Acetylcholine Detection at Micromolar Concentrations with the Use of

Dougherty, D. A.; Stauffer, D. A. Science (Washington, DC, U.S.) 1990, 250, ...... Yang Liu , Magi Mettry , Adam D. Gill , Lizeth Perez , Wenwan Zhong...
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Acetylcholine Detection at Micromolar Concentrations with the Use of an Artificial Receptor-Based Fluorescence Switch Nina Korbakov,† Peter Timmerman,*,‡,§ Nina Lidich,† Benayahu Urbach,| Amir Sa’ar,| and Shlomo Yitzchaik*,† The Chemistry Institute and the Farkas Center for Light Induced Processes and Racah Institute of Physics, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel, Pepscan Therapeutics BV, P.O. Box 2098, 8203 AB Lelystad, The Netherlands, and Van’t Hoff Institute for Molecular Sciences, Faculty of Science, UniVersity of Amsterdam, Amsterdam, The Netherlands ReceiVed September 29, 2007. In Final Form: December 11, 2007 An inclusion complex between water-soluble p-sulfocalix[n]arene (Cn, n ) 4, 6, 8) and the chromophore trans4-[4-(dimethylamino)styryl]-1-methylpyridinium-p-toluenesulfonate (D) formed the basis for a highly sensitive sensor for the selective detection of neurotransmitter acetylcholine (ACh). Formation of the [Cn‚D] complex (Ka ) ∼105 M-1) was accompanied by a drastic increase (up to 20-60-fold) in the chromophore relative quantum yield and by a large hypsochromic shift of the emission band maximum. The observed optical effects are fully reversible: ACh displaces the chromophore molecules from the calixarene cavity as shown by the reappearance of the free chromophore emission band. Formation and dissociation of the complex were studied by fluorescence, 1H NMR, and UV-vis absorption spectroscopies. The [Cn‚D] complex is capable of sensing ACh selectively in solution at sub-micromolar concentrations. Immobilization of monocarboxyl p-sulfocalix[4]arene (C4m) on an oxide-containing silicon surface is in keeping with its properties, such as chromophore binding and the ability of the immobilized inclusion complex to detect ACh. The unique [Cn‚D] complex optical switching paves the way for application in ACh imaging and optoelectronic sensing.

Introduction Acetylcholine (ACh) plays a modulatory role in the central nervous system and is involved in the translation of chemicals into electrical signals (membrane action potential). After exocytosis and binding to ACh receptors, ACh is rapidly degraded by the enzyme acetylcholinesterase (AChE) from the synaptic cleft. ACh hydrolysis to choline and acetic acid prevents overstimulation of the postsynaptic membrane, while AChE inhibitors such as nerve gases (and organophosphorus compounds in general) delay ACh degradation. The accumulation of ACh in synaptic clefts causes severe health problems, and so the reliable sensing of ACh and AChE inhibitors is of obvious importance. AChE-containing biosensors, possessing a high sensitivity, are commonly based on measuring the activity or the inhibition of the enzyme by electrochemical and spectroscopic methods. Electrochemical methods using AChE-modified electrodes represent a detection limit of ACh of nanomolar or micromolar concentrations, using a floating gate ion-sensitive field-effect transistor (FG ISFET)1 or ISFET,2,3 respectively. The ACh detection limit of micromolar concentrations was observed in a case of modified electrodes,4,5 when deposition of nanoparticles * Corresponding authors. E-mail: [email protected]; sy@ cc.huji.ac.il. † The Chemistry Institute and the Farkas Center for Light Induced Processes, The Hebrew University of Jerusalem. ‡ Pepscan Therapeutics BV. § University of Amsterdam. | Racah Institute of Physics, The Hebrew University of Jerusalem. (1) Goykhman, I.; Korbakov, N.; Bartic, C.; Borghes, S.; Spira, M. E.; Shappir, J.; Yitzchaik, S. Sens. Actuators, B, in press. (2) Hai, A.; Ben-Haim, D.; Korbakov, N.; Cohen, A.; Shappir, J.; Oren, R.; Spira, M. E.; Yitzchaik, S. Biosens. Bioelectron. 2006, 22, 605-612. (3) Kharitonov, A. B.; Zayats, M.; Lichtenstein, A.; Katz, E.; Willner, I. Sens. Actuators, B 2000, 70, 222-231. (4) Shibli, S. M. A.; Beenakumari, K. S.; Surma, N. D. Biosens. Bioelectron. 2006, 22, 633-638. (5) Snejdarkova, M.; Svobodova, L.; Evtugyn, G.; Budnikov, H.; Karyakin, A.; Nikoelis, D. P.; Hianik, T. Anal. Chim. Acta 2004, 514, 79-88.

creates a favorable surface for the enzyme attachment that increases the sensor sensitivity.6,7 Polymeric membranes with immobilized AChE demonstrate a good selectivity and sensitivity to pH change at micromolar concentration ranges of ACh or acetylthiocholine (ATCh).8-10 Immobilization of AChE on a multiwalled carbon nanotube resulted in the creation of a sensitive amperometric system for rapid detection of ATCh.11 The electrochemical sensor containing a modified glassy carbon electrode and microdialysis sampling probe showed a detection limit of nanomolar concentration that is comparable to the ACh detection limit achieved by high-performance liquid chromatography (HPLC) -electrochemical methods.12,13 However, the complexities of the enzyme-based sensors such as preservation of the enzyme, high cost, and short lifetime confine their application. In this respect, mimicking the binding process of neurotransmitters in artificial receptor systems has attracted scientific interest for more than 20 years. Synthetic receptors for ACh provide some of the bestdocumented examples in the literature.14-17 Previous work (6) Shulga, O.; Kirchhoff, J. R. Electrochem. Commun. 2007, 9, 935-940. (7) Istamboulie, G.; Andreescu, S.; Marty, J.-L.; Noguer, T. Biosens. Bioelectron. 2007, 23, 506-512. (8) Peng, L. B.; Heng, L. Y.; Hasbullah, S. A.; Ahmad, M. J. Anal. Chem. 2007, 62, 884-888. (9) Reher, S.; Lepka, Y.; Schwedt, G. Microchim. Acta 2002, 140, 15-20. (10) Ozturk, G.; Alp, S.; Timur, S. J. Mol. Catal. B: Enzym. 2007, 47, 111116. (11) Du, D.; Huang, X.; Cai, J.; Zhang, A.; Ding, J.; Chen, S. Anal. Bioanal. Chem. 2007, 387, 1059-1065. (12) Yamamoto, K.; Sato, K.; Chikuma, T.; Kato, T. Anal. Chim. Acta 2004, 521, 209-213. (13) Guerrieri, A.; De Benedetto, G. E.; Palmisano, F.; Zambonin, P. G. Analyst 1995, 120, 2731-2736. (14) Dhaenens, M.; Lacombe, L.; Lehn, J.-M.; Vigneron, J.-P. J. Chem. Soc., Chem. Commun. 1984, 1097-1098. (15) Schneider, H.-J.; Guttes, D.; Schneider, U. Angew. Chem., Int. Ed. Engl. 1986, 25, 647-649. (16) Schneider, H.-J.; Schneider, U. J. Org. Chem. 1987, 52, 1613-1615. (17) Dougherty, D. A.; Stauffer, D. A. Science (Washington, DC, U.S.) 1990, 250, 1558-1563.

10.1021/la703010z CCC: $40.75 © 2008 American Chemical Society Published on Web 02/12/2008

Acetylcholine Detection at Micromolar Concentration

established that water-soluble p-sulfocalix[n]arenes display high affinities for ACh and are able to bind other quaternary ammonium-based cations.18-23 Ligand binding is based on multiple π-interactions and electrostatic and hydrophobic interactions between the cationic quaternary ammonium terminus and the electron-rich p-sulfocalix[n]arene cavity.24,25 The ability of p-sulfocalix[n]arenes to form inclusion complexes with N-alkylpyridinium dyes provides an opportunity to follow ACh interactions using emission or optical absorption spectroscopy.19,21,23,26-31 Most of the optical sensors reported to date are able to detect ACh only at relatively high (i.e., millimolar) concentrations. This is mainly due to the limited spectroscopic changes observed upon the formation of the receptor-chromophore complexes (approximately 2-fold intensity change).21-23,32 Hemicyanine chromophores have both electron-donating and -accepting properties.33-36 Upon excitation, the chromophore undergoes internal rotation, leading to a twisted intramolecular charge-transfer state that is nonfluorescent.33,34 The observed increase in the fluorescence intensity of 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide upon inclusion in β-cyclodextrin (β-CD) having a pendant pyrenyl group was explained by stabilization of the chromophore inside the β-CD cavity.37,38 The association of a number of hemicyanine dyes with watersoluble p-sulfocalix[n]arenes was accurately studied by UVvis spectroscopy and 1H NMR measurements for the analytical determination of the cationic-surfactant concentration in water by Shinkai and co-workers.35 To develop a highly sensitive system for ACh detection, we studied the interactions of trans-4-[4-(dimethylamino)styryl]1-methylpyridinium p-toluenesulfonate (D) with water-soluble p-sulfocalix[n]arenes (Cn) in neutral and acidic media. Upon formation of the [Cn‚D] complex, an intense fluorescence band appeared that was hypsochromically shifted relative to the free chromophore emission band. The presence of ACh in a solution of the complex completely destroyed the new emission band of the caged chromophore. These significant alterations in spectroscopic properties allowed for the detection of the complex (18) Lehn, J.-M.; Meri, R.; Vigneron, J.-P.; Cesario, M.; Guilhem, J.; Pascard, C.; Asfari, Z.; Vicens, J. Supramol. Chem. 1995, 5, 97-103. (19) Shinkai, S.; Koh, K. N.; Araki, K.; Ikeda, A.; Otsuka, H. J. Am. Chem. Soc. 1996, 118, 755-758. (20) Atwood, J. L.; Barbour, L. J.; Junk, P. C.; Orr, G. W. Supramol. Chem. 1995, 5, 105-108. (21) Arena, G.; Casnati, A.; Mirone, L.; Sciotto, D.; Ungaro, R. Tetrahedron Lett. 1997, 38, 1999-2002. (22) Arena, G.; Casnati, A.; Contino, A.; Lombardo, G. G.; Sciotto, D.; Ungaro, R. Chem.sEur. J. 1999, 5, 738-744. (23) Arena, G.; Casnati, A.; Contino, A.; Gulino, G. F.; Sciotto, D.; Ungaro, R. J. Chem. Soc., Perkin Trans. 1 2000, 419-423. (24) Zacharias, A.; Dougherty, D. A. Trends Pharm. Sci. 2002, 23, 281-287. (25) Ma, C. A.; Dougherty, D. A. Chem. ReV. 1997, 97, 1303-1324. (26) Inouye, M.; Hashimoto, K.; Isagawa, K. J. Am. Chem. Soc. 1994, 116, 5517-5518. (27) Tan, S.-D.; Chen, W.-H.; Satake, A.; Wang, B.; Xu, Z.-L.; Kobuke, Y. Org. Biomol. Chem. 2004, 2, 2719-2721. (28) Backirci, H.; Koner, A. L.; Nau, W. M. Chem. Commun. (Cambridge, U.K.) 2005, 5411-5413. (29) Backirci, H.; Koner, A. L.; Nau, W. M. J. Org. Chem. 2005, 70, 90609066. (30) Backirci, H.; Nau, W. M. AdV. Funct. Mater. 2006, 16, 237-242. (31) Jin, T. J. Inclusion Phenom. Macrocyclic Chem. 2003, 45, 195-201. (32) Buston, J. E. N.; Young, J. R.; Anderson, H. L. Chem. Commun. (Cambridge, U.K.) 2000, 905-906. (33) Kim, J.; Lee, M. J. Phys. Chem. A 1999, 103, 3378-3382. (34) Cao, X.; Tolbert, R. W.; McHale, J. L.; Edwards, W. D. J. Phys. Chem. A 1998, 102, 2739-2748. (35) Nishida, M.; Ishii, D.; Yoshida, I.; Shinkai, S. Bull. Chem. Soc. Jpn. 1997, 70, 2131-2140. (36) Kubinyi, M.; Bra´ta´n, J.; Grofcsik, A.; Biczo´k, L.; Poo´r, B.; Bitter, I.; Gru¨n, A.; Boga´ti, B.; To´th, K. J. Chem. Soc., Perkin Trans. 2 2002, 1784-1789. (37) Park, J. W.; Lee, S. Y.; Kim, S. M. J. Photochem. Photobiol., A 2005, 173, 271-278. (38) Suzuki, I.; Nakayama, C.; Ui, M.; Hirose, K.; Yamauchi, A. Anal. Sci. 2007, 23, 249-251.

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Figure 1. Molecular structure of p-sulfocalix[n]arene (Cn), chromophore (D), and acetylcholine (ACh).

and, in turn, ACh sensing at sub-micromolar concentrations. The ACh detection limit of 5 × 10-8 M is 3-4 orders of magnitude higher than was observed for previously reported photonic artificial receptor-based systems.26,30,31 In addition to this, it was found that monocarboxyl p-sulfocalix[4]arene, immobilized onto a silicon surface via EDC coupling, perfectly reproduced the interaction with the chromophore and subsequent binding of ACh as observed in solution, thus paving the way toward the development of a solid state-based optoelectronic biosensor for ACh. Experimental Procedures Interactions of a variety of water-soluble p-sulfocalix[n]arenes (Cn) with trans-4-[4-(dimethylamino)styryl]-1-methylpyridiniump-toluenesulfonate (D) (Sigma-Aldrich Co.) and ACh (Sigma-Aldrich Co.) have been studied in neutral and acidic solutions. C4 was purchased from Sigma-Aldrich Co., p-sulfocalix[6]arene (C6) and p-sulfocalix[8]arene (C8) were synthesized following a published procedure,39 and 25-monokis(hydroxycarbonylmethoxy)-5,11,17,23-tetrasulfocalix[4]arene (C4m) was synthesized according to the procedure described next. All synthesized Cn compounds were purified by preparative reversed-phase HPLC. The molecular structures of Cn, D, and ACh are shown in Figure 1. Phosphate buffer (PB, pH 7.4) was prepared from 0.1 M NaH2PO4 (Merck) and 0.1 M NaOH (Frutarom Ltd.) solutions. Triply distilled water (TDW, 18 MΩ) was used throughout these studies. MeOH (Merck) and all purchased reagents were used as received without further purification. Synthesis of (C4m). The synthesis of C4m was carried out as follows: 200 mg (0.471 mmol) of calix[4]arene (Sigma-Aldrich Co.) was monoalkylated by treatment with 1.25 equiv of methyl bromoacetate (54.6 µL, 0.589 mmol) and 0.5 equiv of K2CO3 (32.6 mg, 0.236 mmol) in refluxing acetonitrile (5 mL) for 1.5 h following the procedure for monoalkylation of calix[4]arenes as developed by Groenen et al.40 The monocarboxylic acid ester was saponified by the addition of 1 mL of a 1 M K2CO3 solution to the reaction mixture, followed by heating for 30-60 min at reflux until HPLC-ESMS analysis showed complete hydrolysis. The reaction mixture was then acidified by 1 M HCl until pH 9. Then, the solution was brought to pH 50 µM) than in PB/MeOH. This, in turn, means that the concentration of ACh needed to break down the [Cn‚D] complex is also significantly higher. Thus, at 50 µM [C6‚D], [ACh]50 increases up to 70 µM (data is not shown). The experimentally observed ACh titration curves for the [C6‚D] complex were investigated in more detail via simulation studies with the use of an AB/AC complexation model (see Supporting Information). ACh Sensing by the Immobilized [C4m‚D] Complex. Changes in the chromophore emission and wavelength maximum upon formation of the [Cn‚D] inclusion complex provide interesting perspectives toward the creation of an optoelectronic solid state-based ACh sensor.47-49 For this purpose, water-soluble monocarboxyl-functionalized p-sulfocalix[4]arene (C4m) was immobilized on an amino-terminated silicon surface by EDC coupling. EDC (in the presence of NHS) facilitated the formation of an amide bond between the carboxylic acid of C4m and the amine functionality at the silicon surface via formation of an active ester intermediate.50 Attachment of C4m was monitored by spectroscopic ellipsometry and by advancing contact angle measurements. The contact angle of the oxide silicon was about 15°, which indicated a termination of the surface by hydrophilic hydroxyl groups. The silicon oxide thickness was ca. 19 ( 0.4 Å. Upon a deposition of the APT coupling layer (Scheme 1, step 1), the water contact angle increased from 15 to 60°. The APT thickness determined by VASE was 7 ( 0.4 Å. Covalent binding of C4m (Scheme 1, step 2) reduced the contact angle from 60 to 50°, which was due to the hydrophilic character of the sulfonate groups, while a non-significant change demonstrates the hy(47) Mlika, R.; Ben Ouada, H.; Jaffrezic-Renault, N.; Dumazet, I.; Lamatrine, R.; Gamoudi, M.; Guillaud, G. Sens. Actuators, B 1998, 47, 43-47. (48) Mlika, R.; Ben Ouada, H.; Ben Chaabane, R.; Gamoudi, M.; Guillaud, G.; Jaffrezic-Renault, N.; Lamatrine, R. Electrochim. Acta 1998, 43, 841-847. (49) Sudholter, E. J.; van der Wal, P. D.; Skworonska-Ptasinska, M. S.; van der Berg, A.; Bergveld, P.; Reinhoudt, D. N. Recl. TraV. Chim. Pays-Bas 1990, 109, 222-225. (50) Sehgal, D.; Vijay, I. K. Anal. Biochem. 1994, 218, 87-91.

Figure 5. (A) Emission spectra of physically adsorbed D on Si before (1) and after (2) being rinsed in MeOH and (3) Si/[C4m‚D] sample after being rinsed in MeOH. (B) Emission spectra of the Si/C4m sample without D (1) and with D (2) after being rinsed in MeOH and (3) after subsequent exposure of this sample to an ACh solution.

drophobic character of the calixarene molecule. Spectroscopic ellipsometry analysis gave a layer thickness of ca. 9 ( 0.4 Å, whereas the calculated value was approximately 8-12 Å (for tilt angles in the range of 45-90°). The ellipsometric thickness remained the same following sonication in MeOH. The samples of bare silicon, Si, APT-modified silicon, (Si/ APT), and C4m-modified silicon, (Si/C4m), show no emission on excitation of their surfaces. These samples were exposed to a 1 mM solution of D in MeOH and dried at room temperature, and the emission spectra of their surfaces were recorded. As shown in Figure 5A, trace 1, the emission band of the physically adsorbed chromophore on the silicon surface (Si/D) was hypsochromically shifted to 584 nm from the band maximum at 604 nm for the free chromophore. Subsequent to the Si/D sample washing with MeOH, the emission band of the chromophore disappeared (Figure 5A, trace 2), a demonstration of the exclusively physical nature of the chromophore binding on the silicon surface. In contrast to this, the chromophore emission band on the calixarene layer remained strong after being rinsed in MeOH, while the band maximum shifted to 575 nm (Figure 5A, trace 3), relative to the band maximum of the physically adsorbed chromophore (Figure 5A, trace 1). Exposing the Si/C4m/D sample (Figure 5B, trace 2) to a 10 mM ACh solution caused a sharp decrease in the intensity of the

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new emission band (Figure 5B, trace 3) close to the intensity of the reference Si/C4m sample without D (Figure 5B, trace 1). The changes observed in the chromophore emission upon interaction with immobilized C4m are similar to the changes observed upon interaction with free calixarene in solution. This phenomenon indicates the formation of an immobilized complex on the semiconductor surface. ACh is capable of displacing the chromophore molecules from the calixarene cavity, as can be seen from the decrease in the emission intensity of the immobilized complex, similar to what was observed in solution.

Discussion In our search for a system that is highly sensitive to ACh, we introduced the hemicyanine chromophore, trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium p-toluenesulfonate as a molecular probe for host-guest complex formation. This chromophore is water-soluble and preserves its fluorescence in buffer solution. Moreover, the molecule of the chromophore has a strong internal dipole. As established by UV-vis and 1H NMR experiments, D forms a 1:1 inclusion complex [C4,6‚D] in neutral media (the dimension for the elliptical lower rim of C4 is ca. 2 Å in diameter and C6 is 3 Å × 7.6 Å44). Upon titration with Cn, the quantum yield increased sharply about 20-60-fold for C4 and C6 in PB/MeOH (pH 7.2) and MeOH (pH 2.4) solutions and 7-fold for C8 in the PB (pH 7.4) solution, while the emission band maximum shifted significantly to shorter wavelengths. Such effects were not observed in previous studies of calixarene-dye systems,24,30,31 but indeed occurred upon the interaction of the hemicyanine chromophore with β-CD.37,38 In the PB/MeOH solution, a binding constant of Ka ) 3.5 × 105 M-1 was calculated for D with C6. In the case of D with C4, Ka ) 1 × 105 M-1. Upon interaction with C8, the fluorescence of D changed linearly, but in two parts with different slopes. This indicates the ability of C8 to include one or two chromophore molecules in the cavity (the dimension for the elliptical lower rim of C8 is 3 Å × 11.6 Å44). An estimation of the binding constant in acidic media shows a non-1:1 interaction. Indeed, protonation of the dimethylamino group of D that was observed in the titration of D with a 10 mM HCl solution caused binding of D by methylpyridinium or dimethylamino groups, while the distance between the two methyl moieties of the dimethyamino group was ca. 5.5 Å. In neutral PB/MeOH solution, the fluorescence lifetime of the [C6‚D] complex increased to τ ) 0.95 ns as compared to that of free D (τ ) 0.13 ns). The observed increases in IF, quantum yield, and lifetime of the inclusion complex are due to stabilization of the chromophore inside the calixarene cavity rather than being the result of a change in the polarity of the medium around the D molecule.33,34,49 Electrostatic interactions of the cationic pyridinium moiety of D either with the negatively charged sulfonate groups on the upper rim or with the (partially) negatively charged phenolic rings of Cn may stabilize the chromophore molecule inside the cavity and in this way block free internal rotation.33,34 Differences in the increase in the fluorescence intensity can be explained by the driving forces of the different interactions and dimensions of the molecules. It has been established that the inclusion of guest molecules inside C4 is mainly enthalpically driven and is accompanied by strong electrostatic interactions, while hydrophobic interactions dominate the formation of the inclusion complexes with larger C6 and C8, which are both driven by entropy. The C4 cavity is far too shallow to accommodate the chromophore completely. For this reason, structural fixation of the chromophore will take place only to a minor extent, and therefore, the observed increases in quantum yield of [C4‚D] and [C4m‚D] complexes are much less than for the [C6‚D] complex.

KorbakoV et al.

On the other hand, the C8 cavity is large and flexible and is able to accommodate one or two chromophore molecules. This may be indicative of the flexibility of the C8 cavity and may also cause internal quenching of the chromophore fluorescence. Consequently, the C6 cavity offers the best compromise in terms of cavity depth and structural rigidity of D inside the cavity and therefore shows the largest increases in IF and quantum yield and a higher association constant. The high affinity18 of Cn for ACh (about 4 × 105 M-1) enables ACh to displace the chromophore molecule inside the Cn cavity. Thus, adding a 1.58 mM ACh solution to a solution of the [Cn‚D] complex resulted in a decrease in the emission intensity of the complex and the disappearance of the emission band of the caged chromophore. The system under investigation operates as a fluorescence switch: the chromophore quantum yield is greatly enhanced as the concentration of the complex increases and, upon adding ACh to the complex, the quantum yield of the free chromophore returns to its initial low value. The ACh detection limit of 5 × 10-8 M is 3-4 orders of magnitude higher than what was observed for previously reported artificial receptor-based systems26,28-31 and is comparable to the ACh detection limit achieved by AChE-based sensors.10,12,13 The [Cn‚D] complex shows a high selectivity for ACh. This was examined by titration of the complex in a PB/MeOH solution (pH 7.2) with a solution of neurotransmitters (tryptamine, glycine, aspartic acid, taurine, and noradrenaline). Changes in the fluorescence intensity of the complex were about 8-30%. In addition, the complex was titrated with the stable ACh analogue, carbamylcholine. This caused a decrease in emission of about 80% of that resulting from the titration with ACh. This emphasizes the high affinity of the inclusion complex to ACh and also shows sensitivity selectivity to other choline derivatives. The emission spectrum of D upon its interaction with immobilized monocarboxyl p-sulfocalix[4]arene is an indication of the inclusion complex formation on the semiconductor surface. ACh is capable of removing D from the cavity of immobilized calixarene in a similar manner as was observed in solution. In this case also, the interaction was accompanied by a decrease in the emission intensity. Iglesia and co-workers51 found that the interaction of the aromatic molecules adsorbed from aqueous solution on the grafted calixarenes was stronger than with free calixarene in solution. The absence of solubilizing groups near the cavity increased the hydrophobic properties of the cavity and resulted in increasing van der Waals interactions of the cavity with the guest molecule at a stoichiometric ratio of 1:1. This phenomenon opens the way to sense ACh optically and electrically by the immobilized [C4m‚D] complex on the semiconductor surface.

Conclusion A sharp increase in the fluorescence quantum yield and significant blue-shift of the emission band of the trans-4-[4(dimethylamino)styryl]-1-methylpyridinium-p-toluenesulfonate chromophore occurs upon inclusion of this chromophore into the cavity of free and immobilized p-sulfocalix[n]arenes. The selectivity and high affinity of this system for ACh permits substitution of the chromophore molecule by ACh in the cavity with the appearance of the initial emission intensity of free chromophore. These changes are so significant that the complex can be detected at low concentrations by fluorescence spectroscopy. This, in turn, makes it well-suited for the detection of the neurotransmitter ACh at sub-micromolar concentrations. (51) Notestein, J. M.; Katz, A.; Iglesia, E. Langmuir 2006, 22, 4004-4014.

Acetylcholine Detection at Micromolar Concentration

Modification of the semiconductor surface with C4m, subsequent formation of the immobilized inclusion complex with D, and dissociation of this complex by ACh were accompanied by optical changes in emission similar to those observed in solution. The use of the hemicyanine chromophore as the molecular probe for the formation of the complex with water-soluble p-sulfocalix[n]arenes for subsequent ACh sensing opens broad prospects for the creation of highly sensitive optical and optoelectronic sensors. Acknowledgment. Prof. J. Huskens is thanked for helpful discussions and assistance with the simulation studies, and Dr.

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E. Vaganova is thanked for fluorescence lifetime measurements. The EU is gratefully acknowledged for financial support (ECContract 510574 “Golden Brain”). Supporting Information Available: Model-simulation studies of formation of inclusion complexes at a variety of concentrations; absorbance spectra of chromophore on formation of [Cn‚D] and effect of ACh; estimation of the association constant and stoichiometry of [C8‚D]; and selectivity of the complex. This material is available free of charge via the Internet at http://pubs.acs.org. LA703010Z