Magnesium-Selective Ion-Channel Mimetic Sensor with a Traditional

Anal. Chem. 2010, 82, 436–440

Magnesium-Selective Ion-Channel Mimetic Sensor with a Traditional Calcium Ionophore Jingwei Zhu,† Yu Qin,*,‡ and Yunhong Zhang† Institute for Chemical Physics, Beijing Institute of Technology, Beijing, China, 100081, and Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China, 210093 A novel magnesium-selective ion-channel mimetic sensor based on a well-known calcium ionophore, ETH 5234, was demonstrated. Due to different stoichiometry of the ionophore with Mg2+ and Ca2+, the sensor exhibits high selectivity to Mg2+ by controlling ionophore concentration on the electrode surface, which allows the measurement of Mg2+ in a 1 mM Ca2+ background solution. Calcium and magnesium ions are important blood electrolytes and the most abundant divalent cations in cells that play important roles in the structure and function of the human body.1,2 Electrochemical detection of Ca2+ and Mg2+ with ion-selective electrodes (ISEs) has advantages of simple instrumentation, fast response, and easy miniaturization.3 Highly selective ISEs are constructed based on ionophores that are lipophilic complexing ligands capable of reversibly binding ions from sample solution. The selectivity of such a sensor is related to the equilibrium constants of exchange reactions for target and interfering ions between the organic membrane and sample aqueous phases.4 In that way, it strongly depends on the binding stoichiometry with the ionophores for different ions. Therefore, the design and synthesis of highly selective ionophores for Ca2+ and Mg2+ are of great importance for direct measurements in complicated samples.5 Research shows that compounds with diamide subunits and ether oxygens have preference for Ca2+ over monovalent cations.6,7 One example from such a diamide is calcium ionophore ETH 129 which has the most favorable selectivities toward Ca2+.7-9 The structure of a Ca2+ salt of this ionophore shows a stable 3:1 complex in which nine oxygen atoms from the diamides form a cavity with the ideal size for Ca2+. However, it was also found that ETH * Corresponding author. E-mail: [email protected]. Tel: +86(25) 83592562. Fax: +86(25) 83592562. † Beijing Institute of Technology. ‡ Nanjing University. (1) Nielsen, F. H.; Lukaski, H. C. Magnesium Res. 2006, 19, 180–189. (2) Trump, B. F.; Berezesky, I. K. FASEB J. 1995, 9, 219–228. (3) Bakker, E.; Pretsch, E. Angew. Chem., Int. Ed. 2007, 46, 5660–5668. (4) Bakker, E.; Buhlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083–3132. (5) Suzuki, K.; Watanabe, K.; Matsumoto, Y.; Kobayashi, M.; Sato, S.; Siswanta, D.; Hisamoto, H. Anal. Chem. 1995, 67, 324–334. (6) Lee, M. H.; Yoo, C. L.; Lee, J. S.; Cho, I.-S.; Kim, B. H.; Cha, G. S.; Nam, H. Anal. Chem. 2002, 74, 2603–2607. (7) Pretsch, E.; Ammann, D.; Osswald, H. F.; Guggi, M.; Simon, W. Helv. Chim. Acta 1980, 63, 191–196. (8) Oh, B. K.; Kim, C. Y.; Lee, H. J.; Rho, K. L.; Cha, G. S.; Nam, H. Anal. Chem. 1996, 68, 503–508. (9) Buhlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593–1687.

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129 could hold Mg2+ stably with a fixed 2:1 ratio.7,10 Furthermore, the dependence of the potentiometric selectivity on the concentration of anionic sites results confirm that the same stoichiometry occurs in ISE membranes.11 At the same time, the successful design of Mg2+-selective ionophores remains a challenge, and the major problem lies in the preference of Ca2+ in complex samples for many applications.12,13 A lot of research has been done in terms of suppressing Ca2+ during Mg2+ measurements. Presently, the best suppression of Ca2+ is reported with double-armed diazacrown ether ionophores.5 Systematic studies have shown that the malonamide type side chains substituted with adamant groups are most favorable to Mg2+ and the logarithmic selectivity coefficient of Mg2+ to Ca2+ was -2.4. Unfortunately, the interferences from alkali metal ions increase as well, especially for K+. Efforts have also been made by creating Mg2+ binding sites in a cross-linked polymer matrix with imprinting techniques.14,15 However, the selectivity improvement was limited, and the rigid polymer was undesirable for sensor fabrication. The concept of ion-channel mimetic sensor (ICS) was introduced in 1987,16 and its mechanism is based on the chargedanalyte-gated permeability changes when redox markers cross self-assembled monolayers (SAMs). The binding of target ions to the receptors modified electrode surface can facilitate opposite charged marker access to the surface, as a result of electrostatic interaction, and subsequently assist electron transfer between the electrode and the markers.17 An ion-channel mimetic sensor with a thioctic acid monolyer was used to measure different metal ions, especially trivalent ion La3+, at pH of 7.4.18 However, the discrimination between divalent ions, i.e. Ca2+ and Mg2+, was unsatisfactory. In this work, we prepared the ion-channel mimetic sensors with immobilized ionophores and reported for (10) Schefer, U.; Ammann, D.; Pretsch, E.; Oesch, U.; Simon, W. Anal. Chem. 1986, 58, 2282–2285. (11) Zhang, W.; Jenny, L.; Spichiger, U. E. Anal. Sci. 2000, 16, 11–18. (12) Gupta, V. K.; Chandra, S.; Mangla, R. Sens. Actuators, B 2002, 86, 235– 241. (13) Bakker, E.; Willer, M.; Lerchi, M.; Seiler, K.; Pretsch, E. Anal. Chem. 1994, 66, 516–521. (14) Rosatzin, T.; Andersson, L. I.; Simon, W.; Mosbach, K. J. J. Chem. Soc., Perkin Trans. 2 1991, 1261–1265. (15) Peper, S.; Qin, Y.; Bakker, E. In U.S. Pat. Appl. Publ.; US, 2003; Vol. US 20030213691. (16) Sugawara, M.; Kojima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1987, 59, 2842–2846. (17) Umezewa, Y.; Aoki, H. Anal. Chem. 2004, 76, 320 A326 A. (18) Zugle, R.; Kambo-Dorsa, J.; Gadzekpo, V. P. Y. Talanta 2003, 61, 837– 848. 10.1021/ac901570u  2010 American Chemical Society Published on Web 12/03/2009

the first time that by controlling the concentration and distribution of ionophores in the SAM modified gold electrode surface, a completely different selectivity pattern of carrier-based ionchannel mimetic sensors can be obtained. EXPERIMENTAL SECTION Reagents. ETH5234, sodium tetrakis[3,4-bis(trifluoromethyl)phenyl]-borate (NaTFPB), bis(2-ethylhexyl) sebacate (DOS), high molecular weight polyvinyl chloride (PVC), and tetrahydrofuran (THF) were purchased in Selectophore or puriss quality from Fluka (Switzerland). 11-Mercapto-1-undecanol (97%) was purchased from Aldrich. Dimethyl sulfoxide (DMSO) was obtained from Sangon (Shanghai, China). All reagents were of the highest grade commercially available and were used without further purification. Aqueous solutions were freshly prepared from Nanopure-purified (18.2MΩcm) deionized water and were purged with nitrogen for at least 15 min prior to use. Buffer solution was prepared from 0.5 M Tris and adjusted to pH 7.4 with HNO3. Potentiometric Measurements. The complex formation constants of ETH 5234 with Ca2+ and Mg2+ were determined by the sandwich membrane method.19 Ion-selective electrode membranes were cast by dissolving ETH 5234 (20 mmol/kg, if used) and NaTFPB (5 mmol/kg), together with PVC and the plasticizer DOS to give a total cocktail mass of 140 mg, in 1.5 mL of THF and pouring it into a glass ring (2.2 cm i.d.) affixed with rubber bands onto a microscope glass slide. The solvent THF was allowed to evaporate overnight. The parent membranes were then removed from the glass and conditioned overnight in a 1 mM CaCl2 or MgCl2 solution to ensure complete conditioning of the ionophore-free membrane segments. Preparation of Ion-Channel Mimetic Sensors. Gold electrodes (2 mm in diameter) were polished with 0.3 µm alumina suspensions on a flet pad for at least 10 min, then rinsed several times with water, and finally sonicated in deionized water and ethanol for 10 min, successively. The polished electrodes were further immersed in a 0.5 M KOH aqueous solution and electrochemically cleaned by scanning the potential between -0.4 and -1.2 V using CHI660C electrochemical workstation in cyclic voltammetry mode. It was scanned repeatedly until the obtained voltammograms no longer changed. The electrodes were then soaked in a 5 mM 11-mercapto-1-undecanol (MU) solution in DMSO for different amounts of time (2-20 h) at room temperature. After washing with DMSO and ethanol, the electrodes were finally soaked in a 1 mM ETH 5234 solution in ethanol. All of the electrodes were stored in deionized water in a refrigerator until use. Electrochemical Measurement. A CHI660C Electrochemical Workstation (ChenHua instruments, Inc./ Shanghai, P.R. China) was used for all electrochemical measurements. A conventional three-electrode setup was employed, with the chemical modified gold electrode as the working electrode, platinum-wire auxiliary, and Ag/AgCl electrode as the reference. All potentials were measured versus the Ag/AgCl electrode. Cyclic voltammetry (scan rate of 100 mV s-1, range from -1200 to -400 mV) and square wave voltammetry (step potential of 5 mV, square wave frequency of 20 Hz, and square wave amplitude of 50 mV) were (19) Qin, Y.; Mi, Y.; Bakker, E. Anal. Chim. Acta 2000, 421, 207–220.

used for analysis. The solutions were prepared with Tris-HNO3 buffer (pH ) 7.4) containing 1 mM K3Fe(CN)6 worked as redox marker and 0.1 M KNO3 as supporting electrolyte. All solutions were deoxygenated by purging nitrogen for at least 15 min prior to the measurements. Electrochemical impedance spectroscopy (EIS) was performed in a 0.01 M Tris-HNO3 buffer solution (pH 7.4) containing equal concentrations of oxidized and reduced forms of the Fe(CN)64-/ Fe(CN)63- couple, with 0.1 M KNO3 as supporting electrolyte. A 5 mV amplitude sine wave was applied to the electrode at the formal potential of the redox couple, with a frequency range from 0.01 Hz to 100 kHz. RESULTS AND DISCUSSION Oxapentanediamide type of compounds, ETH 129 and more lipophilic ETH 5234 (Figure 1A), are excellent calcium ionophores.7,9,20,21 The structures of Ca2+ and Mg2+ salts of ETH 129 show 1:3 and 1:2 complexes, respectively.7,10 Further measurements of selectivity dependence on the concentration of cation exchangers indirectly confirm that the same stoichiometry of cation-ionophore complexes is retained in ISE membranes. With the sandwich membrane method,22 we determined the logarithm of complex formation constants of ETH 5234 with Ca2+ and Mg2+ in PVC-DOS membranes to be 22.49 ± 0.04 and 16.87 ± 0.02, respectively. As expected, the binding between Mg2+ and ionophore is relatively weaker than that with Ca2+. However, it is still a significantly stronger interaction than those other ion-ionophore complexes such as valinomycin and K+ (logβK+L ) 10.10).19 When the binding capability of oxapentanediamide derivatives with Mg2+ was noticed, efforts had been made to design sensors for Mg2+ by suppressing Ca2+ recognition of the ionophores. Rosatzin et al.14 reported the preparation of Ca2+ and Mg2+ imprinted polymers from N,N′dimethyl-N,N′-bis(4-vinylphenyl)3-oxapentanediamide. The resulting polymers were analyzed for their ability to extract calcium or magnesium ions from methanolic water. Unfortunately, such Mg2+-imprinted polymer still preferred Ca2+ more than Mg2+. Similarly, we had prepared imprinting polymers with unsaturated oxapentanediamide derivative AU-1 and applied them to fabricate ISEs.15 The Mg2+ imprinted polymer exhibited improved selectivity to Mg2+; however, due to the low binding capacity of the polymer, the dynamic range was narrow. In addition, the cross-linked polymer powders were undesirable for sensor fabrication. In this work, the ion-channel mimetic sensors were fabricated by immobilization of ionophore into self-assembled thiol monolayer on gold electrode. Figure 1B is the scheme for the preparation of functional ICS. The embedment method involved two steps. First, the electrodes were modified by MU to form an ordered monolayer on Au surface. Then, the ionophores possessing lipophilic side chains were immobilized via hydrophobic interactions to obtain a stable bilayer.23 The electrodes were washed carefully after each incubation step. A similar procedure to obtain stable bilayers by embedment of molecules with a long (20) (21) (22) (23)

Ceresa, A.; Pretsch, E.; Bakker, E. Anal. Chem. 2000, 72, 2050–2054. Gemene, K. L.; Bakker, E. Anal. Chim. Acta 2009, 648, 240–245. Mi, Y.; Bakker, E. Anal. Chem. 1999, 71, 5279–5287. Aoki, H.; Umezawa, Y.; Vertova, A.; Rondinini, S. Anal. Sci. 2006, 22, 1581– 1584.

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Figure 1. (A) Chemical structure of ETH 5234. (B) Ion-channel mimetic sensors prepared by embedment of ionophore in SAM. The response mechanism was suggested as an electrostatic ion-ion interaction induced signal (I), rather than coextraction/coadsorption of target ions with the anionic redox marker (II).

Figure 2. (A) Square wave voltammograms (SWVs) of a bare gold electrode (a), MU-Au electrode (b), and ETH 5234/MU-Au electrode (c) in 1 mM [Fe(CN)6]3- solutions with 0.1 M KNO3 as supporting electrolyte. Scan rate 100 mV · s-1, step potential 5 mV, square wave frequency 20 Hz, square wave amplitude 50 mV. (B) Complex impedance plots for a bare gold electrode (a), MU-Au electrode (b), and ETH 5234/MU-Au electrode (c) in 0.01 M Tris-HNO3 buffer solution (pH 7.4) containing 5 mM Fe(CN)64- + 5 mM Fe(CN)63- with 0.1 M KNO3 as supporting electrolyte; 0.01 Hz -100 kHz frequency range with a 5 mV rms signal.

side chain had also been reported.24,25 Such embedment modification can provide good flexibility of the prepared bilayer. The square wave voltammetry (SWV) has been proved to have better signal-to-noise ratio and resolution than cyclic voltammetry (CV) and was, therefore, selected for the following experiments. Figure 2A shows the square wave voltammograms on a bare gold electrode (a), MU (b), and MU-ETH5234 modified electrode (c) with [Fe(CN)6]3- as redox marker. The results demonstrated that the redox reaction of the marker on the electrode surface was almost completely suppressed due to the steric hindrance of the thiol modified layer that prevented the marker approaching the electrode surface. (24) Radecki, J.; Szymanska, I.; Bulgariu, L.; Pietraszkiewicz, M. Electrochim. Acta 2006, 51, 2289–2297. (25) Krajewska, A.; Smet, M.; Dehaen, W.; Radecka, H. Supramol. Chem. 2009, 21, 520–531.

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Electrochemical impedance spectroscopy (EIS) is an effective method to probe the interface properties of surface-modified electrodes. The impedance spectrum includes a semicircle part at high frequencies, corresponding to the electron transfer limited process and a linear part at lower frequencies, resulting from the diffusion limiting step of the electrochemical process. The semicircle diameter equals the electron transfer resistance, which indicates the blocking behavior of the electrode surface for the redox couple and, therefore, can be used as a signal for characterizing the modification for each step. Figure 2B shows the results of impedance spectra on a bare gold electrode (a), the MU modified gold electrode before embedding ETH 5234 (b), and after embedding ETH 5234 (c). For the bare electrode, due to a large heterogeneous electron transfer rate constant of the Fe(CN)63-/4- couple, only a unit-sloped Warburg line was obtained (Figure 2B-a). It indicated that the electrode reaction is mainly controlled by the diffusion process. After the gold electrode was immersed in 0.5 mM MU-DMSO solution, a semicircle appeared at high frequency and the diameters elevated with increasing adsorption time. It is clear that the electron transfer resistance increased with longer adsorption time, suggesting that an insulate layer of alkylthiol was formed on the electrode surface. The initial and rapid adsorption of thiols leads to an imperfect monolayer. A slower assembly process with longer adsorption time, expulsion of solvent, and removal of contaminants from the monolayer is essential to reduce the defects and acquire a denser packed monolayer. In this work, the density and orientation of the alkylthiol monolayer are crucial for the insertion of ionophores in the next step. For the electrodes assembled in MU solution for less than 8 h, further incubation in the ionophore solution has small effect on the EIS spectrum after washing the electrodes. At the same time, such electrodes did not show any response to the target ions. In the proposed sensor, the ionophore with a long alkyl chain was anchored on a two-dimensional SAM layer through hydrophobic interaction, low density of MU on electrode surface would result in too weak of an interaction to

Figure 3. (A) SWVs of ETH 5234/MU-Au electrodes (incubation time: 10 h in MU and 12 h for ionophore). Mg2+ (main) and Ca2+ (inset) were measured respectively in 1 mM [Fe(CN)6]3- buffered solutions (pH 7.4) with 0.1 M KNO3 as supporting electrolyte containing different concentrations (in M) of cations (a) 0; (b) 10-6; (c) 10-5; (d) 10-4; (e) 5 × 10-4; (f) 10-3; (g) 5 × 10-3; (h) 10-2; (i) 5 × 10-2; (j) 0.1. (B) Calibration curves of Mg2+-selective ICS measured in Ca2+ (9) and Mg2+ ([) solutions. The peak currents obtained from SWVs.

immobilize the ionophores, while a much denser SAM layer may also hinder the embedment of ETH 5234. With appropriate assembly time of the alkylthiol monolayer (9 to 14 h) in DMSO solution, we were able to observe the decrease of the semicircle diameter on the EIS spectrum after the ETH 5234 was immobilized (shown in Figure 2 B-c). The smaller charge transfer resistance after ionophore immobilization might be caused by the following reasons: first, positive charges accumulate on the surface because of the weak interaction between ETH 5234 and K+ (0.1 M as supporting electrolyte), or the hydrophobic interaction between ionophores and MU may change the surface group orientation and make the layer more penetrable for redox markers. After the MU modified electrode was immersed in ETH 5234 solution for 12 h, the ion-channel mimetic sensor exhibited excellent response to magnesium ions. As shown in Figure 3A main, the peak currents in the obtained square wave voltammograms increased when Mg2+ concentration changed from 10-5 to 0.1 M. With the complexation of the magnesium ions with ETH 5234, positive charge is accumulating on the surface, so that the anionic marker can approach the electrode surface more easily and the redox reaction is, therefore, facilitated to generate signals. In Figure 3A, the cathodic peak potentials for the reduction of [Fe(CN)6]3- shifted positively with the increase of Mg2+ concentration. Similar results had been reported,18 and it is assumed to be caused by the change of the electrochemical kinetics of the anionic redox marker when the potential drop across the surface layer is changed as the metal ions bind to the receptors.18,26 Our results confirm that introducing lipophilic perchlorate anion into the solution did not change the sensor response, which suggested that the contribution from the coextraction of magnesium ions with anionic redox marker is negligible (Figure 1B-(II)). The response mechanism is mainly based on the electrostatic attraction of [Fe(CN)6]3- ions to the electrode surface and generate current (Figure 1B-(I)). However, there was nearly no response to Ca2+ up to 0.01 M concentration for the same electrode (Figure 3A inset). These results indicated that ETH 5234 immobilized in MU monolayer complexes of Mg2+ stably, which changes the total charge of the layer and its permeability for marker ions. On the contrary, it is much more (26) Cheng, Q.; BrajtelcToth, A. Anal. Chem. 1995, 67, 2767–2775.

Figure 4. Calibration curve (main) and SWVs response (inset) of Mg2+-selective ETH 5234/MU-Au electrode to Mg2+ in the background of 1 mM Ca2+. Mg2+ concentrations (in M): (a) 0; (b) 10-5; (c) 10-4; (d) 5 × 10-4; (e) 10-3; (f) 5 × 10-3; (g) 10-2; (h)0.05; (i) 0.1.

difficult to form a 3:1 Ca2+-ionophore complex when the ETH 5234 was immobilized in the two-dimensional alkylthiol layer, because of either the insufficient ionophore concentration or the steric inhibition on the electrode surface. Figure 3B showed the dependence of peak currents with different Mg2+ and Ca2+ concentrations. The slope of a Mg2+ response curve is 8.8 µA/ decade with a lower detection limit at 10-4.3 M. By increasing Mg2+ concentrations, the surface becomes more positively charged and increased currents can be observed. In the present system, it is possible that metal ions bind reversibly to the ionophores on the sensor surface so that the saturation issue could be partially circumvented. Our results suggested that the ionophore immobilized sensor were not saturated even in 0.1 M MgCl2 solutions. Furthermore, Figure 3 clearly showed that the ion-channel mimetic sensor with immobilized ETH 5234 could selectively recognize Mg2+ over Ca2+. At the same time, other alkali metal ions such as Na+ and K+ are significantly suppressed due to the instinct selectivity of ETH 5234 (data not shown). We further determined the selectivity factor of Mg2+ over Ca2+ in 1 mM CaCl2 background solutions. The method is similar to the fixed interference method used in obtaining selectivity coefficients for ion-selective electrodes, which defines the selectivity as the logarithm of detection limit of the main ion in the solution containing interfering ions. Figure 4 shows the calibration curve (main) of Mg2+ in the presence of 1 mM Ca2+ and the corresponding SWVs (inset). The selectivity factor of Mg2+ over Ca2+ with proposed ion-channel mimetic sensors was -2. As far as we know, it is the first report that a traditional calcium ionophore can be used to measure Mg2+ with good selectivity. The ionophores in traditional ion-selective membranes are mobile, and they could automatically bind with calcium ions since the 3:1 Ca2+-ETH 5234 complex is highly stable. In our ion-channel mimetic sensor design, the ionophores are immobilized onto the electrode via hydrophobic interaction with modified SAMs and, therefore, are prone to form the Mg2+ complex with lower stoichiometric ratio rather than a 3:1 complex with Ca2+. The prepared sensors could be used repeatedly and measured over hundreds of voltammograms. They were stored in 10 mM Tris-HNO3 buffer solution at 4 °C when not in use. Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

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alkali metal ions (Na+ and K+) is consistent with traditional potentiometric sensors prepared with ETH 5234. We assume that larger amount of ionophores would further block the surface from the marker and a higher concentration of Ca2+ or Mg2+ is necessary to generate signals. The results suggested that higher ionophore density would allow the favorable complexation of ETH 5234 with Ca2+ at relatively high concentration.

Figure 5. Calibration curves of Ca2+-selective ICS (incubation time: 10 h in MU and 36 h for ionophore) measured in Mg2+ (9) and Ca2+ (0) solutions. The peak currents were obtained from SWVs. Inset: SWVs of Ca2+-selective ICS in 1 mM [Fe(CN)6]3- buffered solutions (pH 7.4) with 0.1 M KNO3 as supporting electrolyte containing different concentrations of Ca2+. Step potential 5 mV, square wave frequency 20 Hz, square wave amplitude 50 mV.

We also observed that the electrodes with higher ionophore density (incubation time longer than 36 h in ETH 5234 solution) only had a small Mg2+ response at relatively high concentration (10-2 M), as shown in Figure 5, and such an electrode responded to Ca2+ more readily and selectively (Figure 5 main and inset). The slope of Ca2+ response curve is 14.9 µA/decade with a lower detection limit at 10-3 M. The Ca2+ selectivity over Mg2+ and

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CONCLUSIONS In summary, by dispersing ETH 5234 in a self-assembled alkylthiol layer via hydrophobic interaction, we developed the first magnesium ion selective ion-channel mimetic sensor with a traditional calcium ionophore. With longer immobilizing time in ionophoric solutions, the MU-based ion-channel mimetic sensor can regain Ca2+ selectivity. Such a new sensing strategy based on complex stoichiometry and immobilization conditions can be applied to other ionophore-based sensors and further extend their sensing capability. ACKNOWLEDGMENT The authors thank the National Natural Science Foundation of China (20890021) and 973:2007CB936404 for financial support of this research.

Received for review July 15, 2009. Accepted November 15, 2009. AC901570U