the DPV technique is appropriate for the electrochemical characterization of the genosensors. Thus, different conditions of immobilization time of the probe (Hep1) and the hybridization time with the complementary target (Hep2) were verified to maximize biological recognition and potentially increase the sensitivity of the genosensor (Fig. 2).
In Fig. 2A and B, a study on the immobilization of the Hep1 probe is presented, considering the direct detection of the immobilized biological material. For this, the peak at + 0.98 V vs. Ag/AgCl/Cl − (KCl 3 mol L −1 ) was considered, which can be attributed to the oxidation process of guanine present in the probe [27]. The intensity of this oxidation peak is proportional to the amount of guanine immobilized on the electrode surface and provides immobilization of Hep1 ( Fig. 2A). We can observe in the time of 30 min that a maximum increase was obtained at the peak of the guanine oxidation current, which was two times bigger in relation to the initial immobilization of 5 min. The current values obtained from the guanine oxidation peak remained stable after 30 min, indicating that it has the maximum optimum time for coating the electrode with Hep1 in these conditions. Figure 2C and D illustrates the assay in which EB was tested on the bioelectrode for the indirect detection of the target Hep2 oligonucleotide using EB with an electrochemical indicator. It is an efficient electroactive intercalator to the DNA hybridization due to the oxidation peak around + 0.65 V vs. Ag/AgCl/Cl − (KCl 3 mol L −1 ) [19,20], as the peak current intensity is proportional to the accumulation of genetic material on the electrode surface. This response occurs by the intercalation of BE with double-stranded DNA, which is considered the result of the interaction of a hydrophobic aromatic molecule with the hydrophobic environment of the DNA nitrogenous base pairs [29]. The average value of the oxidation intensity of the experimental control, that is, the bioelectrode containing only the Hep1 probe (0 min), was around 8 μA. An increase in the oxidation intensity of EB is observed with the increase in the time of Hep2 hybridization with the bioelectrode probe, obtaining a maximum value after 30 min of incubation. This difference is statistical, and we can consider that EB was retained on the surface due to the presence of the hybridized product. However, between the studied times, there were no considerable differences. Therefore, the time used in the next experiments was 5 min -sufficient time to guarantee the best result in the exposure time of the DNA hybridized with EB. Studies with oligonucleotides were conducted prior to tests with pure genomic DNA and blood plasma due to the possibility of defining the optimization of the developed platform to validate with more complex samples.

Calibration Curve
The genosensor calibration was conducted, varying the concentration of HBV genomic DNA and monitoring the oxidation peak of EB using differential pulse voltammetry (Fig. 3). Sensitivity is considered an important property of a biosensor that allows the target analyte detection in minimal concentrations, favoring early monitoring and detection.
As evident from the voltammograms indicated in Fig. 3A, the response signal of EB increases with the genomic DNA target concentration. The increase in the oxidation current response of EB with target concentration occurs due to the pairing process that forms the duplex DNA, which allows the intercalation of increasing quantities of ethidium bromide. EB binds to DNA by intercalation, that is, insertion between the stacked base pairs of the double helix [30]. According to Fig. 3A, there is an increase in resistance with an increase in the formation of double-stranded DNA caused by the accumulation of the ethidium bromide onto the surface and displacement of the EB oxidation peak to anodic potential more, reinforcing that the hybridization reaction occurs on the surface of the modified electrode. Figure 3B was constructed based on the data obtained in Fig. 3A, where the charge values (µC) were determined by integrating the area of current × time (potential/scan rate) generated by the EtBr oxidation. The behavior of the obtained calibration curve demonstrated that the interleaving speed of ethidium bromide increases linearly in relation to the number of hybrids formed (Fig. 3B). The genosensor reached a detection limit of 35.69 ng mL −1 (4.63 IU ml −1 ) and a quantification limit of 118.90 ng mL −1 (15.42 IU ml −1 ). The linearity (r) between the load and the DNA concentration is 0.999, and the sensitivity is 3.502 × 10 −5 µg mL −1 /µC or 2.700 × 10 −7 IU ml −1 /µC. These values can also be expressed by copies.ml −1 , for it is necessary to use a conversion factor, where 1 IU ml −1 is equivalent to 5.6 copies.ml −1 [31,32]. Therefore, we can still define the detection limit and quantification limit values, respectively, 25.93 copies.ml −1 and 86.35 copies.ml −1 , demonstrating to be more sensitive than the techniques explored in the literature [26].
Traditional methods of molecular analysis of the hepatitis B virus occur through realtime PCR assays, whose average detection limit is 10 IU ml −1 [33][34][35]. This technique for diagnosis is expensive, once requires qualified labor, and takes a long time, facts that hinder its point-of-care application. Thus, this new genosensor presents attractive features for the application in diagnostics due to the higher sensitivity, quickness, and lower manufacturing cost. Mainly in manufacturing simplicity, it follows a modular model.

Electrochemical Impedance Spectroscopy of the Genosensor Platform
Electrochemical impedance spectroscopy (EIS) studies are a powerful and sensitive tool for studying the charge transfer processes occurring at the electrode-solution or modified electrode-solution interfaces [36]. The EIS technique was used to verify the immobilization of the DNA probe (Hep1) on a surface of the poly(4-AMP)-modified graphite electrode, as well the subsequent hybridization with the HBV genomic DNA. Figure 4 shows the Nyquist plots of the EIS data of the modified electrode before and after the DNA probe immobilization, as well as the different concentrations of the HBV genomic DNA (DNAgen). In addition to the electrical parameters obtained through an equivalent electrical circuit.
Analyzing the behavior at high frequencies (Fig. 4A), it is possible to see the formation of a semicircle in the complex plane with the addition of the Hep1 DNA probe. The immobilization of the Hep1 probe on the surface of the modified graphite electrode creates a thin film which makes difficult the electron transfer from the redox pair ferro/ferricyanide to the electrode, increasing the charge transfer resistance of the system. In Fig. 4B, the behavior of the impedance spectra at high frequencies shows great coherence with the expected, which is a process of hybridization of the DNA single-strand Hep1 probe with the DNAgen. The increase in the resistivity of the genosensor with the increase in the concentration of the DNAgen was related to the intercalation of DNAgen inside the probe and, in the sequence, a probable combination between the nitrogenous bases to form the double helix of DNA. This layer of DNA double helix formed on the electrode makes it less porous, behaving like a passive film, making the passage of electric current difficult.
The equivalent circuit shown in Fig. 4C and D was chosen because it presents more coherent and reproducible results for the genosensor. The results obtained with the simulation for each DNAgen concentration are consistent with their intercalation and, consequently, hybridization with Hep1. The effects of hybridization are observed in the exponential increase in the resistance of the hybridized film and in the charge transfer resistance of the Fe 2+ /Fe 3+ system, and in the decrease in the capacitance of the hybridized film and the capacitance of the double layer of the genosensor. The data obtained for each DNAgen concentration are shown in Table 1.
Analyzing the results of the modified electrode with the hybridized film that appears at high frequencies, it was observed that the R f increased exponentially with the increase in the concentration of the genomic DNA target (DNAgen). The increase in R f is related to where R Ω is the ohmic resistance of the electrolyte, Q f and R f□ capacitance and resistance of the Hep1 probe or hybridized Hep1-DNAgen, respectively, R ct is the charge transfer resistance, and Q dl is the double layer capacitance where ρ is the resistivity of the hybridized film, l film is the thickness of the hybridized film, and A substrate is the uncovered area of the graphite electrode modified with poly(4-AMP). The opposite effect was observed for the film capacitance, Q f , being consistent with Eq. (2): where ε denotes the dielectric constant relative to the DNA probe (Hep1) and the hybrid formed with the complementary target of genomic DNA (positive DNAgen), and ε 0 is the permissiveness of the vacuum (8.85 10 −14 F cm −1 ).
As listed in Table 1, the Q f values are related to the decrease in the dielectric constant because of the elimination of porosity in the film, causing a reduction of the exposed geometric area of the graphite electrode and greater thickness of the hybridized film. The interpretation of the electrical behavior of the components R f and Q f is closely related to the time constant at low frequencies, described by the electrical components R ct and Q dl . The decrease in Q dl with the simultaneous increase in R ct indicates that there was complete coverage of the graphite electrode with the poly(4-AMP) substrate, the effective adsorption of the Hep1, and the greater hybridization with the increase in the concentration of the DNAgen.

Specificity Using Genomic DNA in Real Samples and Microscopy Analysis
The detection of the target analyte in patient samples is an extremely important step to verify the accuracy of the diagnosis [37]. It is known that human plasma is a fluidic mixture composed of several components that can interfere with the reading of the signal, and therefore, it is necessary to evaluate the device's ability to distinguish the target molecule from the contaminants to have a more accurate diagnosis [38]. Figure 5A shows the specificity study of the detection system in which the sensor was tested with plasma from a patient diagnosed with HBV, thus containing the virus genetic material (positive control), and plasma from a healthy patient, used as the negative control. The sensor response was evaluated in a sample of 6 patients, comparing the results obtained with blood plasma and with DNAgen. For this analysis, the plasma samples were diluted, keeping the same genomic DNA concentrations, in SSC buffer, shown in Fig. 5B. Figure 5A shows the comparison of the electrochemical signal before the formation of the duplex (curve a) and in the presence of DNAgen (curve c), positive plasma (curve d), and healthy plasma (curve b). In the presence of the target DNAgen (curve c), there was an increase in the electrochemical response of EB, indicating that the molecular recognition occurred efficiently since the signal is proportional to the concentration of interlayer fixed on the surface [4]. In addition, there was a displacement of the potential for anodic regions, from + 0.69 to + 0.74 V, showing an increase in resistance to charge transfer, thus confirming the formation of the duplex (Hep1: DNAgen) in agreement with electrochemical impedance spectroscopy data. This same shift is also seen in Fig. 3A, reinforcing the idea of genetic, molecular recognition. (1) When working with blood plasma samples (Fig. 5A -curves a and d), we can also observe a shift in the oxidation potential of EB to anode potentials. With the complexity that plasma samples represent to electronic systems, we can suggest that the shift and consequently the increase in resistance are related to some non-specific interactions that may occur between the complex sample and the platform. We can still believe that due to the size of the genetic molecule, it is possible to form hairpin loop structures, where two regions of the same molecule can interact and increase resistance [28,39].
Another observed fact is the increase in the oxidation intensity of EB. The addition of negative plasma (Fig. 5A -curve b) does not considerably influence the oxidation potential and the response of the electrical indicator (EB). Even so, it promotes a small increase in the current; this may be related to the constituents present in the plasma sample that interacted not specifically with the Hep1 probe and, consequently, a small amount of EB can be captured and oxidized; however, this accumulation does not impair the response of the genosensor to the presence of viral genetic material. As in the negative sample, it promoted an insignificant increase in the electrochemical signal, strengthening the proposal that the presence of plasma or the BSA blocking agent does not influence the final response, suggesting that a platform is an excellent option for real-time diagnosis. Real samples (blood, plasma, and other biological fluids) are rich in possible interfering substances, which can vary in composition and concentration and affect diagnostic sensitivity. BSA is important for blocking the modified electrode surface, minimizing non-specific interactions of Hep2, genomic DNA, or EB with the bare electrode surface [40][41][42][43][44][45]. Previous assays using real samples were performed without the use of BSA or another blocking component, and the results were unsatisfactory (unpublished data).
In the presence of positive plasma (Fig. 5A -curve d), an intense peak of EB oxidation is observed, indicating that the Hep1 probe was efficient in recognizing and promoting hybridization with the complementary region of the genomic DNA present in the sample, allowing the connection of EB in the duplex formation. When comparing Fig. 5A -curve c in relation to the Fig. 5A -curve d, the current values obtained are similar; however, the positive plasma sample shows a slight increase in intensity and a slightly more pronounced displacement when compared to the probe, + 0.69 V to + 0.76 V, due to possible non-specific interactions [19]. This behavior is followed for samples from 5 sick patients and is shown in Fig. 5B. We observed the same behavior for all samples. Secondary genetic structures and non-specific interactions can act as anchors and retain some EB on the surface. With a washing of the surface and the thermal process by which the sample was submitted, these interactions are minimized to the point of not causing significant interference in the electrochemical analysis. Therefore, only the genetic material of the virus would be retained on the surface.
The system can distinguish the concentration of genomic DNA in the absence and presence of blood plasma, and the biosensor response is not significantly affected in the presence of plasma, showing a promising device for the diagnosis of hepatitis B. Analysis by scanning electron microscopy was carried out with modified graphite electrodes (Fig. 5A -images  a-d). The images suggest that the modification was carried out successfully and that they have very different characteristics between them. Highlighting indicated in Fig. 5A -image d, which in the presence of plasma caused the most significant change, was presenting some globular structures.

Conclusion
The genosensor developed in this work demonstrated high reproducibility for the target recognition and duplex formation of Hep1 and Hep2 using ethidium bromide as an electrochemical mediator. Furthermore, the genosensor was efficient in detecting genomic DNA with high sensitivity, a low wide dynamic detection range, detection limit better than a reference method used, and could effectively discriminate complementary target sequence without the need for prior purification of the sample. This novel platform also has a short assay time (36 min), and the ease of synthesis of the low-cost polymer by electrosynthesis directly on the electrode surface allows the translation of the platform to portable devices. The agreement of the results obtained with the different techniques utilized indicates a promising application of the DNA genosensor developed for the point-of-care diagnosis. Funding The authors are grateful for the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.