Alphatoxin Nanopore Detection of Aflatoxin, Ochratoxin and Fumonisin in Aqueous Solution

Mycotoxins are toxic and carcinogenic metabolites produced by groups of filamentous fungi that colonize food crops. Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are among the most relevant agricultural mycotoxins, as they can induce various toxic processes in humans and animals. To detect AFB1, OTA and FB1 in the most varied matrices, chromatographic and immunological methods are primarily used; however, these techniques are time-consuming and expensive. In this study, we demonstrate that unitary alphatoxin nanopore can be used to detect and differentiate these mycotoxins in aqueous solution. The presence of AFB1, OTA or FB1 inside the nanopore induces reversible blockage of the ionic current flowing through the nanopore, with distinct characteristics of blockage that are unique to each of the three toxins. The process of discrimination is based on the residual current ratio calculation and analysis of the residence time of each mycotoxin inside the unitary nanopore. Using a single alphatoxin nanopore, the mycotoxins could be detected at the nanomolar level, indicating that alphatoxin nanopore is a promising molecular tool for discriminatory analysis of mycotoxins in aqueous solution.


Introduction
The growth of filamentous fungal species in varied foods and animal feeds results in the production of metabolic substances that, when ingested, can be toxic to humans and animals. These substances are termed mycotoxins, and the toxification process is referred to as mycotoxicosis [1][2][3]. Certain environmental conditions, such as regarding temperature and humidity, are favorable to fungal growth; there is a higher incidence of fungal growth in countries with tropical climates. In particular, species of the genera Aspergillus, Penicillium and Fusarium produce some of the most studied mycotoxins groups, namely, aflatoxins, ochratoxins and fumonisins [1][2][3][4][5][6][7]. Aflatoxin B 1 (AFB 1 ) is the most studied due to its high toxicity and wide presence in food; according to the International Cancer Research Agency, this toxin is a group 1 carcinogenic substance [8,9]. This makes AFB 1 a significant object of scientific interest in the identification of toxicological pathways for humans and the development of new analytical tools to detect its presence in food crops [10]. Ochratoxins are metabolic products of Aspergillus species and some strains of Penicillium, and ochratoxin A (OTA) is prevalent in foods [7,11]. Among the homologs of more than gradient induces electrostatic focusing resulting in an increase in the capture rate of the deoxyribonucleic acid and peptide capture [50,51]. In this study, we demonstrate that single alphatoxin nanopore can be used for the detection and differentiation of the mycotoxins ochratoxin A, aflatoxin B 1 and fumonisin B 1 in a solution with of high potassium chloride concentration and absence of a saline gradient.

Mycotoxins Induce Reversible and Characteristic Blockage of Ionic Current in Single Alphatoxin Nanopore
Previous studies denote the ability of nanopores to detect toxins in aqueous solutions. For example, the conductance fluctuation analysis and determination of the all-points histograms allowed the detection and differentiation of microcystin variants by the single alphatoxin nanopore [48]. Here, we performed a residual current and dwell time analysis and demonstrated that the unitary alphatoxin nanopore is able to detect and discriminate three mycotoxins in solution with high KCl concentration.
Voltage-clamp conditions were used to investigate the interaction of individual mycotoxins molecules with a single alphatoxin nanopore. In a 4 M KCl solution (pH 7.5) and a transmembrane potential of 40 mV, the unitary alphatoxin nanopore presents a constant current of approximately 150 pA and 4 nS conductance; these parameter values are in agreement with those reported in several previous studies [42,48,52,53]. Adding ochratoxin A, aflatoxin B 1 or fumonisin B 1 to the KCl solution in the trans side of unitary alphatoxin nanopore resulted in reversible and characteristic blockage events of the ionic current. A representative single nanopore ionic current profiles for interaction of the alphatoxin nanopore with the ochratoxin A, aflatoxin B 1 and fumonisin B 1 are shown in Figure 1A,D,G, respectively. Note that blockage events are indicated by dashed black rectangles. Figure 1B,E,H shows the blockage events can be observed as fluctuation discrete in the ionic current. This behavior is similar to that reported in studies of other analytes, indicating that this nanopore can be used in mycotoxin detection [48,49]. Figure 1C,F,I depicts the respective histograms of dwell times for the ochratoxin A, aflatoxin B 1 and fumonisin B 1. Mean dwell time values (τ off ) and mean interevent interval values (τ on ) were obtained from the dwell histograms, and the interevent histograms were fitted to single exponential functions [54,55].
The capacity of the single alphatoxin nanopore can be used in directly detecting these mycotoxins, reinforcing the potential to use this nanopore for analyzing small molecules, as demonstrated in previous studies [43][44][45][46][47].
Furthermore, simultaneous addition of three mycotoxins to the solution in the trans side of the unitary alphatoxin nanopore (at concentrations of 10 µM for OTA or AFB 1 and 3.5 µM for FB 1 ) induced three distinct patterns of ionic current blockage ( Figure 3A). The amplitude of these three blockage profiles is similar, as can be seen in Figures 1 and 2. Therefore, the single alphatoxin nanopore facilitates differentiating between these mycotoxins when simultaneously present in aqueous solution. The discrimination process is based on the calculation of the residual current ratio and the residence time of each mycotoxin in the unitary alphatoxin nanopore ( Figure 3B).  Blockage events were characterized based on the mean dwell time (τoff) and residual current ratio (I/I0; I denotes the magnitude of current decrease, and I0 denotes open nanopore current) [54,55]. Analysis of ionic current tracings reveal the presence of three I/I0 and τoff values for each of the mycotoxins: ochratoxin A, I/I0 = 0.15 ± 0.001, 0.0311 ± 0.006 ms (n = 3), respectively; aflatoxin B1, I/I0 = 0.326 ± 0.025, 0.059 ± 0.007 ms (n = 3), respectively; and fumonisin B1, I/I0 = 0.443 ± 0.025, 0.0996 ± 0.011 ms (n = 3), respectively ( Figures  1 and 2).
The capacity of the single alphatoxin nanopore can be used in directly detecting these mycotoxins, reinforcing the potential to use this nanopore for analyzing small molecules, as demonstrated in previous studies [43][44][45][46][47]. Analysis of the ionic current tracings obtained from the single alphatoxin nanopore in the simultaneous presence of the three mycotoxins resulted in the following (I/I 0 ) and τ off values: 0.145 ± 0.021, 0.028 ± 0.004 ms (n = 3) for OTA; 0.364 ± 0.005, 0.061 ± 0.007 ms (n = 3) for AFB 1 ; and 0.476 ± 002, 0.095 ± 0.006 ms (n = 3) for FB 1 . These values are in accordance with the results obtained for the single alphatoxin nanopore in the presence of only one type of mycotoxin (Figures 1 and 2).
These results, together with recent studies demonstrating that unitary alphatoxin nanopore detection can be used to discriminate between microcystins, reinforce the concept of using this nanopore as a potential molecular tool for the discrimination of mycotoxins in aqueous solution [48]. Figure 4 demonstrates the residence times of mycotoxins in the single alphatoxin nanopore. There were no differences between the values of the mycotoxins' residence times in the nanopore when in the presence of only one type of mycotoxin or in the presence  Figure 4). Therefore, the unique residence time for each type of mycotoxin in the unitary alphatoxin nanopore allows for their identification and serves as a second discriminant for multivariate analysis of these toxin classes in aqueous media [48,56,57]. Furthermore, simultaneous addition of three mycotoxins to the solution in the trans side of the unitary alphatoxin nanopore (at concentrations of 10 µM for OTA or AFB1 and 3.5 µM for FB1) induced three distinct patterns of ionic current blockage ( Figure 3A). The amplitude of these three blockage profiles is similar, as can be seen in Figures 1 and 2. Therefore, the single alphatoxin nanopore facilitates differentiating between these mycotoxins when simultaneously present in aqueous solution. The discrimination process is Analysis of the ionic current tracings obtained from the single alphatoxin nanopore in the simultaneous presence of the three mycotoxins resulted in the following (I/I0) and τoff values: 0.145 ± 0.021, 0.028 ± 0.004 ms (n = 3) for OTA; 0.364 ± 0.005, 0.061 ± 0.007 ms (n = 3) for AFB1; and 0.476 ± 002, 0.095 ± 0.006 ms (n = 3) for FB1. These values are in accordance with the results obtained for the single alphatoxin nanopore in the presence of only one type of mycotoxin (Figures 1 and 2). These results, together with recent studies demonstrating that unitary alphatoxin nanopore detection can be used to discriminate between microcystins, reinforce the concept of using this nanopore as a potential molecular tool for the discrimination of mycotoxins in aqueous solution [48]. Figure 4 demonstrates the residence times of mycotoxins in the single alphatoxin nanopore. There were no differences between the values of the mycotoxins' residence times in the nanopore when in the presence of only one type of mycotoxin or in the presence of a mixture of mycotoxins ( Figure 4). Therefore, the unique residence time for each type of mycotoxin in the unitary alphatoxin nanopore allows for their identification and serves as a second discriminant for multivariate analysis of these toxin classes in aqueous media [48,56,57].

Augmentation of the Salt Concentration Increases the Frequency of Ionic Current Blockage in the Unitary Alphatoxin Nanopore
Recent studies have updated methodologies to improve the electrophysiologica measurements generated from biosensors that use synthetic or biological nanopores Changes in the saline concentration or the making of concentration gradients on the ex perimental solution are ways to improve the sensitivity of this biosensors [50][51][52][53][54][55]. Amon

Augmentation of the Salt Concentration Increases the Frequency of Ionic Current Blockage in the Unitary Alphatoxin Nanopore
Recent studies have updated methodologies to improve the electrophysiological measurements generated from biosensors that use synthetic or biological nanopores. Changes in the saline concentration or the making of concentration gradients on the experimental solution are ways to improve the sensitivity of this biosensors [50][51][52][53][54][55]. Among these studies, some used the first approach, demonstrating that the detection capacity and sensitivity of the nanopore can be strongly influenced not only by the composition of the ionic solution, but also by the action of solutions with high ionic concentration [48,53].
In this case, Figure 5 illustrates typical single nanopore ionic current traces of the aflatoxin B 1 interaction with alphatoxin nanopore; with larger blockage frequency values for 4 M KCl solution, indicating that increasing the potassium chloride concentration from 1 to 4 M increases the frequency of ionic current blockage events by approximately five times. Previous data and the results of this work clearly demonstrate that increasing the salt concentration enhances the capability of stochastic detection using protein pores, such as the alphatoxin nanopore [53,58]. Other studies have demonstrated that an increasing salt gradient favorably influences the capture of molecules with residual charge (DNA, for example) via the protein or synthetic nanopore, but only if the analyte is placed where the salt concentration is lowest. This behavior is due to electrostatic focusing [50,51]. Our results were obtained from independent experiments carried out with symmetric KCl solutions, that is, at concentrations of 1 M or 4 M of KCl on both sides of the single alphatoxin nanopore, therefore, we do not have the presence of a saline gradient. Thus, the increase in the frequency of ionic current blocking events denoted here is probably due to the salting-out effect, as already reported in previous studies [42,52,53,57].

Sensitivity of the Unitary Alphatoxin Nanopore to Mycotoxins
The search for alternative and promising tools for mycotoxin detection results in the expansion of studies in the field of analysis [29][30][31][32][33][34]. Studies denote the emergence and advancement of methodologies based on biosensors (mainly immunosensors) used to detect this class of toxins [59,60]. Application of the single alphatoxin nanopore as a recognition element has shown satisfactory results in the stochastic sensing of toxins in aqueous solution [48]. Their properties of high sensitivity, low detection limit values and high dynamic range values indicate that sensors based on the unitary alphatoxin nanopore have beneficial characteristics for the detection of single molecules [35,37,42,61]. Gold-standard techniques are constantly being updated with dynamic range in the nano to picomolar order [28]. The amplitudes between the highest and lowest concentrations in which analytes can be discriminated represents the dynamic range of a sensor [62,63]. A recent study used mycotoxin-specific aptamers to demonstrate use of the single alphatoxin nanopore in detecting ochratoxin A at levels recommended by control agencies [49]. In this study, the increase in KCl concentration allowed use of the single alphatoxin nanopore in dis-

Sensitivity of the Unitary Alphatoxin Nanopore to Mycotoxins
The search for alternative and promising tools for mycotoxin detection results in the expansion of studies in the field of analysis [29][30][31][32][33][34]. Studies denote the emergence and advancement of methodologies based on biosensors (mainly immunosensors) used to detect this class of toxins [59,60]. Application of the single alphatoxin nanopore as a recognition element has shown satisfactory results in the stochastic sensing of toxins in aqueous solution [48]. Their properties of high sensitivity, low detection limit values and high dynamic range values indicate that sensors based on the unitary alphatoxin nanopore have beneficial characteristics for the detection of single molecules [35,37,42,61]. Gold-standard techniques are constantly being updated with dynamic range in the nano to picomolar order [28]. The amplitudes between the highest and lowest concentrations in which analytes can be discriminated represents the dynamic range of a sensor [62,63]. A recent study used mycotoxin-specific aptamers to demonstrate use of the single alphatoxin nanopore in detecting ochratoxin A at levels recommended by control agencies [49]. In this study, the increase in KCl concentration allowed use of the single alphatoxin nanopore in discriminating and detecting the studied mycotoxins at the nanomolar level. The dynamic range was estimated at approximately 0.71 to 15 nM for ochratoxin A and aflatoxin B 1 and, 0.71 to 3500 nM for fumonisin B 1 (Figures 6 and S1). It is important that strategies are underway for the development of portable instrumentation [49] and microfluidic systems [44] that will allow the use of detection systems to advance based on the applicability of biosensors. Given the success of the alphatoxin nanopore biosensor, it is important to expand the technique for detecting new compounds and evaluate parameters that improve the stability and sensitivity of these analysis [44,[48][49][50]53,54]. Previous studies show the usefulness of the alphatoxin nanopore biosensor in combination with aptamers in the analysis of ochratoxin A from corn samples [49]. The sensitivity values of the technique were in the range of 1.69 pM under the experimental conditions proposed in the study, allowing the analysis of real samples with concentrations starting from 0.2 nM OTA [49]. The use of aptamers combined to the alphatoxin The slope value of the calibration curve of a sensor indicates its sensitivity [62]. Therefore, the slope of the calibration curve was calculated to determine the sensitivity of the single alphatoxin nanopore for each mycotoxin. The single alphatoxin nanopore sensitivity values (nM s −1 ) are 0.148 ± 0.001 (ochratoxin A), 0.09 ± 0.010 (aflatoxin B 1 ) and 0.257 ± 0.016 (fumonisin B 1 ) ( Figure 6). The frequency of blockage events increases linearly with the mycotoxin concentration ( Figure 6). Therefore, it was possible to determine the concentration of mycotoxins in solution using the unitary alphatoxin nanopore; the dependence between the frequency of blockage events and the mycotoxin concentration was used to calculate the detection limit. The detection limit is the lowest analyte concentration value detectable by the single alphatoxin nanopore for a certain frequency value of ionic current blocking events [52,53]. Figure 6 shows the ability of the nanopore to detect mycotoxins in aqueous solution (assessment at practical 0.2 Hz of events), estimated at 0.8 nM for aflatoxin B 1 ( Figure 6D) and 0.45 nM for fumonisin B 1 ( Figure 6G). Likewise, adopting a 1 Hz frequency, it was possible to estimate the limit detection of the ochratoxin A as approximately 1.0 nM ( Figure 6A). This value is within the 0.3 to 16 nM range of the maximum allowable levels for this class of toxins in food [59].
It is important that strategies are underway for the development of portable instrumentation [49] and microfluidic systems [44] that will allow the use of detection systems to advance based on the applicability of biosensors. Given the success of the alphatoxin nanopore biosensor, it is important to expand the technique for detecting new compounds and evaluate parameters that improve the stability and sensitivity of these analysis [44,[48][49][50]53,54]. Previous studies show the usefulness of the alphatoxin nanopore biosensor in combination with aptamers in the analysis of ochratoxin A from corn samples [49]. The sensitivity values of the technique were in the range of 1.69 pM under the experimental conditions proposed in the study, allowing the analysis of real samples with concentrations starting from 0.2 nM OTA [49]. The use of aptamers combined to the alphatoxin nanopore has also been used for the detection of other chemical substances, such as cocaine [44]. However, this type of analysis is limited to the specificity of the aptamer and also makes the technique more laborious [44,49]. Other new techniques based on the applicability of chemiluminescence immunosensors and competitive magnetic immunodetection provide quite satisfactory detection limits for AFB 1 in the order of picomolar [34,60]. Therefore, it is necessary to find alternatives to the use of aptamers and antibodies, since the use of these techniques for mycotoxins analysis requires more steps and costs. We present here the perspective of the alphatoxin biosensor for the detection and discrimination of mycotoxins representing the three main groups of fungi (AFB 1 , OTA, and FB 1 ). The values of 0.8 nM (AFB 1 ), 0.45 nM (FB 1 ) and 1 nM (OTA) obtained in this study are lower than those recommended by legislation for different foods [59]. Studies carried out for the detection of other toxins, such as microcystins, have already demonstrated the capacity of the biosensor for multianalytical detection in the absence of molecular adapters [48]. This shows that the alphatoxin nanopore biosensor is able to be used in the future for the analysis of samples extracted from food.
In addition, the method presented in this study considerably simplifies assay procedures and reduces analysis time compared with standard methods, as it is not necessary to mark, modify or conjugate the mycotoxins with other types of molecules (aptamers or antibodies, for example) or nanoparticles. Additionally, the capacity of unitary alphatoxin nanopore to detect mycotoxins can be improved using several strategies, such as changing the ionic composition of the lipid membrane bathing solution and application of a saline gradient.

Conclusions
In this study, we demonstrate that increasing the KCl concentration in solution causes an increase in the frequency of blockage of the ionic current induced by the presence of mycotoxins in aqueous lumen of the single alphatoxin nanopore. Consequently, the stochastic sensing capacity of unitary alphatoxin nanopore is improved, enabling the discrimination of three relevant agricultural mycotoxins: ochratoxin A, aflatoxin B 1 and fumonisin B 1 . In addition, the sensitivity is increased, allowing a single alphatoxin nanopore to be used in the detection of these mycotoxins at the nanomolar level, which is comparable to the standard techniques used for quantification of these molecules. Finally, its ability to detect and discriminate mycotoxins at a nanomolar level indicates that the unitary alphatoxin nanopore is a promising molecular tool for discriminatory analysis of mycotoxins in aqueous solution.

Planar Lipid Bilayer Formation, Insertion of Single Alphatoxin Nanopore and Data Analysis
All experiments to obtain the solvent-free planar bilayer lipid membranes with a capacitance of 40 pF were performed using the lipid monolayer apposition technique and DPhPC in hexane at 25 ± 1 • C, as previously described [52,64]. The lipid bilayers were mounted in the orifice (diameter of approximately 50 µm) a Teflon film serving as a divider for two Teflon hemi-chambers. The membrane bathing solution was composed of 1 M KCl or 4 M KCl and 5 mM Tris−HCl buffered to pH 7.5. We chose to use mainly high concentrations of KCl in each Teflon hemi-chamber, since previous studies have demonstrated improvements in the detection capacity of the single alphatoxin nanopore [48,53]. If not mentioned specially, the applied transmembrane potential was 40 mV. After membrane formation, approximately 0.5 µL of alphatoxin stock solution (0.07 mg/mL) was added to the bath solution on the cis side of the experimental hemi-chamber for a final concentration of approximately 2 ng/mL, an amount sufficient to promote the incorporation of a single alphatoxin nanopore. The Ochratoxin A, Aflatoxin B 1 , and Fumonisin B 1 were added to the trans side of the hemi-chamber, from stock solutions at concentrations ranging from 1 mM to 1 nM.
An Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA) in voltage-clamp mode was used to measure the current travelling through the lipid bilayer membrane. Transmembrane potential was sustained using Ag/AgCl electrodes in 3 M KCl 2% agarose bridges montaged in standard 200 mL pipette tips. Currents were acquired using an Axopatch 200B amplifier with a Bessel filter at 10 kHz and sampled at 50 kHz using an IBM computer connected to Digidata 1440 A (Molecular Devices, San Jose, CA USA).
Analysis of the single nanopore ionic current traces and determination of the sojourn time, amplitude and frequency of blockage events and kinetic constants of mycotoxinnanopore interactions were performed as reported in previous studies [45,46]. Briefly, the characteristic time, τ on , was obtained from the collected time intervals between the end of one blockage event and the onset of the next. The residence time, τ off , was obtained from the collected duration time of blockage events. Finally, Clampfit 10.7 (Molecular Devices, San Jose, CA, USA) was used to analyze single nanopore ionic current trace, and Origin 8.1 (Origin, Version 8.1. OriginLab Corporation, Northampton, MA, USA) was used to generate histograms and for plotting and curve fitting.