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Langmuir Monolayer and Langmuir-Blodgett Film Studies of an Amphiphilic Coumaryl Crown Ether Pe´ter Kele, Jhony Orbulescu, Tiffany L. Calhoun, Robert E. Gawley,* and Roger M. Leblanc* Department of Chemistry and NIEHS Marine and Freshwater Biomedical Sciences Center, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33124 Received June 7, 2002. In Final Form: August 5, 2002 As a potential compound for use in optical fiber fluorescence sensor for rapid detection of saxitoxin, 4-(monoaza-18-crown-6-methyl)-7-octadecanoylaminocoumarin (ODAC) was synthesized and the interfacial and spectroscopic properties of its Langmuir monolayers and Langmuir-Blodgett (LB) films were studied. The surface pressure- and surface potential-area isotherms were obtained on a pure water subphase. In situ fluorescence of the ODAC monolayer at the air-water interface showed a fluorescence band centered around 425 nm (λex ) 332 nm) decreasing in intensity with increasing surface pressure. This observation is due to the self-quenching of the ODAC molecules as the surface concentration per unit area increases and aggregates are formed. To reduce the aggregation phenomenon, mixed monolayers of ODAC with C18GlyGlyAlaGlyNH2 peptidolipid (PL) (PL:ODAC, 20:1 and 100:1) were used to dilute the fluorophore molecules at the air-water interface and diminish the self-quenching. LB films of pure ODAC and PL: ODAC mixed monolayers (100:1) were prepared and tested on saxitoxin dissolved in a phosphate buffer (pH 7.4). Each LB film showed fluorescence increase in the presence of saxitoxin.
Introduction Saxitoxin (STX) (Figure 1) and its hydroxylated and sulfonated structural analogues1 are marine toxins appearing in many kinds of shellfish. Consumption of saxitoxin-containing shellfish produces symptoms known as paralytic shellfish poisoning (PSP).2 The most severe symptom of PSP is respiratory paralysis with severe symptoms occurring in humans after ingestions of 124 µg and death occurring from less than 0.5 mg.3 Since mortality takes place within the first 12-24 h, rapid detection of the disease entity is a clinical necessity. Among a number of STX detection techniques,4 mouse bioassay is the current benchmark technique.5 We have undertaken a program to develop sensors for saxitoxin using fluorescence signaling as the way of detection. These fluorophore crown ether molecules bind to STX in solution and cause strong fluorescence enhancement that is detectable by a conventional spectrofluorimeter.6,7 The long-term goal of our project is to develop an optical fiber based sensor device that can be used in situ for rapid detection of saxitoxin. In this case the optical fiber would contain a monolayer of the sensing fluorophore covalently attached to the fiber. Prior to * Authors to whom correspondence should be addressed. Telephone: (305) 284-2194. Fax: (305) 284-6367. E-mail: rgawley@ miami.edu (R.E.G);
[email protected] (R.M.L.). (1) Kao, C. Y. In Algal Toxins in Seafood and Drinking Water; Falconer, I. R., Ed.; Academic Press: London, 1993; pp 75-104. (2) Shimizu, Y. In Handbook of Natural Toxins; Tu, A. T., Ed.; Marcel Dekker: New York, 1988; Vol. 3, pp 63-85. (3) New Engl. J. Med. 1973, 288, 1126-1127. (4) Sullivan, J. J.; Wekell, M. M.; Hall, S. In Handbook of Natural Toxins; Tu, A. T., Ed.; Marcel Dekker: New York, 1988; Vol. 3, pp 87-106. (5) Fernandez, M.; Cembella, A. D. In Manual on Harmful Marine Microalgae; Hallegraef, G. M., Anderson, D. M., Cembella, A. D., Eds.; IOC Manuals and Guides 33; UNESCO: Paris, 1995; pp 213-228. (6) (a) Gawley, R. E.; Zhang, Q.; Higgs, P. I.; Wang, S.; Leblanc, R. M. Tetrahedron Lett. 1999, 40, 5461-5465. (b) Gawley, R. E.; Pinet, S.; Cardona, C. M.; Datta, P. K.; Ren, T.; Guida, W. C.; Nydick, J.; Leblanc, R. M. J. Am. Chem. Soc., in press. (7) Wang, S.; Zhang, Q.; Datta, P. K.; Gawley, R. E.; Leblanc, R. M. Langmuir 2000, 16, 4607-4612.
Figure 1. Structure of saxitoxin.
fabrication of such a device the monolayer behavior at the air-water interface and the Langmuir-Blodgett (LB) film of these sensor molecules should be studied. We have recently developed two new candidate molecules,8 one of which was transformed into an amphiphilic derivative (Figure 2; 4-(monoaza-18-crown-6-methyl)-7octadecanoylaminocoumarin, ODAC in brief). Langmuir monolayers and LB films of ODAC were successfully prepared. The interfacial and spectroscopic properties of these films are reported here. Experimental Section The long-chain coumaryl crown ether derivative, ODAC, was synthesized as described below. Solutions were prepared with spectroscopic grade solvents purchased from Fischer Scientific (Fair Lawn, NJ). The pure ODAC solution was prepared using chloroform solvent at a concentration of 10-3 M. For the preparation of the mixed peptidolipid (PL)/ODAC monolayers, the peptidolipid was dissolved in CHCl3/MeOH (5:1 v/v) at a concentration of 10-3 M and mixed with a 10-3 M ODAC solution to reach a ratio of PL and ODAC of 20:1 and 100:1 (v/v), respectively. Pure water used as the subphase was provided from a Modulab 2020 water purification system (Continental Water Systems Corporation, San Antonio, TX). The resistance and surface tension of pure water were 18 MΩ‚cm and 72.6 mN m-1 at 20.0 ( 0.5 °C, respectively. The pH of the subphase for all experiments was in the range 5.5-6.0. Following the deposition of the spreading solution, we left the film at zero surface pressure 15 min before compression to allow the solvent to evaporate. The slides were cleaned by dipping them in a chromic-sulfuric acid (8) Kele, P.; Orbulescu, J.; Calhoun, T. L.; Gawley, R. E.; Leblanc, R. M. Tetrahedron Lett. 2002, 43, 4413-4416.
10.1021/la020531w CCC: $22.00 © 2002 American Chemical Society Published on Web 10/02/2002
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solution followed by sonication in pure water for 30 min. Rinsing with ultrapure water 25 times was followed by sonication in water for 50 min. Activation of the quartz slides was carried out using HNO3 (15%). The activated quartz slides were made hydrophobic by silanization with octadecyltrichlorosilane (OTS, 20% in cyclohexane, 30 min). The hydrophobic slides were sonicated in cyclohexane to remove the excess of OTS and stored under cyclohexane prior to use. Methods. All isotherm measurements were carried out in a class 1000 clean room at a constant temperature of 20.0 ( 0.5 °C. For surface pressure-area isotherm determinations we used a KSV minitrough model 2000 (KSV Instrument Ltd., Helsinki, Finland). The trough is supplied with an electronic balance that uses a Wilhelmy plate as surface pressure sensor and has a sensitivity of 0.02 mN m-1. For monolayer compression, two symmetrically movable computer controlled barriers were used. A typical compression speed was 10 Å2 molecule-1 min-1. The volume spread at the air-water interface varied from 20 to 50 µL. The trough has a quartz window fitted in the middle for in situ spectroscopic measurements. Y-type deposition of the monolayer on quartz was achieved by dipping the quartz slides vertically through the monolayer at a speed of 1.0 mm min-1. The deposition ratio was approximately 0.8. Surface potential measurements were obtained in the KSV trough using a Kelvin probe. It consists of a capacitor-like system, which has a vibrating plate set approximately 1 mm above the surface of the monolayer and a counter electrode dipped into the clean subphase. The clean subphase was taken as the zero reference. Solution absorption spectra were obtained from a UV-visNIR Perkin-Elmer spectrometer Lambda 900, using a quartz cuvette of 0.5 cm path length. The in situ UV-vis absorption spectra of the Langmuir monolayer were performed with an HP spectrophotometer Model 8452A, set on a rail close to the KSV trough, suitable for approach toward the quartz window. The fluorescence emission spectra were recorded from a Spex Fluorolog 1680 spectrophotometer. For monolayer at air-water interface and LB film studies, an optical fiber probe was put 1 mm above the surface and the excitation and emission light were transmitted through the optical fiber. Energy-minimized molecular modeling was made using Hyperchem 6.0. To determine the theoretical molecular area, the polar headgroup was approximated as ellipsoidal. Synthesis. The peptide amphiphile was synthesized using standard fluorenylmethyloxycarbonyl (Fmoc) based solid phase peptide synthesis.9 For couplings, Fmoc amino acids were used activated by 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. Cleavage from resin was performed by treatment of the support bounded amphiphiles by CH2Cl2:trifluoroacetic acid (1:1 v/v) for 20 min. Solvent was filtered and evaporated. The peptidolipid was precipitated with diethyl ether. The precipitate was collected using a centrifuge and washed three times with diethyl ether and then three times with water. The product was recrystallized from isopropyl alcohol. 1H NMR (CDCl3/CF3COOD vs TMS) δ ) 0.88 (3H, t, J ) 6.57 Hz), 1.20-1.33 (28H, m), 1.44 (3H, d, J ) 7.45 Hz), 1.52-1.62 (2H, m), 2.33 (2H, t, J ) 7.45 Hz), 3.95-4.17 (6H, m), 4.43-4.54 (1H, m). Elemental analysis calcd for C27H51N5O5: C, 61.68; H, 9.78; N, 13.32. Found: C, 61.39; H, 9.70; N, 13.05. (4-Chloromethyl-2-oxo-2H-chromen-7-yl)carbamic Acid Ethyl Ester, 1. 3-Ethoxycarbonylaminophenol (1 g, 5.52 mmol) and 4-chloroacetoacetate (750 µL, 5.52 mmol) were stirred at 80 °C for 10 min. Concentrated sulfuric acid (100 µL) was added, and the mixture was stirred for 30 min. After cooling the resulting mass was stirred in dichloromethane (50 mL). The solution was washed with water three times; then the organic portions were combined and concentrated. The resulting white solid was dried and then washed with diethyl ether (2 × 20 mL). The product was collected by filtration (680 mg, 45%) and used without further purification. 1H NMR ((CD3)2SO vs TMS) δ ) 1.27 (3H, t, J ) 7.01 Hz), 4.18 (2H, q, J ) 7.01 Hz), 4.98 (2H, s), 6.52 (1H, s), 7.42 (1H, dd, J ) 8.77 Hz, J ) 2.19 Hz), 7.6 (1H, d, J ) 2.19 Hz), 7.76 (9) Fields, G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990, 35, 161-214.
Kele et al. (1H, d, J ) 8.77 Hz), 10.21 (1H, s). 13C NMR ((CD3)2SO vs TMS) δ ) 14.3, 41.4, 60.7, 104.6, 111.6, 112.7, 114.3, 125.7, 143.1, 150.5, 153.2, 154.2, 159.8. HRMS: (HM+) calcd for C13H13ClNO4, 282.0533; found, 282.0533; Elemental analysis calcd for C13H12ClNO4: C, 55.43; H, 4.29. Found: C, 55.42; H, 4.29. [2-Oxo-4-(1,4,7,10,13-pentaoxa-16-aza-cyclooctadec-16-ylmethyl)2H-chrome n-7-yl]carbamic Acid Ethyl Ester, 2. (4-Chloromethyl2-oxo-2H-chromen-7-yl)carbamic acid ethyl ester (150 mg, 0.6 mmol) and 1-monoaza-18-crown-6 (170 mg, 0.64 mmol) in acetonitrile (10 mL) were refluxed for 24 h in the presence of triethylamine (0.4 mL). The solvent was then evaporated, and the product was purified by chromatography on silica using DCM: MeOH (95:5 v/v) as eluent. The product was dissolved in 5 mL of aqueous TFA solution (pH 3). Evaporation of the solvent resulted 220 mg (66%) product as TFA salt. 1H NMR (CD3OD vs TMS) δ ) 1.32 (3H, t, J ) 7.01 Hz), 3.49-3.73 (20H, m), 3.94 (4H, t, J ) 4.82 Hz), 4.22 (2H, q, J ) 7.01 Hz), 4.80 (2H, s) 6.66 (1H, s), 7.43 (1H, dd, J ) 2.19 Hz, J ) 8.77), 7.65 (1H, d, J ) 2.19 Hz), 7.85 (1H, d, J ) 8.77 Hz). 13C NMR (CD3OD vs TMS) δ ) 14.9, 55.7 62.6, 71.3, 71.5, 71.6, 106.6, 113.9, 116.1, 126.9, 144.5, 145.5, 155.5, 156.4, 162.1. HRMS: (HM+) calcd for C25H37N2O9, 509.2498; found, 509.2499. Octadecanoic Acid [2-oxo-4-(1,4,7,10,13-pentaoxa-16-azacyclooctadec-16-ylmethyl)-2H-chromen-7- yl]amide, ODAC, 4. Compound 2 (100 mg, 0.19 mmol) was refluxed with 2 M KOH (10 mL) for 1 h. After cooling the solution was neutralized to pH 5 with concentrated sulfuric acid. Water was removed by rotary evaporator, and the product was purified by chromatography on silica (DCM:MeOH, 95:5 v/v). The amine product was dissolved in chloroform (5 mL) containing triethylamine (0.4 mL); stearoyl chloride (50 µL) was added and the solution was stirred at room temperature for 24 h. The solvent was evaporated and ODAC was purified by chromatography (DCM:MeOH, 98:5 v/v). Evaporation of the solvent gave 50 mg of product (36% yield after two steps). 1H NMR (CDCl3 vs TMS) δ ) 0.88 (3H, t, J ) 6.57 Hz), 1.18-1.39 (28H, m), 1.74 (2H, m), 2.43 (2H, t, J ) 7.01 Hz), 2.86 (4H, t, J ) 5.26 Hz), 3.60-3.74 (20H, m), 3.90 (2H, s), 6.57 (1H, s), 7.63-7.72 (2H, m), 7.84 (1H, d, J ) 8.77 Hz), 8.00 (1H, bs). 13C NMR (CDCl vs TMS) δ ) 14.5, 26.7, 30.3, 30.5, 30.6, 30.7, 3 30.8, 35.2, 56.0, 57.2, 70.7, 71.4, 71.6, 71.7, 107.7, 115.9, 116.8, 123.0, 126.9, 143.6, 155.7, 156.4, 163.5, 175.0. HRMS: (HM+) calcd for C40H67N2O8, 703.4897; found, 703.4897.
Results and Discussion The synthesis of ODAC was carried out in a multistep reaction that is shown in Scheme 1. Ethyl 4-chloroacetoacetate and 3-ethoxycarbonylaminophenol in the presence of concentrated sulfuric acid gave 1 in 30% yield. This molecule alkylated 1-monoaza-18-crown-6 in the presence of Et3N in acetonitrile when refluxed for 24 h, forming 2 (66%). In the next step the ethoxycarbonyl protecting group was removed by saponification with KOH and the resulting amine, 3, was acylated with stearoyl chloride to give ODAC, 4 (36% after two steps). The surface pressure (π)- and surface potential (∆V)area isotherms of ODAC at the air-water interface are shown in Figure 2 (the two measurements were done simultaneously). Compressing the amphiphile resulted in a surface pressure-area isotherm that showed nil surface pressure (110-90 Å2 molecule-1), followed by a lift at 90 Å2 molecule-1 and a knee (80-40 Å2 molecule-1) indicating a condensed phase. Further compression resulted in a steep rise (40-25 Å2 molecule-1) and then the collapse of the monolayer at around 20 Å2 molecule-1 or at 53 mN m-1 surface pressure. The limiting molecular area (extrapolated area from the linear portion of the π-A curve, characteristic of the smallest molecular area before reaching the collapse of the monolayer) is 38 Å2 molecule-1, which is in close agreement with the energy-minimized molecular model (35.8 Å2 molecule-1) of the amphiphilic coumaryl ether. The surface potential-area isotherm of pure ODAC shows an increase that occurs at 185 Å2 molecule-1, and this rise was continued until 90 Å2
Amphiphilic Coumaryl Crown Ether Monolayers
Langmuir, Vol. 18, No. 22, 2002 8525
Scheme 1. Synthesis Scheme for Octadecanoic Acid [2-Oxo-4-(1,4,7,10,13-pentaoxa-16-azacyclooctadec-16-ylmethyl)-2H-chromen-7-yl]amide.
Table 1. Absorbance and Emission Maxima of the ODAC in Solution (10-3 and 10-6 M Solutions for Absorption and Fluorescence Measurements, Respectively) and in Monolayers (λexcitation) 328 and 332 nm for Solution and Monolayers, Respectively)
absorption emission a
Figure 2. Surface pressure- and surface potential-area isotherms of ODAC.
molecule-1. After this, only a steady rise was observed, indicating that further compression of the monolayer caused moderate changes in the orientation of the molecular dipoles. The surface potential-area isotherm shows an earlier increase, i.e., at 185 Å2 molecule-1, which occurs at a larger molecular area compared to the surface pressure-area isotherm, i.e., 90 Å2 molecule-1. This is due to the fact that the surface pressure-area isotherm is related to the van der Waals interactions between the hydrocarbon chains of the ODAC molecules, while the surface potential-area isotherm is related to the dipole-dipole interactions between the polar headgroups in the water subphase. Since dipole-dipole interactions can occur at larger intermolecular distances, the molecules “feel” each other and start to interact earlier than seen in the surface pressure-area isotherm where the van der Waals interactions are at short range.10 UV-vis absorption spectra of ODAC were studied in chloroform solution (10-3 M) and at the air-water interface (Table 1). The absorption spectrum at the air-water
solution (10-3 M in CHCl3) (nm)
air-water interface (nm)
Langmuir-Blodgett film (air-quartz)a (nm)
328 ( 1 415 ( 1
332 ( 1 425 ( 1
420 ( 1
One layer on quartz.
interface showed a maximum at 332 nm that is red-shifted relative to the maximum in solution situated at 328 nm. The red shift is interpreted in terms of (i) the difference in polarity of the surrounding media as chloroform represents a nonpolar environment, whereas at the airwater interface the chromophore polar headgroup is submerged in the water subphase, and (ii) the formation of aggregates as the monolayer is compressed.11 The absorption spectroscopic data cannot discriminate between these two interpretations although the formation of aggregates as the film is compressed is supported by the fluorescence emission data (vide infra). Fluorescence spectra of ODAC were also studied both in solution (10-6 M in chloroform) and at the air-water interface (Table 1). The fluorescence maximum is also red-shifted at the air-water interface (425 nm) relative to the one in solution (415 nm). After spreading the amphiphiles at the air-water interface, an intense fluorescence is observed. As the monolayer is compressed, the fluorescence intensity dramatically drops (Figure 3). This phenomenon supports the interpretation that the ODAC molecules form aggregates. Efficient energy transfer from fluorescent monomeric species to the aggregates that are most likely nonfluorescent and decay via nonradiative processes results in fluorescence quenching.12 (10) Gaines G., Jr. In Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publisher: New York, 1966; p 188. (11) Kele, P.; Orbulescu, J.; Mello, S. V.; Mabrouki, M.; Leblanc, R. M. Langmuir 2001, 17, 7286-7290.
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Figure 3. Fluorescence quenching of pure ODAC monolayer at different surface pressures (0, 1, 3, 5, 10, and 30 mN/m in the order indicated, λexcitation ) 332 nm).
To limit the formation of aggregates, other nonfluorescent amphiphilic molecules are often used to act as a two-dimensional solvent. The coumaryl crown ether being a photoinduced electron transfer (PET) sensor molecule13 shows strong pH dependence regarding its fluorescence intensity. This property dictates that a neutral species should be used as a two-dimensional solvent. We have recently reported the use of peptidolipids (PL) for this purpose.14 A neutral PL (stearoyl-Gly-Gly-Ala-Gly-NH2) was synthesized and used to make mixed monolayers with ODAC. Two ratios of PL:ODAC (20:1 and 100:1) were tested for air-water interface fluorescence. The mixed monolayer 20:1 (PL:ODAC) showed increase in fluorescence as the monolayer was compressed. This increase was continued until reaching 10 mN m-1. Further compression resulted in fluorescence quenching. The mixed monolayer with 100:1 (PL:ODAC) ratio showed an increase of fluorescence up to 15 mN m-1 followed by selfquenching at higher surface pressures. These results are consistent with the dilution by the peptidolipid reducing the aggregation process and made it possible to reach higher surface pressures before observing any fluorescence quenching. One layer of pure ODAC and PL:ODAC (100:1), respectively, was transferred as LB films to quartz slides that were made hydrophobic prior to use. The transfer was carried out at 10 mN m-1 in the case of the pure ODAC monolayer and 15 mN m-1 when PL:ODAC (100:1) was transferred. For pure ODAC monolayer at 10 mN m-1 we observe a self-quenching of the fluorescence emission due to the presence of aggregates. The mixed monolayer, however, had its maximum of fluorescence at 15 mN m-1, so the presence of the peptidolipid reduced the number of aggregates in the monolayer. Both LB films showed fluorescence maximum at 420 nm, lower than the monolayer at the air-water interface. The small shift may originate from differences in the refractive index of the matrix where the molecules are located.15 The LB films were tested for the detection of STX. The toxin was dissolved (10-4 M) in a 0.01 M phosphate buffer (12) Dutta, A. K.; Salesse, C. Langmuir 1997, 13, 5401-5408. (13) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515-1566. (14) Orbulescu, J.; Mello, S. V.; Huo, Q.; Sui, G.; Kele, P.; Leblanc, R. M. Langmuir 2001, 17, 1525-1528. (15) McRae, E. G.; Kasha, M. In Physical Processes in Radiation Biology Augenstein, L., Mason, R., Rosenberg, B., Eds.; Academic Press: New York 1964.
Kele et al.
Figure 4. Fluorescence enhancement of LB films in the presence of saxitoxin.
(pH 7.4, [NaCl] ) 0.1370 M, [KCl] ) 0.0027 M). The slides were placed under a bifurcated optical fiber. For each slide the fluorescence spectra of the LB films in the presence of the pure buffer and in the presence of the STX solution were compared. The presence of the toxin caused enhancement in the fluorescence signals in each case, indicating that even one layer of the PET sensor molecules detects STX (Figure 4). It is noteworthy that these enhancements were much larger than those in solution (4-fold compared to 1.3-fold). This is probably due to much larger excess of saxitoxin as the LB film represents only one layer of the sensing species. The enhancements are measured by the ratio F/F0 values, where F is the fluorescence intensity of the LB film in the presence of saxitoxin and F0 is that when only the pure buffer was used. For both LB films we observed similar enhancement ratios (4.0 and 4.1 for pure and mixed LB films, respectively). This suggests that the presence of aggregates does not affect the detection of STX, as they are not fluorescent and probably only the monomers interact with saxitoxin in the binding process. Conclusions Langmuir monolayers and Langmuir-Blodgett films of ODAC were successfully prepared, and surface pressure-area and surface potential-area isotherms for Langmuir monolayers were obtained. In situ fluorescence measurements at the air-water interface showed that ODAC forms nonfluorescent aggregates that quench the fluorescence signal. The use of a C18 peptidolipid diminished the formation of aggregates and made it possible to reach suitable surface pressures for LB deposition with a partly quenched fluorescence. The enhanced fluorescence of the LB films observed in the presence of saxitoxin showed that even one layer of the sensing fluorophore species is sufficient for detection. These results have brought us closer to our final goal, to construct a fluorescence-based optical device in which the sensing molecules are covalently attached on an optical fiber. Acknowledgment. This work was supported by the United States Department of Agriculture (Grant 0191270) and the National Institute of Environmental Health Sciences, NIH, through Grant ES05705 to the University of Miami Marine and Freshwater Biomedical Sciences Center. We are grateful to Dr. Sherwood Hall (FDA, Office of Seafood) for generous gifts of saxitoxin. LA020531W
