Amphiphilic Anthracyl Crown Ether. A Langmuir and Langmuir

Langmuir−Schaefer films of CAC16/C20 showed better anthracene emission ... Langmuir Monolayer and Langmuir−Blodgett Film Studies of an Amphiphilic...
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Langmuir 2000, 16, 4607-4612

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Amphiphilic Anthracyl Crown Ether. A Langmuir and Langmuir-Schaefer Films Study Shaopeng Wang, Qianhui Zhang, Probal K. Datta, Robert E. Gawley,*,| and Roger M. Leblanc*,⊥ Marine and Freshwater Biomedical Science Center, Center for Supramolecular Science, and Department of Chemistry, University of Miami, Coral Gables, Florida 33124 Received October 13, 1999. In Final Form: February 2, 2000 As a potential material for use in optical fiber fluorescence sensors for rapid detection of saxitoxin, 9-(monoaza-18-crown-6-methyl)-10-hexadecylanthracene (CAC16) was synthesized, and the interfacial and spectroscopic properties of the Langmuir monolayers and Langmuir-Schaefer films of CAC16 were studied. The surface pressure-area and surface potential-area isotherms of CAC16 on different subphases were obtained. An increased limiting molecular area was observed on a pH 2 subphase. In situ fluorescence emission spectra (λex ) 366 nm) of the CAC16 monolayer showed a broad fluorescence band on a pH 2 subphase but none on a pure water subphase. Mixed monolayers of CAC16/C20 (arachidic acid) on a pure water subphase showed an increased fluorescence emission intensity of anthracene with an increase in the proportion of C20. This suggests that the low fluorescence activity of the pure CAC16 monolayer could be caused by self-quenching due to the high concentration of CAC16 at the interface. In a mixed monolayer, C20 acts as a two-dimensional solvent to dilute CAC16 and diminish the self-quenching, thus recovering the fluorescence activity of CAC16. When CAC16 was mixed with C18OH (stearyl alcohol), the monolayers showed no fluorescence signal, regardless of the C18OH content. Analysis of the surface pressure-area isotherms showed that CAC16 is not miscible with C18OH. Langmuir-Schaefer films of CAC16/C20 showed better anthracene emission spectra than the monolayers at the air-water interface.

Introduction Fluorescence sensing of organic and inorganic cations using the principles of host-guest chemistry has received considerable attention.1-6 Utilization of fluorescence signaling in sensing devices has a number of advantages, including high sensitivity, on-off switchability, subnanometer spatial resolution with submicron visualization, and submillisecond temporal resolution. This is a quite large and active research field. An excellent overview is provided in a recent review.4 Saxitoxin (STX) and its hydroxylated and sulfonated structural analogues7 are marine toxins appearing in many kinds of shellfish. Consumption of saxitoxincontaining shellfish produces a syndrome known clinically as paralytic shellfish poisoning (PSP), which has been known in North America since the 1700s.8 The most severe symptom of PSP is respiratory paralysis with severe symptoms occurring in humans after ingestion of 124 µg | ⊥

E-mail: [email protected]. E-mail: [email protected]. Fax: 305-284-4571.

(1) Czarnik, A. W. ACS Symposium Series; American Chemical Society: Washington, DC, 1992; No. 538, p 235. (2) Bissell, R. A.; de Silva, A. P.; Gunaratne, H. Q. N.; Lynch, P. L. M.; Maguire, G. E. M.; Sandanayake, K. R. A. S. Chem. Soc. Rev. 1992, 187-195. (3) Bissell, R. A.; de Silva, A. P.; Gunaratne, H. Q. N.; Lynch, P. L. M.; Maguire, G. E. M.; McCoy, C. P.; Sandanayake, K. R. A. S. Top. Curr. Chem. 1993, 168, 223-264. (4) 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. (5) de Silva, A. P.; Samankumara Sandanayake, K. R. A. Angew. Chem., Int. Ed. Engl. 1990, 29, 1173-1175. (6) de Silva, A. P.; Nimal Gunaratne, H. Q.; McVeigh, C.; Maguire, G. E. M.; Maxwell, P. R. S.; O’Hanlon, E. Chem. Commun. 1996, 21912192. (7) Kao, C. Y. In Algal Toxins in Seafood and Drinking Water; Falconer, I. R., Ed.; Academic Press: London, 1993; pp 75-104. (8) Shimizu, Y. In Handbook of Natural Toxins; Tu, A. T., Ed.; Marcel Dekker: New York, 1988; Vol. 3, pp 63-85.

and death occurring from less than 0.5 mg.9 Since mortality usually occurs within the first 12-24 h, rapid detection of the disease entity is a clinical necessity. Amoung a number of STX detection techniques,10 mouse bioassay is the current benchmark technique.11 We have undertaken a program to develop fluorescence sensors for STX and have reported our successful solution work recently.12 Anthracyl crown ethers bind to STX in solution and cause a strong fluorescence enhancement that is detectable by a normal spectrofluorimeter. The long-term plan for our project is eventually to construct a fiber optical fluorescence sensor that can be used in situ for rapid detection of STX. The best way to construct the sensing part of a fiber optic sensor involves the use of a thin film, such as a Langmuir-Schaefer film (LS film) or a self-assembly-monolayer (SAM). As an initial approach, 9-(monoaza-18-crown-6-methyl)-10-hexadecylanthracene (Figure 1; CAC16 in brief) was synthesized and the Langmuir monolayers and LS films of CAC16 were successfully prepared. The interfacial and spectroscopic properties of these films are reported herein. Material and Methods Synthesis of CAC16. (a) General Information. All reactions were run under an atmosphere of nitrogen. All ethereal solvents were distilled from sodium benzophenone under a nitrogen atmosphere immediately before use. Commercially available reagents were used as received unless otherwise noted. Proton NMR spectra were recorded at either 300 or 400 MHz, and carbon (9) New Engl. J. Med. 1973, 288, 1126-1127. (10) 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. (11) Fernandez, M.; Cembella, A. D. In Manual on Harmful Marine Microalgae; Hallegraeff, G. M., Anderson, D. M., Cembella, A. D., Eds.; IOC Manuals and Guides, No. 33; UNESCO: Paris, 1995; pp 213-228. (12) Gawley, R. E.; Zhang, Q.; Higgs, P. I.; Wang S.; Leblanc, R. M. Tetrahedron Lett. 1999, 40, 5461-5464. See also: Corrigendum, p 6135.

10.1021/la991343h CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000

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Wang et al. Scheme 1. Synthesis of 9-(Monoaza-18-crown-6methyl)-10-hexadecylanthracene

Figure 1. Structure of CAC16: 9-(monoaza-18-crown-6-methyl)-10-hexadecylanthracene.

J ) 8.5). Anal. Calcd for C43H67NO5: C, 76.22; H, 9.90. Found: C, 76.14; H, 9.76. Interfacial and Spectroscopic Studies. All organic solvents used in this study were of HPLC grade (Fisher Scientific Co., Pittsburgh, PA). CAC16 was dissolved in chloroform at a concentration of 1.0 mM. The water used for the monolayer study was purified by a Modulab 2020 water purification system (Continental Water Systems Corp., San Antonio, TX). The water has a resistivity of 18 MΩ cm and a surface tension of 72.6 mN m-1 at 20 °C. The surface tension was measured with a K10 digital tensiometer (KRU ¨ SS GmbH Wissenschaftliche Labogera¨te, Hamburg, Germany). Surface pressure-area isotherms were measured on a KSV minitrough system at room temperature (20.0 ( 0.5 °C) with a relative humidity of 40 ( 5%. The trough had an area of 225 cm2 (7.5 × 30 cm), and the pressure sensor had a resolution of 0.02 mN m-1. For each experiment, 20-50 µL of the 1.0 mM stock sample was spread on the water surface, followed by a 10 min wait for solvent evaporation before compression. The barrier was compressed at a speed of 4.0 mm min-1. Surface potential-area isotherms were measured on a homemade trough. Two symmetrically movable barriers controlled by the computer were used to regulate the surface area. The area of this trough was 100 × 12 cm2. The surface potential was measured by using the ionizing electrode method as described previously.15 A reference platinum electrode was immersed in the reference trough compartment, and an americium electrode (241Am) was placed about 1-2 mm above the water subphase. In situ UV-vis spectra of the monolayers were measured through a quartz window in the center of the KSV trough. They were recorded on a modified Hewlett-Packard 8452A diode-array spectrophotometer that could slide to the top of the quartz window. In situ fluorescence spectra of the monolayer at the air-water interface were measured by an optical fiber detector connected to a SPEX Fluorolog II fluorospectrometer. The tip of the fiber was placed about 1-2 mm above the monolayer. The spectra were blank-subtracted and corrected. The excitation wavelength was 366 nm. Fluorescence spectra of the LS films on glass slides were measured by placing the slide into a standard quartz cell vertically, with an angle of 45° toward the excitation and emission optical paths. Langmuir-Schaefer (LS) films were prepared on the KSV trough by using hydrophobically treated glass slides, which were prepared by depositing five layers of arachidic acid LS films using a homemade trough. The deposition was performed at a surface pressure of 30 mN/m. The subphase was 10-4 M CdCl2, pH 7.5. All equipment for the surface chemistry experiments were kept in a class 1000 clean room.

NMR spectra were recorded at either 75 or 100 MHz; chemical shifts are reported in ppm downfield from TMS using residual solvent as the reference line. (b) 9-Hexadecylanthracene. The following procedure is based on that of Kumada et al.13 A solution of hexadecyl bromide (0.89 g, 2.9 mmol) in 20 mL of ether was added slowly to a dry flask containing 0.10 g (4.3 mmol) of Mg and then refluxed for 4 h. In a separate flask, 32 mg of [1,3-bis(diphenylphosphino)propane]dichloronickel(II), Ni(dppp)Cl2, and 0.5 g (1.9 mmol) of 9-bromoanthracene were dried under high vacuum for 1 h and then dissolved in 5 mL of ether. The Grignard solution was transferred to the latter solution, and the reaction mixture was refluxed overnight. After cooling, the reaction was quenched by addition of 5 mL of 3% HCl. The aqueous layer was separated from the mixture and extracted with three 20 mL portions of ether. The combined organic extracts were washed sequentially with saturated sodium carbonate and brine, followed by drying over MgSO4. Removal of the solvent and purification by flash chromatography (2.5-5.0% EtOAc/hexane) gave the greenishyellow product. Yields ranged from 62 to 83%. 1H NMR (CDCl3): 0.86 (3H, t, J ) 6.5); 1.24 (22H, m); 1.39 (2H, m); 1.56 (2H, m); 1.79 (2H, m); 3.57 (2H, t, J ) 8.2); 7.43-7.50 (4H, m); 7.98 (2H, d, J ) 7.5); 8.25 (2H, d, J ) 8.8); 8.30 (1H, s). 13C NMR (CDCl3): 14.1; 22.7; 28.1; 29.4; 29.7 (9C); 30.4; 31.4; 31.9; 124.5 (2C); 124.7 (2C); 125.3 (2C); 125.4; 129.2 (2C); 129.5; 131.6 (2C); 135.5 (2C). Anal. Calcd for C30H42: C, 89.55; H, 10.45. Found: C, 89.62; H, 10.45. (c) 9-(Monoaza-18-crown-6-methyl)-10-hexadecylanthracene, CAC16. This compound was made in two steps: the chloromethylation of 9-hexadecylanthracene,14 and then substitution by monoaza-18-crown-6. A mixture of 0.12 g (0.29 mmol) of 9-hexadecylanthracene and 0.1 mL of chloromethyl methyl ether in 8 mL of acetic acid was heated at 60-65 °C for 3.5 h. After cooling, 2 mL of water was added and the mixture was filtered. The solid 9-(chloromethyl)-10-hexadecylanthracene was washed with three 5 mL portions of water and then dried in a vacuum desiccator. It was not purified further and was used directly in the next step. A mixture of the crude 9-(chloromethyl)10-hexadecylanthracene (0.11 g), 0.048 g of monoaza-18-crown6, 5 mg of KI, and 0.2 mL of triethylamine in 2 mL of benzene was refluxed for 3 h. After cooling, the mixture was diluted with 5 mL of ethyl acetate and the diluted mixture was filtered through Celite. The Celite was washed with 3 mL of ethyl acetate. Concentration afforded the crude product, which was purified by flash chromatography (0-10% MeOH in CH2Cl2 or CHCl3), giving variable yields of recovered hexadecylanthracene and the desired product. The 9-(monoaza-18-crown-6-methyl)-10-hexadecylanthracene fraction was dissolved in ethyl acetate, and the solution was washed with saturated sodium carbonate and dried over MgSO4, followed by concentration in vacuo. Yields ranged from 30 to 50%. 1H NMR (C6D6): 1.02 (3H, t, J ) 6.9); 1.35-1.65 (28H, m); 1.89 (2H, m); 3.09 (4H, t, J ) 5.5); 3.50-3.68 (20H, m); 4.71 (2H, s); 7.49 (4H, m); 8.42 (2H, d, J ) 8.3); 8.91 (2H, d,

Synthesis of CAC16. Scheme 1 shows the synthetic path for CAC16. Coupling of hexadecylmagnesium bromide

(13) Kumada, M.; Tamao, K.; Sumitani, K. Organic Syntheses; Wiley: New York, 1988; Collect. Vol. VI, p 407. (14) Badger, G. M.; Cook, J. W. J. Chem. Soc. 1939, 802.

(15) Lamarche, F.; Max, J. J.; Leblanc, R. M. In Surface Characterization of Biomaterials; Ratner, B. D., Ed.; Elsevier Science Publishers BV: Amsterdam, 1988; pp 117-133.

Results and Discussion

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Figure 2. Surface pressure-area isotherms of the CAC16 monolayer on different subphases.

with 9-bromoanthracene using Kumada’s procedure13 and [1,3-bis(diphenylphosphino)propane]nickel(II) chloride (Ni(dppp)Cl2) as the catalyst afforded 9-hexadecylanthracene in yields ranging from 62 to 83%. Chloromethylation at the 10-position,14 followed by alkylation with monoaza18-crown-6 (Aldrich), afforded CAC16 in 30-50% yields for the two steps, along with recovered hexadecylanthracene. Interfacial Study of Pure and Mixed CAC16 Langmuir Monolayers. (a) The Langmuir Monolayer of pure CAC16. Surface pressure-area isotherms of the pure CAC16 monolayer on different subphases are shown in Figure 2. On a pure water subphase (pH 5.8), the limiting molecular area16 is 58 Å2 molecule-1 and the collapse surface pressure is 32.5 mN m-1. For an acidic subphase (pH ) 2), the limiting area jumps to 83 Å2 molecule-1 and the collapse surface pressure increases to 36 mN m-1. In addition, surface potential-area isotherms of CAC16 show more fluctuations on the pure water subphase than on the pH 2.0 subphase in a molecular area range of 70-150 Å2 molecule-1. This means that, in this area range, the phase status of CAC16 depends on the subphase pH. On a pure water subphase, CAC16 is still in a coexisting gas/ liquid phase and the surface is only partially covered by the CAC16 monolayer. However, on the pH 2 subphase, CAC16 is in a liquid expanded phase and the surface is fully covered by the CAC16 monolayer. The area expansion could be due to the protonation of the nitrogen in CAC16, causing Coulombic repulsion between CAC16 molecules and increasing the effective surface area of the monolayer. When the same concentration of a sodium cation is present in a subphase (pH 5.8, 0.01 M NaCl), the area expansion is much smaller than that of same concentration (0.01 M) of protons in the subphase. The limiting molecular area is increased to 62 Å2 molecule-1, and the collapse surface pressure is increased to 37 mN m-1. The area expansion could be due to the binding or attachment of sodium cations to the crown ether of CAC16. This effect is similar to our previous observation17 on the Langmuir monolayers of crown ether-fullerene derivatives, which show area expansions when subphase cations are present. A recent report on similar crown ether moieties by Plehnert et al.18 also stated that the surface pressure-area isotherms of these amphiphiles depend on the type and (16) The term “limiting molecular area” means the average molecular area of the most compact monolayer (condensed phase) at a surface pressure of 0 mN/m. It is measured by linearly extrapolating the highest slope (lowest compressibility) of the isotherm to 0 mN/m. (17) Wang, S.; Leblanc, R. M.; Arias, F.; Echegoyen, L. Langmuir 1997, 13, 1672-1676. (18) Plehnert, R.; Schroter, J. A.; Tschierske, C. Langmuir 1998, 14, 5245-5249.

Figure 3. Surface pressure-area isotherms of different mixed CAC16/C20 monolayers on a pure water subphase.

concentration of subphase cations. A control experiment showed that, at pH 10, the limiting molecular area of a CAC16 monolayer is the same as that at pH 5.8 and there is only a slightly increased collapsed surface pressure (35 mN m-1). These results are in agreement with solution work showing that a proton has its maximum binding constant when it binds with anthracyl crown ether.19 (b) Mixed Langmuir Monolayers of CAC16 and Arachidic Acid (C20). To prevent possible fluorescence self-quenching, as discussed later, a matrix lipid was needed to mix with CAC16 to decrease the concentration of CAC16 on the surface. Two simple amphiphilic molecules were selected: arachidic acid (C20) and stearyl alcohol (C18OH). C20 is a commonly used matrix lipid because of its stability and miscibility; however, in our case, the acidic head group of C20 may affect the charge status of the nitrogen of CAC16, and so the less acidic C18OH was also tried. The miscibilities of these molecules with CAC16 were studied as follows. The surface pressure-area isotherms of different mixed CAC16/C20 monolayers on a pure water subphase (pH 5.8; Figure 3) and an acidic subphase (pH 2.0; not shown) were obtained. Similar to the case of pure CAC16, an increased limiting molecular area was observed for the pH 2.0 subphase and the same mixture. To analyze the miscibilities, we plotted the molecular areas of mixed CAC16/C20 monolayers as functions of the proportion of CAC16 at different surface pressures, as shown in Figure 3. For an immiscible monolayer, the molecular area of the mixture should follow the additivity rule20

A12 ) χ1A1 + χ2A2 where A12 is the mean molecular area expected at a given surface pressure in the two-component monolayer, χ1 and χ2 are the mole fractions of the components in the mixed monolayer, and A1 and A2 are the molecular areas of the pure components at the same surface pressure. Thus, a linear correlation between the molecular area and the mole fraction of one component could mean either immiscibility or miscibility with nearly ideal behavior. For a pure water subphase, large positive deviations of the molecular areas from the additivity rule are observed (19) de Silva, A. P.; de Silva, S. A. J. Chem. Soc., Chem. Commun. 1986, 1709-1710. (20) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Interscience: New York, 1966.

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Figure 5. Surface pressure-area isotherms of different mixed CAC16/C18OH monolayers on a pure water subphase.

Figure 4. Molecular areas of mixed CAC16/C20 monolayers as functions of CAC16 content at different surface pressures: (A) on pure water, pH 5.8; (B) on HCl, pH 2.0.

(Figure 4A). Slightly positive deviations are also observed for the pH 2.0 subphase (Figure 4B). These indicate that CAC16 is miscible with C20. The collapse surface pressures of the CAC16/C20 mixed monolayers also show a nearly linear relationship with the mole fractions of CAC16, which is further evidence of the miscibility. (c) Mixed Langmuir Monolayers of CAC16 and Stearyl Alcohol (C18OH). The surface pressure-area isotherms of different mixed CAC16/C18OH monolayers on a pure water subphase (pH 5.8; Figure 5) and an acidic subphase (pH 2.0; not shown) were obtained. Similar to the situation with pure CAC16, an increased limiting molecular area was observed for the pH 2.0 subphase for the same mixture. However, different from the case of the CAC16/C20 mixture, the miscibility analysis of CAC16/ C18OH monolayers showed immiscibility on the pure water subphase. The molecular areas of the mixtures are well fit to the additivity law (Figure 6A), and the collapse surface pressures of the mixtures are the same as those for CAC16. For the acidic subphase (pH 2), the molecular areas of the mixtures show negative deviations from the additivity rule (Figure 6B). The collapse surface pressures of the mixtures are higher than those for pure CAC16 at low molar fractions of CAC16. These observations indicate that, at pH 2, CAC16 and C18OH may be miscible. However, considering the possible hydrogen bonding between the hydroxyl group of C18OH and surrounding water molecules, it is unlikely that CAC16 and C18OH are well mixed; they may form large domains or be just partially miscible.

Figure 6. Molecular areas of mixed CAC16/C18OH monolayers as functions of CAC16 content at different surface pressures: (A) on pure water, pH 5.8; (B) on HCl, pH 2.0.

Although self-repulsion of CAC16 at low pH may ease the mixing, the fluorescence measurements on this binary system suggest a low miscibility of the two components (see below). In Situ UV-Vis Spectroscopy of the CAC16 Langmuir Monolayer. In situ UV-Vis spectra of the pure CAC16 monolayer at different surface pressures are shown in Figure 7. The subphase was pure water (pH 5.8). The intensity of the absorbance increases with the surface pressure. The peak at 265 nm resembles the peak in the solution absorption spectrum of N-(9-anthracyl)methyl-

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Figure 7. In situ UV-vis spectra of the CAC16 monolayer. Subphase: pure water, pH 5.8.

Figure 9. Fluorescence emission spectra of a 1:1 (molar ratio) mixed CAC16/C20 monolayer at different surface pressures. Subphase: pure water, pH 5.8. λex ) 366 nm.

Figure 8. Fluorescence emission spectra of the CAC16 monolayer at different surface pressures. Subphase: HCl, pH 2.0. λex ) 366 nm.

monoaza-18-crown-6. Other small peaks in the solution spectrum, including those at 366 and 386 nm (corresponding to the 0 f 0 and 0 f 1 transitions), were not observed in the monolayer spectra due to the weak signal of the single molecular layer. The absorbance at 265 nm increases linearly with the increase of surface pressure, which is a good indication of a perfect monolayer, i.e., one without any large domain formation. The absorption spectra of CAC16 on a pH 2 subphase and a subphase with 0.01 M NaCl show virtually no difference compared to the spectra on the pure water subphase. In Situ Fluorescence Spectroscopy of Pure and Mixed CAC16 Langmuir Monolayers. (a) The Pure CAC16 Monolayer. On the pure water subphase at pH 5.8, the pure CAC16 monolayer showed no measurable peaks in the in situ fluorescence emission spectra (λex ) 366 nm). Since anthracyl crown ethers have strong fluorescence emissions in aqueous solutions, the low fluorescence activity of the pure CAC16 monolayer could be due to the quenching effect. On a pH 2.0 subphase, a broad peak at about 480 nm appeared and the intensity increased with an increase of surface pressure (Figure 8). This is not a typical anthracene emission spectrum. The origin of this peak is not clear, but it may be due to an emission of multiple close-packed molecules in the monolayer rather than to the emission of a single molecule (a π-π* stacking effect). (b) Mixed Langmuir Monolayers of CAC16 and Arachidic Acid (C20). To test whether the low fluores-

Figure 10. Fluorescence emission spectra of a 1:9 (molar ratio) mixed CAC16/C20 monolayer at different surface pressures. Subphase: pure water, pH 5.8. λex ) 366 nm. The disappearance of the peak 435 nm is due to an artifact of the instrument, which is caused by a strong background peak in the same position at nil surface pressure (0 mN m-1).

cence activity of the pure CAC16 monolayer is caused by a self-quenching effect, arachidic acid was added to the CAC16 monolayer to decrease the concentration of CAC16 at the air-water interface. Figure 9 shows the in situ fluorescence emission spectra of a 1:1 (molar ratio) mixed Langmuir monolayer on a pure water subphase. When the surface pressure was 0 mN m-1, typical anthracene emission peaks at 410, 435, and 460 nm were recorded. The peaks disappeared when surface pressures increased to 10 mN m-1. This is strong evidence for the quenching effect. In the mixed monolayer, C20 acts as a twodimensional solvent to dilute CAC16 and prevent selfquenching, thus recovering part of the fluorescence activity of CAC16. Figure 10 shows the in situ fluorescence emission spectra of a mixed Langmuir monolayer on a pure water subphase with more C20 added (the CAC16:C20 molar ratio is 1:9). The anthracene peaks are clearer than those for the 1:1 mixture, and the maximum intensity of the emission appears at a surface pressure of 10 mN/m.

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Figure 12. Fluorescence emission spectrum of a 1:49 (molar ratio) mixed CAC16/C20 LS film on a hydrophobic glass slide. Subphase: pure water, pH 5.8. Langmuir-Schaefer transfer was performed at 30 mN/m. λex ) 366 nm.

Figure 11. Molecular packing orders and fluorescence quenching effects of pure and mixed CAC16 monolayers.

Furthermore, when the C20 molar ratio is increased to 1:49, the maximum intensity of the peak appears at 30 mN/m. No peak shift was observed regardless of the composition of the monolayer or the surface pressure. For a pH 2 subphase, no anthracene peaks appear for the mixture, regardless of the molar ratio of the components. The intensity of the broad peak appearing in the spectrum of the pure CAC16 monolayer at pH 2 decreases as the amount of C20 increases. It still can be seen in the spectrum of the 1:1 mixed monolayer but completely disappears in the spectra of the 1:9 and 1:49 mixtures. (c) Mixed Langmuir Monolayers of CAC16 and Stearyl Alcohol (C18OH). Mixed CAC16/C18OH Langmuir monolayers have no fluorescence activity regardless of the mole fraction of CAC16 and the subphase pH value. This is understandable in light of the results of the miscibility studies. On a pure water subphase, CAC16 is not miscible with C18OH, so the addition of C18OH cannot prevent contact between CAC16 molecules and thus cannot prevent quenching. At low pH, although the analysis of the surface pressure-area isotherm of CAC16/C18OH shows evidence of miscibility, no fluorescence enhancement was observed. The possible reason could be that CAC16 and C18OH molecules form domains instead of truly molecular-level mixtures; therefore, most of the CAC16 molecules are still not diluted and the self-quenching remains in effect. Figure 11 shows the relationship between the fluorescence activity and the two-dimensional dilution status of the monolayer. Pure CAC16 has no fluorescence because of the denser packing of the monolayer. In mixtures with C20, CAC16 was diluted, therefore, fluorescence can be detected. In mixtures with C18OH, the two kinds of molecules are not miscible. Thus, CAC16 molecules are still packed together and no fluorescence can be detected. (d) A Langmuir-Schaefer Film of CAC16 Mixed with Arachidic Acid. The LS film was successfully prepared from the CAC16/C20 mixed monolayer by the Langmuir-Schaefer transfer method. Figure 12 shows

the fluorescence emission spectrum (λex ) 366 nm) of a 1:49 (molar ratio) mixed CAC16/C20 LS film on a hydrophobic glass slide. The film, transferred at 30 mN m-1, shows a very nice and intense anthracene-like emission spectrum. The intensity of the peaks is much stronger than that for the corresponding Langmuir monolayers at the air-water interface. This is because the LS films were measured directly in the chamber of the fluorometer, like a regular solution sample, but the Langmuir monolayer was excited and detected through an optical fiber, in which a large portion of the signal intensity was lost. Conclusion Langmuir monolayers and Langmuir-Schaefer films of CAC16 were successfully prepared. The surface pressure-area and surface potential-area isotherms of CAC16 on different subphases show that protons and cations in the subphase may bind or attach to the crown ether part of CAC16. An increased limiting molecular area was observed for a pure CAC16 monolayer on a pH 2 subphase, which could be due to the Coulombic repulsion between the charged CAC16 molecules. In situ fluorescence emission spectra (λex ) 366 nm) of a CAC16 monolayer show a broad fluorescence band on the pH 2 subphase but none on the pure water subphase. Mixed monolayers of CAC16/C20 on a pure water subphase show increased fluorescence emission intensity of anthracene with an increase in the C20 mole fraction. This is interpreted to mean that the low fluorescence activity of the pure CAC16 monolayer is caused by a self-quenching effect. Analysis of the surface pressure-area isotherms indicates that CAC16 is not miscible with C18OH at the air-water interface; therefore, the monolayers have no fluorescence signal, regardless of the amount of C18OH. Langmuir-Schaefer films of CAC16/ C20 show stronger anthracene-like emission peaks on a substrate compare to the corresponding Langmuir monolayers at the air-water interface, which is a very positive result for our planned biosensor applications. Acknowledgment. Financial support from the U.S. Department of Agriculture (Grant 98-35201-6209) is gratefully acknowledged. LA991343H