Immobilized Fluorescent Cyclodextrin on a Cellulose Membrane as a

Anal. Chem. 2001, 73, 3126-3130

Immobilized Fluorescent Cyclodextrin on a Cellulose Membrane as a Chemosensor for Molecule Detection 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

Dansylglycine-modified cyclodextrin (DnsC4-β-CD) was prepared as a fluorescent host that is capable of being immobilized on a cellulose membrane (DnsC4-β-CD membrane). DnsC4-β-CD immobilized on the cellulose membrane decreased its fluorescence intensity 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, which is similar to DnsC4-β-CD in solution; thereby, the DnsC4-β-CD membrane is useful as a novel chemosensor for detecting molecules. This result demonstrates that the cellulose membrane is useful as a practical supporting material for various chromophoremodified cyclodextrins. Biosensors using antibodies or enzymes have been studied extensively.1-4 Because these biosensors have characteristics of high selectivity and sensitivity, biosensors have been applied mainly to the medical field;5,6 however, biosensors are fragile under practical conditions. On the other hand, chemosensors using artificial receptors are relatively stable and may overcome this drawback of biosensors. Namely, chemosensors using artificial receptors are expected to be practically useful.7 Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six or more D-(+)-glucopyranose units, and they have the ability to include various organic molecules in their central cavitys.8-10 Heretofore, many fluorescent cyclodextrins have been prepared for use as molecular sensors in solution;11-16 however, there has * Phone: +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) Karube, I. Nippon Rinsho 1992, 50, 1670-1678. (6) Bluesttein, B. I.; Walczak, I. M.; Chen, S. Y. Trends. Biotechnol. 1990, 8, 161-168. (7) Czarnik, A. W. Nature, 1998, 394, 417-418. (8) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; Springer-Verlag: New York, 1997. (9) Rekharsky, M. V.; Inoue, Y.; Chem. Rev. 1998, 98, 1875-1917. (10) Szejtli, J.; Osa, T. Comprehensive Supramolecular Chemistry; Pergamon: 1996, Vol. 3.

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been no attempt to immobilize fluorescent cyclodextrins on the solid support. From a practical point of view, the immobilization of modified CDs must be required to construct CD-based sensory devices. Dansyl moiety was chosen as a fluorophore in this study, because its properties were well characterized by the extensive studies on dansyl-modified cyclodextrins.17-25 Dansylglycinemodified cyclodextrin (DnsC4-β-CD) was prepared as a chemosensor (Figure 1a). A cellulose membrane was chosen as the supporting material, because cellulose is less costly and widely available. Additionally, it is possible to prepare a disposable molecular sensor with a cellulose membrane. The aim of our work is to construct the chemosensor for detecting the molecule. We report here a method for immobilization of a fluorescent cyclodextrin onto a cellulose membrane and show how the DnsC4-β-CD membrane (Figure 1b) acts as a novel sensor for molecular detection. EXPERIMENTAL SECTION Materials. β-cyclodextrin (β-CD) was kindly donated by Nihon Shokuhin Kako Co., Ltd., and was used as received. All of 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 (11) Ueno, A. Supramol. Chem. 1996, 3, 31-36. (12) Dunbar, R. A.; Bright, F. Supramol. Chem. 1994, 3, 93-99. (13) Narita, M.; Hamada, F. J. Chem. Soc. Perkin. Trans. 2 2000, 2, 823-832 (14) Matsumura, S.; Sakamoto, S.; Ueno, A.; Mihara, H. Chem. Eur. J. 2000, 10, 1781-1788. (15) Hossain, A.; Matsumura, S.; Kanai, T.; Hamasaki, K.; Mihara, H.; Ueno, A. J. Chem. Soc., Perkin. Trans. 2 2000, 7, 1527-1533. (16) Yu, L.; Bao-Hang, H.; Shi-Xin, S.; Takehiko, W.; Yoshihisa, I. J. Org. Chem. 1999, 64, 1487-1493. (17) Corradini, R.; Dossena, A.; Marchelli, R.; Panagia, A.; Sartor, G.; Saviano, M.; Lombardi, A.; Pavone, V. Chem. Eur. J. 1996, 4, 373-381. (18) Hamasaki, K.; Usui, S.; Ikeda, H.; Ikeda, T.; Ueno, A. Supramol. Chem. 1997, 8, 125-135. (19) Corradini, R.; Dossena, A.; Galaverna, G.; Marchelli, R.; Panagia, A.; Sator, G. J. Org. Chem. 1997, 62, 6283-6289. (20) 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. (21) Ikunaga, T.; Ikeda, H.; Ueno, A. Chem. Eur. J. 1999, 5, 2698-2704. (22) Ikeda, H.; Michiei, N.; Ise, N.; Fujio, T.; Ueno, A. J. Org. Chem. 1997, 62, 1411-1418 (23) Ueno, A.; Ikeda, A.; Ikeda, H.; Ikeda, T.; Toda, F. J. Org. Chem. 1999, 64, 382-387. (24) Ikeda, H.; Michiei, N.; Ise, N.; Naomi, O.; Nakamura, A.; Ikeda, T.; Fujio, T.; Ueno, A. J. Am. Chem. Soc. 1996, 118, 10980-10988. (25) Ju ¨ rgen, B.; Nico, S.; Antonie, V.; Arie, H.; Roeland, N.; Johan, E.; David, R. J. Am. Chem. Soc. 1999, 121, 28-33. 10.1021/ac001386z CCC: $20.00

© 2001 American Chemical Society Published on Web 05/10/2001

Figure 1. (a) Synthetic scheme of DnsC4-β-CD and (b) The Synthetic scheme for the immobilization of DnsC4-β-CD to the cellulose membrane.

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 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 using caffeic acid as the matrix. Thin-layer chromatography (TLC, n-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 using a membrane sandwiched between quartz glass plates and allocated a sharpcut filter (HOYA L-39, 390-nm sharp-cut filter) on the emission side (Figure 2). Binding Analysis of the DnsC4-β-CD Membrane. In the presence of an excess amount of guest, the binding constants (K) of the DnsC4-β-CD membrane were estimated by nonlinear curvefitting analysis using following equation to the guest-induced fluorescence variations of the membrane.26,27

∆I )

K[G]0

∆I 1 + K[G]0 max

(1)

where [G]0 represents the initial concentration of the guest, ∆I

Figure 2. Schematic representation for membrane fluorescence measurements of DnsC4-β-CD immobilized membrane.

represents the difference in the fluorescence intensity at 531 nm between the DnsC4-β-CD membrane alone and in the presence of the guest, and ∆Imax is the difference in the fluorescence intensity at 531 nm between the DnsC4-β-CD membrane alone and the complex of the DnsC4-β-CD membrane and the guest. According to eq 1, K values can be determined without knowing the exact amount of DnsC4-β-CD immobilized to a cellulose membrane. Synthesis of DnsC4-β-CD. DnsC4-β-CD was synthesized by the condensation reaction of 6-deoxy-6-(4-aminobutylamino)-β(26) Kuwabara, T.; Takamura, M.; Matsushita, A.; Ikeda, H.; Nakamura, A.; Ueno, A.; Fujio, T. J. Org. Chem. 1998, 63, 8729-8735. (27) Ikunaga, T.; Ikeda, H.; Ueno, A. Chem. Eur. J. 1999, 5, 2698-2704.

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Figure 3. Fluorescence spectra of DnsC4-β-CD (10 µM) in phosphate buffer (pH 7.0) at 25 °C at various concentrations of 1-adamantanol.

Figure 4. Fluorescence spectra of the DnsC4-β-CD membrane and a blank cellulose membrane in phosphate buffer (pH 7.0) at 25 °C. Solid line is the DnsC4-β-CD membrane, and broken line is a blank cellulose membrane. The peak ∼430 nm is the scattering light that reflects at the cellulose surface.

cyclodextrin25 (C4-β-CD) and dansylglycine. To a cooled solution (0 °C) of dansylglycine (20 mg, 65 µmol) in 500 µL of N,Ndimethylformamide (DMF) was added dicychlohexylcarbodiimide (13 mg, 65 µmol) and 1-hydroxybenzotriazole hydrate (9.8 mg, 64 µmol). The reaction mixture was stirred at 0 °C for 1 h. To the stirred solution was added C4-β-CD (36 mg, 30 µmol), and the solution was stirred at room temperature for 5 h. The reaction mixture was poured into 10 mL of acetone, giving a crude product (49 mg). This product was purified by ion-exchange column chromatography on a CM Sephadex C-25 column and eluted with 500 mL of 0.1 N ammonium hydroxide. The eluent was concentrated by evaporation and then poured into 10 mL of acetone, giving 42 mg (28 µmol, yield 94%) of DnsC4-β-CD. The product was characterized by TLC (n-butanol:ethanol:water ) 5:4:3, Rf ) 0.24) and 1H NMR (D2O, 500 MHz): δ 2.89 (s, 6H, NMe2), 3.204.05 (m), 4.90-5.10 (m, 7H, C1H), 7.32-7.38 (d, 1H, aromatic) 7.55-7.60 (t, 1H, aromatic), 7.68-7.75 (t, 1H, aromatic), 8.048.15 (d, 1H, aromatic), 8.20-8.26 (d, 1H, aromatic), 8.38-8.55 (d, 1H, aromatic); MALDI-TOFMS: MW calcd for C60H94O37N4S, 1496. Found, 1519 (M + 23). Immobilization of DnsC4-β-CD to the 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 agitated 3128 Analytical Chemistry, Vol. 73, No. 13, July 1, 2001

Figure 5. Time course of the fluorescence intensity of the DnsC4β-CD membrane in phosphate buffer (pH 7.0) at 25 °C at the addition of UDCA 100 µM. I, the fluorescence intensity at 533 nm; I0, the initial fluorescence intensity at 533 nm.

Figure 6. Fluorescence spectra of the DnsC4-β-CD membrane in phosphate buffer (pH 7.0) at 25 °C at various concentrations of 1-adamantanol.

at room temperature for 2 h (80 rpm), then the membrane was washed with deionized water four times and with phosphate buffer solution (500 mM, pH 6.0) three times. The oxidized membrane was soaked in 5 mL of phosphate buffer solution (500 mM, pH 6.0) containing 1 mM DnsC4-β-CD and 5 mM of NaCNBH3 and agitated at room temperature for 3 h (80 rpm). After the reaction, the membrane was washed with DMF, saturated NaCl solution, and deionized water at least three times each until no fluorescence was detected from the filtrate. RESULTS AND DISCUSSION Fluorescence Spectra of DnsC4-β-CD. Figure 3 shows the fluorescence spectra of DnsC4-β-CD (10 µM) in phosphate buffer solution (pH 7.0) excited at 340 nm. The spectra have a peak ∼545 nm in the absence of guest. Upon the addition of 1-adamantanol as a guest, fluorescence intensity of DnsC4-β-CD decreased along with a red shift of the emission peak (548 nm in the presence of 200 µM 1-adamantanol). This result is explained in terms of the exclusion of the dansyl moiety from the hydrophobic CD cavity to bulk water solution. Thus, DnsC4-β-CD works as a chemosensor for detecting molecules in a manner similar to that in the previously reported literature.21,22 Fluorescence Spectra of the DnsC4-β-CD Membrane. Figure 4 shows the fluorescence spectra of the DnsC4-β-CD membrane, excited at 340 nm, in phosphate buffer solution (pH 7.0). The solid line is the spectrum of the DnsC4-β-CD membrane

Figure 7. Structure of guest molecules.

Figure 8. Fluorescence titration of the DnsC4-β-CD membrane in phosphate buffer (pH 7.0) at 25 °C as a function of steroid concentration; 2, UDCA; b, HDCA; 1 DCA; 9, CDCA; O, CA.

Figure 9. ∆I/I0 values of the DnsC4-β-CD membrane for various guests in phosphate buffer (pH 7.0) at 25 °C: b, UDCA; O, 1-adamantanol; 0, cyclohexanol.

in the absence of guest. The spectrum has a peak ∼533 nm with a blue shift, as compared to the case of the solution of DnsC4-βCD. The broken line is the spectrum of a blank cellulose membrane that was measured by the same system. Most of the scattering light, which is generated on the cellulose membrane, was avoided by the sharp-cut filter (>390 nm) in the emission side. Guest-Induced Variations in Fluorescence Spectra of the DnsC4-β-CD Membrane. Figure 5 shows the time course for the fluorescence intensity of DnsC4-β-CD membrane at 533 nm. Immediately after the injection of ursodeoxycholic acid (UDCA, 100 µM), the fluorescence intensity of the DnsC4-β-CD membrane started to decrease, and the fluorescence intensity was equilibrated after 10 min. This result suggests that the response time of this system is ∼10 min. Figure 6 shows the variations in fluorescence spectra of the DnsC4-β-CD membrane upon the addition of 1-adamantanol as a guest. The fluorescence spectra of the DnsC4-β-CD membrane exhibit a slight red shift (peak at 536 nm in the presence of 200 µM of 1-adamantanol) and a decrease in the fluorescence intensity,

which is similar to the fluorescence spectra of the DnsC4-β-CD in solution. Thus, the DnsC4-β-CD membrane can act as a chemosensor for molecular detection. When the added guest was washed out with phosphate buffer solution from the DnsC4-β-CD membrane after the fluorescence measurements, the fluorescence intensity of the membrane was recovered. On the other hand, no fluorescence was detected from the solution used for the wash of the DnsC4-β-CD membrane. This result implies that the DnsC4-β-CD was not eluted from the cellulose membrane by the washing with the buffer solution. These results confirm that the DnsC4-β-CD is stably immobilized on a cellulose membrane. Binding Constants. Nine compounds were chosen as guests for the evaluation of guest-binding abilities of the DnsC4-β-CD membrane soaked in aqueous solution. The chosen guests included five steroids, two terpenes, 1-adamantanol, and cyclohexanol, and all of them are well-known as typical guests for β-CD. Their structures are shown in Figure 7. The data obtained for the DnsC4-β-CD membrane are finely fitted to the theoretical Analytical Chemistry, Vol. 73, No. 13, July 1, 2001

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Table 1. Binding Constants and Sensitivity Parameters (∆I/I0) of the DnsC4-β-CD Membrane for Various Guestsa DnsC4-β-CD membrane guest

∆I20/I0b

K/M-1

cyclohexanol (-)-borneol (+)-borneol 1-adamantanol ursodeoxycholic acid (UDCA) hyodeoxycholic acid (HDCA) deoxycholic acid (DCA) chenodeoxycholic acid (CDCA) cholic acid (CA)

CDCA > (+)borneol, (-)-borneol, 1-adamantanol, CA, DCA > cyclohexanol.

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The guest selectivity of the DnsC4-β-CD membrane is roughly parallel to that of the DnsC4-β-CD in solution. Figure 9 indicates the selectivity of the DnsC4-β-CD membrane for a particular compound in a mixture of several substances. These results show that the DnsC4-β-CD membrane can detect the strength by which the guest is bound, such as UDCA selectively in a concentration up to 50 µM. CONCLUSION Fluorescence measurements and binding analyses of DnsC4β-CD membranes demonstrate that the cellulose membrane is a useful supporting material for the immobilization of chromophoremodified cyclodextrins. As an application of this method, disposable sensor chips or papers may be developed for molecular detection. 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 November 28, 2000. Accepted March 28, 2001. AC001386Z