Water-mediated NOE: a promising tool for interrogating interfacial clay–xenobiotic interactions
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The sorption of anthropogenic compounds on clay minerals is a complex molecular process with important implications for the fate of agrochemicals and organic pollutants in the environment.
The present study illustrates the use of a water-mediated NOE approach to study clay binding interactions. This method exploits the interfacial water layer on clay surfaces as a hydrogen reservoir for magnetization transfer. The interactions of four different xenobiotics with clay suspension were investigated through this method to demonstrate its capability to screen for the clay–xenobiotic molecular affinity. Further, based on the NOE build-up rates, epitope map of clay–xenobiotic interactions can be generated, explaining the orientation and mechanism of the interactions.
KeywordsNuclear Magnetic Resonance Nuclear Magnetic Resonance Spectrum Clay Particle Magnetization Transfer Nuclear Magnetic Resonance Signal
nuclear magnetic resonance
nuclear overhauser effect
soluble organic matters
small-angle neutrons scattering
x-ray powder diffraction
saturation transfer difference
cross polarization magic angle spinning
Water Ligand Observed via Gradient Spectroscopy
The enormous number of different xenobiotic substances in our environment is becoming a significant health concern [1, 2, 3]. These xenobiotic substances, including hormones, antibiotics and numerous pesticides, are commonly released to the environment through human activities [2, 3, 4]. As these substances accumulate in our environment, the question of their bioavailability becomes increasingly important. For instance, the occurrence of hormones, particularly estrogens, has recently gained attention due to their toxicological significance and their influence on biological activities in aquatic organisms [2, 4]. Although the acute health effects of these xenobiotics are well documented, the effect of chronic exposure remains an open question [1, 2, 3, 5, 6]. The intrinsic interactions between these xenobiotics and soils hold the key to understanding both their environmental accumulation and their bioavailability [7, 8, 9, 10, 11]. Therefore, a better understanding as to how xenobiotics are sequestered into complex rock, soil, and sediments is required [12, 13, 14].
Soil is represented by a complex mixture consisting of mineral and organic constituents that are in solid, gaseous, and aqueous states . Amongst the inorganic constituents in soil, clays constitute the major component (often around 30% w/w) and are important in the preservation of labile organic compounds. Clays are inorganic materials composed prevalently of layers of SiO4 and/or AlO4 . Because of their anionic nature, the surfaces of clays are covered with small cations. For instance, a host of cations, such as Na+, Ca2+, Mg2+, K+, and NH4 + are capable of binding to clay surfaces, giving them different colloidal property . In addition to sequestering cations from solution, clays are capable of adsorbing water molecules on their surfaces through hydrogen bonding, allowing them to expand and swell several times their dry mass [17, 18]. Due to this excellent colloidal property, clay is often been employed in a range of applications, from mining to agriculture .
So far, most soil–organic interaction studies mainly concerned the association of xenobiotic with humic substances [13, 20, 21]. These studies reveal that the xenobiotics are sequestered into soils through weak interactions driven synergistically by both hydrophobic and hydrophilic interactions . Despite the inorganic nature of clay, it is able to interact with a number of organic molecules such as fatty acids and aromatic compounds [22, 23]. In addition, recent studies have demonstrated that soluble organic matters (SOM) from terrestrial source are able to bind to clay particles in solution [12, 13]. Although the binding between clay and organic matter has already been demonstrated, the exact mechanism of interactions at the interface remains an open question due to the lack of suitable high-resolution structure determination techniques. Since these organic compounds exhibit a range of binding affinity to clay particles, traditional high-resolution diffraction techniques, such as SANS and powder XRD, are ill-suited to tackle such problems [19, 24]. In fact, this requires a technique that can interrogate molecular interactions over a wide range of timescales.
All chemicals, including pesticides, were purchased from Sigma-Aldrich. The mica-montmorillonite was purchased from the Clay society (www.clay.org). The montmorillonite was carefully treated according to the procedure outlined in the subsequent section before use.
Preparation of colloidal clay particles
Synthetic mica-montmorillonite (<2 µm, NL Industries) was subject to sodium exchange and saturation in order to form a stable suspension. A centrifuge tube was filled to approximately 1.5 cm with clay, and to 8 cm with 1 M sodium chloride solution. The centrifuge tube was continuously inverted until the clay was thoroughly suspended. The mixture was centrifuged for 5 min at 2000 rpm. The supernatant was decanted, and the procedures stated above were repeated two additional times. After the last decantation, the centrifuge tube was filled to 8 cm with distilled water and the clay was re-suspended. This mixture was centrifuged for 5 min at 2000 rpm. The supernatant was decanted, and the distilled water wash was repeated two more times in order to obtain a supernatant which appeared turbid. The Na-saturated sediment was then re-suspended in distilled water and two drops of 5 M sodium chloride solution. The mixture was centrifuged for 6 min at 1000 rpm. The turbid supernatant was pipetted into a 500-mL beaker with caution to prevent disturbing the sediment. This process was repeated seven more times until the supernatant was clear. The bulk supernatant containing Na-saturated clay was freeze dried.
Preparation of NMR samples
5 mg of pesticide and 10 mg of colloidal clay were dissolved in 500 μL of H2O/D2O (90:10). The mixture was stirred on a vortexer for 30 min and was subsequently transferred to a 5-mm NMR tube. The pH of the solution was tested via litmus paper.
The NMR experiments were acquired on a Bruker Avance III NMR spectrometer operating at 11.7 T, observing 1H at 499.98 MHz. The 1H field strength used was 25 kHz, with an acquisition length of 4 k data points and 15 ppm spectral width. The 1H waterLOGSY spectra were averaged over 128 scans with a recycle delay of 5 s. All NMR spectra were recorded at 297 K using a 4-channel 5-mm QXI inverse detection probe tuned to 1H, 13C, 15N, and 19F. Typical parameters used for waterLOGSY experiment were as follows: 4-ms 180° selective pulse (Φ 2) with Gaussian shape along with a 5-ms sinc-shaped 90° pulse (Φ 5) (water flipback) were used for selecting the water resonance; 1 ms squared gradient pairs were used at 40% of the maximum gradient strength (~54 G/cm) to select the water signal; and 2-ms square pulses (Φ 6 and Φ 8) along with gradients at 31 and 11% to dephase the water during water suppression. A gradient at 0.2% was applied throughout the mixing time to dephase water magnetization. A gradient recovery of 200 μs was used. The typical mixing times used in the waterLOGSY experiments were between 10 ms to 1.5 s. The mixing times used should be less than or equal to that of the longitudinal (T 1) of water.
Numerical simulation of NOE based on clay–bound interactions
1H waterLOGSY NMR spectra of four different xenobiotics in the absence and presence of clay particles
The changes in the sign of the NOE depending on clay’s presence confirm that interactions occur for the case of Nicotine and Imazapyr. On the other hand, there is no change in the sign of the NOE for the case of Diflufenzopyr and DMSO; this indicates that their interactions with the clay particle were transient at best. Furthermore, the aromatic 1H peaks of Diflufenzopyr remain inverted despite the absence of clay, while the amide proton (~9 ppm) has an opposite phase due to fast exchange. The different intensities for hydrogen in the NMR spectrum can be attributed to the clay interactions leading to differences in the NOE build-up rate and can be confirmed through experiments with varying mixing times. Epitope maps, which provide information on the binding orientation, can be generated from the data in Fig. 6 as well as from NOE build-up curves. Both these approaches are discussed later in the paper. In conclusion, the results of these experiments confirm our numerical simulations that indeed, clay–bound water-mediated NOE can be used to discriminate binding interactions on clay surfaces.
Strength of clay–bound interaction revealed through NOE build-up
Water NOE-driven epitope map of clay–xenobiotic interactions
In summary, NOE-based approaches outlined here should provide a key tool to study the dynamic interactions of organic molecules with mineral surfaces. Further studies using a wider array of structures, different clay, cations, and pH will be needed to fully elucidate the relative influence of functional groups and structural motifs on clay binding. Many of the NMR applications currently present in the literature mainly focus on the irreversible binding of anthropogenic compounds with clay particles. However, the water-mediated NOE approach introduced in this study should represent a further complimentary approach to enable interrogation of systems involving reversible binding in the fast exchange regime. In addition, it allows a better understanding of the role played by interfacial water in the thermodynamics of xenobiotic interactions. Importantly, it has been suggested over the years that this interfacial water, namely vicinal water, provides a favorable environment for organic compounds to partition from bulk aqueous environments . Although none of these models have been verified through high-resolution spectroscopic means, a comprehensive interrogation into these models will provide a better understanding of how clay particles facilitate the partitioning of organics from bulk water environments, which is fundamental to the question of transport, bioavailability, and bioaccessiblity of anthropogenic contaminants and agrochemicals .
The water-mediated NOE approach was successfully applied to study the interactions established between several xenobiotics and a model clay. This approach takes advantage of the differences in the NOE build-up rate developing when a compound is in contact with the clay vicinal water, which leads to a change in the sign of NOE. The latter effect manifests itself only when the molecule resides on clay surfaces for longer than 2 ns. Therefore, this is a tool to screen compounds as a function of their affinity with clay. Importantly, the sorption of anthropogenic compounds on clay minerals is a complex multivariable problem with intricate interacting parameters that warrant an in-depth investigation. The water-mediated NOE approach can provide insights into the role that vicinal water played in the binding process whether it is kinetically or thermodynamically driven. Therefore, this further validates the incorporation of this approach in the current repertoire of NMR experiments used in organo-mineral research.
RS carried out the experiments and drafted the manuscript. AB assisted in carrying out the experiments and JW prepared the clay particles. HF and DCM assisted in drafting the manuscript. AJS conceived this study and drafted the manuscript. All authors read and approved the final manuscript.
AJS thanks NSERC, (Strategic and Discovery Programs), the Canada Foundation for Innovation (CFI), the Ministry of Research and Innovation (MRI), and Krembil Foundation for providing funding. AJS also thanks the Government of Ontario for an Early Researcher Award. AJS would like to thank Dr. Rudraksha Dutta Majumdar for reading the manuscripts and his helpful suggestions.
The authors declare that they have no competing interests.
Consent of publication
The publisher has our consent to publish this manuscript including figures and associated data.
This study is supported by Grants from NSERC (Strategic and Discovery Programs), the Canadian Foundation for innovation (CFI), the Ministry of Research and Innovation (MRI), and Krembil Foundation.
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