Article pubs.acs.org/ac
Smart Detection of Toxic Metal Ions, Pb2+ and Cd2+, Using a NMR-Based Sensor
129
Xe
Nawal Tassali,† Naoko Kotera,‡ Céline Boutin,† Estelle Léonce,† Yves Boulard,†,∥ Bernard Rousseau,*,‡ Emmanuelle Dubost,‡ Frédéric Taran,‡ Thierry Brotin,§ Jean-Pierre Dutasta,§ and Patrick Berthault*,† †
CEA Saclay, IRAMIS, NIMBE, UMR CEA/CNRS 3299, Laboratoire Structure et Dynamique par Résonance Magnétique, 91191 Gif sur Yvette, France ‡ CEA Saclay, iBiTec-S, SCBM, Building 547, PC No. 108, 91191 Gif sur Yvette, France § Laboratoire de Chimie, CNRS, Ecole Normale Supérieure de Lyon, 46 Allée d’Italie, 69364 Lyon Cedex 07, France ABSTRACT: An approach for sensitive magnetic resonance detection of metal cations is proposed. Combining the use of hyperpolarized 129Xe NMR and of a cage-molecule functionalized by a ligand able to chelate different cations, we show that simultaneous detection of lead, zinc, and cadmium ions at nanomolar concentration is possible in short time, thanks to fast MRI sequences based on the HyperCEST scheme.
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in its local environment induce significant modification of NMR parameters. This property has been used in various situations: to differentiate the outer and inner compartments of biological cells using dissolved hyperpolarized xenon in 129Xe NMR spectroscopy22 and to propose a molecular MRI approach based on xenon encapsulated in host systems functionalized to reach defined biological targets.23 In this approach, cryptophanes are ideal candidates to accommodate xenon, as (i) they have a large affinity for the noble gas, (ii) their aromatic rings give a large shielding effect to the caged xenon signal, and (iii) a permanent in−out exchange enables the constant refreshment of the cage into hyperpolarization.24,25 DNA fragments,26 enzymes,27−29 and cell surface receptors30,31 have been targeted using this approach. Here, we show that the grafting of a ligand able to chelate various metal cations, such as NTA (nitrilotriacetic acid), on a cryptophane core can lead to fast multiplexed detection of nanomolar concentrations of metal ions through dedicated 129Xe NMR sequences. The reversible chelation of the metal by the ligand leads to a chemical shift variation of caged xenon that not only witnesses the presence of the metal, but also identifies it. Using a hydrophilic cryptophane bearing a NTA group (compound 1M, Chart 1) and xenon polarized at a level higher than 10% we recently detected32 in a few seconds 100 nM Zn2+, a threshold 2 orders of magnitude lower than that detected with recent gadolinium contrast agents.33−38 This sensor was also selective to Zn2+ with respect to Ca2+ and Mg2+ ions, addition of which gave rise to no change in the hyperpolarized 129Xe NMR spectrum.
he chronic and severe exposure to heavy metal ions, such as Pb2+, Cd2+, and Hg2+, can exert strong impact on human health and is linked to major human diseases, such as cancer and cardiovascular disorders.1,2 For example, human exposure to lead is estimated to account for 143 000 deaths every year.3 Similarly, cadmium poses severe harm to human health, as it accumulates mainly in the kidneys with a half-life in humans of 10−35 years. As a result, there is a great demand for highly sensitive analytical methods aiming at the selective detection of these metal ions. Techniques such as fluorescence spectroscopy, atomic absorption spectrophotometry, and electrochemical analysis are daily used to detect metal ions present in biological or environmental samples.4−10 Among these methods, fluorescence spectroscopy is particularly useful, because of its high sensitivity, simple operation, and its ability to provide real-time detection.11−18 However, fluorescencebased methodologies are limited by low tissue penetration and therefore are not suitable for in vivo imaging in deep tissues. Recent advances in the field of molecular magnetic resonance imaging (MRI) have led to the development of new strategies based on the design and synthesis of responsive contrast agents.19,20 Designing contrast agents that possess high sensitivity and chemical selectivity is a formidable challenge. The purpose of this Article is to show that NMR of hyperpolarized xenon allows for a sensitive and multiplexed detection of these metal ions. For the first time Pb2+ is detected at the nanomolar range by NMR. In addition, our method allows selective detection of Cd2+ in the presence of Zn2+, a difficult challenge because of the similarity of these ions.21
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RESULTS AND DISCUSSION Among the hyperpolarized noble gases used for sensitive MRI (3He, 129Xe, 83Kr), xenon is of special interest. The large polarizability of its electron cloud is such that even tiny changes © 2014 American Chemical Society
Received: November 12, 2013 Accepted: January 16, 2014 Published: January 16, 2014 1783
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Chart 1. Structure of the 129Xe NMR-Based Sensor of Metal Ions: (+)-MM-Cryptophane-(L)-NTA, Compound 1M
the ion concentration. The required concentrations of 1M are anyway well below the water solubility threshold. The sensitivity of the method is so high that the 129Xe NMR spectrum even shows spurious peaks at ∼68.2 and 69.7 ppm, which maybe correspond to trace amounts of other cations. Other cations were tested. Noting that two metals from the d-block of the periodic table (Cd2+ and Zn2+) gave a response on the hyperpolarized 129Xe spectrum, we undertook the study of the interaction of 1M with Hg2+. Though, addition of Hg2+ did not give rise to a new xenon signal. Only one paramagnetic cation, Co2+, was tried. Addition of Co2+ to a solution of 1M did not lead to additional peaks on the 129Xe NMR spectrum. However, the presence of cobalt ions could be detected using the other diastereoisomer ((−)-PP-cryptophane-(L)-NTA, that is, compound 1P) through a high-field shift of the caged xenon signal by −2.4 ppm whatever the cobalt concentration (Figure 2). The angular dependence of the paramagnetic shift and the
Here, we propose a new concept enabling detection of several cations present in a solution using this unique sensor. Figure 1 displays the 129Xe NMR spectra obtained with
Figure 2. One-scan 129Xe NMR spectrum performed with the sensor 1P at a concentration of 300 μM in the presence of 150 μM Co2+ ions. It was recorded at 11.7 T and 298 K with a Bruker 129Xe/1H 5 mm probe head.
lack of knowledge of the 3D structure of the complex impede any interpretation of this effect. Be that as it may, the metal sensor 1P is able to probe the presence of low amounts of Co2+ ions. This study was limited to some diamagnetic dications (or paramagnetic ones with low induced relaxation). Various experimental conditions such as the nature of the buffer, the pH and the temperature were tested. An important observation was made: whereas the chemical shifts of xenon free in solution and of xenon caged in 1M may vary significantly, the frequency difference between the signal of xenon in the sensor free of cation (Xe@1M) and that of xenon in the cage chelating a given cation (Xe@1M-Cation2+) remains almost constant. This gives a way to detect the cation whatever the medium or the sensor environment. To detect trace amounts of cobalt, cadmium, lead, or zinc cations, sensitivity is a major concern and the direct detection method can be supplanted by the HyperCEST method, provided that the xenon magnetization level is stable.40 Using the xenon exchange in and out of the cryptophane cavity, the presence of a biosensor is translated into a depletion of the bulk xenon signal after saturation at the caged xenon frequency. Simultaneous detection of these ions at nanomolar concentration is straightforward on a z-spectrum. And when localization of the ions is desired, the HyperCEST scheme can be added to various imaging sequences, in particular those dedicated to short T*2 samples. We decided to test this approach. It has recently been shown that the HyperCEST scheme can easily be combined with Echo
Figure 1. High-field region of 129Xe NMR spectra obtained at 298 K in one scan with the noble gas encapsulated in the sensor 1M alone at 970 μM in D2O (a), and after addition of 5% Zn2+ (48.5 μM) b), 5% Cd2+ (c) and 5% Pb2+ (d). The asterisk denotes a signal due to xenon encapsulation in the residual impurity (cryptophane-222-hexacarboxylate).
successive addition of 5% Zn2+, 5% Cd2+, and 5% Pb2+ to a solution of 1M in HEPES buffer. Each of these cations gives rise to a specific downfield chemical shift splitting for encapsulated xenon: 1.5 ppm for Zn2+, 0.3 ppm for Cd2+, and 4.5 ppm for Pb2+. To our knowledge, this is the first simultaneous detection of these metal cations at such low quantities by NMR. Dissolved xenon (its solubility is near 4.3 mM per atm in water at room temperature) is usually in large excess with respect to the cryptophane concentration, and the xenon binding constant on the order of 6000 M−1 (estimated from a competition experiment with cryptophane-222-hexacarboxylate) is not expected to be modified by the chelation of the metal cation. Therefore, all the cryptophane cages are filled with a noble gas atom and the areas of the 129Xe signals at high field represent the respective concentrations of 1M, 1M-Cd2+, 1M-Zn2+, and 1M-Pb2+ from high field to low field. Obviously the affinity constant of 1M for different cations can vary (more than 2 orders of magnitude are reported in the literature for the NTA headgroup39), and the responses have to be tabulated according to it. Also, to keep the quantitative character of the signals the cage concentration must be in excess with respect to 1784
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Experimental Section). Here, for the sake of clarity, the zspectra obtained using only two slices are represented. Such a protocol enables us to assess the efficacy of the saturation and its spectral selectivity. From Figure 3c, it is clear that the presence of lead ions can easily be established from the z-spectra, and that the relative signal intensities are respected. Also it is noteworthy that increasing the temperature would have largely improved our results by speeding up the in−out xenon exchange. Compound 1M constitutes a powerful 129Xe NMR-based sensor that enables sensitive and simultaneous detection of lead, zinc, and cadmium cations (the latter ones when the magnetic field value and homogeneity are sufficient). To get a more precise idea of the sensitivity threshold reachable through this method (although it largely depends on the xenon polarization level and delivery method), dilution experiments were performed. Figure 4 displays the evolution of the HyperCEST response as a function of the ion concentration, from 3 μM to 9 nM in
Planar Imaging (EPI) where a large part of or the whole k space can be spanned in a single acquisition.41 We therefore chose this scheme. In the present case, it was mandatory to implement an adequate, band-selective, saturation so as to irradiate one caged xenon frequency (e.g., xenon in the cage chelating Pb2+ ions) without affecting the other one (e.g., xenon in the “free” cage). For this purpose, a saturation step using multiple pulse inversion elements42 was developed. Figure 3a displays the
Figure 3. Fast MRI detection of lead cations. (a) Example of HyperCEST-EPI axial images recorded at 298 K with a sample containing 3 μM Pb2+ and a 3-fold excess of 1M. Left image: Saturation for 4.5 s with a B1 value of 9 μT at the Xe@1M-Pb2+ frequency (at an offset Δ from the signal of free xenon). Middle image: Off-resonance saturation (at an offset −Δ). Right image: Subtraction “on−off”. On the left image, the dashed circles represent the contour of the area integrated for the z-spectra (black = signal; red = noise) displayed in c. (b) For each point of these z-spectra, 5 axial slices of 1 mm separated by 1 mm were acquired (the numbers give the chronological order, slice 0 being an off-resonance HyperCEST experiment). (c) z-spectra recorded by integration of the images at various offsets (these data were obtained with 10.7 μM Pb2+ and 30 μM 1M). Only slices 2 and 4 are represented, corresponding to 9 and 18 s of saturation, respectively.
Figure 4. Hyper-CEST contrast as a function of the concentration in Pb2+ ions (after integration of the areas drawn in Figure 2a). The same saturation scheme and strength as in Figure 3 were used, for 4.5 s. The data points were fitted by an exponential function. See Experimental Section.
aqueous solution. The result obtained at this last concentration shows that we are able to detect 1.6 × 1012 Pb2+ ions in a few seconds (the slice volume is 34 μL). Whereas the data show a linear behavior of the HyperCEST effect for low Pb2+ concentrations, the point at 3 μM strongly deviates from it. As dissected in the Experimental Section, this arises from too low a concentration ratio of hyperpolarized xenon to cage. Anyway, this experiment highlights the strength of the use of hyperpolarized 129Xe MRI for the detection of trace quantities of metal cations. Indeed, nanomolar concentrations of metal cation can be revealed using only one bolus of xenon. The feasibility of such an approach for cell samples or living tissues can then obviously be questioned and not only for the specific absorption rate. To evaluate the difficulty of the experiment in the worth case in term of magnetic field inhomogeneity and presence of paramagnetism we did an experiment in whole, nonoxygenated blood, where the xenon relaxation time is short (because of deoxyhemoglobin, the xenon relaxation time falls to ∼5 s) and the susceptibility effects degrade the field homogeneity. On the one-scan 129Xe NMR spectrum of the sample loaded with Pb2+ ions (Figure 5a), from low field to high field, five signals were observed, corresponding to xenon in the red blood cells (1) and in the bulk (2) near 200 ppm,43,22 both Xe@1M-Pb2+ (3) and Xe@1M (4) near 70 ppm, and gaseous xenon (5). We checked that the presence of
axial images of an 8 mm NMR tube containing a solution of Pb2+ ions at 3 μM with an excess of 1M, recorded at 298K in 4.8 s with a HyperCEST-EPI sequence. The first image was obtained with saturation centered on the Xe@1M-Pb2+ signal, the second one with saturation off-resonance (at an offset symmetric with respect to the signal of dissolved xenon). The difference between the two images enables detection of the Pb2+ ions. Note the slight spatial shift (one pixel) that leads to bright and dark areas in the periphery of the solution. An integration region with a size smaller than the sample diameter (black circle) enabled us to get rid of these artifacts. To evaluate the HyperCEST effect as a function of the saturation time, for each saturation frequency several HyperCEST-EPI images were acquired (with a single xenon batch) exciting different slices of the sample (Figure 3b). As the saturation is not spatially selective, the resulting images corresponded to different saturation periods. Figure 3c displays the z spectra extracted from these images (details in the 1785
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EXPERIMENTAL SECTION
Production of Polarized Xenon. 83%-Enriched 129Xe from Euriso-Top was polarized by spin-exchange optical pumping of rubidium45 using a recently installed home-built setup based on laser diodes. The photons exiting from a duoFAP system (2 × 30W) and circular polarizer from Coherent illuminated a Pyrex cell placed at the center of a 100G magnetic field. The bandwidth of the laser diodes being about 2 nm, pressure-broadening was necessary. Therefore, the pressure in the cell rose 3 atm (measured at room temperature) with a mixture of 2% Xe−10% N2−88% He. The pumping cell was heated for 2 min to 410 K via a flow of hot N2 in an external envelope, in a fashion similar to what was developed for our previous experimental setup.46 Then xenon was condensed in a coldfinger inside a 3 kG solenoid immersed in liquid nitrogen, and thus separated from helium and nitrogen. The average polarization value with this experimental setup was 15%, measured for the gaseous phase in the NMR spectrometer. Sample Preparation. Prior to xenon introduction, the solutions in the NMR tubes were degassed via helium bubbling followed by evacuation. Human blood was obtained from EFS according to the established contract and with the required authorization. NMR and MRI Experiments. Hyperpolarized xenon was introduced on top of the solution in the upper part of the screwed NMR tube via a vacuum line in the fringe field of the NMR magnet. Then vigorous shaking of the tube followed by a 10 s delay ensured complete dissolution and equilibration of both gaseous and dissolved phases. Unless otherwise indicated, the 129Xe NMR spectra and images were recorded at 11.7 T and 298K with a dual 129Xe/1H 5 mm probehead and TopSpin 3.0 for the spectra and a micro5 Bruker probehead (dual 129Xe/1H 8 mm insert) and ParaVision 5.1 for the images. Each NMR tube received ∼1 atm of hyperpolarized xenon on top of the solution. The axial images were obtained via an EPI sequence with double sampling. The field-of-view was 19.2 mm, the raw matrices were constituted by 64 × 38 points (Fourier acceleration factor in the phase dimension: 1.68). For the experiment of Figure 3c, five successive slices were acquired: the first one was an EPI sequence, the other ones were HyperCEST-EPI sequences with a saturation of 4.5 s with a peak amplitude of 9 μT via a train of 80 D-SNOB pulses of 56 ms each, followed by a spoil gradient. As the saturation is not space-selective, it can be considered that the saturation period was 4.5 s for the first image, 9 s for the second image, etc. The slices were not acquired contiguously; the numbers indicated in Figure 3b give the chronological order. For the dilution experiments, a first 1-mm slice EPI image served as reference for the following 8-mm slice HyperCESTEPI image. There was no time delay between the EPI and HyperCEST-EPI experiments. Each data point was obtained from the ratio of the integral of the region encircled in black to the integral encircled in red, normalized with the ratio of the same regions recorded in the prior EPI experiment. Description of the Protocol for the HyperCEST Data Presented in Figure 4. To take into account the fluctuant hyperpolarization level, 10 s after having shaken the NMR tube to introduce new polarized xenon into the solution, we chained an axial EPI experiment (slice 1 mm) with an axial HyperCEST-EPI experiment (slice 8 mm shifted by 1 mm from the previous image, saturation time 4.5 s). This procedure
Figure 5. One-scan 129Xe NMR spectra performed at 11.7 T and 298 K on samples of whole human blood loaded with (a) the sensor 1M and Pb2+ ions and (b) the sensor 1M without any addition of metal ion. The numbers indicate the xenon location (see main text).
the two signals (3 and 4) was not due to two different environments for the cage, such as in the inner compartment of red blood cells and in the plasma, for instance, a situation already encountered in other cases.30,44 In an experiment performed in the absence of Pb2+ ions, only one signal appeared in this spectral region (Figure 5). Figure 5 shows that the lead cations can straightforwardly be detected.
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CONCLUSION The usual approach for multiplexed detection would require the synthesis of several cryptophane sensors, each of them able to bind a specific target. This would require a tremendous amount of time synthesizing these elaborate molecules. Here we have shown another promising approach based on the synthesis of a cation-responsive cryptophane sensor optimized for the simultaneous detection of several targets. The wide frequency range of the xenon response enables us to propose a new concept with a host system bearing a ligand that provides a unique and well-separated spectral signature of each ion. This approach is flexible, since several metal chelating groups, among which EDTA (ethylenediaminetetraacetic acid), DTPA (diethylene triamine pentaacetic acid), NTA, and cyclen, are available and can be grafted on a cryptophane core. It is applicable to the study of cell samples and is also expected to be amenable to ex vivo or in vivo experiments. Conversely to methods using fluorescent sensor arrays, the 129Xe NMR-based approach does not require a statistical analysis, the ions being straightforwardly recognized on the spectrum. Development of new sensors based on the same principle of xenon host bearing a generic ligand but with a shorter tether, which hopefully will enhance the xenon chemical shift effect, will be the subject of future work in our laboratories. 1786
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Analytical Chemistry was repeated ten times. Between each pair of images, the offset for the saturation pulse was changed from on-resonance to offresonance and vice versa. Each HyperCEST image was normalized with respect to the precedent EPI image (same integration regions as in Figure 2). Then we computed the pairwise difference between two successive normalized HyperCEST images (S = SON − SOFF). The standard deviation between the five values gave the error bar. Figure 4 reveals that the point at 3 μM cryptophane deviates from a linear slope joining the other points. Given that the characteristic time for the encapsulation of xenon in a cryptophane 2.2.2 cavity is close to 10 ms, if one also considers a saturation efficiency factor of 0.3 (ref 47) it means that during the saturation period (4.5 s) one cryptophane molecule would need 4500/(0.3 × 10) = 1500 hyperpolarized xenon atoms in its vicinity. When the cage concentration reaches 3 μM, it can be observed that a xenon concentration close to 4.3 mM (1 atm) can be insufficient. The HyperCEST experiment amounts in increasing the apparent relaxation of all xenon atoms entering in the cryptophane cavity (by rf saturation). Neglecting the natural xenon relaxation (in the absence of rf saturation on a xenon resonance) the signals arising from HyperCEST experiments on- and off-resonance are therefore
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REFERENCES
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SOFF proportional to [Xe]t f (tsat)
where [Xe]t is the total xenon concentration and f(tsat) a function of the saturation time. As mentioned by Kilian and co-workers,47 the factor k contains contributions from the isotopic enrichment, from the efficacy of the saturation and from the cage occupation factor koc, given by 1/k OC = [C]t /[XeC] = 1 + 1/(K[Xe]) = 1 + 1/(K[Xe]t − [XeC])
where K is the binding constant and [XeC] the concentration of cages filled with a xenon atom K = [XeC]/([Xe][C])
From this equation, it can be seen that when the total xenon concentration is not far higher than the concentration of caged xenon (most of the xenon is in the cryptophane cavities), koc seriously decreases. The data points of Figure 4 can be fitted by an exponential.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Present Address ∥
ACKNOWLEDGMENTS
The authors thank Dr. X. Millot (CEA/SST) for his help and expertise. Support from the French Ministry of Research (project ANR-12-BSV5-0003) and from the Fondation pour la Recherche Médicale (project DCM20111223065) is acknowledged.
SON proportional to [Xe]t f (tsat)exp(−ktsat)
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Yves Boulard: CEA Saclay, iBiTec-S, SB2SM.
Author Contributions
N.T. and N.K. contributed equally. Notes
The authors declare no competing financial interest. 1787
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(29) Chambers, J. M.; Hill, P. A.; Aaron, J. A.; Han, Z.; Christianson, D. W.; Kuzma, N. N.; Dmochowski, I. J. J. Am. Chem. Soc. 2009, 131, 563−569. (30) Boutin, C.; Stopin, A.; Lenda, F.; Brotin, T.; Dutasta, J.-P.; Jamin, N.; Sanson, A.; Boulard, Y.; Leteurtre, F.; Huber, G.; BogaertBuchmann, A.; Tassali, N.; Desvaux, H.; Carrière, M.; Berthault, P. Bioorg. Med. Chem. 2011, 19, 4135−4143. (31) Palaniappan, K. K.; Ramirez, R. M.; Bajaj, V. S.; Wemmer, D. E.; Pines, A.; Francis, M. B. Angew. Chem., Int. Ed. 2013, 52, 4849−4853. (32) Kotera, N.; Tassali, N.; Léonce, E.; Boutin, C.; Berthault, P.; Brotin, T.; Dutasta, J.-P.; Delacour, L.; Traoré, T.; Buisson, D.-A.; Taran, F.; Coudert, S.; Rousseau, B. Angew. Chem., Int. Ed. 2012, 51, 4100−4103. (33) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Chem. Soc., Perkin Trans. 2001, 2, 1840−1843. (34) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Narazaki, M.; Yokawa, T.; Sakamoto, S.; Yamaguchi, K.; Nagano, T. Chem. Biol. 2002, 9, 1027− 1032. (35) Major, J. L.; Parigi, G.; Luchinat, C.; Meade, T. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 13881−13886. (36) Major, J. L.; Boiteau, R. M.; Meade, T. J. Inorg. Chem. 2008, 47, 10788−10795. (37) Esqueda, A. C.; Lopez, J. A.; Andreu-de-Riquer, G.; AlvaradoMonzon, J. C.; Ratnakar, J.; Lubag, A. J. M.; Sherry, A. D.; De LeonRodriguez, L. M. J. Am. Chem. Soc. 2009, 131, 11387−11391. (38) Zhang, X. A.; Lovejoy, K. S.; Jasanoff, A.; Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10780−10785. (39) (a) Chaberek, S.; Courtney, R. C.; Martell, A. E. J. Am. Chem. Soc. 1952, 74, 5057−5060. (b) Anderegg, G. Pure Appl. Chem. 1982, 54, 2693−2758. (40) Schröder, L.; Lowery, T. J.; Hilty, C.; Wemmer, D. E.; Pines, A. Science 2006, 314, 446−449. (41) Kunth, M.; Döpfert, J.; Witte, C.; Rossella, F.; Schröder, L. Angew. Chem., Int. Ed. 2012, 51, 8217−8220. (42) Meldrum, T.; Bajaj, V.; Wemmer, D.; Pines, A. J. Magn. Reson. 2011, 213, 14−21. (43) Wolber, J.; Cherubini, A.; Leach, M. O.; Bifone, A. Magn. Reson. Med. 2000, 43, 491−496. (44) Sloniec, J.; Schnurr, M.; Witte, C.; Resch-Genger, U.; Schröder, L.; Hennig, A. Chem.Eur. J. 2013, 19, 3110−3118. (45) Walker, T. G.; Happer, W. Rev. Mod. Phys. 1997, 69, 629−641. (46) Desvaux, H.; Gautier, T.; Le Goff, G.; Pétro, M.; Berthault, P. Eur. Phys. J. D 2000, 12, 289−296. (47) Kilian, W.; Mitschang, L.; Freund, C.; Schlundt, A. Proceedings of the Joint Annual Meeting ISMRM-ESMRMB,Stockholm, Sweden, 1−7 May 2010; International Society for Magnetic Resonance in Medicine: B e r k e le y , C A , 20 1 0; h t tp : / / c d s. i s m r m . o r g / p r o t e c t e d / 10MProceedings/files/1_program2.htm.
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Smart Detection of Toxic Metal Ions, Pb2+ and Cd2+, Using a NMR-Based Sensor
129
Xe
Nawal Tassali,† Naoko Kotera,‡ Céline Boutin,† Estelle Léonce,† Yves Boulard,†,∥ Bernard Rousseau,*,‡ Emmanuelle Dubost,‡ Frédéric Taran,‡ Thierry Brotin,§ Jean-Pierre Dutasta,§ and Patrick Berthault*,† †
CEA Saclay, IRAMIS, NIMBE, UMR CEA/CNRS 3299, Laboratoire Structure et Dynamique par Résonance Magnétique, 91191 Gif sur Yvette, France ‡ CEA Saclay, iBiTec-S, SCBM, Building 547, PC No. 108, 91191 Gif sur Yvette, France § Laboratoire de Chimie, CNRS, Ecole Normale Supérieure de Lyon, 46 Allée d’Italie, 69364 Lyon Cedex 07, France ABSTRACT: An approach for sensitive magnetic resonance detection of metal cations is proposed. Combining the use of hyperpolarized 129Xe NMR and of a cage-molecule functionalized by a ligand able to chelate different cations, we show that simultaneous detection of lead, zinc, and cadmium ions at nanomolar concentration is possible in short time, thanks to fast MRI sequences based on the HyperCEST scheme.
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in its local environment induce significant modification of NMR parameters. This property has been used in various situations: to differentiate the outer and inner compartments of biological cells using dissolved hyperpolarized xenon in 129Xe NMR spectroscopy22 and to propose a molecular MRI approach based on xenon encapsulated in host systems functionalized to reach defined biological targets.23 In this approach, cryptophanes are ideal candidates to accommodate xenon, as (i) they have a large affinity for the noble gas, (ii) their aromatic rings give a large shielding effect to the caged xenon signal, and (iii) a permanent in−out exchange enables the constant refreshment of the cage into hyperpolarization.24,25 DNA fragments,26 enzymes,27−29 and cell surface receptors30,31 have been targeted using this approach. Here, we show that the grafting of a ligand able to chelate various metal cations, such as NTA (nitrilotriacetic acid), on a cryptophane core can lead to fast multiplexed detection of nanomolar concentrations of metal ions through dedicated 129Xe NMR sequences. The reversible chelation of the metal by the ligand leads to a chemical shift variation of caged xenon that not only witnesses the presence of the metal, but also identifies it. Using a hydrophilic cryptophane bearing a NTA group (compound 1M, Chart 1) and xenon polarized at a level higher than 10% we recently detected32 in a few seconds 100 nM Zn2+, a threshold 2 orders of magnitude lower than that detected with recent gadolinium contrast agents.33−38 This sensor was also selective to Zn2+ with respect to Ca2+ and Mg2+ ions, addition of which gave rise to no change in the hyperpolarized 129Xe NMR spectrum.
he chronic and severe exposure to heavy metal ions, such as Pb2+, Cd2+, and Hg2+, can exert strong impact on human health and is linked to major human diseases, such as cancer and cardiovascular disorders.1,2 For example, human exposure to lead is estimated to account for 143 000 deaths every year.3 Similarly, cadmium poses severe harm to human health, as it accumulates mainly in the kidneys with a half-life in humans of 10−35 years. As a result, there is a great demand for highly sensitive analytical methods aiming at the selective detection of these metal ions. Techniques such as fluorescence spectroscopy, atomic absorption spectrophotometry, and electrochemical analysis are daily used to detect metal ions present in biological or environmental samples.4−10 Among these methods, fluorescence spectroscopy is particularly useful, because of its high sensitivity, simple operation, and its ability to provide real-time detection.11−18 However, fluorescencebased methodologies are limited by low tissue penetration and therefore are not suitable for in vivo imaging in deep tissues. Recent advances in the field of molecular magnetic resonance imaging (MRI) have led to the development of new strategies based on the design and synthesis of responsive contrast agents.19,20 Designing contrast agents that possess high sensitivity and chemical selectivity is a formidable challenge. The purpose of this Article is to show that NMR of hyperpolarized xenon allows for a sensitive and multiplexed detection of these metal ions. For the first time Pb2+ is detected at the nanomolar range by NMR. In addition, our method allows selective detection of Cd2+ in the presence of Zn2+, a difficult challenge because of the similarity of these ions.21
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RESULTS AND DISCUSSION Among the hyperpolarized noble gases used for sensitive MRI (3He, 129Xe, 83Kr), xenon is of special interest. The large polarizability of its electron cloud is such that even tiny changes © 2014 American Chemical Society
Received: November 12, 2013 Accepted: January 16, 2014 Published: January 16, 2014 1783
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Chart 1. Structure of the 129Xe NMR-Based Sensor of Metal Ions: (+)-MM-Cryptophane-(L)-NTA, Compound 1M
the ion concentration. The required concentrations of 1M are anyway well below the water solubility threshold. The sensitivity of the method is so high that the 129Xe NMR spectrum even shows spurious peaks at ∼68.2 and 69.7 ppm, which maybe correspond to trace amounts of other cations. Other cations were tested. Noting that two metals from the d-block of the periodic table (Cd2+ and Zn2+) gave a response on the hyperpolarized 129Xe spectrum, we undertook the study of the interaction of 1M with Hg2+. Though, addition of Hg2+ did not give rise to a new xenon signal. Only one paramagnetic cation, Co2+, was tried. Addition of Co2+ to a solution of 1M did not lead to additional peaks on the 129Xe NMR spectrum. However, the presence of cobalt ions could be detected using the other diastereoisomer ((−)-PP-cryptophane-(L)-NTA, that is, compound 1P) through a high-field shift of the caged xenon signal by −2.4 ppm whatever the cobalt concentration (Figure 2). The angular dependence of the paramagnetic shift and the
Here, we propose a new concept enabling detection of several cations present in a solution using this unique sensor. Figure 1 displays the 129Xe NMR spectra obtained with
Figure 2. One-scan 129Xe NMR spectrum performed with the sensor 1P at a concentration of 300 μM in the presence of 150 μM Co2+ ions. It was recorded at 11.7 T and 298 K with a Bruker 129Xe/1H 5 mm probe head.
lack of knowledge of the 3D structure of the complex impede any interpretation of this effect. Be that as it may, the metal sensor 1P is able to probe the presence of low amounts of Co2+ ions. This study was limited to some diamagnetic dications (or paramagnetic ones with low induced relaxation). Various experimental conditions such as the nature of the buffer, the pH and the temperature were tested. An important observation was made: whereas the chemical shifts of xenon free in solution and of xenon caged in 1M may vary significantly, the frequency difference between the signal of xenon in the sensor free of cation (Xe@1M) and that of xenon in the cage chelating a given cation (Xe@1M-Cation2+) remains almost constant. This gives a way to detect the cation whatever the medium or the sensor environment. To detect trace amounts of cobalt, cadmium, lead, or zinc cations, sensitivity is a major concern and the direct detection method can be supplanted by the HyperCEST method, provided that the xenon magnetization level is stable.40 Using the xenon exchange in and out of the cryptophane cavity, the presence of a biosensor is translated into a depletion of the bulk xenon signal after saturation at the caged xenon frequency. Simultaneous detection of these ions at nanomolar concentration is straightforward on a z-spectrum. And when localization of the ions is desired, the HyperCEST scheme can be added to various imaging sequences, in particular those dedicated to short T*2 samples. We decided to test this approach. It has recently been shown that the HyperCEST scheme can easily be combined with Echo
Figure 1. High-field region of 129Xe NMR spectra obtained at 298 K in one scan with the noble gas encapsulated in the sensor 1M alone at 970 μM in D2O (a), and after addition of 5% Zn2+ (48.5 μM) b), 5% Cd2+ (c) and 5% Pb2+ (d). The asterisk denotes a signal due to xenon encapsulation in the residual impurity (cryptophane-222-hexacarboxylate).
successive addition of 5% Zn2+, 5% Cd2+, and 5% Pb2+ to a solution of 1M in HEPES buffer. Each of these cations gives rise to a specific downfield chemical shift splitting for encapsulated xenon: 1.5 ppm for Zn2+, 0.3 ppm for Cd2+, and 4.5 ppm for Pb2+. To our knowledge, this is the first simultaneous detection of these metal cations at such low quantities by NMR. Dissolved xenon (its solubility is near 4.3 mM per atm in water at room temperature) is usually in large excess with respect to the cryptophane concentration, and the xenon binding constant on the order of 6000 M−1 (estimated from a competition experiment with cryptophane-222-hexacarboxylate) is not expected to be modified by the chelation of the metal cation. Therefore, all the cryptophane cages are filled with a noble gas atom and the areas of the 129Xe signals at high field represent the respective concentrations of 1M, 1M-Cd2+, 1M-Zn2+, and 1M-Pb2+ from high field to low field. Obviously the affinity constant of 1M for different cations can vary (more than 2 orders of magnitude are reported in the literature for the NTA headgroup39), and the responses have to be tabulated according to it. Also, to keep the quantitative character of the signals the cage concentration must be in excess with respect to 1784
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Experimental Section). Here, for the sake of clarity, the zspectra obtained using only two slices are represented. Such a protocol enables us to assess the efficacy of the saturation and its spectral selectivity. From Figure 3c, it is clear that the presence of lead ions can easily be established from the z-spectra, and that the relative signal intensities are respected. Also it is noteworthy that increasing the temperature would have largely improved our results by speeding up the in−out xenon exchange. Compound 1M constitutes a powerful 129Xe NMR-based sensor that enables sensitive and simultaneous detection of lead, zinc, and cadmium cations (the latter ones when the magnetic field value and homogeneity are sufficient). To get a more precise idea of the sensitivity threshold reachable through this method (although it largely depends on the xenon polarization level and delivery method), dilution experiments were performed. Figure 4 displays the evolution of the HyperCEST response as a function of the ion concentration, from 3 μM to 9 nM in
Planar Imaging (EPI) where a large part of or the whole k space can be spanned in a single acquisition.41 We therefore chose this scheme. In the present case, it was mandatory to implement an adequate, band-selective, saturation so as to irradiate one caged xenon frequency (e.g., xenon in the cage chelating Pb2+ ions) without affecting the other one (e.g., xenon in the “free” cage). For this purpose, a saturation step using multiple pulse inversion elements42 was developed. Figure 3a displays the
Figure 3. Fast MRI detection of lead cations. (a) Example of HyperCEST-EPI axial images recorded at 298 K with a sample containing 3 μM Pb2+ and a 3-fold excess of 1M. Left image: Saturation for 4.5 s with a B1 value of 9 μT at the Xe@1M-Pb2+ frequency (at an offset Δ from the signal of free xenon). Middle image: Off-resonance saturation (at an offset −Δ). Right image: Subtraction “on−off”. On the left image, the dashed circles represent the contour of the area integrated for the z-spectra (black = signal; red = noise) displayed in c. (b) For each point of these z-spectra, 5 axial slices of 1 mm separated by 1 mm were acquired (the numbers give the chronological order, slice 0 being an off-resonance HyperCEST experiment). (c) z-spectra recorded by integration of the images at various offsets (these data were obtained with 10.7 μM Pb2+ and 30 μM 1M). Only slices 2 and 4 are represented, corresponding to 9 and 18 s of saturation, respectively.
Figure 4. Hyper-CEST contrast as a function of the concentration in Pb2+ ions (after integration of the areas drawn in Figure 2a). The same saturation scheme and strength as in Figure 3 were used, for 4.5 s. The data points were fitted by an exponential function. See Experimental Section.
aqueous solution. The result obtained at this last concentration shows that we are able to detect 1.6 × 1012 Pb2+ ions in a few seconds (the slice volume is 34 μL). Whereas the data show a linear behavior of the HyperCEST effect for low Pb2+ concentrations, the point at 3 μM strongly deviates from it. As dissected in the Experimental Section, this arises from too low a concentration ratio of hyperpolarized xenon to cage. Anyway, this experiment highlights the strength of the use of hyperpolarized 129Xe MRI for the detection of trace quantities of metal cations. Indeed, nanomolar concentrations of metal cation can be revealed using only one bolus of xenon. The feasibility of such an approach for cell samples or living tissues can then obviously be questioned and not only for the specific absorption rate. To evaluate the difficulty of the experiment in the worth case in term of magnetic field inhomogeneity and presence of paramagnetism we did an experiment in whole, nonoxygenated blood, where the xenon relaxation time is short (because of deoxyhemoglobin, the xenon relaxation time falls to ∼5 s) and the susceptibility effects degrade the field homogeneity. On the one-scan 129Xe NMR spectrum of the sample loaded with Pb2+ ions (Figure 5a), from low field to high field, five signals were observed, corresponding to xenon in the red blood cells (1) and in the bulk (2) near 200 ppm,43,22 both Xe@1M-Pb2+ (3) and Xe@1M (4) near 70 ppm, and gaseous xenon (5). We checked that the presence of
axial images of an 8 mm NMR tube containing a solution of Pb2+ ions at 3 μM with an excess of 1M, recorded at 298K in 4.8 s with a HyperCEST-EPI sequence. The first image was obtained with saturation centered on the Xe@1M-Pb2+ signal, the second one with saturation off-resonance (at an offset symmetric with respect to the signal of dissolved xenon). The difference between the two images enables detection of the Pb2+ ions. Note the slight spatial shift (one pixel) that leads to bright and dark areas in the periphery of the solution. An integration region with a size smaller than the sample diameter (black circle) enabled us to get rid of these artifacts. To evaluate the HyperCEST effect as a function of the saturation time, for each saturation frequency several HyperCEST-EPI images were acquired (with a single xenon batch) exciting different slices of the sample (Figure 3b). As the saturation is not spatially selective, the resulting images corresponded to different saturation periods. Figure 3c displays the z spectra extracted from these images (details in the 1785
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EXPERIMENTAL SECTION
Production of Polarized Xenon. 83%-Enriched 129Xe from Euriso-Top was polarized by spin-exchange optical pumping of rubidium45 using a recently installed home-built setup based on laser diodes. The photons exiting from a duoFAP system (2 × 30W) and circular polarizer from Coherent illuminated a Pyrex cell placed at the center of a 100G magnetic field. The bandwidth of the laser diodes being about 2 nm, pressure-broadening was necessary. Therefore, the pressure in the cell rose 3 atm (measured at room temperature) with a mixture of 2% Xe−10% N2−88% He. The pumping cell was heated for 2 min to 410 K via a flow of hot N2 in an external envelope, in a fashion similar to what was developed for our previous experimental setup.46 Then xenon was condensed in a coldfinger inside a 3 kG solenoid immersed in liquid nitrogen, and thus separated from helium and nitrogen. The average polarization value with this experimental setup was 15%, measured for the gaseous phase in the NMR spectrometer. Sample Preparation. Prior to xenon introduction, the solutions in the NMR tubes were degassed via helium bubbling followed by evacuation. Human blood was obtained from EFS according to the established contract and with the required authorization. NMR and MRI Experiments. Hyperpolarized xenon was introduced on top of the solution in the upper part of the screwed NMR tube via a vacuum line in the fringe field of the NMR magnet. Then vigorous shaking of the tube followed by a 10 s delay ensured complete dissolution and equilibration of both gaseous and dissolved phases. Unless otherwise indicated, the 129Xe NMR spectra and images were recorded at 11.7 T and 298K with a dual 129Xe/1H 5 mm probehead and TopSpin 3.0 for the spectra and a micro5 Bruker probehead (dual 129Xe/1H 8 mm insert) and ParaVision 5.1 for the images. Each NMR tube received ∼1 atm of hyperpolarized xenon on top of the solution. The axial images were obtained via an EPI sequence with double sampling. The field-of-view was 19.2 mm, the raw matrices were constituted by 64 × 38 points (Fourier acceleration factor in the phase dimension: 1.68). For the experiment of Figure 3c, five successive slices were acquired: the first one was an EPI sequence, the other ones were HyperCEST-EPI sequences with a saturation of 4.5 s with a peak amplitude of 9 μT via a train of 80 D-SNOB pulses of 56 ms each, followed by a spoil gradient. As the saturation is not space-selective, it can be considered that the saturation period was 4.5 s for the first image, 9 s for the second image, etc. The slices were not acquired contiguously; the numbers indicated in Figure 3b give the chronological order. For the dilution experiments, a first 1-mm slice EPI image served as reference for the following 8-mm slice HyperCESTEPI image. There was no time delay between the EPI and HyperCEST-EPI experiments. Each data point was obtained from the ratio of the integral of the region encircled in black to the integral encircled in red, normalized with the ratio of the same regions recorded in the prior EPI experiment. Description of the Protocol for the HyperCEST Data Presented in Figure 4. To take into account the fluctuant hyperpolarization level, 10 s after having shaken the NMR tube to introduce new polarized xenon into the solution, we chained an axial EPI experiment (slice 1 mm) with an axial HyperCEST-EPI experiment (slice 8 mm shifted by 1 mm from the previous image, saturation time 4.5 s). This procedure
Figure 5. One-scan 129Xe NMR spectra performed at 11.7 T and 298 K on samples of whole human blood loaded with (a) the sensor 1M and Pb2+ ions and (b) the sensor 1M without any addition of metal ion. The numbers indicate the xenon location (see main text).
the two signals (3 and 4) was not due to two different environments for the cage, such as in the inner compartment of red blood cells and in the plasma, for instance, a situation already encountered in other cases.30,44 In an experiment performed in the absence of Pb2+ ions, only one signal appeared in this spectral region (Figure 5). Figure 5 shows that the lead cations can straightforwardly be detected.
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CONCLUSION The usual approach for multiplexed detection would require the synthesis of several cryptophane sensors, each of them able to bind a specific target. This would require a tremendous amount of time synthesizing these elaborate molecules. Here we have shown another promising approach based on the synthesis of a cation-responsive cryptophane sensor optimized for the simultaneous detection of several targets. The wide frequency range of the xenon response enables us to propose a new concept with a host system bearing a ligand that provides a unique and well-separated spectral signature of each ion. This approach is flexible, since several metal chelating groups, among which EDTA (ethylenediaminetetraacetic acid), DTPA (diethylene triamine pentaacetic acid), NTA, and cyclen, are available and can be grafted on a cryptophane core. It is applicable to the study of cell samples and is also expected to be amenable to ex vivo or in vivo experiments. Conversely to methods using fluorescent sensor arrays, the 129Xe NMR-based approach does not require a statistical analysis, the ions being straightforwardly recognized on the spectrum. Development of new sensors based on the same principle of xenon host bearing a generic ligand but with a shorter tether, which hopefully will enhance the xenon chemical shift effect, will be the subject of future work in our laboratories. 1786
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Analytical Chemistry was repeated ten times. Between each pair of images, the offset for the saturation pulse was changed from on-resonance to offresonance and vice versa. Each HyperCEST image was normalized with respect to the precedent EPI image (same integration regions as in Figure 2). Then we computed the pairwise difference between two successive normalized HyperCEST images (S = SON − SOFF). The standard deviation between the five values gave the error bar. Figure 4 reveals that the point at 3 μM cryptophane deviates from a linear slope joining the other points. Given that the characteristic time for the encapsulation of xenon in a cryptophane 2.2.2 cavity is close to 10 ms, if one also considers a saturation efficiency factor of 0.3 (ref 47) it means that during the saturation period (4.5 s) one cryptophane molecule would need 4500/(0.3 × 10) = 1500 hyperpolarized xenon atoms in its vicinity. When the cage concentration reaches 3 μM, it can be observed that a xenon concentration close to 4.3 mM (1 atm) can be insufficient. The HyperCEST experiment amounts in increasing the apparent relaxation of all xenon atoms entering in the cryptophane cavity (by rf saturation). Neglecting the natural xenon relaxation (in the absence of rf saturation on a xenon resonance) the signals arising from HyperCEST experiments on- and off-resonance are therefore
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SOFF proportional to [Xe]t f (tsat)
where [Xe]t is the total xenon concentration and f(tsat) a function of the saturation time. As mentioned by Kilian and co-workers,47 the factor k contains contributions from the isotopic enrichment, from the efficacy of the saturation and from the cage occupation factor koc, given by 1/k OC = [C]t /[XeC] = 1 + 1/(K[Xe]) = 1 + 1/(K[Xe]t − [XeC])
where K is the binding constant and [XeC] the concentration of cages filled with a xenon atom K = [XeC]/([Xe][C])
From this equation, it can be seen that when the total xenon concentration is not far higher than the concentration of caged xenon (most of the xenon is in the cryptophane cavities), koc seriously decreases. The data points of Figure 4 can be fitted by an exponential.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Present Address ∥
ACKNOWLEDGMENTS
The authors thank Dr. X. Millot (CEA/SST) for his help and expertise. Support from the French Ministry of Research (project ANR-12-BSV5-0003) and from the Fondation pour la Recherche Médicale (project DCM20111223065) is acknowledged.
SON proportional to [Xe]t f (tsat)exp(−ktsat)
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Yves Boulard: CEA Saclay, iBiTec-S, SB2SM.
Author Contributions
N.T. and N.K. contributed equally. Notes
The authors declare no competing financial interest. 1787
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