Orientational Effect of Surface-Confined Cyclodextrin on the Inclusion

Sensing Capabilities of Colloidal Gold Monolayer Modified with a Phenylboronic Acid-Carrying Polymer Brush. Hiromi Kitano, Yasutaka Anraku, and Hiroak...
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Langmuir 2005, 21, 1314-1321

Orientational Effect of Surface-Confined Cyclodextrin on the Inclusion of Bisphenols Hiroshi Endo, Tadashi Nakaji-Hirabayashi, Shinta Morokoshi, Makoto Gemmei-Ide, and Hiromi Kitano* Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan Received June 7, 2004. In Final Form: November 16, 2004 The molecular recognition of various kinds of bisphenols (BPs) and a bisphenol A-polymer conjugate (BPA-polymer) by a self-assembled monolayer (SAM) of thiolated β-cyclodextrin (CD) on a gold electrode was examined using cyclic voltammetry (CV). Based on the inhibitory effect of BPs on the inclusion of hydroquinone (HQ) as a probe by the surface-confined CD, the association constants (Kassoc) of BPs with the immobilized β-CD were estimated. The Kassoc values for BPs with the SAM of 3-dithiobis(undecanoylamido)-3-deoxy-β-cyclodextrin (DTUA-β-CD) were smaller than those in the free β-CD system reported previously. A similar tendency was obtained when 6-(lipoylamido)-6-deoxy-β-cyclodextrin (LPβ-CD) was used in place of DTUA-β-CD. The Kassoc values for all the BPs except for bisphenol B with the SAM of LP-β-CD were always larger than those with the SAM of DTUA-β-CD, due to a difference in the orientation of the β-CD moiety in the SAMs. Furthermore, adsorption and desorption processes of the BPA-polymer from the surface-confined β-CD was followed using local surface plasmon resonance spectroscopy.

Introduction Recently, environmental pollution has been spread almost everywhere on the earth by endocrine-disrupting chemicals such as p-nonylphenol, phthalic acid esters, and bisphenol A, etc. Among them, bisphenol A (BPA, 2,2bis(4-hydroxyphenyl)propane; Figure 1) is widely used as a monomer for the production of epoxy resins and polycarbonate, and its toxicity and estrogenic activity have very often been reported (BPA is sometimes used as a fungicide).1 Furthermore, it has been pointed out that BPA is released not only in surface water but also into the natural environment during manufacturing processes and by leaching from final products.2a-d In fact, BPA has been detected in plastic waste,2a reusable containers,2b and polycarbonate baby bottles,2c and in aquatic environments and in air.2d Therefore, technical development for the immediate detection and removal of BPA is a problem of great urgency. Cyclodextrin (CD) has been studied in various research fields by taking advantage of its nonpolar and chiral cavity.3 Especially, self-assembled monolayers (SAMs) of thiolated CD derivatives have often been prepared for the construction of model systems of membrane receptors and chemical sensors.3d-l,4 Colloidal silver and gold electrodes have been chosen as a substrate to introduce the SAM of CD, and inclusion complexation of surface-confined CD with azo dyes has been investigated using surfaceenhanced resonance Raman spectroscopy (SERRS)4a,b and cyclic voltammetry (CV).4c-g The stereoselective complexation of the azo dyes by the surface-confined CD was * To whom correspondence should be addressed. Telephone: +81-76-445-6868. Fax: +81-76-445-6703. E-mail: kitano@ eng.toyama-u.ac.jp. (1) Dodds, E. C.; Lawson, W. Proc. R. Soc. London B 1938, 125, 222. (2) (a) Yamamoto, T.; Yasuda, A. Chemosphere 1999, 38, 2569. (b) Biles, J. E.; McNeal, T. P.; Begley, T. H.; Hollifield, H. C. J. Agric. Food. Chem. 1997, 45, 3541. (c) Takao, Y.; Lee, H. C.; Ishibashi, Y.; Kohra, S.; Tominaga, N.; Arizono, K. J. Health. Sci. 1999, 45, 39. (d) Staples, C. A.; Dom, P. B.; Klecka, G. M.; O’Block, S. T.; Branson, D. R.; Harris, L. R. Chemosphere 1998, 36, 2149.

Figure 1. Chemical structure of bisphenols.

observed directly by SERRS4b and indirectly by CV using hydroquinone (HQ) as a probe.4c The stereoselective complexation of dihydroxyphenylalanine (DOPA) derivatives by the surface-confined CD was previously observed directly by CV.4d Recently, the complexation of phthalic acid esters4e and bisphenols (BPs)4f by the SAM of R-CD has been investigated using the CV technique. (3) (a) Okada, M.; Harada, A. Macromolecules 2003, 36, 9701. (b) Kawaguchi, Y.; Harada, A. J. Am. Chem. Soc. 2000, 122, 3797. (c) Culha, M.; Lavrik, N. V.; Shell, F. M.; Tipple, C. A.; Sepaniak, M. J. Sens. Actuators B 2003, 92, 171. (d) Beulen, M. W. J.; Bugler, J.; Lemmerink, B.; Geurts, F. A. J.; Biemond, E. M. E. F.; van Leedam, K. G. C.; van Veggel, F. C. J. M.; Engbersen, J. F. J.; Reinhoudt, D. N. Langmuir 1998, 14, 6424. (e) Qian, J.; Hentschke, R.; Knoll, W. Langmuir 1997, 13, 7092. (f) Weisser, M.; Nelles, G.; Wohlfart, P.; Wenz, G.; MittlerNeher, S. J. Phys. Chem. 1996, 100, 17893. (g) Henke, C.; Steinem, C.; Janshoff, A.; Steffan, G.; Luftmann, H.; Sieber, M.; Galla, H.-J. Anal. Chem. 1996, 68, 3158. (h) Odashima, K.; Kotato, M.; Sugawara, M.; Umezawa, Y. Anal. Chem. 1993, 65, 927. (i) Nelles, G.; Weisser, M.; Back, R.; Wohlfart, P.; Wenz, G.; Mittler-Neher, S. J. Am. Chem. Soc. 1996, 118, 5039. (j) Suzuki, I.; Murakami, K.; Anzai, J.; Osa, T.; He, P.; Fang, Y. Mater. Sci. Eng. C 1998, 6, 19. (k) Rojas, M. T.; Ko¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336. (l) Michalke, A.; Janshoff, A.; Steinem, C.; Henke, C.; Sieber, M.; Galla, H.-J. Anal. Chem. 1999, 71, 2528. (4) (a) Maeda, Y.; Kitano, H. J. Phys. Chem. 1995, 99, 487. (b) Yamamoto, H.; Maeda, Y.; Kitano, H. J. Phys. Chem. B 1997, 101, 6855. (c) Maeda, Y.; Fukuda, T.; Yamamoto, H.; Kitano, H. Langmuir 1997, 13, 4187. (d) Fukuda, T.; Maeda, Y.; Kitano, H. Langmuir 1999, 15, 1887. (e) Kitano, H.; Taira, Y.; Yamamoto, H. Anal. Chem. 2000, 72, 2976. (f) Kitano, H.; Taira Y. Langmuir 2002, 18, 5835. (g) Chi, Q.; Zhang, J.; Nielsen, J. U.; Friis, E. P.; Chorkendorff, I.; Canters, G. W.; Anderson, J. E. T.; Ulstrup, J. J. Am. Chem. Soc. 2000, 122, 4047.

10.1021/la048595p CCC: $30.25 © 2005 American Chemical Society Published on Web 01/08/2005

Surface-Confined CD Effect on Bisphenol Inclusion

Figure 2. Chemical structure of poly(BPAM-co-AAm). Chart 1. Chemical Structure of DTUA-β-CD

Chart 2. Chemical Structure of LP-β-CD

In this report, the inclusion complexation of various kinds of BPs (Figure 1) and BPA-polymer conjugate (poly(BPAM-co-AAm); Figure 2) with surface-confined β-CD derivatives, 3-dithiobis(undecanoylamido)-3-deoxy-β-cyclodextrin (DTUA-β-CD; Chart 1) and 6-(lipoylamido)-6deoxy-β-cyclodextrin (LP-β-CD; Chart 2), was investigated using the CV technique and compared with that in the free β-CD system.5 The effect of the orientation of the β-CD moiety in the SAMs on the Kassoc value for various BPs was discussed. Furthermore, adsorption and desorption processes of a bisphenol A-polymer conjugate from the surface-confined β-CD were followed using localized surface plasmon resonance (SPR) spectroscopy. Experimental Section Materials. Bisphenol A and β-estradiol were obtained from Nacalai Tesque, Kyoto, Japan. Bisphenol B, bisphenol S, and bisphenol F were from Tokyo Kasei, Tokyo, Japan. All other reagents used were commercially available. Milli-Q grade water was used for the preparation of sample solutions. 3-Dithiobis(undecanoylamido)-3-deoxy-β-cyclodextrin (DTUA-β-CD; Chart 1).6 Mono-2-O-(p-toluenesulfonyl)β-cyclodextrin (2-TsO-β-CD, 1.68 g)7 was prepared by the reaction of β-CD (24.17 g, 21.3 mmol) with the same equivalent of m-nitrophenyl tosylate in the mixture of N,N-dimethylformamide (DMF, 400 mL) and 0.2 M carbonate buffer (200 mL, pH 9.9), while being vigorously stirred at 60 °C for 1 h. Then, the mixture was neutralized with 1 N HCl and added to acetone (1 L). The filtered precipitate was purified by passing through a column packed with porous polystyrene resins (Diaion HP-20, Mitsubishi (5) Kitano, H.; Endo, H.; Gemmei-Ide, M.; Kyogoku, M. J. Inclusion Phenom. 2003, 47, 83. (6) Baugh, S.; Yang, Z.; Leung, D. K.; Wilson, D. M.; Breslow, R. J. Am. Chem. Soc. 2001, 123, 12488. (7) Ueno, A.; Breslow, R. Tetrahedron Lett. 1982, 23, 3451.

Langmuir, Vol. 21, No. 4, 2005 1315 Chemicals, Tokyo). The degree of substitution (DS, the number of substituents in one β-CD molecule) of the tosyl group was determined to be 1.0 by a matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometer (Voyager RP, PerSeptive): MS (MALDI-TOF), m/z 1312 [[M + Na]+; calcd for C49H76O37S (DS ) 1), 1289], 1328 [[M + K]+; calcd for C49H76O37S (DS ) 1), 1289]; Mw ) 1275; Mw/Mn ) 1.01. Anal. Calcd for C49H76O37S: C, 45.64; H, 5.95. Found: C, 44.55; H, 5.89. In the TOF mass spectra, several subpeaks, which come from a lower or higher number of substitution of “legs”, were detected. However, the peak intensities were small. Therefore, it is thought that these small numbers of substituents will not affect the construction of the SAM nor inclusion of BPs. The 2-TsO-β-CD (DS ) 1.0, 1.68 g, 1.30 mmol) was converted to a 2,3-mannoepoxide according to the conventional method.8 Then, the 2,3-mannoepoxide was treated with a 28% aqueous ammonia solution at room temperature for 24 h to give mono3-(deoxyamino)-β-cyclodextrin (3-NH2-β-CD, 0.72 g). Next, 11,11′-dithiobis(undecanoic acid) (DTUA, 0.04 g, 0.09 mmol)9 and hydroxybenzotriazole (HOBt, 0.040 g, 0.27 mmol) were dissolved in DMF (3 mL). Dicyclohexylcarbodiimide (DCC, 0.060 g, 0.28 mmol) dissolved in DMF (1.0 mL) was added to the solution mixture using a dropping funnel, and the reaction solution was continuously stirred for 1 h in ice, and afterward 1 h at room temperature. Furthermore, 3-NH2-β-CD (0.30 g, 0.27 mmol) dissolved in DMF (30 mL) was added, and the reaction solution was stirred for 48 h at room temperature. The product was isolated by silica gel column chromatography (DTUA-β-CD; 50 mg). Anal. Calcd for [β-CD-(NHCO(CH2)10S1.0)2: C, 47.74; H, 6.80; N, 1.05. Found: C, 46.01; H, 6.70; N, 1.38. MS (MALDITOF): m/z 2690 [[M + Na]+]; Mw ) 2666; Mw/Mn ) 1.15. 6-(Lipoylamido)-6-deoxy-β-cyclodextrin (LP-β-CD; Chart 2).10 6-Amino-6-deoxy-β-cyclodextrin (DS ) 4, 0.25 g, 0.22 mmol) prepared by the method of Henke et al.3g was dissolved in a water-tetrahydrofuran (THF) mixture (1:2, 30 mL), and incubated with lipoic acid (0.73 g, 3.56 mmol) in the presence of 1-ethyl3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC; 0.76 g, 3.98 mmol) at 0 °C. The reaction mixture was kept at pH 5 for 0.5 h, and afterward, the reaction solution was continuously stirred at room temperature for 24 h. After evaporation, cold water was poured into the flask. The yellow-colored precipitate was filtered, washed several times with ethyl acetate, and dried in vacuo (LP-β-CD, 65 mg). Anal. Calcd for β-CD-(NHCO(CH2)4CH(CH2)2S2)4.0: C, 47.17; H, 6.53; N, 2.97. Found: C, 46.63; H, 6.76; N, 2.32. MS (MALDI-TOF): m/z 1736 [[M + K]+; calcd for C66H109O35N3S6 (DS ) 3), 1697], 1921 [[M + K]+; calcd for C74H122O35N4S8 (DS ) 4), 1884], 2108 [[M + K]+; calcd for C82H135O35N5S10 (DS ) 5), 2071]; Mw ) 1835; Mw/Mn ) 1.05. Copolymer of Bisphenol A Methacrylate and Acrylamide (BPA-Polymer; Figure 2). Bisphenol A (11.8 g, 51.7 mmol), triethylamine (8 mL, 57.7 mmol), and di-tert-butyl-pcresol (10 mg, 0.045 mmol, polymerization inhibitor) were dissolved in dry chloroform (110 mL). The reaction solution was continuously stirred for 5 min in ice under a N2 gas atmosphere. Methacryloyl chloride (1 mL, 20.7 mmol) dissolved in dry chloroform (10 mL) was slowly added using a dropping funnel. The reaction solution was continuously stirred for 1 h in ice, and afterward for 24 h at room temperature. After evaporation, the reaction mixture (about 50 mL) was washed with water, and the organic phase was dehydrated under an anhydrous Na2SO4. The product was isolated by silica gel column chromatography (bisphenol A methacrylate; 3.69 g, yield 46.9%). 1H NMR (400 MHz FT-NMR, DX-400, JEOL, Tokyo, Japan): δ 1.63 (s, 6H, methyl group in BPA), 2.05 (s, 3H, methyl group in methacrylate), 5.74 (s, 1H, vinyl group in methacrylate), 6.33 (s, 1H, vinyl group in methacrylate), 6.67-6.74 (d, 2H, phenyl group in BPA), 6.987.10 (d-d, 4H, phenyl group in BPA) and 6.95-7.24 (d, 2H, phenyl group in BPA). Bisphenol A methacrylate, acrylamide (AAm) and 2,2′-azobis(isobutyronitrile) (AIBN) (1:40:0.4) were incubated in methanol (8) Breslow, R.; Czarnik, A. W. J. Am. Chem. Soc. 1983, 105, 1390. (9) (a) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (b) Kitano, H.; Saito, T.; Kanayama, N. J. Colloid Interface Sci. 2002, 250, 134. (10) (a) Hayes, W. A.; Shannon, C. Langmuir 1996, 12, 3688. (b) Rojas, M. T.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 5883.

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(MeOH) at 65 °C for 24 h. The precipitated polymer was filtrated and washed several times with methanol and 2-propanol. After drying in vacuo, the polymer product was dissolved in water. The polymer solution was applied to an ultrafiltration apparatus (Amicon; membrane, YM 10; exclusion limit, 104), and lyophilized (poly(BPAM-co-AAm), BPA-polymer; 1.50 g; BPAM:AAm ) 1:213). The weight-averaged molecular weight (Mw) of the polymer was estimated to be 2.66 × 104 (Mw/Mn ) 2.00) using GPC (Waters HPLC System; column, Wako Gel G-30, Wako Pure Chemicals, Osaka, Japan; mobile phase, 0.1 M NaBr; standard sample, pullulan, Showa Denko, Tokyo, Japan). The number of BPA moieties in the polymer molecule was calculated to be 2.1. Self-Assembled Monolayer of Thiolated β-CD on a Gold Electrode. A gold electrode (surface area of Au, 2.2 mm2) was polished with diamond (diameter, 6.0 µm) and alumina powders (diameter, 1.0 and 0.05 µm) and repeatedly rinsed with pure water. The electrode was further washed by dipping in an aqueous 0.1 N H2SO4 and subsequently scanned from -0.4 to +1.5 V more than 10 times (scan rate, 100 mV/s) with a potentiostat (HA-301, Hokuto-Denko, Tokyo, Japan) and a function generator (HB-104, Hokuto-Denko). The total area of the gold electrode was determined from the average of anodic and cathodic peak currents of aqueous K3Fe(CN)6 solution (5 mM, scanned from +0.6 to -0.3 V at 10 mV/s) by using the Randles-Sevcik equation.11 For the immobilization of thiolated β-CD, the gold electrode was immersed in a 1.0 mM solution of DTUA-β-CD or LP-β-CD (solvent, MeOH) for 24 h under a N2 gas atmosphere. The modified electrode obtained was washed alternately more than five times with water and MeOH. Cyclic voltammetric measurements were performed with the potentiostat and the function generator. Outputs of the potentiostat were converted by an A/D converter and collected by a microcomputer (PC-9821V166, NEC, Tokyo, Japan). Data analyses were carried out with an in-home program. A Pt electrode and KCl-saturated calomel electrode (SCE) were used as the counter and reference electrodes, respectively. The electrochemical cell was thermostated at 25 °C using a circulating water bath (RM6, Lauda, Postfach, Germany). Before the redox reaction of the probe, HQ, in the presence and absence of BP, the modified electrode was incubated at 25 °C for 5 min in the sample solution, which had been prepared under a N2 gas atmosphere for the removal of dissolved oxygen. Evaluation of Association Constant for Free β-CD with BPA-Polymer. The association constant for β-CD with BPApolymer (Kassoc) in a mixture of 10% (v/v) methanol-phosphate buffer (pH 7.0, 9 mM) was determined fluorimetrically using 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) as a probe with a fluorescence spectrophotometer (Model FP-777, Japan Spectroscopic Co., Tokyo, Japan). 2,6-ANS, which is well-known as a “hydrophobicity” probe, showed a much stronger fluorescence in the CD cavity than in the free state (Figure 3a).5,12 The association constant for β-CD with 2,6-ANS (K1) was determined by the double reciprocal plots of the increase in fluorescence intensity of 2,6-ANS (∆F) and the total concentration of β-CD ([CD]o). With the coexistence of BPA-polymer, the ∆F value was diminished (Figure 3b), suggesting that the BPA residues in the polymer competitively inhibited the inclusion of 2,6-ANS in the CD cavity. Using eq 1, the Kassoc value for the β-CD-BPApolymer complex was determined by the curve-fitting method.

Kassoc )

{[CD]o - [X/(1 - X)K1] - [2,6-ANS]oX}

[X/(1 - X)K1]{[BP]o - [CD]o + [X/(1 - X)K1] + [2,6-ANS]oX} (1) where K1 and Kassoc are the association constant for β-CD with 2,6-ANS and that with BPA-polymer, respectively. X is defined as ∆F′/∆F∞, where ∆F∞ is the ∆F value at [CD]o ) ∞ and ∆F′ is the ∆F value in the presence of both β-CD and BPA-polymer (total concentration of bisphenol residues, [BP]o). (11) Bard, A. J.; Faukner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley & Sons: New York, 1980. (12) (a) Catena, G. C.; Bright, F. B. Anal. Chem. 1989, 61, 905. (b) Hirasawa, T.; Maeda, Y.; Kitano, H. Macromolecules 1998, 31, 4480.

Endo et al.

Figure 3. (a) Changes in fluorescence intensity of 2,6-ANS in the presence of β-CD ([ANS]o ) 90 µM), in 9 mM phosphate buffer (pH 7.0) containing 10% (v/v) MeOH at 25 °C. (b) Relationship between the ∆F′ value and the concentration of bisphenol A-polymer in the presence of β-CD ([ANS]o ) 90 µM; [β-CD]o ) 135 µM). Scheme 1. Schematic of the Steps Involved in the Fabrication of an Immobilized Colloidal Gold Sensor Chip on Glass

Preparation and Characterization of Gold Colloid. All glassware used for preparation of colloids was thoroughly washed with aqua regia (3:1 HCl-HNO3) and rinsed extensively with water. Gold colloids were prepared by the reduction of HAuCl4 with sodium citrate in water at 100 °C.13 The average hydrodynamic diameter of the gold colloids and a dispersion were estimated to be 40 nm and 30-50 nm, respectively, by the dynamic light scattering technique (DLS-7000, Otsuka Electronics, Hirakata, Osaka, Japan; light source, He-Ne laser, 632.8 nm). Preparation of Colloidal Gold Monolayer on a Glass Substrate and Its Functionalization by CD-SAM (Scheme 1). A glass substrate (thickness ) 160 µm, 9.5 × 24 mm) was cleaned by sonication for 2 h in water containing a 3% detergent (Contaminon N, Wako Pure Chemicals) and washed thoroughly (13) Kawaguchi, T.; Tagawa, K.; Senda, F.; Matsunaga, T.; Kitano, H. J. Colloid Interface Sci. 1999, 210, 290.

Surface-Confined CD Effect on Bisphenol Inclusion

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Table 1. Association Constants (Kassoc) for the Complexation of Bisphenols and Estradiol with β-CD of Both Free and SAM States at 25 °C Kassoc (103 M-1) freea

SAM guest

3-DTUA-β-CD

6-lipoyl-β-CD

β-CD

BPA BPB BPF BPS estradiol BPA-polymer

14 18 5.7 3.0 17 750

22 13 10 4.7 13 150

35 40 5.0 4.0 25 9.0

a

From ref. 5.

with water. The substrate was further cleaned in concentrated HNO3 for 4 h, washed thoroughly with water, and, after cleaning by sonication for 2 min, dried in a drying oven at 70 °C overnight. To prepare an amine-terminated glass surface, the substrate was immersed in a 10% (v/v) 3-(aminopropyl)triethoxysilane aqueous solution for 20 min, rinsed with water, sonicated for 20 s, and finally dried at 70 °C for 3 h. The amine-terminated glass substrate was subsequently immersed in a colloidal gold solution overnight to form the colloidal gold monolayer on the glass substrate. The colloidal gold-fixed glass substrate was immersed in a 1.0 mg/mL thiolated β-CD (3-DTUA-β-CD or 6-lipoyl-β-CD) solution for 24 h and extensively rinsed with water. The self-assembled monolayer-carrying glass substrates were stored in water. Absorption Measurements. A UV-visible-near-infrared spectrophotometer (Lambda 19 UV/vis/near-IR spectrometer, Perkin-Elmer) was used to measure the absorbance of the immobilized gold colloids on a glass substrate placed in a quartz cell. Spectra were obtained in transmission mode over a range of 350-850 nm. The temperature of the cell was kept at 25 °C by using a Peltier device.

Results and Discussion A. Evaluation of the Kassoc Value in the Free System. The complexation of BPA-polymer (2.1 BPA moieties in one polymer molecule on average) with β-CD was examined fluorimetrically using 2,6-ANS as a probe. The fluorescence intensity of 2,6-ANS at 450 nm was increased by the addition of β-CD (Figure 3a),12b due to the formation of an inclusion complex between 2,6-ANS and β-CD. From the double reciprocal plot of the initial concentration of β-CD ([CD]o) versus the increase in fluorescence intensity (∆F), the association constant for the β-CD-ANS complex (K1) was determined to be 8.8 × 102 M-1. This value is smaller than those reported previously (in water,12a 1.9 × 103 M-1, and in a M/30 phosphate buffer (pH 7.2),12b 2.7 × 103 M-1), due to the presence of 10% (v/v) methanol which was added to dissolve the BPA-polymer. The ∆F value was decreased by the addition of BPApolymer into the 2,6-ANS solution containing β-CD (Figure 3b), suggesting that BPA-polymer competitively inhibited β-CD from inclusion of 2,6-ANS. On the other hand, the addition of polyacrylamide (PAAm, Mw ) 7.1 × 103), which has no BPA moieties, did not affect the ∆F value at all. These results clearly showed the formation of an inclusional complex between β-CD and BPA residues in the BPA-polymer. The association constant of β-CD with the BPA-polymer (Kassoc) was unequivocally determined using eq 1. The Kassoc value for the BPA-polymer with free β-CD was smaller than that for BPA itself (Table 1). This is probably because the bisphenol moiety in the polymer with a larger steric hindrance (due to the neighboring AAm residues) compared to free BPA could not be deeply included into the cavity of β-CD. Moreover, the dispersion

Figure 4. Voltammetric response of 5 mM K3[Fe(CN)6] on a bare (curve a) or modified (DTUA-β-CD, curve b; LP-β-CD, curve c) Au electrode dipped in 0.5 M KCl at 25 °C. Scan rate ) 10 mV/s.

Figure 5. Voltammetric response for reductive desorption of SAM in a 0.5 M KOH on the Au electrode: (a) DTUA-β-CD SAM and (b) LP-β-CD. Scan rate ) 100 mV/ s.

of polymer main chain might also slightly affect the inclusion behavior of the bisphenol moiety into the CD cavity, although the dispersion of the BPA-polymer we used in this work was not so large. B. Formation of CD SAM on a Gold Electrode. The formation of LP-β-CD SAM on a gold electrode was confirmed by the increase in the potential difference (∆Ep) of Fe(CN)64-/3- using cyclic voltammetry (CV).4c The ∆Ep value for the modified electrode (96 mV) was larger than that of a bare electrode (65 mV) (Figure 4c) due to the presence of the CD ring between the bulk solution and the Au surface. Furthermore, DTUA-β-CD SAM completely blocked the electrochemical reaction of Fe(CN)64-/3(Figure 4b). The previous studies on a barrier effect of β-CD SAM to Fe(CN) 64-/3- showed that Fe(CN)64-/3- is sterically not permeable through the β-CD cavity, but is permeable only through the space remaining between the surface-confined β-CD residues.14 Therefore, the figure indicates that the electrochemical reaction of Fe(CN)64-/3was inhibited by the densely packed DTUA-β-CD SAM. An immersion of the SAM-modified electrode in 0.5 M KOH solution for 10 min did not affect the potential difference (∆Ep) of Fe(CN)64-/3-, indicating a remarkable stability of the SAM. For the reductive desorption of DTUA-β-CD or LP-βCD from the electrode, a scanning from 0 to -1.2 V at 100 mV/s in an aqueous 0.5 M KOH solution was carried out (Figure 5). The peak around -0.9 V in the figure has been attributed to the reductive desorption of thiolated compounds that are chemisorbed to Au.3h,15 This result supports that both SAMs were sorbed to the gold electrode with the cleavage of the S-S bond and following Au-S bond formation. With the assumption that all disulfide (14) He, P.; Ye, J.; Fang, Y.; Osa, T. Electroanalysis 1997, 9, 68. (15) (a) Walczak, M. M.; Popenone, D. D.; Deinhammer, R. S.; Lamp, B. D.; Chung, C.; Porter, M. D. Langmuir 1991, 7, 2687. (b) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335. (c) Zhong, C. J.; Porter, M. D. J. Am. Chem. Soc. 1994, 116, 11, 616.

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Table 2. A Values for Various SAMs SAM

A (nm2)

3-DTUA-β-CD 6-lipoyl-β-CD

0.89 2.24

groups of both DTUA-β-CD (DS ) 1.0) and LP-β-CD (DS ) 4.0) form Au-S bonds, integration of the cathodic peak around -0.9 V gives the molecular surface area (abbreviated as A hereafter) of these SAMs (Table 2). The A value for the DTUA-β-CD SAM (0.89 nm2) was smaller than that for the LP-β-CD SAM (2.24 nm2), probably because the DTUA-β-CD SAM was fixed to the gold surface only with a single anchor, and the CD moiety was to some extent at tilt to the gold surface. The long anchor group of DTUA-β-CD seemed to be closely packed by the intermolecular interaction. Moreover, using the maximum density of β-CD (diameter, 1.53 nm) immobilized on the electrode (0.82 × 10-10 mol/cm2), the percentage of coverage for the LP-β-CD SAM was also calculated. On the basis of the DS value ()4.0) of the disulfide group in the LP-β-CD molecule, the surface coverage was calculated to be 94% of the theoretical value. Previously, we reported that the surface coverage of 6-(lipoylamido)-6-deoxy-R-cyclodextrin (LP-R-CD) was 98% of the theoretical value.4f Similarly, Suzuki et al. reported that the surface coverage of LP-R-CD on a gold wire electrode was 98%, when the concentration of LP-R-CD at the modification was 1.0 mM.3g C. CV Measurements of HQ. In the solutions of HQ, the modified Au electrodes showed a voltammetric response (Figure 6). The larger oxidation peak in the figure (+0.12 V vs SCE, curve a) corresponds to the response of HQ on the bare gold electrode, whereas, the smaller ones (+0.51 V vs SCE, curve b; and +0.43 V vs SCE, curve c) correspond to that on the electrode modified with DTUAβ-CD and LP-β-CD, respectively. The peak shift to higher voltage and the decrease in peak currents suggest that HQ molecules formed an inclusion complex with the surface-confined β-CD molecule and were oxidized in the β-CD cavity. Both cathodic and anodic peak currents for HQ increased linearly with an increase in the scan rate as expected for a redox reaction of surface-confined molecules (Figure 7a, b),3j,4c-e which was in contrast with the nonlinear relationship for the bare Au electrode (Figure 7c).4f The relationship between the peak current and the scan rate did not go through the zero point in both cases, probably due to the small error associated with subtraction of the voltammetric peaks from the base-line charging current as suggested by Stevenson et al.16 A similar result was reported previously for ferrocene in the thiolated CD-SAM electrode system.3k The results in Figure 7 show that the observed voltammetric response is due to HQ molecules included in the cavity of β-CD attaching to the surface of Au and that HQ molecules diffusing in the solution have little contribution to the voltammetric response. To confirm that the faradaic currents came solely from the surface-confined HQ molecules, we investigated the dependence of the peak currents on the concentration of HQ in the solution (see Supporting Information). The peak current was saturated at high HQ concentration and obeyed the following eq 2 derived from Langmuir isotherm.

[HQ]o/I ) 1/K1Imax + [HQ]o/Imax

(2)

where I is the peak current when the initial concentration (16) Stevenson, K. J.; Hatchett, D. W.; White, H. S. Langmuir 1997, 13, 6824.

Figure 6. Voltammetric response of 3.0 mM HQ on a bare (curve a) or modified (DTUA-β-CD, curve b; LP-β-CD, curve c) Au electrode dipped in 10 mM phosphate buffer (pH 7.0) containing 10% (v/v) MeOH at 25 °C. Scan rate ) 100 mV/s.

Figure 7. Scan rate dependence of anodic peak current of 0.1 mM HQ on the electrode dipped in 0.1 M Na2SO4 at 25 °C: (a) DTUA-β-CD SAM and (b) LP-β-CD SAM Au electrode; (c) bare Au electrode. The broken line is drawn by using the RandlesSevcik equation (I is proportional to the square root of the scan rate).11

of HQ is [HQ]o, Imax is the maximum peak current, and K1 is the association constant of HQ with surface-confined β-CD. The K1 values in the DTUA-β-CD system and the LP-β-CD system were calculated to be 1.55 × 102 and 1.75 × 102 M-1, respectively, from the x-intercept of the linear line in the plot of [HQ]o/I versus [HQ]o. D. Inclusion of Bisphenols by CD SAM. When BPs were added to the solution of HQ in the LP-β-CD and DTUA-β-CD SAM systems, the peak current was decreased (see Supporting Information). A similar result was obtaind when the BPA-polymer was added. On the other hand, the addition of PAAm, which has no BPA moieties, did not affect the peak current at all. Moreover, when the bare Au electrode was used, the existence of free BP had no significant influence on the peak currents of the reaction. These voltammetric results indicate that BPs or BPA moieties in the polymer as well as HQ form inclusion complexes with the surface-confined β-CD on the Au electrode and that the redox reaction of HQ is competitively inhibited by BPs, as shown in Scheme 2. As is expected for the competitive inhibition, the difference between the peak currents of the redox reaction of HQ in the presence and absence of BPs (∆I) on both anode and cathode increased with an increase in the concentration of BPs (Figure 8) and followed the equations below.

[BP]/∆I ) A{(K1[HQ]o + 1)/Kassoc + [BP]}

(3)

A ) (1 + K1[HQ]o)/c[CD]o

(4)

where Kassoc is the association constant of BP with surfaceconfined CD and c is the current per 1 mol of HQ molecule. The association constants for four kinds of BPs calculated from the x-intercepts (-{(K1[HQ]o + 1)/Kassoc}) of

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Scheme 2. Schematic Drawing of Inclusional Complexes of HQ and BP with LP-β-CD SAM

the lines drawn by a linear regression analysis of the experimental points are compiled in Table 1. The Kassoc values for BPA and BPB in both SAM systems were smaller than those for the free β-CD system.5 On the other hand, the Kassoc values for BPF and BPS in both SAM systems were similar to those for the free β-CD system. The former tendency could be ascribed to the relatively larger steric obstruction of substituents at the center of BPA and BPB molecules among four kinds of BPs, prohibiting them from penetrating deeply into the CD cavity on the gold electrode. The latter was because both BPF and BPS could be more deeply included into the CD cavity than BPA and BPB due to the relatively smaller steric hindrance. Previously, we examined the geometry of the complexation between β-CD and BPs for the free system by 1H NMR method and the Corey-PaulingKoltun (CPK) model.5 It is possible for BPs to penetrate into the cavity of β-CD from both the primary and secondary hydroxy faces, whereas it is impossible into that of the surface-confined β-CD, which results in the difference in the Kassoc values between the free and SAM systems.

Figure 8. Langmuir plots for cathodic peak current of 3.0 mM HQ on the Au/DTUA-β-CD electrode dipped in 10 mM phosphate buffer (pH 7.0) containing 10% (v/v) MeOH at 25 °C: (3) BPS; (0) BPF; (O) BPA; (4) BPB. Scan rate ) 100 mV/s. The ∆I corresponds to the difference in the peak currents.

The Kassoc values for four kinds of BPs in the DTUAβ-CD system were in the order of BPB > BPA > BPF > BPS, which is ascribable to the difference in the hydrophobic interaction between the cavity of β-CD and bisphenols. The Kassoc values for four kinds of BPs in the LP-β-CD systems were in the order of BPA > BPB > BPF > BPS. The Kassoc value for BPA was larger than that for BPB in this system, and the difference in the Kassoc values for them was more significant compared to the DTUA-β-CD system, probably due to the preferential penetration of BPA from the secondary hydroxyl face of the β-CD cavity. As compared with the shape of BPs penetrated from the primary hydroxyl face of the surface-confined β-CD, that from the secondary one might be more diverse because of the difference in the size of the inlet between the secondary and primary hydroxyl faces. Among them, furthermore, the guest molecule would be most tightly fixed into the host molecule in the BPA-β-CD system as suggested in the 1H-1H rotating frame Overhauser effects (ROESY) of the free β-CD-BP systems.5 In the case of the surface-confined R-CD reported previously,4f on the contrary, the Kassoc values for BPS were the largest among four kinds of BPs, probably due to the hydrogen bonding between the sulfonyl group of BPS and the secondary OH groups at the rim of the surfaceconfined R-CD. Therefore, as intuitively acceptable, the inclusional behavior of BPs to the surface-confined CD is largely affected by the difference in a cavity size between R-CD (the inside diameter, 0.45 nm) and β-CD (the inside diameter, 0.70 nm). Furthermore, the Kassoc values for three kinds of BPs except for BPB in the DTUA-β-CD system were smaller than those for the LP-β-CD system, probably due to the preferential penetration of BPs from the secondary hydroxyl face of the β-CD cavity compared to the primary hydroxyl face. In the case of BPB, the inclusion from the primary hydroxyl face of β-CD seemed to be limited by the steric hindrance of the bulky ethyl group.

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It should be mentioned here that the Kassoc value for β-estradiol, a potent mammalian estrogenic hormone produced by the ovary, in the DTUA-β-CD system was larger than that in the LP-β-CD system (Table 1), indicating that β-estradiol could be deeply included into the DTUA-β-CD with a relatively longer spacer. In the case of BPA-polymer, the Kassoc values with two kinds of β-CD SAMs were larger than that with free β-CD (Table 1), probably because of a cooperative inclusion of a plural number of bisphenol moieties in the BPA-polymer molecule (2.1 BPA moieties in the polymer molecule on average) into neighboring surface-confined β-CDs, though a stepwise inclusion could not be clearly observed. Furthermore, the Kassoc value of BPA-polymer in the DTUA-β-CD system was larger than that in the LP-β-CD system. The mobility of the bisphenol moiety in the polymer is much smaller than that of a bisphenol itself, and the inclusional complex from the narrow secondary hydroxyl face of β-CD cavity might be more advantageous for a ligand with a smaller mobility, which might result in the larger association constant. Moreover, we think the hydrogen bonding array in the DTUA-β-CD cavity is destroyed by the substitution of the anchor group to the third hydroxyl group, which reduces the rigidity of the cavity. Generally, the CD molecule assumes a rigid structure through the formation of a belt of intermolecular hydrogen bonding between hydroxyl groups at the second and third positions of adjacent glucose units.17 The substitution-induced distortion of the circular shape might cause a decrease of volume in the cavity.17 Since the inlet of the cavity from the primary hydroxyl face is smaller than that from the secondary one, the substitution of anchor groups might have a more serious effect on the inclusion in the DTUA-β-CD SAM system than in the LP-β-CD SAM. Therefore, it is understandable that the Kassoc values for three kinds of BPs except for BPB and β-estradiol in the former system are smaller than those for the latter system. The dehydration of nonpolar moieties of BP and CD might also be changed by the difference in the shape of the cavity. We think the contribution of the dehydration in the LP-β-CD to enhance the inclusion is more dominative than in DTUA-β-CD, because the amount of dehydration in the LP-β-CD cavity upon the inclusion of a guest molecule will be larger than that in DTUA-β-CD due to the difference in the number of hydroxyl groups directing to the solution phase (LP-β-CD, 14 OH groups at the second and third positions; DTUA-β-CD, 7 OH groups at the sixth position). Therefore, the Kassoc values for most of the BPs in the LP-β-CD system were larger than those for the DTUA-β-CD system. E. SPR Measurements. Finally, adsorption and desorption processes of the bisphenol A-polymer conjugate from the surface-confined β-CD were followed using localized SPR spectroscopy. Recently, Nath et al.18 showed that a colloidal Au monolayer can be prepared on glass by self-assembly from Au sol and functionalized with thiol derivatives. They further demonstrated a new label-free optical sensor, which can observe biomolecular interactions at the surface in real time. Herein, we prepared an optical biosensor (β-CD-modified colloidal gold on glass) using a similar methodology and examined the recognition of BPA residues in the BPA-polymer by the β-CD SAM spectrophotometrically. Quite recently, we have examined (17) Khan, R. A.; Forgo, P.; Stine, J. K.; D’Souza, T. V. Chem. Rev. 1998, 98, 1977. (18) Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504.

Endo et al.

Figure 9. Time evolutions of absorbance at 550 nm for the DTUA-β-CD-immobilized sensor chip after immersion (A) in BPA-polymer solution and (B) in 10% (v/v) methanolphosphate buffer (pH 7.0, 9 mM) solution. [BPA-polymer] ) 1.0 mg/mL.

the specific binding of concanavalin A using the similar optical biosensor modified with glucose-carrying polymer.19 Using the CD-modified sensor chip, the adsorption and desorption steps of BPA-polymer adsorption onto the CD SAM were investigated by monitoring the absorbance change at 550 nm in real time. As shown in Figure 9, upon immersion into the BPA-polymer solution, the absorbance for the DTUA-β-CD-modified colloidal gold on glass chip gradually increased and leveled off. After rinsing with water, furthermore, this sensor chip was incubated in 10% (v/v) methanol-phosphate buffer (pH 7.0, 9 mM) solution. The absorbance at 550 nm slowly decreased. These absorptive changes seem to be attributed to the adsorption and desorption of the polymer onto the sensor chip surface. From the relaxation curves, the association and dissociation rate constants (kassoc and kdiss, respectively) were calculated using eqs 5 and 6.20,21

∆Absassoc ) Abst - Abso ) [polymer]kassoc∆Absmax{1 - exp[-([polymer]kassoc + kdiss)t]}/{[polymer]kassoc + kdiss} (5) ∆Absdiss ) Abst - Abstf∞ ) (Abso - Abstf∞) × exp(-kdisst) + Abstf∞ (6) where Abso, Abst, and Abstf∞ are the absorbance at time ) 0, t, and infinity, respectively. The kassoc and kdiss values for DTUA-β-CD SAM were calculated to be 7.9 × 102 M-1 min-1 and 1.8 × 10-2 min-1, respectively, and the apparent association constant (Kassoc ) kassockdiss-1) was 4.4 × 104 M-1. By the same way, the kassoc, kdiss, and Kassoc values for LP-β-CD SAM were estimated to be 8.2 × 103 M-1 min-1, 1.9 × 10-1 min-1, and 4.3 × 104 M-1, respectively. The size of the gold colloids was much larger than those of the CD derivatives and the BPA-polymer. Therefore, the particle size and its dispersion might rarely affect the Kassoc values. The kassoc and kdiss values in the LP-β-CD system were larger than those in the DTUA-β-CD system. This is probably because the steric hindrance for the bisphenol moiety in the polymer to adsorb and desorb from the secondary hydroxyl face of β-CD cavity is smaller than that from the primary hydroxyl face, resulting in the larger association and dissociation rate constants in the LP-β(19) Morokoshi, S.; Ohhori, K.; Mizukami, K.; Kitano, H. Langmuir 2004, 20, 8897. (20) Kitano, H.; Maehara, Y.; Matano, M.; Sugimura, M.; Shigemori, K. Langmuir 1997, 13, 5041. (21) Lookene, A.; Chevreuil, O.; Østergaard, P.; Olivecrona, G. Biochemistry 1996, 35, 12155.

Surface-Confined CD Effect on Bisphenol Inclusion

CD system. Furthermore, the Kassoc values obtained by the localized SPR method were much smaller than those obtained by the CV method. This is mostly due to the small surface coverage of colloidal gold on the glass surface (5% estimated from the image obtained by an atomic force microscope (AFM; see Supporting Information). Only 5% of BPA-polymers making a Brownian collision could approach the surface-confined β-CD on the colloidal gold at the most. The Kassoc value in the DTUA-β-CD system was slightly larger than that in the LP-β-CD system. The same tendency was observed in both CD-modified sensor chip (localized SPR) and CD-modified gold electrode (CV method). We think that the association constants and rate constants are quite useful for clarifying complicated adsorption processes of any polymer as a guest similar to this work, because these values give detailed information of each process in a series of molecular recognition phenomena. In conclusion, it turned out that the selectivity in the inclusion of BPs by the β-CD SAM mainly comes from the difference in the orientation of SAM, since the percents

Langmuir, Vol. 21, No. 4, 2005 1321

of coverage of the SAMs prepared in this study were almost the same. According to the governmental regulations in Japan, the concentration of BPA leached from tableware made of polycarbonate into environmental water has to be less than 2.5 ppm (11 µM), which is about one-third of the minimum concentration of BPA examined here. Therefore, the present system does not seem to be applicable directly to a practical assay of BPA. However, such a procedure will be useful in constructing various sensing systems using surface-confined artificial receptors. Acknowledgment. This work was supported by a Grant-in-Aid (16205015) from the Japan Society for the Promotion of Science. Supporting Information Available: (1) The dependence of peak current on the concentration of HQ for the DTUAβ-CD-modified electrode, (2) the dependence of peak current of HQ on the concentration of addd BPA for the DTUA-β-CDmodified electrode, and (3) photograph of the colloidal Au-fixed glass substrate taken by AFM. This material is available free of charge via the Internet at http://pubs.acs.org. LA048595P