Fluorescent Cyclodextrin Immobilized on a Cellulose Membrane as a

Anal. Chem. 2001, 73, 1877-1880

Fluorescent Cyclodextrin Immobilized on a Cellulose Membrane as a Chemosensor System for Detecting Molecules Tetsuya Tanabe, Kazuhiro Touma, Keita Hamasaki, and Akihiko Ueno*

Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Dansyl glutamate-modified cyclodextrin (DnsGlu-β-CD) was prepared as a fluorescent host, which is capable of being immobilized on a cellulose membrane (DnsGlu-βCD-membrane). The fluorescence intensity of DnsGlu-βCD decreased with increasing concentration of guest molecules, indicating that the host changes the location of the dansyl group from inside to outside the cyclodextrin cavity upon guest accommodation. Similar guest-induced decrease in the fluorescence intensity was observed for DnsGlu-β-CD immobilized to a cellulose membrane. This result demonstrates that the cellulose membrane may be used as a practical supporting material of various chromophore-modified cyclodextrins and that DnsGlu-β-CDmembrane is useful as a novel disposable chemosensor for molecules. Biosensors using antibody or enzyme have been studied extensively.1-4 Since these biosensors have characteristics of high selectivity and sensitivity, biosensors have been mainly applied to the medical field. However, the biosensors have several demerits that may cancel the advantages because they are easily inactivated in a short time under practical conditions such as high salt, strong acid or base, and drying. From the viewpoint of overcoming the demerits of biosensors, chemosensors using artificial receptors are of practical use.5 There have been various studies on the use of fluorescent cyclodextrins for detecting molecules.6-10 Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six or more of D-(+)* Corresponding author: (tel) +81-45-924-5757; (fax) +81-45-924-5833; (e-mail) [email protected] (1) Benkert, A.; Scheller, F.; Scho¨ssler, W.; Hentschel, C.; Micheel, B.; Behesing. O.; Scharte, G.; Sto ¨cklein, W.; Warsinke, A. Anal. Chem. 2000, 72, 916921. (2) Anzai, J.; Takeshita, H.; Kobayashi, Y.; Osa, T.; Hoshi, T. Anal. Chem. 1998, 70, 811-817. (3) Bu, H.; Mikkelsen, S. R.; English, A. M. Anal. Chem. 1998, 70, 43204325. (4) Katz, E.; Heleg-Shabtai, V.; Willner, I.; Rou, H. K.; Haehnel, W. Angew. Chem., Int. Ed. 1998, 37, 3253-3256. (5) Czarnik, A. W. Nature 1998, 394, 417-418. (6) Ueno, A. Supramol. Chem. 1996, 3, 31-36. (7) Dunbar, R. A.; Bright, F. Supramol. Chem. 1994, 3, 93-99. (8) Narita, M.; Hamada, F. J. Chem. Soc., Perkin. Trans. 2 2000, 2, 823-832. (9) Matsumura, S.; Sakamoto, S.; Ueno, A.; Mihara, H. Chem. Eur. J. 2000, 10, 1781-1788. (10) Hossain, A.; Matsumura, S.; Kanai, T.; Hamasaki, K.; Mihara, H.; Ueno, A. J. Chem. Soc., Perkin. Trans. 2 2000, 7, 1527-1533. 10.1021/ac001062a CCC: $20.00 Published on Web 03/14/2001

© 2001 American Chemical Society

glucopyranose units and are stable under practical conditions. They form inclusion complexes with various organic compounds in aqueous solution.11-13 Heretofore, many fluorescent cyclodextrins were prepared for use as molecular sensors in solution. However, there has been no attempt to immobilize florescent cyclodextrins on the solid support. From a practical point of view, immobilization of modified CDs may be required to construct CDbased sensory devices. The dansyl moiety is chosen as a fluorophore in this study, because its properties are well characterized from extensive studies on dansyl-modified cyclodextrins.14-21 Dansyl L-glutamate-modified cyclodextrin (DnsGlu-β-CD) was prepared as a chemosensor, and cellulose membrane was chosen as the supporting material with the view of constructing the disposable sensor (Figure 1). We report here a novel method for immobilization of the fluorescent cyclodextrin to a cellulose membrane and the character of the DnsGlu-β-CD-membrane as the sensor paper for detecting molecules. EXPERIMENTAL SECTION Materials. β-CD was kindly donated by Nihon Shokuhin Kako Co., Ltd., and was used as received. All the guest compounds were purchased from Tokyo Kasei and were used without further purification. Cellulose membrane was obtained from Adventec Co. The water used for photometry and deuterium oxide used for NMR measurements were obtained from Cica-Merck and Merck, respectively. Measurements. 1H NMR spectra were measured on a Varian VXR 500S spectrometer (500 MHz). HDO (δ ) 4.70) was used (11) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; Springer-Verlag: New York, 1997. (12) Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875-1917. (13) Szejtli, J.; Osa, T. Comprehensive Supramolecular Chemistry; Pergamon: New York, 1996; Vol. 3. (14) Corradini, R.; Dossena, A.; Marchelli, R.; Panagia, A.; Sartor, G.; Saviano, M.; Lombardi, A.; Pavone, V. Chem. Eur. J. 1996, 4, 373-381. (15) Hamasaki, K.; Usui, S.; Ikeda, H.; Ikeda, T.; Ueno, A. Supramol. Chem. 1997, 8, 125-135. (16) Corradini, R.; Dossena, A.; Galaverna, G.; Marchelli, R.; Panagia, A.; Sator, G. J. Org. Chem. 1997, 62, 6283-6289. (17) Nelissen, H. F. M.; Venema, F.; Uittenbogaard, R. M.; Feiters, M. C.; Nolte, R. J. M. J. Chem. Soc., Perkin. Trans. 2 1997, 10, 2045-2053. (18) Ikunaga, T.; Ikeda, H.; Ueno, A. Chem. Eur. J. 1999, 5, 2698-2704. (19) Ikeda, H.; Michiei, N.; Ise, N.; Fujio, T.; Ueno, A. J. Org. Chem. 1997, 62, 1411-1418. (20) Ueno, A.; Ikeda, A.; Ikeda, H.; Ikeda, T.; Toda, F. J. Org. Chem. 1999, 64, 382-387. (21) Ikeda, H.; Michiei, N.; Ise, N.; Naomi, O.; Nakamura, A.; Ikeda, T.; Fujio, T.; Ueno, A. J. Am. Chem. Soc. 1996, 118, 10980-10988.

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represents the initial concentration of DnsGlu-β-CD, ∆I represents the difference in the fluorescence intensity at 540 nm between DnsGlu-β-CD alone and in the presence of the guest, and ∆Imax is the difference in the fluorescence intensity at 540 nm between DnsGlu-β-CD alone and the one totally complexed with the guest. Binding Analysis of DnsGlu-β-CD-membrane. In the case of DnsGlu-β-CD-membrane, when the guest concentration is excess ([G]0 . [H]0), free guest concentration is almost the same as that of initial guest concentration. Thus the binding constant (K) is approximated to the following equation.

K≈

Figure 1. Structures of DnsGlu-β-CD and DnsGlu-β-CD-membrane.

[HG] ([H]0 - [HG])[G]0

where [H], [G], and [HG] represent free host, free guest, and host-guest complex, respectively. The concentration of the complex, [HG], is reflected in the magnitude of ∆I. In this case, ∆I is the difference in the fluorescence intensity at 525 nm. Using the values of [H]0, [G]0, ∆I, and ∆Imax, eq 2 leads to the following representation.23

∆I )

Figure 2. Schematic representation for membrane fluorescence measurements of DnsGlu-β-CD immobilized membrane.

as an internal standard. Matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry (MALDI-TOFMS) was performed on a Shimadzu Kratos Kompact MALDI III mass spectrometer with caffeic acid as the matrix. Thin-layer chromatography (TLC, 1-butanol/ethanol/water 5:4:3) was carried out with silica gel 60 F254 (Merck Co.). Fluorescence spectra were measured on a Hitachi fluorescence spectrometer F-2500. Fluorescence of the membrane was measured with a membrane sandwiched between quartz glass plates and allocated a sharp cut filter (Hoya L-39: 390-nm sharp cut filter) on the emission side (Figure 2). Binding Analysis of DnsGlu-β-CD. The binding constants (K) of DnsGlu-β-CD were estimated by nonlinear curve-fitting analysis using the following equation to the guest-induced fluorescence variations:22

∆I ) ((K[G]0 + 1 + K[H]0 -

x(K[G]0 + 1 + K[H]0)2 - 4K2[H]0[G]0 )/2K[H]0)∆Imax (1) where [G]0 represents the initial concentration of guest, [H]0 1878

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(2)

K[G]0 1 + K[G]0

(3)

According to eq 3, which does not include [H]0, K values can be estimated without knowing the amount of DnsGlu-β-CD immobilized to a cellulose membrane. Syntheses. DnsGlu-β-CD was synthesized by the condensation reaction of 6-deoxy-6-amino-β-CD24 and Boc-L-Glu(Bzl)-OH in the presence of 1,3-dicyclohexylcarbodiimide (DCC) followed by deprotection. DnsGlu-β-CD was immobilized to a cellulose membrane. A cellulose membrane was oxidized by NaIO4, and the reaction of aldehyde derived on cellulose with 1,6-hexanediamine gave 1,6-hexanediamine-modified cellulose. Then, DnsGlu-β-CDmembrane was prepared by the condensation reaction of DnsGluβ-CD and 1,6-hexanediamine-modified cellulose in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Synthesis of Mono-6-(O5-Bzl-N-Boc-glutamylamino)-6deoxy-β-CD (Boc-Glu(Bzl)-β-CD). Boc-Glu(Bzl)-β-CD was synthesized by the condensation reaction of 6-deoxy-6-amino-β-CD and Boc-Glu(Bzl)-OH. Boc-Glu(Bzl)-OH (245 mg, 0.617 mmol), DCC (382 mg, 1.85 mmol), and 1-hydroxybenzotriazole hydrate (284 mg, 1.85 mmol) were dissolved in 1 mL of DMF and the reaction mixture was stirred for 1 h at 0 °C. Then, 6-deoxy-6-aminoβ-CD (700 mg, 0.617 mmol) was added and the resultant mixture was stirred at room temperature for another 2 h. After filtration, the filtrate was poured into 20 mL of acetone. The precipitates were collected by filtration, giving a white powder (815 mg, yield 92.0%). The purity of the product was confirmed by TLC (1butanol/ethanol/water 5:4:3, Rf ) 0.57) as single spot. Deprotection of Boc-Glu(Bzl)-β-CD. Boc-Glu(Bzl)-β-CD (170 mg, 0.118 mmol), palladium on charcoal (Pd/C, 17 mg), and (+)(22) Kuwabara, T.; Nakamura, A.; Ueno, A.; Toda, F. J. Phys. Chem. 1994, 98, 6297. (23) Kuwabara, T.; Takamura, M.; Matsushita, A.; Ikeda, H.; Nakamura, A.; Ueno, A.; Fujio, T. J. Org. Chem. 1998, 63, 8729-8735. (24) Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno A.; Toda, F.; Suzuki, I.; Osa, T. J. Am. Chem. Soc. 1993, 115, 5035-5340.

Figure 3. Fluorescence spectra of (A) DnsGlu-β-CD (20 µM) and (B) DnsGlu-β-CD-membrane in phosphate buffer (pH 7.3) at 25 °C at various concentrations of 2-adamantanol.

borneol (55 mg, 0.357 mmol) were dissolved in 20% methanol aqueous solution (40 mL) in 400-mL scale of the autoclave, and then stirred for 15 h at room temperature in the presence of 4 atm of hydrogen gas. After the reaction, the hydrogen was vented from the autoclave and Pd/C was removed by filtration. The methanol was removed by evaporation, the residue was dissolved in DMF, and then the solution was poured into 20 mL of acetone. The precipitate was collected by filtration and dried. The product was obtained as a white powder (130 mg, yield 96%). The crude Boc-Glu-β-CD was dissolved in TFA (10 mL), and the solution was stirred at 0 °C for 30 min. To the reaction mixture, 1,4-dioxane (100 mL) was added, and the solution was removed by evaporation. The product was lyophilized and obtained as a white powder (120 mg, yield 98.0%). The product was characterized by TLC (1butanol/ethanol/water 5:4:3, Rf ) 0.13): 1H NMR (D2O, 500 MHz) δ 2.08-2.20 (m, 2H), 2.40-2.48 (m, 2H), 3.30-3.92 (m), 4.05 (t, 1H, COCH), 4.95-5.05 (m, 7H, C1H); MALDI-TOFMS MW calculated for C47H78O37N2, 1263; found, 1286 (M + 23). Synthesis of Mono-6-(N-dansylglutamylamino)-6-deoxyβ-CD (DnsGlu-β-CD). DnsGlu-β-CD was synthesized by the reaction of Glu-β-CD and dansyl chloride. Glu-β-CD (140 mg, 0.111 mmol), dansyl chloride (34 mg, 0.133 mmol), and N,N-diisopropylethylamine (129 µL, 0.777 mmol) were dissolved in 10 mL of DMF, and then the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was poured into 100 mL of acetone. The precipitate was collected on a membrane filter, giving a yellow powder. This crude product was purified by HPLC, and the fractions containing the desired compound were collected. The product was obtained as lyophilized white powder (41.5 mg, yield 25%). The product was characterized by TLC (1-butanol/ ethanol/water 5:4:3, Rf ) 0.53): 1H NMR (D2O, 500 MHz) δ 1.582.21 (m), 3.21 (s, 6H, NMe2), 3.40-4.10 (m), 4.85-5.10 (m, 7H, C1H), 7.70-7.82 (m, 3H, aromatic), 8.29 (d, 1H, aromatic), 8.508.58 (dd, 2H, aromatic);, MALDI-TOFMS MW calculated for C59H89O39N3S, 1496; found, 1520 (M + 23). Immobilization of DnsGlu-β-CD to Cellulose Membrane. A cellulose membrane (1.2 g, diameter 11 cm, thickness 0.3 mm) was soaked in 0.5 N NaIO4 solution (20 mL) and shaken at room temperature for 1 h (80 rpm), and then the membrane was washed

with deionized water four times and with phosphate buffer (500 mM, pH 6.0) three times. The oxidized membrane was soaked in 5 mL of phosphate buffer (500 mM, pH 6.0) containing 50 mM 1,6-hexanediamine and 5 mM NaCNBH3 and shaken at room temperature for 3 h (80 rpm). Then, DnsGlu-β-CD was immobilized by the condensation reaction with the amino group of 1,6-hexanediamine-modified cellulose membrane. The membrane was soaked in 5 mL of aqueous solution (pH 4.7; adjusted with 1 N HCl) containing 1 mM DnsGlu-β-CD and 0.1 M EDC and shaken at room temperature for 3 h (80 rpm). After the reaction, the membrane was washed by DMF, saturated NaCl solution, and deionized water until no fluorescence was detected from the filtrate. RESULTS AND DISCUSSION Fluorescence Spectra of DnsGlu-β-CD. Figure 3A show the fluorescence spectra of DnsGlu-β-CD (20 µM) in phosphate buffer (pH 7.3) measured by excitation at 340 nm. The spectra have a peak around 530 nm in the absence of guest. Upon addition of 2-adamantanol as a guest, the fluorescence intensity of DnsGluβ-CD decreased with a red shift (peak at 550 nm in the presence of 200 µM 2-adamantanol). This result is explained in terms of exclusion of the dansyl moiety from the hydrophobic CD cavity to bulk water solution. Thus, DnsGlu-βCD in aqueous solution works as a chemosensor for detecting molecules as similar to the previously reported ones.19,20 Fluorescence Spectra of DnsGlu-β-CD-membrane. Figure 3B shows the fluorescence spectra of DnsGlu-β-CD-membrane in phosphate buffer (pH 7.3) and measured by excitation at 345 nm. The spectra have a peak around 520 and 430 nm. The peak around 520 nm is derived from the dansyl of immobilized DnsGlu-β-CD and the peak around 430 nm is scattering excitation light. Though most of scattering light that reflects on the cellulose membrane was cut by the sharp cut filter (>390 nm) on the emission side, the remainder of the scattering light was detected. Upon the addition of 2-adamantanol as a guest, the fluorescence spectra of DnsGlu-β-CD-membrane exhibit a red shift (peak at 530 nm in the presence of 200 µM 2-adamantanol) and decrease in the fluorescence intensity, similar to the fluorescence spectra of Analytical Chemistry, Vol. 73, No. 8, April 15, 2001

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Table 1. Binding Constants of DnsGlu-β-CD and DnsGlu-β-CD-membrane for Various Guestsa

a

guest

DnsGlu-β-CD,b K/M-1

DnsGlu-β-CD-membrane, K/M-1

(-)-borneol (+)-borneol 2-adamantanol ursodeoxycholic acid (UDCA) hyodeoxycholic acid (HDCA) deoxycholic acid (DCA) chenodeoxycholic acid (CDCA)

11000 ( 1700 3700 ( 90 14000 ( 300 23000 ( 180 19000 ( 6000 330 ( 200 1500 ( 440

6100 ( 490 9200 ( 1400 13000 ( 870 25000 ( 1200 21000 ( 2000 9200 ( 2500 8700 ( 360

Measured in phosphate buffer (pH 7.3) at 25 °C. b The concentration of DnsGlu-β-CD is 20 µM.

Figure 4. Structures of guest molecules.

DnsGlu-β-CD in solution. Thus, DnsGlu-β-CD-membrane can act as a chemosensor for detecting molecules on the cellulose membrane. When added guest was washed out with phosphate buffer from the DnsGlu-β-CD-membrane, the fluorescence intensity of the membrane was recovered, even after the detection of guests. On the other hand, no fluorescence was detected from the solution when the DnsGlu-β-CD-membrane was removed. This result implies that DnsGlu-β-CD was not removed from the cellulose membrane by washing. Namely, DnsGlu-β-CD is immobilized on a cellulose membrane. Binding Constants. Seven compounds were chosen as guests for evaluation of the guest-binding abilities of DnsGlu-β-CD in solution and on the cellulose membrane. The selected guests were four steroids, two terpenes, and 2-adamantanol, and all of them are well known as typical guests for β-CD. Their structures are shown in Figure 4. The data obtained for DnsGlu-β-CD and its immobilized membrane are nicely fitted to the theoretical curves obtained by eqs 1 and 3. The binding constants are summarized

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in Table 1. The guest selectivity of DnsGlu-β-CD-membrane is somewhat different from that of DnsGlu-β-CD in solution. The binding constants of DnsGlu-β-CD in solution for UDCA, HDCA, CDCA, and DCA are 23 000, 19 000, 1800, and 330 M-1, respectively, with the order of the values of UDCA > HDCA . CDCA > DCA. On the other hand, the binding constants of DnsGlu-βCD-membrane for UDCA, HDCA, CDCA, and DCA are 25 000, 21 000, 8700, and 9200 M-1, respectively, with the order of the values of UDCA > HDCA > DCA > CDCA. The binding constants for CDCA and DCA of DnsGlu-β-CD-membrane are much larger than those of DnsGlu-β-CD in solution. On the other hand, the binding constants of (-)- and (+)-borneol of DnsGlu-β-CDmembrane were also different from those of DnsGlu-β-CD solution. Since there is a free carboxyl group in DnsGlu-β-CD, it is suggested that these differences in guest binding are due to the presence of the carboxyl group of DnsGlu-β-CD. CONCLUSION Fluorescence measurements and binding analyses of the DnsGlu-β-CD-membrane demonstrate that the cellulose membrane can be a useful supporting material for immobilization of various chromophore-modified cyclodextrins. As application of this method, sensor chips or sensor papers such as pH indicator papers may be produced. The study for preparing moleculeresponsive color-change paper is now underway. ACKNOWLEDGMENT We are grateful to Nihon Shokuhin Kako Co., Ltd., for the generous gift of β-CD. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan. Received for review September 7, 2000. Accepted January 23, 2001. AC001062A