Continuous Glucose Detection Using Boronic Acid-Substituted

Graham J. Worsley , Guilhem A. Tourniaire , Kathryn E. S. Medlock , Felicity K. Sartain , Hazel E. Harmer , Michael Thatcher , Adrian M. Horgan , John...
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Langmuir 2006, 22, 9067-9074

9067

Continuous Glucose Detection Using Boronic Acid-Substituted Viologens in Fluorescent Hydrogels: Linker Effects and Extension to Fiber Optics Soya Gamsey, Jeff T. Suri,† Ritchie A. Wessling, and Bakthan Singaram* Department of Chemistry and Biochemistry, UniVersity of California, Santa Cruz, California 95064 ReceiVed June 13, 2006. In Final Form: July 28, 2006 A fluorescent anionic dye and a viologen appended with boronic acids, which serve as glucose receptors, have been synthesized and immobilized into a poly(2-hydroxyethyl methacrylate) hydrogel for use as a continuous glucose monitor. The fluorescence of the dye is modulated by the quenching efficiency of the viologen-based receptor, which in turn is dependent on the glucose concentration. Two monomeric versions of the quencher/receptor unit were prepared and their performance within the hydrogel evaluated. By tethering the quencher/receptor to the hydrogel matrix using a single-point attachment, slightly improved glucose sensing was observed. The hydrogels were tested for their ability to continuously and reversibly detect glucose over the course of several hours. The tests were carried out using a cuvette-based system, as well as a fiber-optic-based configuration. Under physiological conditions (0.1 M phosphate buffer, pH 7.4, 37 °C), the fluorescent hydrogels display an excellent dynamic response to glucose concentrations within the biologically significant range (2.5-20 mM).

Introduction The ability to accurately and conveniently monitor blood glucose concentrations has proven essential for the management of diabetes.1 The use of glucose-responsive polymers is one of the most promising methods for attaining a continuous glucose monitor (CGM) for in vivo applications. Many of the polymeric systems reported are enzyme-based biosensors, in which glucose oxidase (GOx) or glucose dehydrogenase (GDH) is immobilized or otherwise entrapped within a glucose-permeable matrix.2 An alternative methodology involves the incorporation of syntheticbased receptors, such as boronic acids, into polymers. Due to their ability to efficiently and reversibly bind diols,3-7 boronic acids have been extensively studied and have been successfully utilized as effective glucose receptors.8 For example, many solution-based glucose detection systems have been developed in which fluorescence spectroscopy is used to monitor changes in a boronic acid-appended fluorophore upon glucose binding.9-11 Far fewer systems exist, however, in which the boronic acid * To whom correspondence should be addressed. Phone: (831) 4593154. Fax: (831) 459-2935. E-mail: [email protected]. † Current address: GluMetrics, Inc., 15375 Barranca Pkwy., Suite I-108, Irvine, CA 92618. (1) (a) Heinemann, L.; Schmelzeisen-Redeker, G. Diabetologia 1998, 41, 848. (b) McNichols, R. J.; Cote, G. L. J. Biomed. Opt. 2000, 5, 5. (c) Khalil, O. S. Clin. Chem. 1999, 165. (d) Koschinsky, T.; Heinemann, L. Diabetes/Metab. Res. ReV. 2001, 113. (2) For reviews see: (a) Wang, J. Sens. Update 2002, 10, 107-119. (b) Wilson, G. S.; Hu, Y. Chem. ReV. 2000, 100, 2693-2704. (c) Abel, P. U.; von Woedtke, T. Biosens. Bioelectron. 2002, 17, 1059-1070. (d) Staiano, M.; Bazzicalupo, P.; Rossi, M.; D’Auria, S. Mol. BioSyst. 2005, 1, 354-362. (3) Lorand, J. P.; Edwards, J. J. Org. Chem. 1959, 24, 769. (4) James, T. D.; Sandanayake, K. R. A. S.; Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995, 117, 8982-8987. (5) Norrild, J. C.; Sotofte, I. J. Chem. Soc., Perkin Trans. 2 2001, 727-732. (6) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291-5300. (7) Rohovec, J.; Maschmeyer, T.; Aime, S.; Peters, J. A. Chem.sEur. J. 2003, 9, 2193-2199. (8) For reviews see: (a) Wang, W.; Gao, X.; Wang, B. Curr. Org. Chem. 2002, 6, 1285-1317. (b) James, T. D.; Shinkai, S. Top. Curr. Chem. 2002, 218, 159200. (c) Striegler, S. Curr. Org. Chem. 2003, 7, 81-102. (d) Cao, H.; Heagy, M. D. J. Fluoresc. 2004, 14, 569-584. (9) For reviews see: (a) Pickup, J. C.; Hussain, F.; Evans, N. D.; Rolinski, O. J.; Birch, D. J. S. Biosens. Bioelectron. 2005, 20, 2555-2565. (b) Moschou, E. A.; Sharma, B. V.; Deo, S. K.; Daunert, S. J. Fluoresc. 2004, 14, 535-547. (c) Fang, H.; Kaur, G.; Wang, B. J. Fluoresc. 2004, 14, 481-489.

receptor moiety is immobilized for use as a CGM. Saccharidesensitive polymers have been made by incorporation of boronic acids into polylysine,12-15 poly(N-vinyl-2-pyrrolidone),16 polyacrylamide,17 poly[[(N,N-dimethylamino)propyl]acrylamide-coN,N-dimethylacrylamide],18 poly[N-vinyl-2-pyrrolidone-co[(N,N-dimethylamino)propyl]acrylamide],19 poly(styrene-comaleic acid),20 polyaniline,21 and hydrogels.22-29 Boronic acidmodified sensor molecules have also been immobilized onto (10) Czarnik, A. W. Fluorescent Chemosensors for Ion and Molecule Recognition; American Chemical Society: Washington, DC, 1993. (11) Yoon, J.; Czarnik; A. W. J. Am. Chem. Soc. 1992, 114, 5874-5875. (12) Nagasaki, T.; Kimura, T.; Arimori, S.; Shinkai, S. Chem. Lett. 1994, 1495. (13) Kimura, T.; Arimori, S.; Takeuchi, M.; Nagasaki, T.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 1995, 1889. (14) Kimura, T.; Takeuchi, M.; Nagasaki, T.; Shinkai, S. Tetrahedron Lett. 1995, 36, 559. (15) Kobayashi, H.; Nakashima, K.; Ohshima, E.; Hisaeda, Y.; Hamachi, I.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 2000, 997-1002. (16) Kitano, S.; Koyama, Y.; Kataoka, K.; Okano, T.; Sakurai, S. J. Controlled Release 1992, 19, 162-170. (17) Kanekiyo, Y.; Sato, H.; Tao, H. Macromol. Rapid Commun. 2005, 26, 1542-1546. (18) Hisamitsu, I.; Kataoka, K.; Okano, T.; Sakurai, Y. Pharm. Res. 1997, 14, 289-293. (19) Kitano, S.; Hisamitsu, I.; Koyama, Y.; Kataoka, K.; Okano, T.; Sakurai, Y. Polym. AdV. Technol. 1991, 2, 261-264. (20) Arimori, S.; Bell, M. L.; Oh, C. S.; Frimat, K. A.; James, T. D. Chem. Commun. 2001, 1836-1837. (21) Pringsheim, E.; Terpetschnig, E.; Piletsky, S. A.; Wolfbeis, O. S. AdV. Mater. 1999, 10, 865-868. (22) Suri, J. T.; Cordes, D. B.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Angew. Chem., Int. Ed. 2003, 42, 5857-5859. (23) Cappuccio, F. E.; Suri, J. T.; Cordes, D. B.; Wessling, R. A.; Singaram, B. J. Fluoresc. 2004, 14, 521-533. (24) Lee, M.-C.; Kabilan, S.; Hussain, A.; Yang, X.; Blyth, J.; Lowe, C. R. Anal. Chem. 2004, 76, 5748-5755. (25) (a) Matsumoto, A.; Yoshida, R.; Kataoka, K. Biomacromolecules 2004, 5, 1038-1045. (b) Matsumoto, A.; Ikeda, S.; Harada, A.; Kataoka, K. Biomacromolecules 2003, 4, 1410-1416. (26) Lee, Y.-J.; Pruzinsky, S. A.; Braun, P. V. Langmuir 2004, 20, 30963106. (27) Alexeev, V. L.; Sharma, A. C.; Goponenko, A. V.; Das, S.; Lednev, I. K.; Wilcox, C. S.; Finegold, D. N.; Asher, S. A. Anal. Chem. 2003, 75, 23162323. (28) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I. J. Phys. Chem. B 2001, 105, 8196-8202. (29) Kataoka, K.; Miyazaki, H.; Bunya, M.; Okano, T.; Sakurai, Y. J. Am. Chem. Soc. 1998, 120, 12694-12695.

10.1021/la0617053 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/15/2006

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solid supports such as cellulose membranes30 and onto various electrodes.31-35 We have developed a two-component sensing system,36-39 comprising a fluorescent anionic dye and a cationic boronic acidcontaining benzyl viologen, in which the fluorescence of the dye is modulated in response to varying glucose concentrations.40-43 The modified benzyl viologen acts dually as a fluorescence quencher and a glucose receptor. We recently demonstrated the feasibility of this system toward potential in vivo use by immobilizing the sensing components in a thin-film poly(2hydroxyethyl methacrylate) (p(HEMA)) hydrogel, which functioned as a reversible CGM under physiological conditions in vitro.22,23 HEMA-based hydrogels are particularly well suited for use in biological systems due to their lack of toxicity44 and resistance to degradation.45 Immobilization of the solution-based sensing system was accomplished by synthetically appending the dye and quencher/receptor components with reactive linkers for copolymerization with the hydrogel network. The nature of these linkers has since been modified, and we now report the development of a glucose-responsive p(HEMA) hydrogel with improved sensing performance. The effect of a single-linker versus a double-linker attachment of the quencher/ receptor to the hydrogel was studied as a way to enhance fluorescence signal modulation and shorten the response time of the sensor. The syntheses of the optimized dye and quencher/ receptor monomers and their use in p(HEMA) hydrogels for real-time glucose detection are described herein. A continuous testing platform for the hydrogel utilizing a fiber-optic-based sensor configuration is also illustrated. Experimental Section Materials. All solvents and reagents used in the synthesis of the monomeric sensing components were purchased from Aldrich, except NH4OH (from Acros) and 4-(2-aminoethyl)pyridine (from TCI America). Dimethylformamide (DMF) was stirred over CaH2 and filtered prior to use. Dichloromethane (DCM) was distilled from CaH2 prior to use. For hydrogel synthesis, HEMA and 3-sulfopropyl methacrylate potassium salt (SPM) were purchased from Aldrich, poly(ethylene glycol) (1000) dimethacrylate (PEGDMA) was purchased from Polysciences, Inc., 2,2′-azobis[2-(2-imidazolin-2yl)propane] dihydrochloride (VA-044) was purchased from Wako (30) Kawanishi, T.; Romey, M. A.; Zhu, P. C.; Holody, M. Z.; Shinkai, S. J. Fluoresc. 2004, 14, 499-512. (31) Kikuchi, A.; Suzuki, K.; Okabayashi, O.; Hoshino, H.; Kataoka, K.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 823-828. (32) Murakami, H.; Akiyoshi, H.; Wakamatsu, T.; Sagara, T.; Nakashima, N. Chem. Lett. 2000, 940-941. (33) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2002, 124, 12486-12493. (34) Takahashi, S.; Kashiwagi, Y.; Hoshi, T.; Anzai, Jun. Anal. Sci. 2004, 20, 757-759. (35) Takahashi, S.; Anzai, J. Langmuir 2005, 21, 5102-5107. (36) For two-component glucose-sensing systems using an Alizarin Red-based competitive assay, see: (a) Arimori, S.; Ward, C. J.; James, T. D. Tetrahedron Lett. 2002, 43, 303-305. (b) Springsteen, G.; Wang, B. J. Chem. Soc., Chem. Commun. 2001, 17, 1608-1609. (c) Reference 6. (37) For a review of E. V. Anslyn’s two-component system for sensing other analytes, see: Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963-972. (38) Arimori, S.; Murakami, H.; Takeuchi, M.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1995, 9, 961-962. (39) DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir 2002, 18, 7785-7787. (40) Camara, J. N.; Suri, J. T.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Tetrahedron Lett. 2002, 43, 1139-1141. (41) Suri, J. T.; Cordes, D. B.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Langmuir 2003, 19, 5145-5152. (42) Cordes, D. B.; Gamsey, S.; Sharrett, Z.; Miller, A.; Thoniyot, P.; Wessling, R. A.; Singaram, B. Langmuir 2005, 21, 6540-6547. (43) Cordes, D. B.; Miller, A.; Gamsey, S.; Sharrett, Z.; Thoniyot, P.; Wessling, R. A.; Singaram, B. Org. Biomol. Chem. 2005, 3, 1708-1713. (44) Cifkova, I.; Brynda, E.; Mandys, V.; Stol, M. J. Controlled Relase 1988, 9, 372. (45) Kalal, J. Makromol. Chem., Suppl. 1984, 7, 31.

Gamsey et al. Pure Chemical Industries, Ltd., and dichlorodimethylsilane was purchased from Acros. All aqueous solutions were prepared with water that was purified by a Barnstead NANOpure system (17.7 MΩ/cm). Phosphate buffer (0.1 M ionic strength, pH 7.4) was freshly prepared from KH2PO4 and Na2HPO4. Instrumentation and General Methods. Synthetic manipulations were performed using standard syringe techniques and carried out in oven-dried glassware under an argon atmosphere. 1H NMR spectra were recorded on a Varian spectrometer at 500 MHz and are reported in parts per million with respect to the peak for TMS (δ ) 0). Proton-decoupled 13C NMR spectra were recorded on a Varian instrument at 125 MHz and are reported in parts per million (due to relaxed 13C-11B spin-spin coupling, signals for carbons directly attached to boron are not observed). 11B NMR spectra were recorded on a Bruker instrument at 80.25 MHz and are reported in parts per million with respect to the peak for BF3:OEt2 (δ ) 0). pH measurements were taken on a Denver Instrument UB-10 pH/mV meter and calibrated with standard buffer solutions (pH 4, 7, and 10 from Fisher). All non-fiber-optic-based fluorescence measurements were taken on a Perkin-Elmer LS50-B luminescence spectrometer controlled using FL WinLab (version 2.0) software. Solution-based experiments were performed using standard quartz fluorescence cuvettes (10 mm path length, 3 mL capacity) and were carried out at 25 °C. For fluorescence measurements on hydrogels mounted in a flow-through cuvette, the cuvette was held in the spectrometer using a front surface accessory, and studies were carried out at 37 °C. Fluorescence measurements on hydrogels mounted on the tip of a fiber-optic cable were taken on an Ocean Optics SF2000 fluorescence spectrophotometer controlled via OOIBase32 (version 2.0) software and were carried out at 37 °C. Since HPTS is sensitive to pH changes, in all fluorescence-based experiments the pH was strictly maintained at 7.4. Synthesis. The syntheses of dye intermediates 5 and 6 have been previously reported.23 The syntheses of quencher/receptor intermediates 7, 10, and 11 have been previously reported.42 NE-Methacryloyl-(S)-lysine (3). 3 was prepared according to the literature procedure.46 See the Supporting Information for details. Polymerizable Dye 1. To a suspension of 3 (1.8 g, 8.4 mmol) in DCM (40 mL) was added chlorotrimethylsilane (1.3 mL, 10.2 mmol). The reaction mixture became clear, and N,N-diisopropylethylamine (1.8 mL, 10.2 mmol) was then added. To this was added a solution of 6 (0.93 g, 1.7 mmol) in DCM (40 mL), after which the reaction mixture turned green and then red. After being stirred at room temperature for 16 h, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in a small amount of methanol and dripped into rapidly stirring 3 M HCl (500 mL). The resulting yellow precipitate was filtered, washed with 1 M HCl and hexanes, and air-dried. Purification by flash column chromatography on silica gel, using a slow gradient of 10-15% concentrated NH4OH in 2-propanol, afforded 1 as an orange powder (0.5 g, 28%): 1H NMR (500 MHz, CH OD) δ 1.30 (m, 12H), 1.64 (m, 6H), 1.87 3 (m, 9H), 2.96 (m, 6H), 3.75 (m, 3H), 5.29, (m, 3H), 5.60, (m, 3H), 8.36 (s, 1H), 8.81 (d, J ) 9.5 Hz, 1H), 9.02 (d, J ) 10 Hz, 1H), 9.08 (d, J ) 9.5 Hz, 1H), 9.26 (d, J ) 9.5 Hz, 1H), 9.28 (s, 1H); 13C NMR (125 MHz, D2O/NaOD) δ 18.9, 19.0, 23.3, 23.5, 23.5, 28.0, 28.5, 28.7, 33.8, 33.8, 34.0, 40.3, 40.6, 60.1, 60.3, 115.5, 119.5, 121.9, 122.0, 122.2, 124.4, 128.6, 128.7, 129.0, 129.7, 129.7, 129.8, 132.6, 134.2, 139.7, 140.1, 140.5, 169.4, 172.1, 172.5, 172.6, 183.1, 183.3, 183.7. N-[2-(Pyridin-4-yl)ethyl]methacrylamide (8). Methacryloyl chloride (6.7 mL, 69.6 mmol) was added dropwise to a cooled (-10 °C) solution of 4-(2-aminoethyl)pyridine (7.0 mL, 58 mmol) in CH2Cl2 (200 mL), and the reaction was stirred at room temperature for 16 h. Saturated Na2CO3 (200 mL) was added, and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (100 mL), the organic layers were combined, washed with 1 M NaOH (2 × 100 mL), dried with Na2SO4, filtered, and evaporated to give (46) Belokon, Y. N.; Tararov, V. I.; Savel’eva, T. F.; Belikov, V. M. Makromol. Chem. 1980, 181, 2183-2197.

Fluorescent Hydrogels for in Vitro Glucose Detection

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8 as an orange oil (7.0 g, 63% yield): 1H NMR (CDCl3, 500 MHz) δ 1.54 (s, 3H), 2.49 (t, J ) 7.0 Hz, 2H), 3.17 (q, J ) 6.5 Hz, 2H), 4.90 (s, 1H), 5.32 (s, 1H), 6.74 (d, J ) 5.5 Hz, 2H), 7.57 (t, J ) 5.5, NH), 7.98 (d, J ) 4.5 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 18.5, 34.7, 39.8, 119.2, 124.2, 139.9, 148.5, 149.2, 168.9. 1-[3-Borono-5-(bromomethyl)benzyl]-4-[(2-methacrylamido)ethyl]pyridinium Bromide (9). Compound 8 (1.3 g, 6.8 mmol) was added to a solution of 7 (9.5 g, 27.3 mmol) in CH2Cl2 (370 mL) and CH3OH (180 mL), and the reaction was stirred at 40 °C for 20 h. The CH2Cl2 was removed in vacuo, and the excess 7 which precipitated out of methanol was filtered off and washed with icecold methanol. The filtrate was concentrated down to ca. 20 mL, and then acetone (ca. 300 mL) was added, followed by the addition of ether until turbidity occurred. Storage at -4 °C for 24 h resulted in the formation of a white precipitate that was collected by centrifugation, washed several times with acetone, and dried under argon to yield 0.91 g of 9 (33% yield): 1H NMR (CD3OD, 500 MHz) δ 1.83 (s, 3H), 3.16 (t, J ) 6.5 Hz, 2H), 3.61 (t, J ) 6.5 Hz, 2H), 4.57 (s, 2H), 5.32 (s, 1H), 5.59 (s, 1H), 5.79 (s, 2H), 7.50-7.79 (m, 3H), 7.98 (d, J ) 6.5 Hz, 2H), 8.92 (d, J ) 6.5 Hz, 2H); 13C NMR (CD3OD, 125 MHz) δ 17.3, 31.8, 35.2, 38.6, 119.4, 128.6, 130.7, 133.1, 133.6, 135.5, 139.0, 139.5, 143.6, 143.8, 169.8; 11B NMR (80 MHz, CD3OD) δ 28.1. Polymerizable Quencher 2a. To a solution of 9 (2.5 g, 6.0 mmol) in DMF (25 mL) was added 4,4′-dipyridyl (0.40 g, 2.6 mmol), and the reaction was stirred at 55 °C for 72 h. The yellow precipitate was collected by centrifugation, washed with DMF and then acetone, and dried under a stream of argon to yield 2a (0.6 g, 20% yield): 1H NMR (CD OD, 500 MHz) δ 1.84 (s, 6H), 3.29 (t, J ) 6.5 Hz, 3 4H), 3.76 (t, J ) 6.5 Hz, 4H), 5.43 (s, 2H), 5.57 (s, 2H), 5.95 (s, 4H), 6.12 (s, 4H), 7.89 (s, 2H), 7.95 (s, 2H), 8.02 (s, 2H), 8.07 (d, J ) 6.5 Hz, 4H), 8.69 (d, J ) 7.0 Hz, 4H), 8.92 (d, J ) 6.5 Hz, 4H), 9.29 (d, J ) 7.0 Hz, 4H); 13C NMR (CD3OD, 125 MHz) δ 17.7, 35.1, 38.8, 63.4, 64.3, 121.1, 127.5, 129.0, 132.0, 133.1, 134.0, 135.5, 135.7, 138.8, 143.7, 145.7, 150.4, 171.6; 11B NMR (80 MHz, CD3OD) δ 28.2. Polymerizable Quencher 2b. Compound 11 (0.47 g, 0.87 mmol) was sonicated in DMF (50 mL) for 10 min, and any insoluble material was filtered off. To the clear yellow filtrate was added compound 9 (0.44 g, 1.0 mmol), and the reaction was stirred at 55 °C for 72 h. Acetone was added (∼200 mL), and the resulting yellow precipitate was collected by centrifugation, washed with acetone, and dried under a stream of argon to yield 2b (0.51 g, 57% yield): 1H NMR (CD3OD, 500 MHz) δ 1.83 (s, 6H), 3.16 (t, J ) 6.5 Hz, 2H), 3.61 (t, J ) 6.5 Hz, 2H), 5.31 (s, 1H), 5.60 (s, 1H), 5.85 (s, 2H), 5.92 (s, 2H), 6.01 (s, 4H), 7.84 (br s, 6H), 7.99 (d, J ) 6.5 Hz, 2H), 8.13 (t, J ) 7.25 Hz, 2H), 8.60 (t, J ) 7.75 Hz, 1H), 8.69 (d, J ) 6.5 Hz, 4H), 8.99 (d, J ) 6.5 Hz, 2H), 9.16 (d, J ) 5.5 Hz, 2H), 9.37 (d, J ) 6.5 Hz, 4H); 13C NMR (D2O, 125 MHz) δ 18.98, 36.39, 40.06, 64.79, 65.45, 65.7, 122.3, 123.9, 127.7, 128.7, 129.9, 130.2, 132.5, 134.2, 135.1, 136.6, 140.1, 144.9, 145.7, 146.9, 147.5, 151.7, 172.9; 11B NMR (80 MHz, D2O) δ 23.9. Fluorescence Measurements in Solution. All experiments were carried out in triplicate, and the errors in the reported quenching and glucose binding constants are based on the standard deviation of three independent determinations. For quenching studies, fluorescence measurements were done in situ by taking the emission spectrum of 1 (λex ) 490 nm, λem ) 540 nm) at a series of quencher concentrations. The emission spectrum of 1 (2 mL of a 4 × 10-6 M solution in buffer) was first obtained, quencher was added (0.510 µL aliquots of a 5 mM solution in buffer), the solution was shaken for 30 s, and the new emission was measured after each quencher addition. Fluorescence intensity was taken as the area under the emission curve between 500 and 630 nm. Stern-Volmer quenching constants were calculated by fitting the data with eq 1, F0/F ) (1 + Ks[Q])eV[Q]

Figure 1. Structures of the monomeric dye and quenchers/receptors. *At pH 7.4 the dye most likely exists as the carboxylate. concentration.47,48 All data were analyzed using Solver (nonlinear least-squares curve fitting) in Microsoft Excel. For glucose-sensing studies, the emission spectrum of 1 (2 mL of a 4 × 10-6 M solution in buffer) was taken, quencher was added (200 µL of a 5 mM solution in buffer) to obtain a quencher:dye ratio of 125:1, the emission was measured, then a sugar solution was added (0.5-10 µL aliquots of a 1 M solution in buffer), the solution was shaken for 30 s, and the new emission was measured after each addition of sugar. Apparent glucose binding constants were calculated by fitting the data with eq 2, F/F0 ) (F0 + FmaxKb[glu])/(1 + Kb[glu])

(2)

where F0 is the fluorescence intensity of the quenched dye, F is the fluorescence intensity after the addition of sugar, Fmax is the intensity at which the fluorescence increase reaches its maximum, Kb is the apparent binding constant, and [glu] is the concentration of glucose.49 Hydrogel Preparation. A 1 mL volumetric tube was charged with HEMA (0.35 g, 2.7 mmol), PEGDMA (111 mg, 0.96 mmol), SPM (28 mg, 0.114 mmol), 1 (0.2 mL of a 0.01 M solution in Nanopure water, 0.002 mmol), 2a (23 mg, 0.02 mmol) or 2b (21 mg, 0.02 mmol), and 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) (2.4 mg, 0.0074 mmol) and filled to the 1 mL mark with Nanopure water. The vial was sealed with a septum, vortexed until a clear red solution was obtained, and then deoxygenated by passing a stream of argon through the solution for about 10 min. The solution was then injected, by syringe, into a polymerization mold, which was constructed in the following manner. Two 3 mm thick glass plates (8 × 8 cm), one containing two small holes for solution injection, were treated with dichlorodimethylsilane (2% solution in toluene) to facilitate removal of the gel after polymerization, and stacked atop each other separated by a 25.4 µm thick Teflon spacer. The plates were clamped together with a metal mold that contained syringe injection ports aligned with the holes

(1)

where F0 is the initial fluorescence intensity, F is the fluorescence intensity after the addition of quencher, V is the dynamic quenching constant, Ks is the static quenching constant, and [Q] is the quencher

(47) de Borba, E. B.; Amaral, C. L. C.; Politi, M. J.; Villalobos, R.; Baptista, M. S. Langmuir 2000, 16, 5900-5907. (48) Frank, I. M.; Vavilov, S. I. Z. Phys. Chem. (Muenchen) 1931, 69, 100. (49) Cooper, C. R.; James, T. D. J. Chem. Soc., Perkin Trans. 1 2000, 963969.

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Scheme 1. Schematic Representation of the Putative Glucose-Sensing Mechanism

Scheme 2. Synthesis of Polymerizable Dye 1a

a Reagents and conditions: (a) CuSO ‚5H O, NaOH, Na CO , 4 2 2 3 methacryloyl chloride, H2O; (b) Dowex 50WX8, NH4OH; (c) Ac2O, reflux; (d) SOCl2, catalytic DMF, reflux; (e) (TMS)Cl, DIEA, DCM.

in the upper glass plate. After the mold was filled with monomer solution, it was sealed in a Ziploc bag that was purged and filled with argon and incubated in a 45 °C oven for 24 h. The glass plates were then removed from the mold, placed in phosphate buffer for 2 h, and separated to expose the thin orange/pink film. The film was cut into 1 × 3 cm pieces and immersed in phosphate buffer at 40 °C for 16 h.

Results and Discussion The glucose-sensing components used in this study are the fluorescent anionic dye 1 and the cationic quencher derivatives 2a,b (Figure 1). In the absence of glucose, 1 and 2 form an essentially nonfluorescent ground-state complex. Complexation is facilitated by electrostatic attraction, and fluorescence quenching occurs due to the electron-receiving ability of the viologen 2.50 It is postulated that when the boron moieties become negatively charged after glucose binding, the electrostatic attraction between the dye and quencher is weakened, resulting

in a loss of quenching efficency and a concomitant increase in fluorescence intensity. A simple representation of this proposed sensing mechanism is shown in Scheme 1. To help substantiate the mechanism, we have monitored the change in charge of boron from neutral (sp2) to anionic (sp3) upon addition of glucose using 11B NMR.42 Design and Synthesis of Sensing Components. The synthesis of pyranine derivative 1, outlined in Scheme 2, makes use of an already established procedure for the preparation of Nmethacryloyl-(S)-lysine (3)46 in which dual protection of the carboxylic acid and R-amino groups of lysine with copper(II) allows for selective acylation of the -amino group with methacryloyl chloride. Subsequent reaction of 3 with 8-acetoxypyrenyl-1,3,6-trisulfonyl chloride (6) to form the desired compound was carried out in dichloromethane after 3 was solublized via transient protection with chlorotrimethylsilane. The two quencher derivatives 2a,b were synthesized to assess whether only a single point of attachment to the polymer network, as in 2b, has a favorable effect on the fluorescence signal modulation and response time due to increased mobility within the hydrogel. The synthesis of 2a was carried out by reaction of N-[2-(pyridin-4-yl)ethyl]methacrylamide (8) with an excess of 2-[3,5-bis(bromomethyl)phenyl][1,3,2]dioxaborinane (7) to afford the monosubstituted derivative 9, which was subsequently reacted with 0.5 equiv of 4,4′-dipyridyl (Scheme 3). The synthesis of 2b was accomplished by monoalkylation of 4,4′-dipyridyl with intermediate 10 to provide compound 11, which was then reacted with intermediate 9 (Scheme 3). Quenching and Glucose Sensing in Solution. The quenching efficency and glucose-sensing ability of 2a and 2b were first assessed in solution. The addition of increasing amounts of 2 to a buffered solution of 1 (4 × 10-6 M) resulted in effective fluorescence quenching of the dye. Figure 2 shows a SternVolmer plot of the quenching data, where F0/F, the ratio of the fluorescence emission of 1 (λex ) 490 nm, λem ) 540 nm) before

Scheme 3. Synthesis of Polymerizable Quenchers 2a and 2b

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Figure 2. Stern-Volmer plot for the quenching of 1 (4 × 10-6 M, λex ) 490 nm, and λem ) 540 nm) with 2a (b) and 2b (2). Table 1. Static (Ks) and Dynamic (V) Quenching Constants of 2a and 2b with 1a and Apparent Glucose Binding Constants (Kb) Determined for 2a and 2bb with 1 quenching 2a 2b

glucose sensing

Ks (M-1)

V (M-1)

Kb (M-1)

F/F0c

4200 ( 200 3900 ( 200

550 ( 90 150 ( 70

140 ( 10 100 ( 10

1.57 1.44

[1] ) 4 × 10-6 M, λex ) 490 nm, λem ) 540 nm. b [2a], [2b] ) 5 × 10-4 M. c Relative fluorescence intensity at a 20 mM glucose concentration. a

and after addition of quencher, is plotted as a function of the quencher concentration. The calculated Stern-Volmer constants Ks and V, indicating the degree of static and dynamic quenching, respectively, are summarized in Table 1. The stronger quenching behavior displayed by the quencher containing two polymerizable arms, 2a, appeared to result mainly from the dynamic component (V) of the quenching mechanism. Thus, the larger sphere of gyration of 2a relative to 2b may allow more collisional interactions with the dye in solution. The stronger quenching displayed by 2a could also arise from increased hydrophobicity.50g To test for glucose sensitivity, the dye was first quenched with 2, and then glucose aliquots (0-30 mM) were added, which resulted in an increase of the fluorescence emission signal. By plotting the relative fluorescence increase (F/F0) versus glucose concentration, binding isotherms were obtained (Figure 3). From these data, the relative affinities of the quenchers toward glucose were determined by calculation of the apparent binding constants, Kb (Table 1). The degree of fluorescence enhancement achieved from the addition of glucose to the dye/2a complex was greater than that obtained for the dye/2b complex. The apparent glucose binding constant for 2a is also slightly higher than that of 2b. The stronger quenching properties of 2a relative to 2b may cause these observed differences in their glucose-sensing profiles. Phenylboronic acids are known to have a high binding affinity for fructose.3 However, since the physiological concentrations (50) For studies involving fluorescence quenching with viologens see: (a) Nakashima, K.; Kido, N. Photochem. Photobiol. 1996, 64, 296-302. (b) Zhao, Z. G.; Shen, T.; Xu, H. J. J. Photochem. Photobiol., A 1990, 52, 47-53. (c) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287-12292. (d) Gaylord, B. S.; Wang, S. J.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2001, 123, 6417-6418. (e) Wang, D. L.; Gong, X.; Heeger, P. S.; Rininsland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 49-53. (f) Wang, D. L.; Wang, J.; Moses, D.; Bazan, G. C.; Heeger, A. J. Langmuir 2001, 17, 1262-1266. (g) Fan, C.; Hirasa, T.; Plaxco, K. W.; Heeger, A. J. Langmuir 2003, 19, 3554-3556. (h) Serpone, N. In Photoinduced Electron Transfer; Fox, M. A., Chanon, M., Eds.; Elsevier: Amsterdam, 1988; Vol. D, p 47.

Figure 3. Fluorescence increase of 1 (4 × 10-6 M, λex ) 490 nm, and λem ) 540 nm) in the presence of 2a (b) and 2b (2) upon addition of glucose ([2a], [2b] ) 5 × 10-4 M). Table 2. Composition of Glucose-Responsive Hydrogels compositiona (mol %) hydrogel HEMA PEGDMA SPM A B

92 92

3.2 3.2

3.9 3.9

1

[1]d 2a 2b % Tb % Hc (mM)

0.07 0.7 0 0.07 0 0.7

52 52

50 50

2 2

a Initiator (VA-044) concentration 7.4 mM. b % T ) [mass of all monomers (g)/volume of the reaction mixture (mL)] × 100. c See the Supporting Information for degree of hydration determination. d [1] ) amount of 1 (mmol)/volume of the swollen hydrogel (L).

Figure 4. Continuous glucose sensing with hydrogel B mounted in a flow-through cuvette. The plot shows the relative fluorescence intensity changes of the hydrogel (λex ) 490 nm, λem ) 540 nm) as a function of time when exposed to varying glucose concentrations (pH 7.4, 37 °C).

of fructose are far lower than those of glucose, it may not pose a significant interference with the accurate detection of glucose. Nonetheless, to achieve selectivity of glucose over fructose, cooperative binding of diboronic acids has proven to be an effective strategy.51 However, for the viologen-substituted diboronic acids 2 used in this study, both boronic acids cannot bind to the same glucose molecule, since the distance between them is too great. When quenchers with diboronic acid configurations identical to that of 2 were tested against other monosaccharides, selectivity ratios of fructose > galactose > glucose were observed.42 To achieve glucose selectivity, we are (51) (a) Shiomi, Y.; Saisho, M.; Tsukagoshi, K.; Shinkai, S. J. Chem. Soc., Perkin Trans. 1 1993, 2111-2117. (b) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem., Int. Ed. Engl. 1994, 33, 2287-2289. (c) Eggert, H.; Frederiksen, J.; Morin, C.; Norrild, J. C. J. Am. Chem. Soc. 1999, 64, 3846-3852. (d) Yang, W.; He, H.; Drueckhammer, D. G. Angew. Chem., Int. Ed. 2001, 40, 1714-1718. (e) Arimori, S.; Ward, C. J.; James, T. D. Tetrahedron Lett. 2002, 43, 303-305.

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Figure 5. Excitation (a) and emission (b) spectra of hydrogel B (λex ) 490 nm, λem ) 540 nm) at 0, 5, and 20 mM glucose concentrations (pH 7.4, 37 °C).

currently working toward the development of diboronic acids that are capable of cooperative binding. Glucose Sensing by Front-Face Illumination of a Hydrogel. Hydrogels are defined as cross-linked polymers capable of imbibing and retaining large quantities of water without loss of their three-dimensional network structure.52,53 To evaluate the glucose-sensing performance of 2a versus 2b with 1 when immobilized in a p(HEMA) hydrogel, two hydrogels, A and B containing 2a and 2b, respectively, were prepared (Table 2). The structures of the monomeric components HEMA, SPM, PEGDMA, and the radical initiator used in these gels are shown in the Supporting Information. Table 2 lists the gel formulations in terms of the relative mole percentage of the monomers (mol %), total monomer concentration (% T), and degree of hydration (% H). The hydrophilic anionic comonomer SPM was used to help enhance the swelling capacity of the gels.54 Aqueous solutions containing the dye and quencher monomers and the other monomers listed in Table 2 were injected between two glass plates separated by a 25.4 µm Teflon spacer (see the Supporting Information for details) and polymerized at 45 °C for 24 h. The resultant thin-film gels were cut into 1 × 3 cm pieces and allowed to swell in phosphate buffer at 40 °C for 16 h. A piece of hydrogel was mounted into a custom-made flow-through fluorescence cuvette (see the Supporting Information for details), and the cuvette was held in the spectrometer using a modified frontsurface accessory. Phosphate buffer (0.1 M ionic strength, pH 7.4) was circulated through the cell while the temperature was kept constant at 37 °C. (52) Park, K.; Shalaby, W. S. W.; Park, H. In Biodegradable Hydrogels for Drug DeliVery; Technomic Publishing: Lancaster, PA, and Basel, Switzerland, 1993. (53) Kim, S. W.; Bae, Y. H.; Okano, T. Pharmacol. Res. 1992, 9, 283-290.

Figure 6. Glucose response of hydrogels A and B (λex ) 490 nm, λem ) 540 nm): (a) binding curves for the average of three independent runs, (b) bar graph representation showing the error associated with the three trials. Table 3. Apparent Glucose Binding Constants (Kb) and Response Times (τ90) Determined for Hydrogels A and B Containing 2a and 2b, Respectivelya glucose sensing

A B

response time (τ90, min)

Kb (M-1)

F/F0b

0-2.5 mM

2.5-5 mM

5-10 mM

10-20 mM

110 ( 10 120 ( 10

1.52 ( 0.09 1.54 ( 0.09

27 ( 5 27 ( 4

17 ( 1 17 ( 2

16 ( 2 15 ( 3

13 ( 1 12 ( 1

a See Table 2 for hydrogel compositions. b Relative fluorescence intensity at a 20 mM glucose concentration.

The film was excited at 490 nm by front-face illumination at a 30° angle,55 and the emission at 540 nm was monitored over time. The gradual infusion of buffered glucose (2.5, 5.0, 10, and 20 mM) into the hydrogel resulted in an increase in fluorescence intensity that was directly related to the glucose concentration (Figure 4). The hydrogel displayed an excellent dynamic response to glucose concentrations within the clinically relevant range of 2.5-20 mM (45-360 mg/dL). Importantly, the reversibility of the system was demonstrated by the infusion of decreasing glucose concentrations, which led to a reduction in the fluorescence signal intensity (Figure 4). The changes in the excitation and emission spectra of the hydrogel after exposure to glucose are shown in Figure 5. To accurately compare hydrogels A and B, containing 2a and 2b, respectively, three different pieces from each hydrogel film (54) Wang, C.; Cao, W. Polym. Int. 1996, 41, 449-451. (55) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum Pess: New York, 1999.

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Figure 7. Schematic representation of the fiber-optic-based hydrogel testing platform. The inset diagram details the attachment of a hydrogel disk to the tip of the fiber-optic cable. The figure is not drawn to scale.

were independently tested. Figure 6 shows the averaged glucose response curves and errors generated from the three pieces of hydrogels A and B. Converse to the glucose response differences between 2a and 2b obtained in solution (Table 1, Figure 3), in this hydrogel formulation, the quencher/receptor 2b (containing only one attachment to the polymer matrix) provided a slightly higher relative fluorescence increase in response to each glucose concentration. The response times (τ90) (defined as the time taken to reach 90% of the maximum fluorescence intensity) of the two hydrogels were essentially the same (Table 3). Although the differences between the two gels in terms of relative fluorescence modulation and response time are very subtle at best, they suggest that 2b might have greater mobility within the hydrogel. We suspect that quenchers/receptors 2a and 2b may exhibit more drastic behavioral differences when immobilized in hydrogels with a higher water content and/or lower cross-link density. Glucose Response of a Hydrogel by Fiber Optics. For an optical sensing device to be practical, excitation by inexpensive visible light sources such as blue light emitting diodes (LEDs) is necessary. Also important is the feasibility of the system to be miniaturized and configured on the distal end of an optical fiber for remote sensing. Therefore, to analyze the hydrogels in a configuration that more closely resembles what might be used in a working medical device, a fiber-optic-based system was fabricated. The distal end of an optical fiber was threaded through an epoxy-filled glass capillary tube to add extra stability to and enhance the surface area of the fiber tip (Figure 7, inset). After curing, the tip was polished until smooth, and a disk of hydrogel B was attached to it with Vetbond (butyl cyanoacrylate), a tissue adhesive used by veterinarians to seal wounds in animals. Other workers have employed Vetbond for use with p(HEMA) hydrogels.56 The end of the fiber containing the hydrogel was then immersed in a flow-through cell through which buffered glucose solutions were circulated, and the opposite end of the fiber was connected to a bifurcated cable and attached to an Ocean Optics spectrophotometer equipped with a 470 nm LED (Figure 7). The real-time data generated using this method are shown in Figure 8, where changes in the intensity of the fluorescence (56) Kermis, H. R.; Rao, G.; Barbari, T. A. J. Membr. Sci. 2003, 212, 75-86.

Figure 8. Fiber-optic-based continuous glucose sensing of hydrogel B. The plot shows the relative fluorescence intensity changes of the hydrogel as a function of time when exposed to varying glucose concentrations (pH 7.4, 37 °C). Table 4. Comparison of Response Times of Hydrogel B Obtained When Tested in the Cuvette-Based Arrangement versus Those Obtained When Tested in the Fiber-Optic-Based Configuration response time (τ90, min)

response time (τ90, min)

range (mM)

cuvette

optical fiber

range (mM)

cuvette

optical fiber

0-2.5 2.5-5

27 17

12 9

5-10 10-20

15 12

7 5

emission signal monitored at 540 nm correspond directly to changes in the glucose concentration. Interestingly, when in the fiber-optic configuration, this hydrogel displayed a significantly faster response time than when it was monitored by front-face illumination in a cuvette (Table 4). We suspect that this may be due to differences in the sizes of the hydrogel pieces relative to the flow cell capacities in the two gel analysis arrangements. The sensor was also evaluated for stability during constant glucose circulation. To test whether the sensor could provide a stable signal throughout prolonged glucose exposure, it was challenged with 5 mM glucose for 36 h. Importantly, the absolute fluorescence intensity did not change to any significant extent during this experiment (Figure 9).

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Figure 9. Stability of the sensor to prolonged glucose exposure. A 30 min period of buffer circulation is followed by 36 h of glucose (5 mM) circulation at 37 °C.

Conclusions A two-component glucose-sensing system, comprised of a fluorescent reporter dye and a boronic acid-substituted viologen (acting as a fluorescence quencher and glucose receptor), was immobilized into a p(HEMA) hydrogel. Two monomeric quencher derivatives, one containing a single linker for attachment to the polymer backbone and the other containing two linkers, were prepared. When first assessed in solution, the two-armed quencher displayed a stronger response to glucose than the onearmed derivative, most likely as a result of the dynamic quenching differences between the two, which are most prevalent in solution. Once incorporated into a hydrogel, however, the singly tethered quencher displayed slightly enhanced glucose-sensing abilities over the doubly tethered derivative. We attribute this phenomenon to the possibility that a single-linker attachment to the hydrogel

Gamsey et al.

allows for increased mobility of the quencher. Future studies designed to test the importance of unrestricted mobility of the sensing components within the hydrogel include the synthesis of sensing components containing increasingly long hydrophilic linkers, as well as optimization of the hydrogel network by varying the monomer compositions and cross-link density to obtain gels with a higher water content. We are currently working to improve the specificity of the system by designing glucose-selective diboronic acid-based quenchers. Analysis of the hydrogels for glucose response was accomplished by using a cuvette-based approach where the gels were mounted into a custom-made flow-through cell and excited via front-face illumination. A more realistic method of analysis was also employed, where the hydrogel was monitored on the tip of a fiber-optic cable using a blue LED excitation source. In this configuration, the sensor displayed excellent glucose sensitivity, reversibility, and short-term stability under physiological conditions (pH, ionic strength, temperature). We believe that much of the information derived from these studies can help to facilitate optimization of other immobilized systems for chemosensor development. Acknowledgment. We thank the BioSTAR Project and the Industry-University Cooperative Research Program with GluMetrics, Inc. for their financial support. We thank Lacie Hirayama for her technical assistance. Supporting Information Available: Experimental procedure for synthesis of 3, 1H and 13C NMR spectra for all compounds reported, detailed procedures for the fabrication and use of the flow-through cuvette and the fiber-optic assemblies, structures of the hydrogel monomers, and degree of swelling determination. This material is available free of charge via the Internet at http://pubs.acs.org. LA0617053