Label-Free Impedimetric Immunosensors for Detection of Snake Venoms Using Polyaniline as a Transducer Substrate

Two label-free impedimetric immunosensors were developed to be employed in the specific diagnosis of snakebite. To produce the devices, a conductive substrate was obtained by electrodeposition of Polyaniline on the surface of a Crofer 22 APU steel. The polymer-based substrates were functionalized either with an affinity purified antibody specific for venom of the Bothrops genus or for the Crotalus genus. The performance of the as-prepared sensors was evaluated by exposing them to different concentrations of the venoms from Bothrops, Crotalus and Micrurus snakes. The capacity to recognize the specific analytes was measure by Electrochemical Impedance Spectroscopy within a frequency range from 10,000 Hz to 0.01 Hz. The increase of the immunosensors’ charge transfer resistance was correlated to the increase in the concentration of the homologous venoms pool with a detection limit of 0.1 μg. m L-1. Heterologous venoms were not detected at any tested concentration, proving the expected high selectivity. Furthermore, the results showed the reusability of the sensor, as it retained the capability of detecting the specific analytes after many regeneration cycles. The use of immunosensors to detect snake venom shows important promise as an aid to snakebite treatment.

this scenario, a precise and reliable method of diagnosis would permit the production of more specific antivenoms for instance, anti-genera serum [8]. Owing to the para specificity of antivenoms against venoms of the same genus, this approach could make available to the health system more efficacious antivenoms. These specific sera would result in the application of smaller volumes in the patient and, hence, lower frequency of adverse reactions would be expected [8][9][10][11] reported a method for the species-specific diagnosis of snake venom for the Bothrops and Lachesis genera using immunoaffinity adsorbed antibodies. That was followed by many publications showing the possibility of developing different specific methods to identify the offending snake through its venom present in body fluids of the patient [12,13]. These diagnostic methods are species-specific or genera specific. Enzyme-linked Immunosorbent Assays (ELISA), however precise, sensitive and reliable, have complex protocols that requires trained staff to perform, laboratory infraestructure and the results are obtained after hours of assay.
A point-of-care diagnostic system permitting the precise diagnosis of snakebites in a time frame of minutes rather than hours would greatly improve snakebite management and clinical treatment by ensuring the use of a more specific antivenom and ancillary measures to counteract the toxic effects of venoms. To meet the necessary requirements for providing a robust and precise diagnostic for snakebite envenoming, the biosensors seem to be a promising technology. Biosensors are devices that translate biological reactions into signals that can be detected and quantified.
Signals can be electrical, optical or mass based [14]. In this group of biological/analytical devices, the impedimetric immunosensors have recently received particular attention since they possess a number of attractive characteristics associated with the use of electrochemical transducers. Indeed, due to these characteristics, Electrochemical Impedance Spectroscopy (EIS) based sensors are considered as promising candidates for use at on-site applications [15]. Antibody-based biosensors (immunosensor) have antibodies as the capture biomolecule and can be label free providing fast diagnostics with few steps and no need of laboratory infraestructure nor trained staff.
Antibody-based sensors can provide robust, sensitive and rapid analysis [16,17]. Immobilization of antibodies to the chip surface can be oriented or non-oriented. Oriented attachment results in increased sensitivity of the immunosensor and specificity [18,19].
The immobilization of the capture biomolecule can be made directly onto the chip surface or attached to a coating polymer with electrical conducting properties. In the last 40 years, considerable attention has been focused on conducting polymers because of their unusual electronic properties and their great potential for biomolecules immobilization [20]. Among the conductor polymers that has attracted greater industrial and scientific attention in the last few decades, polyaniline (PANI) has stood out due to its good chemical stability, easiness to prepare, low cost, precursor monomer largely available, good electrical conductivity and interesting redox properties, leading to a wide range of application possibilities in various areas [21,22]. On its basal form, PANI has its chemical formula as shown in Figure 1. Of PANI's possible applications, the use in biosensors is justified by the fact that its conductivity propitiates the efficiency of the electrical charge exchange produced by biochemical or biological reactions on the sensor's surface. As opposed to other conductive polymers, PANI presents the advantage of being biocompatible and capable to incorporate great quantities of biomolecules, resulting in better characteristics of sensibility and response time of the biosensor [23]. In this study, we prepared genera-specific antibodies that were used in the construction of biosensors for the specific detection of venoms from the Bothrops and Crotalus genera.

Purification and Characterization of Specific Antibodies
Affinity purification of antibodies followed the protocol described by Heneine et al. [10] and was carried out for both anti-bothropic and anti-crotalic plasmas. Briefly, equal amounts of venoms comprising the bothropic immunizing venom pool and venom of Crotalus durissus terrificus and of Micrurus leminiscatus were insolubilized onto glutaraldehyde [24] to prepare three separate immunoadsorbents. Adsorbents were separately placed into 50 mL falcon tubes and volume measured. The antivenom sera were sequentially passed into the heterologous adsorbents to remove cross-reacting antibodies and finally in the homologous adsorbent to harvest the genera specific antibodies.

Characterization of the PANI Transducer during the Functionalization Steps Using Cyclic Voltammetry (CV)
In order to monitor the functionalization steps to obtain the immunosensor, CV was carried out after each step of modification

Sample Preparation
Venoms were dissolved in 0.1 M PBS pH 7.4 and centrifuged at 10,000 g for 30 min. The supernatant was collected, and protein concentration measured by Bradford assay. Aliquots of each venom were separately frozen at -20°C until use.

EIS Measurements
All the EIS measurements were performed using the same conventional three-electrode cell used in the PANI electrodeposition, but with the immunosensor as the working electrode containing the

Specific Venom Recognition
The working electrode containing the specific antibodies was immersed in 2 mL of venom solution and incubated for 20 min.

Antibodies Production and Characterization
The electrophoretic profile of the purified antibothropic antibody is shown in Figure 3.

Electrochemical Characterization of the Immunosensor
During the functionalization of the PANI substrate to detect the snake venoms, each step was monitored by CV. Figure 4

Impedimetric Recognition of the Venom Analyte
In Figure 5, the Bode diagram shows the good correspondence between the experimental and fitted curve indicating that the Randle model is representative of the phsysical interface phenomenoms between the electrodes and the electrolyte. Insert represents the equivalent circuit adopted to fit the impedance data, where Re is the electrolyte resistance; Rct is the charge transfer resistance and CPE the constant phase element. Besides, the fit quality was also ensured considering the chi-squared (χ²) parameter, which was lower than 10 -3 independent of the venom concentration or of testing the bare device. The criterion of χ² < 10 -3 indicates that the chosen equivalent circuit well represents the experimental data and, then, the values of the electrical elements can be employed to interpret the electrochemical phenomena taking place at the interfaces of the sensor and the surrounding electrolyte [31,32].   Thus, greater amounts of antigen-antibody complex increase the resistance of the sensor to transfer charge with the environment.
The graphic in Figure 7 shows    Because of the insulate properties of both biological molecules and the consequent steric effect of the formed protein layer, the immunocomplex hinders the charge transfer processes that previously took place in the PANI backbone and increase the global R ct of the sensor [30,33]. Affinity purification of commercial antibodies for the diagnostic assays of snakebite accidents has been employed, albeit in ELISA strips, with similar results as obtained by us, regarding specificity [34,35]. The degree of purification of the antivenom antibodies was sufficient for application as capture biomolecule in the immunosensor as it was capable to detect the homologous venoms in a range of concentrations varying from 0.1 to 10 µg.mL -1 .

Crotalic Venom Detection
Antibody attachment was non-oriented and could explain the lower limit of detection of only 0.1 µg.mL -1 at the incubation time used.
Nonetheless specificity was not affected as only the homologous venoms were detected across the concentration range tested.
Detection limits reported in the literature for immunoassays are mostly in nanogram level and even pictogram [12]. Actual detection of bothropic venom in serum of patients has been reported to be in the range of 13 ng.m L -1 to 120 ng.m L -1 [36]. Another report quantifying venom in the serum of patients in Peru, which included snakes from the botropic genera, showed the detected venom levels to be in the range of 3 ng.m L -1 to 466 ng.m L -1 [4].
Oriented attachment or increased incubation time could have resulted in lower limit of detection without specificity loss [37]. All

Conclusion
The immunoaffinity purified (anti-Bothrops and anti-Crotalus) antibodies were suitable as capture biomolecule for the application of electrochemical impedance spectroscopy as transduction technique. The functionalization of PANI was monitored by CV. This technique confirmed that the pure and selective antibodies were successfully attached to the sensor electrode, causing successive decreases of the overall current due to the insulator characteristic of the used molecules. The higher variations of R ct were observed at the highest concentrations of homologous venoms, indicating that the device was sensitive to the presence of the target analyte.
Instead, the contact of the immunosensors with heterologous venoms did not significantly change this parameter. Diagnosis of snakebite should primarily identify the venom presence or absence, and as such the immunosensors developed by us could be used in the specific diagnosis of bothropic and crotalic snakebite accidents.