Ion-Channel-Mimetic Sensing of Hydrophilic Anions Based on

Chem. , 1999, 71 (6), pp 1183–1187 .... Shinpei KADO , Ayumi FURUI , Yu AKIYAMA , Yoshio NAKAHARA , Keiichi KIMURA .... Detection of Nitrate and Sul...
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Anal. Chem. 1999, 71, 1183-1187

Ion-Channel-Mimetic Sensing of Hydrophilic Anions Based on Monolayers of a Hydrogen Bond-Forming Receptor Kang Ping Xiao, Philippe Bu 1 hlmann, and Yoshio Umezawa*

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan

Ion-channel-mimetic sensing based on monolayers of a hydrogen bond-forming bis(thiourea) receptor was used to detect inorganic anions. Receptor monolayers were formed at the air-water interface and subsequently contacted with a highly oriented pyrolytic graphite electrode. Horizontal touch cyclic voltammetry was performed with subphase solutions containing various electroinactive analyte anions and [Fe(CN)6]4- as electroactive marker. Binding of analyte anions to the receptor monolayer was found to inhibit [Fe(CN)6]4- oxidation. The influences of the analyte anions on the cyclic voltammograms were largest for HPO42- and decreased in the order of HPO42> F- ≈ SO42- > CH3COO- > Cl-, whereas ion-selective electrodes (ISEs) containing the same receptor respond with a selectivity order of Cl- > SO42- > CH3COO- > H2PO4-/HPO42-. Because the bis(thiourea) receptor does not bind to all potentially hydrogen-bonding sites of most of these anions, it is apparent that several of the larger anions, and in particular phosphate and sulfate, are still substantially hydrated while being bound to the interfacial receptor layer. This distinct feature of interfacial molecular recognition seems to explain why selectivities of these ionchannel-mimetic sensors differ so strongly from the selectivities for the complete anion transfer from aqueous to organic phases, as represented by the ISE selectivity. The results in the present work suggest that ion-channelmimetic sensors are particularly promising for the analysis of very hydrophilic, relatively large analytes, for which hosts that encapsulate the analyte and do not allow the analyte in this complex to be hydrated are difficult to synthesize. Hydrogen bonding plays a very important role in molecular recognition of many biological and artificial systems. For example, X-ray crystal structures show that anion binding in various biological systems results from multiple hydrogen bond formation and a size-selective fit of the anion to its host. Also, numerous artificial systems in which hydrogen bonding allows molecular recognition in bulk solutions were studied. However, only at the beginning of this decade was complementary hydrogen bonding at phase boundaries shown to be effective for interfacial analyte recognition.1,2 Surface pressure-molecular area (π-A) isotherms,3 (1) Paleos, C. M.; Tsiourvas, D. Adv. Mater. 1997, 9, 695-710. (2) Kurihara, K. Colloids Surf. 1997, 123-124, 425-432. 10.1021/ac9809635 CCC: $18.00 Published on Web 02/05/1999

© 1999 American Chemical Society

FT-IR3-5 and UV-visible spectroscopy,6,7 quartz crystal microbalances (QCM),8 XPS elemental analysis,3,9 and atomic force microscopy3 have since then been used to confirm selective hydrogen bond formation at interfaces. Kunitake and co-workers found that hydrogen bonds at lipidwater interfaces are stronger than in bulk water.3 For example, 5′-ATP and 5′-AMP bind to guanidinium-functionalized monolayers 106-107 times more strongly3 than guanidinium to phosphate in aqueous solution (1.3 M-1 for H2PO4- at pH 4; 5.1 M-2 for HPO42at pH 7).10 A quantum-chemical model11 and a classical electrostatic model12 based on the Poisson-Boltzmann equation and the Debye-Hu¨ckel approximation suggest that the high stability of the interfacial complexes results from the low dielectric constant of the monolayer phase, which strongly affects the charge distribution in the receptor groups and their complexes. The results from the calculations agree well with the experimental findings and not only explain that guanidinium-phosphate binding becomes stronger when the receptor binding sites are located very close to the hydrophobic phase but also suggest that the enhancement of intermolecular and ionic binding at the air-water interface is a universal phenomenon. Despite the advances in the understanding of interfacial hydrogen bond formation, so far only few approaches to chemical sensing based on analyte recognition by this type of interaction have been reported. The principle of ion-channel-mimetic sensors, which was reported first in 1987 by Umezawa and co-workers,13 is to mimic biomembranes containing ion-channel proteins. Selective binding of ligands to such proteins opens their ordinarily closed ion channels and allows a large amount of ions or molecules to pass across such membranes. Similarly, analyte binding to (3) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371-378. (4) Shimomura, M.; Nakamura, F.; Ijiro, K.; Taketsuna, H.; Nakamura, H.; Hasebe, K. J. Am. Chem. Soc. 1997, 119, 2341-2342. (5) Weck, M.; Fink, R.; Ringsdorf, H. Langmuir 1997, 13, 3515-3522. (6) Ahuja, R.; Caruso, P.; Mo ¨bius, D.; Paulus, W.; Ringsdorf, H.; Wildburg, G. Angew. Chem., Int. Ed. Engl. 1993, 32, 1033-1036. (7) Berti, D.; Baglioni, P.; Bonaccio, S.; Barsacchi-Bo, G.; Luisi, P. L. J. Phys. Chem. B 1998, 102, 303-308. (8) Ebara, Y.; Itakura, K.; Okahata, Y. Langmuir 1996, 12, 5165-5170. (9) Koyano, H.; Bissel, P.; Yoshihara, K.; Ariga, K.; Kunitake, T. Langmuir 1997, 13, 5426-5432. (10) Springs, B.; Haake, P. Bioorg. Chem. 1977, 6, 181-190. (11) Sakurai, M.; Tamagawa, H.; Inoue, Y.; Ariga, K.; Kunitake, T. J. Phys. Chem. B 1997, 101, 4810-4816. (12) Tamagawa, H.; Sakurai, M.; Inoue, Y.; Ariga, K.; Kunitake, T. J. Phys. Chem. B 1997, 101, 4817-4825. (13) Sugawara, M.; Kojima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1987, 59, 2842-2846.

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Figure 2. Structure formula of H2PO4- complex of anion receptor 1.

Figure 1. Schematic illustration of the marker oxidation at the surface of an oriented monolayer of receptor 1: (a) oxidation of [Fe(CN)6]4- at the electrode surface coated with a monolayer of receptor 1 in the absence of analyte anions; (b) oxidation of [Fe(CN)6]4- is hampered by binding of phosphate ion to the monolayer.

receptor mono- or multilayers on the electrode of an ion-channelmimetic sensor controls access of electroactive species, which are often referred to as markers (shown schematically in Figure 1 for monolayers of the receptor and marker used in this study). Sensing of this type inherently allows signal amplification because a single analyte ion can control the flow of many marker ions or molecules.13,14 For example, in the case of ion-channel-mimetic sensing of nucleotides, neutral receptors that bind guanosine or adenosine nucleotides by multiple hydrogen bonds were used to form monolayers at the air-water interface, and permeabilities of the monolayers were measured by cyclic voltammetry with [Fe(CN)6]4- as marker. Decreases in the rate of [Fe(CN)6]4oxidation were observed in the presence of nucleotides, which can be explained by repulsion between the analyte complexes on the electrode surface and the negatively charged marker.15 In this paper, we report on the use of a hydrogen bond-forming bis(thiourea) compound (1, see Figure 2) to form receptor monolayers on highly oriented pyrolytic graphite (HOPG) electrodes and on their use for channel-mimetic sensing of inorganic anions. This ionophore was designed and synthesized in the course of our previous work with anion sensors.16 1H NMR spectra showed that H2PO4- is bound to these receptors strongly (e.g., (14) Bu ¨ hlmann, P.; Aoki, H.; Xiao, K. P.; Amemiya, S.; Tohda, K.; Umezawa, Y. Electroanalysis, in press. (15) Tohda, K.; Amemiya, S.; Ohki, T.; Nagahora, S.; Tanaka, S.; Bu ¨ hlmann, P.; Umezawa, Y. Isr. J. Chem. 1997, 37, 267-275.

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receptor 1: K11 ) 55 000 M-1 for 1:1 binding in DMSO-d6) and preferentially (H2PO4- > CH3COO- > Cl-) by formation of hydrogen bonds to the thiourea groups of these receptors. So far, no other published neutral receptor forms stronger 1:1 complexes with H2PO4- than these xanthene derivatives. We report here on the selectivities of channel-mimetic sensing based on monolayers of this receptor and compare them to selectivities of ion-selective electrodes (ISEs) based on membranes that contain the same receptor.17 While the selectivities of ion channel sensors result from analyte binding at the electrode surface, the selectivities of ISEs can be explained by equilibration of the sample and the electrode membrane phase18,19 and therefore represent the selectivities of complete anion transfer across the interface of the aqueous sample and a water-immiscible, organic phase. To explain the differences in the selectivities of the two types of sensors, their response mechanisms are compared. EXPERIMENTAL SECTION Reagents. Sodium salts of anions were of the highest grade commercially available and used without further purification. Na4[Fe(CN)6]‚10H2O was purchased from Wako Pure Chemicals (Osaka, Japan). The synthesis of receptor 1 (see Figure 2) was reported previously.16 Deionized and charcoal-treated water (18.2 MΩ cm specific resistance) was prepared with a Milli-Q type I reagent grade water system (Millipore, Bedford, MA). The receptor solutions used for the formation of monolayers were prepared with HPLC-grade chloroform after passing it through basic alumina for purification. Monolayer Formation and Surface Pressure-Molecular Area (π-A) Isotherms. A Langmuir film balance (model HBM, Kyowa Kaimenkagaku, Tokyo, Japan) equipped with a glass Wilhelmy plate and a Teflon-coated trough (14 cm × 70 cm) were used for the formation of monolayers and the measurement of π-A isotherms. The temperature of the aqueous subphase was kept at 20.0 ( 0.1 °C for all experiments. Monolayers were obtained by spreading 150 µL of a 1.0 mM CHCl3 solution of the receptor on the subphase. The monolayers were allowed to stand for 10 min to ensure complete evaporation of the chloroform. The barrier shift velocity used to compress the monolayers to a preset surface pressure was 25.0 cm2/min. Horizontal Touch Cyclic Voltammetry. The electrochemical properties of oriented monolayers at packing densities determined (16) Bu ¨ hlmann, P.; Nishizawa, S.; Xiao, K. P.; Umezawa, Y. Tetrahedron 1997, 53, 1647-1654. (17) Xiao, K. P.; Bu ¨ hlmann, P.; Nishizawa, S.; Amemiya, S.; Umezawa, Y. Anal. Chem. 1997, 69, 1038-1044. (18) Bakker, E.; Bu ¨ hlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132. (19) Tohda, K.; Umezawa, Y.; Yoshiyagawa, S.; Hashimoto, S.; Kawasaki, M. Anal. Chem. 1995, 67, 570-577.

Figure 3. Surface pressure-molecular area (π-A) isotherm of a monolayer of receptor 1 on water as subphase. Measured at 20 °C.

by an externally applied surface pressure can be determined with horizontal touch cyclic voltammetry.20 An HOPG electrode was chosen as the working electrode because of its smooth and easily renewable surface.21 The backside of an HOPG block (area, 12 mm × 12 mm; initial thickness, 2 mm; Union Carbide, Cleveland, OH) was placed in contact with a silver wire using silver paste (Nilaco, Tokyo, Japan). A fresh HOPG surface layer on the topside of the electrode was obtained before every experiment by peeling off a thin layer with adhesive tape. An Ag/AgCl single-junction electrode (Denki Kagaku Keiki, Tokyo, Japan) and a spiral platinum wire were used as reference and auxiliary electrodes, respectively. A computer-controlled electroanalysis system (BASCV 50W, Bioanalytical Systems, West Lafayette, IN) was used to measure all cyclic voltammograms (CVs). The solutions were degassed at reduced pressure prior to use. Before CV measurements, each monolayer was kept for 30 min at a surface pressure of 5 mN/m. The HOPG electrode was lowered with a constant displacement speed of 0.1 mm/min by a motor-driven lifter until the electrode touched the monolayer. CVs were obtained with a sweep rate of 100 mV/s, starting from an initial potential of -100 mV versus Ag/AgCl, scanning to a potential of +700 mV and returning to -100 mV. All reported anion and concentrations indicate total concentrations of the respective Na+ salts as they were used to prepare the subphases; no corrections for protonation equilibria were performed. The pH values of the subphases were 4.5, 9.3, and 8.4 for H2PO4-, HPO42-, and CH3COO- solutions, respectively, and were larger than 6.0 for Cl-, F-, and SO42- solutions. With the exception of the 10-3 M F-, SO42-, CH3COO-, and Cl- solutions, for which CVs were measured with two different monolayers, CVs were measured for each analyte and each analyte concentration with three different monolayers. RESULTS AND DISCUSSION Monolayers of Receptor 1 at the Air-Water Interface. Figure 3 shows a surface pressure-molecular area (π-A) (20) Odashima, K.; Kotato, M.; Sugawara, M.; Umezawa, Y. Anal. Chem. 1993, 65, 927-936. (21) Chang, H.; Bard, A. J. Langmuir 1991, 7, 1143-1153.

Figure 4. Horizontal touch cyclic voltammograms of 1.00 mM [Fe(CN)6]4- in 0.1 M solutions of various analyte anion for an HOPG electrode coated (A) without and (B-G) with a monolayer of receptor 1. Analyte anion: (B) Cl-; (C) SO42-; (D) CH3COO-; (E) F-; (F) HPO42-.

isotherm of a monolayer of receptor 1 on electrolyte-free water. Extrapolation to zero surface pressure gives a molecular area of 0.65 nm2, which agrees well with the molecular area of 0.67 nm2 that is estimated from a Corey-Pauling-Koltun (CPK) spacefilling atomic model by assuming that receptor 1 “stands” at the air-water interface with the xanthene spacer directed toward the air and the thiourea groups toward the aqueous subphase. The collapse pressure of the monolayer is between 12 and 15 mN/m. Kept at a surface pressure of 5 mN/m, the monolayer area decreases only by 8% within a period of 30 min. This shows that the monolayer is stable and suitable for cyclic voltammetry. Anion Responses of Electrodes Coated with a Monolayer of Receptor 1. To determine the influence of anion binding to receptor monolayers, changes in oxidation currents and potentials in CVs measured with [Fe(CN)6]4- as an anionic marker were determined after the monolayers formed at the air-water phase from the air side made contact with an HOPG electrode (horizontal touch in-trough cyclic voltammetry; for details, see Experimental Section). Figure 4 shows CVs for electrodes coated (A) without and (B-G) with monolayers of receptor 1. The subphase solutions contained 1.00 × 10-3 M Na4[Fe(CN)6] and sodium salts of various analyte anions (1.00 × 10-1 M). As compared to CVs for bare electrodes, the CVs for electrodes covered with a receptor monolayer show smaller oxidation currents and positive shifts in oxidation peak potentials. Subsequently, the effect of the concentration of various analyte anions on CVs was determined. To minimize the iR drop in the aqueous subphase without having to use a background electrolyte, the concentration ratio of the Na+ salt of the analyte anions and the electroactive marker [Fe(CN)6]4- was kept at 100 for all analyte anion concentrations. The peak potentials, Ep, shifted considerably toward positive potentials as the analyte anion concentrations increased (Figure 5). The peak shift was largest for HPO42- and for solutions of different anions decreased in the order of HPO42> F- ≈ SO42- > CH3COO- > Cl-. To investigate the effect of the pH on CVs, solutions containing NaH2PO4 were used as subphases. However, because the pH Analytical Chemistry, Vol. 71, No. 6, March 15, 1999

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Figure 5. Dependence of peak potentials in cyclic voltammograms for 1.00 mM [Fe(CN)6]4- samples as obtained with an HOPG electrode coated with a monolayer of receptor 1 and sample solutions containing different anions at varying concentrations.

values of the solutions containing 0.10 M NaH2PO4 was 4.6, and because at this pH ∼30% of the [Fe(CN)6]4- anions are protonated (pKa 4.2),22 the CVs measured at this pH cannot be directly compared to the CVs obtained with the other subphase solutions, which all had pH values of 6.0 or higher. Therefore, CVs were also measured for Cl- solutions of pH 4.6 (pH adjusted with HCl). The peak shift for the NaH2PO4 solutions was 364 ( 13 mV, which is slightly larger than the peak shift of 320 ( 7 mV for the NaCl solutions of the same pH, and shows that not only HPO42- but also H2PO4- are selectively bound to the receptor monolayer. At the analyte concentration of 1.00 × 10-3 M, the peak potentials of CVs measured with bare and with monolayer-covered electrodes do not differ significantly except for the case of the most strongly bound HPO42-. For the latter, a difference of 29.0 ( 4.7 mV between the peak potentials of the bare and monolayercovered electrode was observed, which can be explained by the fact that HPO42- is the only anion that is bound to the monolayer at this low analyte concentration. This shows that the monolayer alone does not inhibit the electron transfer significantly. Furthermore, these CVs do not show any evidence for marker binding to the monolayer. Upon binding of the analyte anions to the monolayer of the electrically neutral receptor, the monolayer becomes negatively charged. The electrostatic repulsion between the monolayer and the anionic marker [Fe(CN)6]4- lowers the local concentration of the latter at the monolayer surface and slows down the electron transfer between [Fe(CN)6]4- and the HOPG electrode (shown schematically in Figure 1), thereby explaining the observed changes in the CVs. To understand the anion selectivity of the electrode modified with receptor 1, it is useful to consider results from studies on the interaction of inorganic salts and surfactants from colloid and interfacial chemistry.23-32 Measuring the effect of the interactions between various anions and charged monolayers without specific (22) Jordan, J.; Ewing, G. J. Inorg. Chem. 1962, 1, 587-591. (23) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888, 24, 247-260. (24) Hsiao, L.; Dunning, H. N.; Lorenz, P. B. J. Phys. Chem. 1956, 60, 657-660.

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functional binding sites, such as alkyltrimethylammonium or dialkyldimethylammonium monolayers, usually always gives the same sequence of interaction strengths.25,26,28,30,32 For example, the strength of interaction between anions and monolayers at the airwater interface decreased in the case of docosyltrimethylammonium monolayers in the order of SCN- > I- > NO3- > Br- > Cl- > F-,26 as determined by surface potential-area measurements, and in the case of dioctadecyldimethylammonium monolayers in the order of I- > Br- > Cl- > F-, as observed by Brewster angle microscopy.30 Also in the case of the free energies of anion adsorption to hexadecyltrimethylammonium monolayers at the nitrobenzene-water interface, a series of Br- > Cl- > Fis observed.28 A similar sequence of anion affinities was also found with measurements of electrophoretic mobilities of liposomes in aqueous anion solutions (ClO4- > I- > SCN- > Br- > NO3- > Cl- ≈ SO42-).32 While minor differences between these rather varied interfacial systems can be observed, the selectivities are always very similar. The selectivity sequence always reflects the free energy of hydration of the analyte ions, and is called the Hofmeister series, honoring its discoverer, who first observed it when studying egg white protein precipitation induced by various salts.23 However, the response order obtained in the present work differs significantly from the Hofmeister series. This clearly shows that the analyte anions bind specifically to the monolayer of receptor 1. Responses are largest for HPO42- as analyte, for which the receptor was originally designed. Sensing Modes of Ion-Selective Electrodes and ChannelMimetic Sensors. We showed previously that ISEs with solvent polymeric membranes containing receptor 1 are chloride selective with little interference from phosphate.17 Even though in dimethyl sulfoxide receptor 1 binds phosphate preferentially, the selectivity of ISEs based on this receptor falls in the order of Cl- > SO42- > CH3COO- > H2PO4-/HPO42-. It is well-known that the selectivities of such solvent polymeric membrane ISEs can be explained on the basis of phase boundary equilibria.18,19 Therefore, the selectivity of the ISE based on 1 can be explained with the free energies of phase transfer of these anions from the aqueous sample into the organic ISE membrane phase. As a consequence of its lower hydration energy, transfer of chloride from the sample into the membrane phase is energetically more favorable than the phase transfer of phosphate. The selectivity of phosphate binding by receptor 1 in the membrane phase is apparently not large enough to counterbalance the large difference in the phasetransfer energies of the two ions. The large difference between the selectivities of ISEs and the selectivities of the monolayer-modified electrodes described in the present work suggests that the HPO42- ions bound to the monolayer of receptor 1 are still substantially hydrated. Binding of phosphate to receptor 1 by four hydrogen bonds16 leaves sufficient interaction sites of phosphate free to interact with water (25) Goddard, E. D.; Kao, O.; Kung, H. C. J. Colloid Interface Sci. 1967, 24, 297-309. (26) Schick, M. J. J. Phys. Chem. 1964, 68, 3585-3592. (27) Schick, M. J. J. Colloid Sci. 1962, 17, 801-813. (28) Kakiuchi, T.; Kobayashi, M.; Senda, M. Bull. Chem. Soc. Jpn. 1988, 61, 1545-1550. (29) Schott, H. J. J. Colloid Interface Sci. 1973, 43, 150-155. (30) Ahuja, R. C.; Caruso, P.-L.; Mo¨bius, D. Thin Solid Films 1994, 242, 195200. (31) Deguchi, K.; Meguro, K. J. Colloid Interface Sci. 1975, 50, 223-227. (32) Tatulian, S. A. Biochim. Biophys. Acta 1983, 736, 189-195.

molecules. Even formation of 1:2 complexes of phosphate and two molecules of receptor 1 would still allow direct interaction of the bound phosphate ions with water. Only full encapsulation of the analyte by the receptor would eliminate direct interactions between the analyte and water.

analyte binding at the interface of electrodes chemically modified with monolayer receptors, which facilitates the design and synthesis of receptors for this type of sensors. It appears therefore that channel-mimetic sensors are particularly suited for the analysis of very hydrophilic, relatively large analytes.

CONCLUSIONS The differences between the selectivities of ISEs and ionchannel-mimetic sensors based on receptor 1 suggest that the design of receptors for the latter type of sensors can take advantage of the particular recognition at the electrode interface. Sufficiently selective receptors for two phase-transfer systems such as ion-selective electrodes may in many cases have to completely enclose highly hydrophilic ions. While such analyte-enclosing receptors can be expected to provide a high selectivity, the design and synthesis of such ionophores for large analyte ions is obviously very laborious and not easy. On the other hand, complete analyte encapsulation seems not required for selective

ACKNOWLEDGMENT This work was supported by the Ministry of Education, Science and Culture, Japan. A research fellowship from the Japan Society for the Promotion of Science (JSPS) to K.P.X. is gratefully acknowledged. Grant-in-Aid for Scientific Research for the Priority Areas of “Electrochemistry of Ordered Interfaces” (10131216) partially supported the present work.

Received for review August 26, 1998. Accepted December 19, 1998. AC9809635

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