Stochastic Sensing of Organotin Compounds with Alpha-Hemolysin Nanopore

Abstract

Organotin compounds (OTCs) are pollutants that affect the reproduction of some marine organisms and can cause autoimmune diseases in humans. Most of the techniques used for detection and monitoring of OTCs in the environment have limitations due to several stages of sample pre-processing. Here, we demonstrated the use of the α-hemolysin (αHL) nanopore as an alternative platform for the detection of the cyhexatin and diphenyltin dichloride (DPhT) in aqueous systems. The detection process is based on the analysis of the residence times of each OTCs within the unitary nanopore, as well as, on the amplitudes of the blockages in the ionic current flowing through it. Cyhexatin and DPhT induced two and three patterns in the amplitude of the blockages in the ionic current, respectively. Residence time value of the cyhexatin inside the nanopore was higher than the value presented by the DPhT. Molecular docking was used to evaluate the interactions between OTCs and nanopore constriction, mainly identifying cation-π and hydrogen interactions. Classical atomistic molecular dynamic simulations confirm moderate aggregative behavior of these OTCs in solution in cationic form, especially for cyhexatin species. The nanopore detects OTCs at nanomolar level and it is possible to use this nanopore to monitor OTCs in aqueous systems.

Keywords:
alpha-hemolysin; organotin compounds; stochastic sensing; cyhexatin; diphenyltin dichloride

Introduction

Environmental pollution has become a major concern worldwide and the impacts caused by human activities have effects on the various ecosystems.¹ Among these activities, the production and using of industrial products containing organotin compounds (OTCs) which are part of the group of chemical substances that pose a risk to the environment.²,³ The OTCs have at least one chemical bond between a tin (Sn) with organic groups.⁴ The general chemical formula is R_(n)SnX_(4-n), where R is an alkyl or aryl group and X is an anionic species (chlorides, hydroxides, oxides, among others).⁵ The application of OTCs-based products largely covers the production of biocides, used in agriculture and marine activity;⁶,⁷ as also in the industry for manufacturing polyvinyl chloride (PVC)⁸ and silicones.⁹ The tricyclohexyltin hydroxide (cyhexatin) (Figure 1a) and diphenyltin dichloride (DPhT) (Figure 1b) are among the organotins used for these purposes, respectively.¹⁰,¹¹ From 1950, the widespread use of these products promoted the elevation of OTCs levels in the environment, mainly with the use of OTC-based antifouling systems on ships.⁵ In 2008, the International Convention on the control antifouling systems prohibits this practice providing a gradual decrease in the levels of these substances in water and sediment in general.¹² However, several studies are reporting the presence and elevation OTCs levels in food and coastal areas in emerging countries, such as China¹³ and Brazil,¹⁴ suggesting that the practices of using these compounds have not been completely abandoned.¹⁵ The worry caused by the presence of these substances in the environment comes from the ability of most triorganotins, such as cyhexatin, to act as endocrine disruptors, causing disorders in various hormone conversion pathways mediated by enzymes.^{16 17 18} Due to its toxic effects, the cyhexatin had its use restricted by regulatory agencies.¹⁹ In 2009, the Brazil National Health Surveillance Agency (ANVISA) banned its use and commercialization.²⁰ The USA, and Europe also banned the use of cyhexatin.²¹ Because of the high efficiency in control of pests and diseases, azocyclotin acaricide is still extremely popular in countries like China. Used as acaricide in fruits and vegetables, the main degradation product of this acaricide is cyhexatin, exposing the environment to its toxic effects.¹³ Although there are no restrictions about use of DPhT, studies clearly show that PVC-based materials may undergo leach and release DPhT in the environment, contaminating drinking water, and consequently, contributing to human exposure.²² The proven toxic effects of OTCs have led to the withdrawal of these compounds from the composition of the biocides, as well as the establishment of environmental quality standards, proposing a maximum limit for organotin residues present in water between 0.0002 and 0.0015 µg L^-1, estimated by the European Union (EU).²³ Given the need to control the levels of these elements based on values established and recommended by the environmental control agencies, it has emerged the perspective for the development of platforms that allow the detection of OTCs at the levels mentioned above.²³ The analytical techniques based on the use of gas chromatography (GC) or liquid chromatography (HPLC), coupled to a detection system, allow the identification of these substances in the environment from several matrices.^{23 24 25} However, some problems make it difficult to apply these techniques routinely, as the high operating costs and exhaustive derivatization steps.^{23 24 25 26} Biosensors based on nanometric pores are gaining prominence in this scenario, particularly due to ability of these structures to detect, identify and characterize molecules.^{27 28 29} The nanopore formed by the alpha-hemolysin (αHL) from Staphylococcus aureus inserted in planar lipid bilayer (Figure 1c), has become a biosensing platform with great importance in this type of approach, allowing the investigation of the single molecules properties in aqueous solution, without destroying them and providing very low limits of detection.^{27 28 29 30 31} Analytes dissolved in electrolytic solution diffuse through the aqueous lumen of the nanopore, causing transient changes (blockages) in the maximum ionic current flowing through the αHL nanopore. The analysis of the amplitudes as well as the dwell times of these blockages reveals the individual characteristics of each organotin allowing its identification.^{27 28 29 30 31} Thus, a wide range of biological processes can be investigated using this biosensor.^{31 32 33 34 35 36} The study also aimed to identify the non covalent interactions involved between OTCs-nanopore complex, and how these processes make a relevant function in ligand and αHL recognition molecular, as reported in previous studies.³⁵,³⁶ Investigation of these interactions and the behavior of current traces of OTCs with protein pores may provide a future study model to elucidate transport mechanisms in cells.³⁷ Also, considering the importance of monitoring cyhexatin and DPhT in the environment and the paucity of studies offering alternative techniques to detect these compounds at low concentrations, the present work aims to identify and detect very low concentrations of two OTCs compounds in aqueous solution by stochastic sensing with the αHL nanopore.

Figure 1
Chemical structures of OTCs and αHL nanopore. (a) Tricyclohexyltin hydroxide (cyhexatin); (b) diphenyltin dichloride (DPhT); (c) representative structure of αHL nanopore in planar lipid bilayer.

Experimental

Chemicals

The wild type Staphylococcus aureus alpha-hemolysin was purchased from Calbiochem (Madison, WI, USA). 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was purchased from Avanti Polar Lipids (Birmingham, AL, USA). The analytical standards of tricyclohexyltin hydroxide (cyhexatin) and diphenyltin dichloride (DPhT) (> 99%) were purchased from Sigma (St. Louis, MO, USA). Methanol was obtained from J. T. Baker (Phillipsburg, NJ, USA). High-purity water was obtained through a Milli-Q Plus purification system (Billerica, MA, USA). 2-Amino-2 hydroxymethyl-1,3-propanediol (Tris) and citric acid were from Fluka (Buchs, Switzerland). Potassium chloride (KCl) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Hexane was purchased from Merck (Darmstadt, Germany). The stock solutions were prepared dissolving the cyhexatin in methanol and DPhT in DMSO at concentrations of 0.3 and 1 mM, respectively.

Single nanopore insertion in planar lipid bilayer and data analysis

The preparation process of the planar lipid bilayer has been reported previously, where the bilayer was formed by the lipid monolayer apposition technique.³⁸ In all experiments, the lipid used was DPhPC. After membrane formation, ca. 0.5 µL of αHL stock solution (0.07 mg mL^-1) was added to the bath solution in cis side of the experimental chamber to a final concentration of ca. 2 ng mL^-1, a sufficient quantity to promote the incorporation of a single nanopore. The bath solution was composed of 4 M KCl, 5 mM Tris-acid citric buffer (pH 7.5) as previously described.³⁹ Then, with the nanopore incorporated in lipid membrane, OTCs were added to the bath solution to the trans side. Experiments were carried out using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) in voltage clamp mode at room temperature (25 ± 1 °C). Membrane potential was maintained using silver/silver chloride (Ag/AgCl) electrodes in 3 M KCl 2% agarose bridges assembled within standard 200 mL pipette tip. Currents were filtered by a low-pass eight-pole Bessel filter at 15 kHz (Model 9002; Frequency Devices, Haverhill, MA, USA) and directly saved into computer memory with a sampling frequency of 50 or 100 kHz. The graphs were created with the OriginLab software.⁴⁰ All stages of data analysis for molecular signature parameters have been described in our previous studies.³⁹,⁴¹

Theoretical studies: molecular docking and atomistic molecular dynamics simulations

The heptameric structure of αHL nanopore was retrieved from PDB ID 7AHL.⁴² Cyhexatin and DPhT were obtained from ChemSpider⁴³ chemical database, while their 3D structures were constructed using Avogadro software⁴⁴ (Jekyll and Minimal Mistakes). The molecular docking calculations were performed in the constriction region of the αHL nanopore, using Dockthor online portal (version 2.0. GMMSB), with the standard program docking parameters.⁴⁵ Using its in-built genetic algorithm, the atomic coordinates of the lowest energy OTCs conformations were clustered based in a root-mean-squared-deviation (RMSD) of 2.0 Å. All the output conformations that presented favorable interaction energies with αHL nanopore were organized in order of increasing energy. The lowest energy docked conformations were retrieved for further analysis. Discovery Studio software was used for 3D visualization of the structural molecules, analysis of the interactions, and generation of images of the complexes between the OTCs and the constriction region of the αHL nanopore.⁴⁶ Subsequently, based on the “Solvent Extractor” tool in “Voss Volume Volexator” website,⁴⁷ volume analyses were performed. Such data were obtained aiming to correlate the space occupied by the OTCs and the residual current observed from single-nanopore experiments. For this purpose, an outer probe radius of 10 and an inner probe radius of 2 were employed.

The behavior of OTCs in solution was investigated using classical molecular dynamics simulations. Simulation boxes containing 2 up to 12 molecules of OTCs corresponding to 0.1 M concentration in aqueous phase and a concentration of 0.15 M of NaCl were built for each compound. If necessary, Cl^- were added to neutralize the simulation boxes. We developed force field parameters for OTCs compatible with AMBER force field⁴⁸ using MCPB methodology.⁴⁹ Electronic structure calculations of full optimization and frequency calculations of the parametrized molecules were carried out using Gaussian 16.B01⁵⁰ using Becke-3 parameter-Lee-Yang-Parr (B3LYP) functional, LaNL2DZ core potential for Sn atoms and 6-31G* basis sets for the other elements. TIP3P water-model was adopted for the solvent molecules.⁵¹ Each simulation box was submitted to 10000 steps minimization with steepest descent method followed by a short NpT thermalization (2 ns length) and equilibration phase of 200 ns. All systems were built and simulated using GROMACS 2022.0⁵² at pressure value of 1 bar and temperature of 298.15 K using a time-step of 2 fs. Van der Waals, electrostatic and neighbor list cut-offs were set up to a value of 1.2 nm. Particle Mesh-Ewald was adopted for the treatment of long range interactions.⁵³ Nose-Hoover⁵⁴ and Parrinello-Rahman⁵⁵ were adopted respectively as thermostat and barostat.

Results and Discussion

Integral effect of OTCs at α-HL nanopore conductance: voltage dependence

The individual αHL nanopore was inserted into a planar lipid bilayer which separates the experimental chamber in two sides called cis and trans (Figure 1). Both sides contained electrolyte solution described in the Experimental section. For this study, we established the standard use of the 4 M KCl solution. Rodrigues et al.³⁹ demonstrated that the kinetic parameters of the analyte-αHL interaction are strongly influenced by solutions with a high concentration of halogenated salts. The degree of hydration of the analytes and consequently the detection of the biosensor is positively affected. Electric potential was applied, flowing an ionic current through the αHL nanopore. In the absence of OTCs, the maximum conductance of the nanopore was ca. 4 nS, as already reported by previous works.³⁹,⁵⁶ However, the addition of one of the compounds (cyhexatin or DPhT) on the trans side caused reduction of the ionic current flowing through the nanopore. Similar studies have demonstrated the ability of the platform formed by αHL nanopore to detect volatile deoxiribonucleic acid (DNA)-bound pesticides,⁵⁷ as well as small organic molecules such as organophosphorus chemical agents,⁵⁸,⁵⁹ microcystins,³⁶,³⁷ pesticides⁶⁰ and mycotoxins.⁶¹ In order to evaluate the influence of transmembrane potential on the partitioning of OTC inside nanopore the relative conductance was calculated. The relative conductance allows to estimate the partitioning of the analytes in the nanopore.⁵⁶,⁶² We observed voltage dependence on relative conductance of the αHL nanopore in the presence of OTCs (Figure 2). Both OTCs cause a significant decrease in nanopore conductance from 140 mV. Under the conditions adopted here, there was a reduction of 9.4 ± 0.14% for cyhexatin and 18 ± 1.76% for DPhT. Furthermore, the relative conductance data indicate that the transmembrane potential which provides ideal conditions for detection of OTCs are 140 to 160 mV. This condition can be explained by the residual positive charge that the molecules can assume suggesting that repulsion favors the flow of OTCs through the lumen of the nanopore when a positive voltage is applied in the same chamber section where the OTCs are added (trans side).⁶³

Figure 2
Effects of OTCs in the integral conductance of the αHL nanopore. Nanopore conductance in the absence of the compounds is considered 100% (control condition). In the presence of one of the OTCs, the nanopore conductance is altered according to the type of organotin. Nanopore conductance in the presence of OTCs was relativized to the control condition. (∆) Control condition; (□) cyhexatin; (○) DPhT. Data are a mean ± SD of three experiments.

Single molecule events of OTCs in the ionic current through αHL nanopore: amplitude of the blockages and residence time

Typical high-resolution recordings of the ionic current through nanopore in the presence of OTCs are shown in Figure 3. Large ionic current flows through a unitary αHL nanopore when its aqueous lumen is free of OTCs. In contrast, the presence of OTCs in the aqueous lumen induces stepwise transition between completely open and blocked states in the ionic current flowing through nanopore (Figure 3). This behavior clearly indicates that this nanopore can detect OTCs, so it works as a molecular counter. According to our previous studies,³⁰,³³ we used the residual current analysis to characterize the amplitudes of the blockages to identify each OTCs (Figures 3a and 3b). This analysis was established as a percentage of the ratio of the values of the current amplitude produced by the blockage (I) and the current of the fully opened nanopore (I_o): I_R = (I/I_o) × 100. Blockages in the ionic current induced by the cyhexatin exhibit principally two levels of the amplitude (I₁ and I₂). The residual current (I/I_o) to each level was: I₁ = 31 ± 0.04% and I₂ = 17 ± 0.1%. We also observed transitions from level I₁ to a deeper blockage current sublevel (I_1’) with residual current of I₁ = 8 ± 1.4% (Figure 3a). In the case of DPhT, it was observed principally three levels of the amplitude of blockages in the ionic current: I₁ = 83 ± 0.18%; I₂ = 53 ± 0.2% and I₃ = 22 ± 0.04%. DPhT also exhibits a deeper blockage sublevel (I_3’) associated to level I₃ with residual current of I_3’ = 6 ± 0.04% (Figure 3b). Small ionic current blockades in the sublevels (I_1’ and I_3’) near zero were ignored in the analysis since they were exceedingly rare. As could be observed, the current residual values of the two OTCs are different from each other, allowing to distinguish them. Perera et al.,³³ with similar analyses and using DNA strands, reported the presence of sublevels associated with strand translocation through the nanopore lumen of αHL. Park et al.⁶⁴ evaluated the interaction of complementary DNA hairpins with the αHL nanopore, an indirect detection method for bacterial spores. Two levels of blockage were observed in current traces, providing an indication of the degree of translocation of the complementary DNA hairpins into the lumen of the nanopore. Presence of several ionic current amplitude levels to both OTCs may be explained by ratio OTC diameter/αHL lumen diameter.²⁷,⁶⁵ As the diameter of the OTC molecule is much smaller than the diameter of the αHL nanopore, two situations can probably occur: (i) the molecule “find space” to diffuse through nanopore in different conformations; (ii) more than one molecule translocate through nanopore at the same time or in aggregated form by forming complexes of OTCs, given the conditions of ionic strength caused by the aqueous solution. For further studies, with purpose attempt to restrict diameter of the αHL lumen, and thus reduce the number of current levels that an analyte may induce, proved relevant the use of molecular adapters such as β-cyclodextrin.⁶⁵ The β-cyclodextrin is very employed in the detection of small organic molecules by αHL nanopore, and its use improves the nanopore sensitivity.⁶⁵

Figure 3
Effects of the OTCs in the ionic current through the single αHL nanopore. (a) The ionic current through αHL nanopore in the presence of 1 μM of cyhexatin. I₁, I₂ and I_1’ indicate the ionic current amplitude levels represented by all-point histograms. (b) The ionic current through αHL nanopore in the presence of 1 μM of DPhT. I₁, I₂, I₃ and I_3’, indicate the current amplitude levels represented by all-point histograms. The compounds were added on the trans side. Solution: 4 M KCl, 5 mM Tris-HCl, pH 7.5. Transmembrane potential applied was 160 mV and time resolution of 0.01 ms.

According to our previous works,³⁶,⁵⁶ to analyze the dynamics of the interaction between OTCs and nanopore, we considered the process a simple bimolecular complexation reaction. In this context, one important parameter to analyze the kinetic process is the association rate constant (k_on). This parameter is correlated with the frequency of blockages caused by analytes.²⁷,³⁰,³⁶,⁵⁶ The k_on was calculated by assuming a simple bimolecular complexation reaction between OTCs and αHL nanopore at the single-molecule level, so that k_on = 1/(τ_on × [OTC]), where [OTC] is the OTCs concentration and t_on is the characteristic time between successive blockages.²⁷,³⁰,³⁶,⁵⁶ In Figure 4 can be seen that the values of τ_on for the OTCs possess dependence on applied transmembrane voltage, exhibiting low values at high voltages. This observation corroborates with the relative conductance data. The τ_on also has dependence on the type of OTCs, for example, at 160 mV cyhexatin has τ_on = 838.00 ± 62.00 ms and DPhT has τ_on = 315.83 ± 55.63 ms.

Figure 4
Effects of OTCs on characteristic time between successive blockages. The OTCs were added on the trans side at concentrations 1 μM of cyhexatin (□) and 50 μM of DPhT (○); solution (4 M KCl, Tris-HCl 5 mM, pH 7.5). Data are a mean ± SD of three experiments.

Another important parameter of kinetics analysis is the τ_off, which represents the dwell time of the analyte inside the nanopore.²⁷,³⁶,⁵⁶ Statistically, this parameter obeys an exponential distribution function of the time duration of the current blockage events, as previous studies have shown.³⁴,⁴¹ To both OTCs, we observed the absence of current blockage events at negative voltages as well as a low frequency at positive voltages below 80 mV (data not shown). Because of this, it was obtained values of τ_off at high positive voltages only. For both OTCs, the analysis of dwell time of the blockage events indicated the presence of three populations of time duration: short (τ_off1), intermediate (τ_off2) and long (τ_off3) (Figures 5a and 5b). Cyhexatin presented values of τ_off1, τ_off2 and τ_off3 ca. 200, 3.8 and 2600 ms, respectively (Figure 5a). For the DPhT the values were ca. 60, 400 and 2.2 ms, respectively (Figure 5b). The data presented by τ_off2 and τ_off3 of cyhexatin (Figure 5a) showed voltage dependence, indicating a higher affinity of the molecules of cyhexatin with the nanopore lumen at high voltages. However, we could not establish a correlation pattern between the levels of current blockage amplitude and the τ_off of cyhexatin. On the other hand, DPhT showed slight voltage dependence only for τ_off3. Another particularity observed was a probable correlation of the levels of current blockage amplitude with the dwell times populations of the DPhT (Figure 5b). In this way, we propose that the current blockages framed in the population profile of τ_off1 represent in the great majority the level I₁ of current amplitude, characterized by short and fast blockages. For presenting an intermediate profile in its both current amplitude and dwell time, the blockages from population τ_off2 represent the level I₂ and the blockages from population τ_off3 represent the levels I₃ with the predominance of great current amplitude and long dwell time.

Figure 5
Voltage dependence of the residence time of OTCs inside αHL nanopore. (a) Dwell time of cyhexatin inside nanopore: (■) short duration; (●) intermediate duration and (▲) long duration (insert). (b) Dwell time of DPhT inside nanopore: (○) short duration; (□) intermediate duration and (∆) long duration. Concentration of OTCs is 1 µM in solution (4 M KCl, Tris-HCl 5 mM, pH 7.5). Data are a mean ± SD of three experiments.

Interaction of OTCs with αHL nanopore constriction: molecular docking and dynamic simulations analysis

Studies at the atomic level have become a reality in several areas.⁶⁶ One of the tools used in these studies is molecular docking. This tool allows to obtain information about the interactions and possible conformations of small molecules at protein binding sites.⁶⁷,⁶⁸ Recently, we use this technique to predict the position and orientation of molecules on the internal surface of the αHL nanopore.³⁶,⁶² Júnior et al.³⁶ identified that electrostatic interactions were considered relevant factors for the biosensor to detect microcystins in aqueous solution. It is assumed that the αHL nanopore constriction (region of smaller diameter) is the region where the greater interaction between analytes and nanopore occurs.³⁵,³⁶,⁴² Chen et al.³⁵ proposed alterations in the constriction because it is a region of primary recognition of the nanopore. The study proposed to create a site for non-covalent interactions by inserting functionalized groups that have the ability to enhance recognition and discriminate the cationic, anionic, aromatic, and hydrophobic molecules. Thus, we performed the docking calculations between OTCs and αHL nanopore constriction using the Dockthor online portal.⁶⁷ Molecular docking results showed that DPhT and cyhexatin may have up to three and two types of conformers interacting with amino acid residues from the constriction region, respectively (Figures 6a-6c). Moreover, the main interactions between the nanopore constriction and the OTC molecules are different between DPhT and cyhexatin. Thus, cation-π interactions predominate between DPhT and αHL amino groups of Lys147. Conversely, hydrogen bonds between cyhexatin and the amino group of Lys147 and carboxylic acid of Glu111 may be observed, allied to hydrophobic interaction between the methyl group of Met113 and cyhexatin cyclohexane groups. The different amount and types of interactions observed may explain the greater dwell time for the cyhexatin molecule when compared to DPhT. In this context, the higher availability of regions susceptible to form hydrogen bonds and hydrophobic interactions provide stronger forces of interaction for cyhexatin than DPhT to bind αHL nanopore constriction region. Experimental findings suggest that each current block level depicts the passage through one or more molecules simultaneously. Docking results suggest, as the responsible for each level, the possibility of forming three different conformers for the DPhT (Figures 6d-6f) and two for the cyhexatin (Figures 6g and 6h).

Figure 6
Docking simulations between OTCs and αHL nanopore constriction. Panorama of αHL nanopore view from the trans side in absence (a) and presence the conformers of cyhexatin (b) or DPhT in constriction of nanopore (c). From the data obtained of Dockthor, it was proposed that blockages profiles in the presence of DPhT can be represented by three possibilities: passing one, two or three molecules (d-f). For the cyhexatin, we propose passage of one or two molecules through αHL (g-h).

Analysis of the poses using the “Solvent Extractor” tool provided the values of the residual volume of the nanopore lumen in the absence or presence of OTCs.⁴⁷ This parameter can be related to the residual current obtained from the experimental analyzes according to previous studies.³⁶ According to docking data, each conformer presented residual current theoretical of 67, 24 and 6%, respectively (Table 1). These values corroborate with the experimental findings where the DPhT exhibits three levels of current amplitudes (Figure 3a). Rarely, it was also observed the presence of a sub-level by the third level, which suggests the hypothesis of the passage of a fourth conformer through the nanopore. Cyhexatin showed residual current theoretical values of 58 and 7% (Table 1). These values suggest that the levels observed experimentally (Figure 3b), refer to the translocation of single conformer (Figure 6g) with values of I₁ = 31 ± 0.04% or I₂ = 17 ± 0.1%. Already the sub-level rarely observed, may be correlated to passage of two molecules (Figure 6h), causing nearly total blockage of the current. These results are in agreement with those of Júnior et al.³⁶ who, from the rankings with the molecular docking method and using these poses for the theoretical calculation of the occupation volume of microcystins in the lumen of the αHL nanopore, obtained values close to the experimentally calculated values for the percentage occupation of the blockages in the ionic current the nanopore. Thus, here, we were able to relate the top ranked poses in the Dockthor online portal of OTCs to their respective values of ionic current recordings from the αHL nanopore biosensor.

Table 1
αHL nanopore constriction region volume as a function of DPhT an cyhexatin interaction

Nanopore state		Volumea / Å3	Residual current theoretical / %
Unoccupied (absence of OTCs)	759	@	−
Occupied by DPhT	one	508	67
	two	178	24
	three	43	6
Occupied by cyhexatin	one	437	58
	two	51	7

• ^a
  The αHL residues 110-113 and 146-148 were considered for this analysis. OTCs: organotin compounds; DPhT: diphenyltin dichloride.

From the hypothesis suggested by the molecular docking analysis of nanopore constriction in the presence of two more molecules of OTCs in the nanopore, atomistic molecular dynamic simulations were carried out in order to investigate the behavior of organotin molecules in solution. Molecular dynamics simulations emerged some decades ago as an important tool to understand and predict molecular structure and dynamics.⁶⁹,⁷⁰ A set of simulation boxes were built containing 2 up to 12 solute molecules (DPhT and cyhexatin) at neutral and cationic species, minimized, thermalized and equilibrated for 200 ns of simulation length. In both simulations solute aggregation was observed after a few nanoseconds of simulation (Figure 7), which was more pronounced for neutral species. Aggregation stabilities for neutral species were confirmed during the remaining simulation time after their formation (Figure S1, ^{Supplementary Information (SI)} section). DPhT molecules in solution formed two aggregates of different sizes, a big one containing 10 units and a small one containing only 2 units. Cyhexatin molecules aggregate in 4 clusters formed by 3 or 4 monomers and one lonely unit (Figure S1). Cationic species of OTCs, (Ph)₂Sn²⁺ and (C₆H₁₁)₃Sn⁺ show a less pronounced aggregation pattern within formation of dimers between species, but with a variable stability. Less chargeable species (C₆H₁₁)₃Sn⁺ shows a more stable dimer aggregation along the whole simulation (Figure 7). These data confirm the possibility of using the αHL nanopore for the analysis of various biological processes. Mereuta et al.³² proposed the use of the biosensor to analyze the kinetic behavior of the folding of different conformations of peptides, by using different conditions of salt concentration and the pH of the solution. Size and number of aggregates can be explained by the shape and volume of each OTC. Smaller aggregates are observed in cyhexatin simulations as a result to accommodate large and flexible cyclohexane rings in aggregate structure. DPhT presents smaller size ligands around tin and can arrange an aggregate with a large number of monomers. The results obtained from the molecular dynamics simulations are consistent with the ability of the biosensor to verify this degree of aggregation experimentally using the records of ionic current traces. Moreover, these results may support future studies on the functional principle of transport of these compounds in ion channels and elucidate some toxic processes associated with OTCs.

Figure 7
Final snapshots of molecular dynamics simulations of (Ph)₂Sn₂₊ (a) and (C₆H₁₁)₃Sn⁺ (b). OTC in van der Waals representation with carbon atoms in green, hydrogen in white and tin in yellow colors. Water molecule are represented in a blue surface. Na⁺ and Cl_- were omitted for clarity.

Sensitivity of the nanopore sensor

Sensitivity of the nanopore was evaluated by calculating the frequency of events (1/τ_on) as a function of OTCs concentration as described in our and others studies.³⁶,⁵⁶,⁵⁹,⁷¹ For this parameter, we observed a linear behavior for cyhexatin and DPhT, providing a basis for quantification of these compounds.³⁶,⁵⁶,⁶¹ The values of sensitivity of the nanopore based method were 0.15 ± 0.02 and 0.14 ± 0.01 nM S^-1 for cyhexatin and DPhT, respectively. The limits of detection obtained for cyhexatin and DPhT were 0.005 nM (i.e., 0.001 μg L^-1) and 0.01 nM (i.e., 0.003 μg L^-1), respectively (Figure 8). Limit of detection values obtained for the detection of DPhT in previous studies⁷² using gas chromatography mass spectrometry (GC-MS) were close to our values. However, for cyhexatin, the αHL nanopore sensor proved to be a sensitive tool, since it was possible to obtain a low limit of detection, including lower than the limit of detection values obtained in other studies that used the GC-MS technique for the detection of this compound.¹³,²⁵,⁷³ In addition, various strategies, such as changing the composition of the ionic solution³²,³⁴,³⁹,⁵⁶ and using molecular adapters,⁶⁰,⁶⁴,⁶⁵ may be employed in future studies to improve the present sensing and discrimination capacity of the αHL nanopore for the OTCs.

Figure 8
Limits of detection of the OTCs. The arrows indicate the detection limit to Cyhexatin (■) and DPhT (○). The bath solution was composed of 4 M KCl and 5 mM Tris-acid citric buffer (pH 7.5). OTCs were added in trans side of the nanopore. Transmembrane potential applied was of 140 mV. Data are a mean ± SD.

Conclusions

In this work, two approaches (experimental and theoretical) were used to analyze the ability of the nanopore to interact and detect the following OTCs: tricyclohexyl tin hydroxide (cyhexatin) and diphenyl tin dichloride (DPhT). Experimentally, it was demonstrated that the presence of these compounds in the aqueous lumen of the nanopore induces different blockage profiles in the ionic current that flows through it. Furthermore, these blockage profiles are correlated with the respective OTCs, being two profiles for cyhexatin and three profiles for DPhT, showing an aggregation pattern of these compounds. In addition, cyhexatin remains for a longer time inside the nanopore, suggesting a greater interaction with the constriction of the αHL nanopore. Theoretically, different bonding modes were demonstrated between each of the OTCs with the narrowest region of the nanopore. The intensity of the interaction force between each of the OTCs with the constriction of the nanopore are correlated with the respective blockage profiles in the ionic current and with the residence time of each of the OTCs inside the nanopore. Molecular dynamics simulations show the ability of these OTCs to aggregate. This argues for the use of the biosensor in studying the degree of aggregation of these molecules and their transport through protein pores in aqueous solution. Finally, the corroboration between the experimental and theoretical results allows us to conclude that it is possible to use the αHL nanopore for the detection and monitoring of OTCs in aqueous systems.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants 470008/2012-1 and 313538/2019-0; Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), Grant APQ-0707-2.09/15, Brazil. This work used computational resources of the Centro Nacional de Processamento de Alto Desempenho em São Paulo (CENAPAD-SP) for computer simulations and electronic structure calculations. The authors dedicate this work to Oleg Vladimirovich Krasilnikov (in memoriam).

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Edited by

Editor handled this article: Paula Homem-de-Mello (Associate)

Publication Dates

• Publication in this collection
  05 Feb 2024
• Date of issue
  Feb 2024

History

• Received
  15 Apr 2023
• Accepted
  03 Aug 2023