- Email: [email protected]
water interface
Thin Solid Films, 243 (1994) 547-552
547
Influence of the phase state of phosphatidylcholine matrix monolayers on the photophysical characteristics of an amphiphilic oxacyanine dye at the air/water interface Ramesh C. Ahuja and Dietmar M6bius Max-Planek lnstitut fi~r biophysikalische Chemie, Am Faflberg, D-37077 Gdttingen (Germany)
Abstract
The photophysical characteristics of an amphiphilic oxacyanine dye embedded in phospholipid matrix monolayers at the air/water interface are reported. The matrix molecule used in the present study was dimyristoyl phosphatidylcholine. Surface pressure-, reflection- and fluorescence- area isotherms along with fluorescence and reflection spectra were measured as a function of dye/lipid molar ratio and subphase temperature. It is observed that the photophysical properties of oxacyanine dye depend sensitively on the molecular organization parameters of the matrix monolayer. The surface density normalized fluorescence intensity shows a strong increase with increasing surface pressure. The reflection-area isotherms along with the reflection spectra show no evidence of dimer formation up to a dye/lipid molar ratio of 1:5. Further increase of dye concentration leads to changes in reflection spectra indicating dimer and higher aggregate formation. The fluorescence intensity varies linearly with dye concentration up to a dye/lipid molar ratio of 1:50. The fluorescence quantum yield increases strongly with decreasing temperature (275-300 K) of the subphase. The dependence of photophysical properties of the dye on various molecular organization parameters is discussed in terms of phase transitions and fluidity of the matrix monolayer.
1. Introduction
Molecular probes have been used extensively to investigate various aspects of the structure, dynamics and molecular organization in membranes and monolayers [ 1]. Among the various photophysical properties of the probe molecules fluorescence has been found to be most sensitive to micro-environment characteristics such as polarity, local pH, viscosity, phase transitions, surface potential and phase separation. Fluorescent probe molecules have also been used for the visualization of various phases of monolayers at the air/water interface [2]. In addition, lipid fluorescent probes have been used to study diffusion phenomenon in two dimensions [3-5]. For diffusion studies and other photophysical applications, it is desirable that the fluorescent probe molecules remain in monomeric form. But what structural features of the lipid matrix and of the probe itself are responsible for the association behavior of the probe molecules? The preference of the probe molecules for a particular phase (basis of fluorescence microscopy of monolayers) leads to the segregation of the probe in that phase if the lipid monolayer shows two coexistent phases. The present investigation was therefore undertaken to characterize the influence of molecular organization parameters on the photophysical properties of an
0040-6090/94/$7.00 SSDI 0040-6090(93)04136-G
amiphiphilic oxacyanine dye in phosphatidylcholine matrix monolayers. Monolayers at the air/water interface are ideally suited for this purpose, as molecular organization parameters such as surface pressure, surface density (dye-lipid molar ratio), chromophore orientation and aggregation, subphase composition (nature of electrolyte and pH) and temperature are easy to control experimentally. Phosphatidylcholine molecules with even chain lengths of 1 0 - 1 8 C H 2 units were used as matrix monolayers. In this investigation, we report results only for dimyristoyl phosphatidylcholine matrix. The temperature of the aqueous subphase was varied between 275 K and 300 K. The dye/lipid molar ratio ranged between 1:1000 and 1:1. Surface pressure- and surface potential-area isotherms along with optical reflection and fluorescence spectroscopy were used for the characterization of mixed monolayers at the air/ water interface.
2. Materials and methods
N,N'-dioctadecyl oxacyanine perchlorate ($9) was synthesized by Sondermann [6]. Dimyristoyl-phosphatidylcholine (DMPC) was obtained from Sigma Chemical Co. and used as received. Chloroform (HPLC;
© 1994- Elsevier Sequoia. All rights reserved
548
R. C. Ahuja, D. M6bius / Association o/ oxacyanine dyes in matrix monalayers
Baker) stabilized with 2% ethanol was used as the spreading solvent. The subphase for all monolayer experiments was Milli-Q filtered water which has a p H of about 5.6 when in equilibrium with the atmosphere. Monolayer compression isotherms were measured on a thermostated Fromherz type round trough enclosed in a dark plastic cabinet which was flushed with nitrogen. The temperature of the subphase was kept constant at 294 K unless otherwise specified. A Wilhelmy balance was used for the surface pressure measurements. A detector head for either optical surface reflection or fluorescence measurements was located above the water surface. All reflection measurements were done with the light beam normal to the water surface using an apparatus as described previously [7]. The reflectance (AR) here is the difference in the reflectivity between the monolayer covered surface and the clean water surface. A chloroform solution of $9 and D M P C in the desired molar ratio was spread at the water surface using the continuous flow technique with the syringe tip barely touching the water surface.
3. R e s u l t s
and discussion
3.1. Characteristics o f $ 9 and D M P C The chemical structures of the amphiphilic oxacyanine dye and the matrix lipid molecule D M P C are shown in Fig. 1. The $9 chromophore is planar and has an additional plane of symmetry normal to the molecular plane. The oxacyanine molecule absorbs in the 300-400 nm region with a double m a x i m u m at 365 nm and 375nm. The absorbance ratio ( 3 6 5 n m / 3 7 5 n m ) depends on the aggregation and is about 0.85 for monomers. Dimers show a blue shifted band at about 362 nm. The emission spectrum shows a Stokes shift of about 30 nm. A C P K model of $9 shows that in the
case of a chromophore part lying flat, the area/molecule is about 1 nm 2. With the molecular plane normal to the interface, the area/molecule is about 0.6 nm 2. Photophysical characteristics of $9 chromophore in pure and mixed monolayers have been investigated [8-12]. Most of these studies have, however, been carried out on transferred monolayers. The matrix molecule D M P C used in the present investigation is a phospholipid having two saturated aliphatic chains and a polar phosphatidylcholine head group. The geometrical area per molecule is about 0.40 nm 2. In most of the previous studies [9-11, 13, 14] involving oxacyanine, arachidic acid or analogous lipids have been used as matrix molecules. Monolayers of arachidic acid show a solid type of isotherm at the air/water interface. Over the past few years, we have investigated mixed monolayers of oxacyanine dyes in many matrix molecules and our experience shows that all the matrix monolayers that show solid type isotherms are not suitable for the homogeneous mixing of the guest molecules. The best matrix molecules for homogeneous mxing are those that show expanded liquid type isotherms. 3.1. I. Sur)Cace pressure area isotherms o f D M P C The surface pressure-area isotherms of D M P C at different subphase temperatures are shown in Fig. 2. It is seen that the ~ - A isotherm at 2 9 7 K is of liquid expanded type and does not show any phase transition until ~ = 4 0 m N m '. The area per molecule at : z - - 4 0 m N m ' is about 0 . 5 n m 2, which is more than the area of two aliphatic chains in the densely packed state. As the temperature is lowered to 275 K (Fig. 2, curve (f)), the isotherm shows liquid phase followed by a liquid/solid coexistence region and the transition to the solid condensed region. The area per molecule in the condensed state is 0.39 nm 2, which is equal to the geometrical area of two aliphatic chains. This indicates
50-
z
(a)
40-
0~ ffl ffl 0
SO-
IX. O O
20-
a
0
II
(b)
0
o,.4", H o i la
~,~/~'...,,O~ u
.o-
I
N+.. / CHs ~Ha
Fig. 1. Chemical structures of (a) amphiphilic $9 and (b) the matrix lipid DMPC.
0-
o'4
o'.s
o'.~
o'7
0'.8
0'9
Area/Molecule [nm 2 ]
Fig. 2. Surface pressure area isotherms of the matrix lipid DMPC at the air/water interface at different temperatures of the subphase: (a) 297 K; (b) 292 K: (c) 288 K: (d) 282 K: (e) 278 K; (f) 275 K.
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
that the phosphocholine group does not contribute to the molecular area. The control on the shape of the n - A isotherm profile through the subphase temperature allows us to study the effect of phase state of the lipid monolayer on the photophysical properties of the oxacyanine dye.
0.70.6=
o~
Aav =
X M •
0.50.4-
~_. 0)
3.2. Oxaeyanine/DMPC mixed monolayers 3.2.1. Surface pressure -area isotherms Figure 3 shows the rc-A isotherms of the S9:DMPC mixed monolayers at various molar ratios of $9. It is seen that the isotherms show expansion due to the presence of $9. The area per $9 molecule at various surface pressures can be calculated from the following relation
549
0.3-
n-" <1
0.20.1
0.0320
i 340
i 360
i 380
i 400
420
Wavelength [nm] Fig. 4. Reflectionspectra of the mixed monolayers S9:DMPC, molar ratio l:x, at various compositions: (a) x = 1; (b) x = 2; (c) x = 5; (d) x = 10; (e) x = 20 (temperature 296 K).
A M + X"D . A D
where Aav is the average area per molecule in the mixed monolayer, X M and XD are the molar fractions and A M and A D a r e the areas per molecule of the matrix and dye molecules at the corresponding surface pressure respectively. The area per $9 molecule thus calculated from the data in Fig. 3 decreases from 1.08 nm2(10 mN m -i) to 0.5 nm2(40 mN m-i). As mentioned above, these area values correspond to the two conformations of the chromophore. The change in area/S9 molecule with increasing surface pressure shows that reorientation of $9 takes place during the compression process with the molecular plane changing from parallel to perpendicular to the air/water interface. Another interpretation of such isotherm data is that the free area on the chromophore may be occupied by the matrix molecules. But this is not likely in our case, as the matrix D M P C molecules are large. Similar conclusions have been reached by other investigators [I I, 12] using matrix molecules such as arachidic acid.
50.EE 40-
3.2.2. Reflection spectroscopy The optical reflection spectroscopy of S9:DMPC mixed monolayers, and reflection-area isotherm measurements, were performed to get information about the orientation and aggregation of $9. Figure 4 shows the reflection spectra at 40 m N m - ' of S9:DMPC mixed monolayers at various compositions of $9. Two reflection maxima corresponding to those in the solution (see Section 3.1) are seen at 363 nm and 378 nm. It is seen that the reflection ratio (AR363[AR378) changes as the concentration of $9 in the mixed monolayer increases. The value of the reflection ratio in mixed monolayers is close to the value in solution up to an S9:DMPC molar ratio of 1:5. As the concentration of $9 is increased furhter, the short wavelength maximum at 363 nm gains more weight and the value of the reflection ratio increases. At the same time, the long wavelength maximum shifts to higher wavelengths, indicating the presence of higher aggregates. The reflection spectra (data not shown) of S9:DMPC mixed monolayers, molar ratio 1:10, at different surface pressures show that the spectra of $9 are of monomer type and that the shape of the spectra does not change as the surface density of $9 is increased by a factor of two through compression.
3.2.3. Reflection-area isotherms 30-
0..
20-
0
~.) 100-
os
1'0
lls
210
2s
Area/DMPC Molecule [nm 2 ] Fig. 3. Surface pressure area i s o t h e r m s o f the m i x e d m o n o l a y e r s o f S 9 : D M P C , m o l a r r a t i o l:x, at various c o m p o s i t i o n s : (a) x = 1; (b) x = 2; (c) x = 5; (d) x = 10; (e) x = 2 0 ; (f) x = 50 ( t e m p e r a t u r e 296 K).
Correlation of the reflection spectra with reflectionarea isotherms provides information about any changes in the orientation of the optical transition axis of the chromophores during the compression process. The optical transition moment is known [ 12] to be parallel to the long molecular axis. Thus in the absence of any aggregation, the surface density normalized reflection (ARN = AR. A) of the probe molecule should remain constant as a function of area/molecule if the probe molecules do not undergo any reorientation during the compression process. The surface density normalized reflection (at 375 n m ) - a r e a isotherms of S9:DMPC
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
550 0.35~ o
0.30-
.~
0.25-
"5
,,~ 0.20-
n-
"O 0.15O N :'~ 0.10-
E O
z
C
d 0.05-
--
e
f ooo
o:6
i 0'.8 1.o 1.2 Area/Molecule [nm 2 ]
1..
Fig. 5. Normalized reflection ( A R . A ) - a r e a isotherms of the mixed monolayers S9:DMPC, molar ratio l:x, at various compositions: (a) x = l; (b) x = 2; (c) x = 5; (d) x = 10; (e) x = 20; (f) x = 50 (temperature 296 K).
mixed monolayers at various concentrations of $9 are shown in Fig. 5. It is seen that for S9:DMPC molar ratios up to 1:10, the value of A R N remains constant during the whole compression process. This result coupled with the fact that reflection spectra do not change with compression shows that the optical transition axis of $9 does not reorient during the compression process. The earlier conclusion from the n - A isotherms about the change of $9 molecular conformation with the molecular plane of $9 going from parallel to perpendicular to the air/water interface during the compression does not contradict this, as the optical transition axis remains parallel to the interface for both the conformations. For higher concentrations of $9, the ARN isotherms are no longer flat and the values of ARN decrease with decreasing area/molecule. This is mainly because of the aggregation of $9 at higher concentrations, as has already been discussed (see Fig. 4 and section 3.2.). The S9:DMPC mixed monolayer, molar ratio 1:1, shows peculiar behavior in that the value of ARN (at 375 nm) shows first a continuous decrease up to A = 0.63 nm 2 and then a sharp increase with further compression. This is due to the fact that at high surface densities and at such higher $9 concentrations, phase separation of $9 and D M P C in the mixed monolayer occurs (see Fig. 3, curve (a), where a sharp change in isotherm slope is seen at high surface pressure values), leading to the formation of higher aggregates which show red shifted absorption spectra. For example, J-aggregates of $9 show a sharp absorption band at 402 nm. Thus for mixed monolayers of S9:DMPC with molar ratios lower than 1:5, we expect to have mixtures of monomers, dimers and higher aggregates. This is also clearly seen by the asymmetrical broadening of the reflection spectra, as shown in Fig. 4. The dependence of reflection at 375 nm on $9 concentration at different surface pressure values shows that for mixed monolayers with
molar ratio up to 1:5, the reflection increases linearly at all surface pressure values. In addition, at surface pressure values below or equal to 20 m N m - i, the reflection signal increases linearly in the whole concentration range investigated. The deviation from linearity for higher concentrations of $9 and at higher surface pressure values is due to dimer formation. These results show that aggregates are not formed immediately after spreading but show up as a result of compression at high surface densities. 3.2.4. Fluorescence spectra The fluorescence emission and excitation spectra of the mixed monolayers of S9:DMPC at different molar ratios of $9 were measured to check for aggregation effects. The surface pressure was kept constant 40 mN m-~ and the temperature was 296 K. The emission spectrum shows a peak at 410 nm and a shoulder at 390 nm. The excitation spectrum taken at the emission wavelength 415 nm shows a shoulder at 364nm and a peak at 378 nm. The excitation and emission spectra do not change with increasing concentration of $9 up to a molar ratio of 1:10. The excitation spectra resemble the reflection spectra as shown in Fig. 4, which shows the corresponding bands at 363.5 nm and 378 nm respectively. 3.2.5. Fluorescence-area isotherms The surface density normalized fluorescence ( F . A ) area isotherms of the S9:DMPC mixed monolayers at different molar ratios of $9 are shown in Fig. 6. It is seen that independent of the concentration of $9, the fluorescence quantum yield shows a strong dependence on surface pressure in that the sursface density normalized fluorescence intensity (F.A) increases with increasing surface pressure. Thus normalized intensity increases by a factor of 1.5-3 as the area/molecule decreases by a
50 ~ "'-2, 40-
~
30-
o ~ 0
20-
ii 10-
0-
0.4
0.6
0.8
1.0
1.2
1.4
Area/Molecule [nm 2 ] Fig. 6. Normalized fluorescence (F. A ) - a r e a isotherms of the mixed monolayers S9:DMPC, molar ratio l:x, at various compositions: (a) x = 5 ; (b) x = 10; (c) x = 2 0 ; (d) x = 5 0 ; (e) x = 100; (f) x = 200; (g) x = 5 0 0 ; (h) x = 1 0 0 0 (temperature 2 9 6 K , 2 e x = 3 6 0 n m , 2e, , = 415 nm).
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
factor of about 3. The data of Fig. 6 have been replotted (data not shown) to study the dependence of fluorescence intensity on S9:DMPC molar ratio at various surface pressure values. These curves show that the fluorescence intensity varies linearly with increasing $9 concentration up to a molar ratio of 1:50. For higher $9 concentrations, the intensity increase is sublinear, showing fluorescence quenching due to dimer formation [9]. In this context it should be mentioned that the reflection spectra do not show any evidence for dimer formation up to an S9:DMPC molar ratio as high as 1:5. Evidently, reflection/absorption spectroscopy is less sensitive in the detection of dimers. 3.2. 6. Effect of temperature To investigate whether or not the $9 fluorescence quantum yield is sensitive to phase changes of the surrounding matrix monolayer, the temperature dependence of the fluorescence characteristics of S9:DMPC (1:100) mixed monolayers was investigated at various subphase temperatures in the 275-298 K range. The dependence of 7t-A isotherms of DMPC on temperature is shown in Fig. 2. These results show that as the temperature is lowered, the isotherm profile of DMPC changes from that of pure liquid phase (T > 295 K) to that showing liquid, liquid-solid coexistence and solid phases. The appearance of solid phase is also reflected in the fluorescence intensity-area isotherms. The fluorescence intensity increases discontinuously in going from liquid to solid phase. $9 shows a preference for the liquid phase in the liquid/solid coexistance region. The fluorescence quantum yield of $9 in the solid phase is much higher than that in the fluid phase. At low temperatures ( T < 2 7 8 K) and within the solid phase, there is another subsolid transition at 0.42 nm 2 (re = 37 mN m-~), indicated by a further change in the slope of fluorescence intensity-area profile. The temperature dependence of the fluorescence intensity of the S9:DMPC, molar ratio 1:100, mixed monolayer at 40 mN m ~ is shown in Fig. 7. Three regions (marked I, II, III) of fluorescence intensity variation with temperature are observed. The break in intensity at 286 K may be identified by the known subtransition temperature for the DMPC matrix monolayer. Below 286 K, a pronounced solid/liquid coexistence region and solid phase regions (see Fig. 2) within the investigated surface pressure range are observed. The increase of fluorescence intensity with decreasing temperature within region III may be related to increased rigidity of the system. Region II in the fluorescence intensity may be attributed to increasing density of solid domains within this temperature range. The second break point may be attributed to another subtransition temperature which has not yet been described in the literature. In addition to the phase state, interfacial viscosity and the
551
11.5-
.'~--~11.0. ¢-
E lo.5. " 10.0. i
9.52
3.35
a.:~o a.:~5 3.~0
3.~5
3.~0
3.65
1000/T
Fig. 7. Temperature dependence of the fluorescence intensity of S9:DMPC mixed monolayer, molar ratio hl00 (surface pressure 40 mN m -I, 2ex = 360nm, ~'em= 415 rim.
change in location of the chromophore may contribute to the temperature dependence of $9 fluorescence.
4. Conclusions It has been observed that the photophysical properties of the amphiphilic oxacyanine dye depend sensitively on the molecular organization state of the matrix phospholipid monolayer. The reflection spectroscopy data, along with surface pressure-area isotherms, show that the chromophore adopts an edge-on conformation, with the long molecular axis parallel to the air/water interface. The reflection spectra show the presence of dimers and higher aggregates for dye/lipid molar ratios lower than 1:10. The fluorescence characteristics are much more sensitive to the matrix monolayer state. The fluorescence intensity of the mixed monolayers depends linearly on dye/lipid molar ratios higher than 1:50. The fluorescence quantum yield of oxacyanine is highly sensitive to the phase state of the monolayer and is higher in the solid phase. The oxacyanine dye shows preference for the liquid phase in the liquid/solid coexistence region. The temperature dependence of the fluorescence intensity shows two breakpoints, at 286 K and 291 K. The break at 286 K coincides with the known subtransition temperature of DMPC.
Acknowledgments Financial support of the project by the Bundesministerium f/Jr Forschung und Technologie through grant 03M40080D is gratefully acknowledged.
References 1 R. P. Haugland, Handbook of Fluorescent Probes and Chemicals, Molecular Probes Inc., Eugene, OR, 1992. 2 H. M6hwald, Annu. Rev. Phys. Chem., 41 (1990) 441. 3 F. Caruso, F. Grieser, A. Murphy, P. Thistlewaite, R. Urquhart, M. Almgren and E. Wistus, J. Am. Chem. Soc., 113 (1991) 4838.
552
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
4 T. Loughran, M. D. Hatlee, L. K. Patterson and J. J. Kozak, J. Chem. Phys., 72 (1980) 5791. 5 R. Peters and K. Beck, Pro c. Natl. A cad. Sci. U.S.A., 80 (1983) 7183. 6 J. Sondermann, Liebigs Ann. Chem., 749 (1971) 183. 7 H. Grfiniger, D. M6bius and H. Meyer, J. Chem. Phys., 79(1983) 3701. 8 K. Lesch, Ph.D. Thesis, University of Marburg, 1959. 9 R. C. Ahuja and D. M6bius, Thin Solid Films, 179(1989) 457.
10 N. Tamai, H. Matuso, T. Yamazaki and I. Yamazaki, J. Phys.
Chem., 96 (1992) 6550. 11 S. Vaidyanathan, L. K. Patterson, D. M6bius and H. R. Gruniger, J. Phys. Chem., 89(1985)491. 12 H. Kuhn, Pure Appl. Chem., 27(1971) 421. 13. D. M6bius and H. Kuhn, Isr. J. Chem., 18 (1979) 375. 14 I. Yamazaki, N. Tamai and T. Yamazaki, J. Phys. Chem., 94(1990) 516.
547
Influence of the phase state of phosphatidylcholine matrix monolayers on the photophysical characteristics of an amphiphilic oxacyanine dye at the air/water interface Ramesh C. Ahuja and Dietmar M6bius Max-Planek lnstitut fi~r biophysikalische Chemie, Am Faflberg, D-37077 Gdttingen (Germany)
Abstract
The photophysical characteristics of an amphiphilic oxacyanine dye embedded in phospholipid matrix monolayers at the air/water interface are reported. The matrix molecule used in the present study was dimyristoyl phosphatidylcholine. Surface pressure-, reflection- and fluorescence- area isotherms along with fluorescence and reflection spectra were measured as a function of dye/lipid molar ratio and subphase temperature. It is observed that the photophysical properties of oxacyanine dye depend sensitively on the molecular organization parameters of the matrix monolayer. The surface density normalized fluorescence intensity shows a strong increase with increasing surface pressure. The reflection-area isotherms along with the reflection spectra show no evidence of dimer formation up to a dye/lipid molar ratio of 1:5. Further increase of dye concentration leads to changes in reflection spectra indicating dimer and higher aggregate formation. The fluorescence intensity varies linearly with dye concentration up to a dye/lipid molar ratio of 1:50. The fluorescence quantum yield increases strongly with decreasing temperature (275-300 K) of the subphase. The dependence of photophysical properties of the dye on various molecular organization parameters is discussed in terms of phase transitions and fluidity of the matrix monolayer.
1. Introduction
Molecular probes have been used extensively to investigate various aspects of the structure, dynamics and molecular organization in membranes and monolayers [ 1]. Among the various photophysical properties of the probe molecules fluorescence has been found to be most sensitive to micro-environment characteristics such as polarity, local pH, viscosity, phase transitions, surface potential and phase separation. Fluorescent probe molecules have also been used for the visualization of various phases of monolayers at the air/water interface [2]. In addition, lipid fluorescent probes have been used to study diffusion phenomenon in two dimensions [3-5]. For diffusion studies and other photophysical applications, it is desirable that the fluorescent probe molecules remain in monomeric form. But what structural features of the lipid matrix and of the probe itself are responsible for the association behavior of the probe molecules? The preference of the probe molecules for a particular phase (basis of fluorescence microscopy of monolayers) leads to the segregation of the probe in that phase if the lipid monolayer shows two coexistent phases. The present investigation was therefore undertaken to characterize the influence of molecular organization parameters on the photophysical properties of an
0040-6090/94/$7.00 SSDI 0040-6090(93)04136-G
amiphiphilic oxacyanine dye in phosphatidylcholine matrix monolayers. Monolayers at the air/water interface are ideally suited for this purpose, as molecular organization parameters such as surface pressure, surface density (dye-lipid molar ratio), chromophore orientation and aggregation, subphase composition (nature of electrolyte and pH) and temperature are easy to control experimentally. Phosphatidylcholine molecules with even chain lengths of 1 0 - 1 8 C H 2 units were used as matrix monolayers. In this investigation, we report results only for dimyristoyl phosphatidylcholine matrix. The temperature of the aqueous subphase was varied between 275 K and 300 K. The dye/lipid molar ratio ranged between 1:1000 and 1:1. Surface pressure- and surface potential-area isotherms along with optical reflection and fluorescence spectroscopy were used for the characterization of mixed monolayers at the air/ water interface.
2. Materials and methods
N,N'-dioctadecyl oxacyanine perchlorate ($9) was synthesized by Sondermann [6]. Dimyristoyl-phosphatidylcholine (DMPC) was obtained from Sigma Chemical Co. and used as received. Chloroform (HPLC;
© 1994- Elsevier Sequoia. All rights reserved
548
R. C. Ahuja, D. M6bius / Association o/ oxacyanine dyes in matrix monalayers
Baker) stabilized with 2% ethanol was used as the spreading solvent. The subphase for all monolayer experiments was Milli-Q filtered water which has a p H of about 5.6 when in equilibrium with the atmosphere. Monolayer compression isotherms were measured on a thermostated Fromherz type round trough enclosed in a dark plastic cabinet which was flushed with nitrogen. The temperature of the subphase was kept constant at 294 K unless otherwise specified. A Wilhelmy balance was used for the surface pressure measurements. A detector head for either optical surface reflection or fluorescence measurements was located above the water surface. All reflection measurements were done with the light beam normal to the water surface using an apparatus as described previously [7]. The reflectance (AR) here is the difference in the reflectivity between the monolayer covered surface and the clean water surface. A chloroform solution of $9 and D M P C in the desired molar ratio was spread at the water surface using the continuous flow technique with the syringe tip barely touching the water surface.
3. R e s u l t s
and discussion
3.1. Characteristics o f $ 9 and D M P C The chemical structures of the amphiphilic oxacyanine dye and the matrix lipid molecule D M P C are shown in Fig. 1. The $9 chromophore is planar and has an additional plane of symmetry normal to the molecular plane. The oxacyanine molecule absorbs in the 300-400 nm region with a double m a x i m u m at 365 nm and 375nm. The absorbance ratio ( 3 6 5 n m / 3 7 5 n m ) depends on the aggregation and is about 0.85 for monomers. Dimers show a blue shifted band at about 362 nm. The emission spectrum shows a Stokes shift of about 30 nm. A C P K model of $9 shows that in the
case of a chromophore part lying flat, the area/molecule is about 1 nm 2. With the molecular plane normal to the interface, the area/molecule is about 0.6 nm 2. Photophysical characteristics of $9 chromophore in pure and mixed monolayers have been investigated [8-12]. Most of these studies have, however, been carried out on transferred monolayers. The matrix molecule D M P C used in the present investigation is a phospholipid having two saturated aliphatic chains and a polar phosphatidylcholine head group. The geometrical area per molecule is about 0.40 nm 2. In most of the previous studies [9-11, 13, 14] involving oxacyanine, arachidic acid or analogous lipids have been used as matrix molecules. Monolayers of arachidic acid show a solid type of isotherm at the air/water interface. Over the past few years, we have investigated mixed monolayers of oxacyanine dyes in many matrix molecules and our experience shows that all the matrix monolayers that show solid type isotherms are not suitable for the homogeneous mixing of the guest molecules. The best matrix molecules for homogeneous mxing are those that show expanded liquid type isotherms. 3.1. I. Sur)Cace pressure area isotherms o f D M P C The surface pressure-area isotherms of D M P C at different subphase temperatures are shown in Fig. 2. It is seen that the ~ - A isotherm at 2 9 7 K is of liquid expanded type and does not show any phase transition until ~ = 4 0 m N m '. The area per molecule at : z - - 4 0 m N m ' is about 0 . 5 n m 2, which is more than the area of two aliphatic chains in the densely packed state. As the temperature is lowered to 275 K (Fig. 2, curve (f)), the isotherm shows liquid phase followed by a liquid/solid coexistence region and the transition to the solid condensed region. The area per molecule in the condensed state is 0.39 nm 2, which is equal to the geometrical area of two aliphatic chains. This indicates
50-
z
(a)
40-
0~ ffl ffl 0
SO-
IX. O O
20-
a
0
II
(b)
0
o,.4", H o i la
~,~/~'...,,O~ u
.o-
I
N+.. / CHs ~Ha
Fig. 1. Chemical structures of (a) amphiphilic $9 and (b) the matrix lipid DMPC.
0-
o'4
o'.s
o'.~
o'7
0'.8
0'9
Area/Molecule [nm 2 ]
Fig. 2. Surface pressure area isotherms of the matrix lipid DMPC at the air/water interface at different temperatures of the subphase: (a) 297 K; (b) 292 K: (c) 288 K: (d) 282 K: (e) 278 K; (f) 275 K.
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
that the phosphocholine group does not contribute to the molecular area. The control on the shape of the n - A isotherm profile through the subphase temperature allows us to study the effect of phase state of the lipid monolayer on the photophysical properties of the oxacyanine dye.
0.70.6=
o~
Aav =
X M •
0.50.4-
~_. 0)
3.2. Oxaeyanine/DMPC mixed monolayers 3.2.1. Surface pressure -area isotherms Figure 3 shows the rc-A isotherms of the S9:DMPC mixed monolayers at various molar ratios of $9. It is seen that the isotherms show expansion due to the presence of $9. The area per $9 molecule at various surface pressures can be calculated from the following relation
549
0.3-
n-" <1
0.20.1
0.0320
i 340
i 360
i 380
i 400
420
Wavelength [nm] Fig. 4. Reflectionspectra of the mixed monolayers S9:DMPC, molar ratio l:x, at various compositions: (a) x = 1; (b) x = 2; (c) x = 5; (d) x = 10; (e) x = 20 (temperature 296 K).
A M + X"D . A D
where Aav is the average area per molecule in the mixed monolayer, X M and XD are the molar fractions and A M and A D a r e the areas per molecule of the matrix and dye molecules at the corresponding surface pressure respectively. The area per $9 molecule thus calculated from the data in Fig. 3 decreases from 1.08 nm2(10 mN m -i) to 0.5 nm2(40 mN m-i). As mentioned above, these area values correspond to the two conformations of the chromophore. The change in area/S9 molecule with increasing surface pressure shows that reorientation of $9 takes place during the compression process with the molecular plane changing from parallel to perpendicular to the air/water interface. Another interpretation of such isotherm data is that the free area on the chromophore may be occupied by the matrix molecules. But this is not likely in our case, as the matrix D M P C molecules are large. Similar conclusions have been reached by other investigators [I I, 12] using matrix molecules such as arachidic acid.
50.EE 40-
3.2.2. Reflection spectroscopy The optical reflection spectroscopy of S9:DMPC mixed monolayers, and reflection-area isotherm measurements, were performed to get information about the orientation and aggregation of $9. Figure 4 shows the reflection spectra at 40 m N m - ' of S9:DMPC mixed monolayers at various compositions of $9. Two reflection maxima corresponding to those in the solution (see Section 3.1) are seen at 363 nm and 378 nm. It is seen that the reflection ratio (AR363[AR378) changes as the concentration of $9 in the mixed monolayer increases. The value of the reflection ratio in mixed monolayers is close to the value in solution up to an S9:DMPC molar ratio of 1:5. As the concentration of $9 is increased furhter, the short wavelength maximum at 363 nm gains more weight and the value of the reflection ratio increases. At the same time, the long wavelength maximum shifts to higher wavelengths, indicating the presence of higher aggregates. The reflection spectra (data not shown) of S9:DMPC mixed monolayers, molar ratio 1:10, at different surface pressures show that the spectra of $9 are of monomer type and that the shape of the spectra does not change as the surface density of $9 is increased by a factor of two through compression.
3.2.3. Reflection-area isotherms 30-
0..
20-
0
~.) 100-
os
1'0
lls
210
2s
Area/DMPC Molecule [nm 2 ] Fig. 3. Surface pressure area i s o t h e r m s o f the m i x e d m o n o l a y e r s o f S 9 : D M P C , m o l a r r a t i o l:x, at various c o m p o s i t i o n s : (a) x = 1; (b) x = 2; (c) x = 5; (d) x = 10; (e) x = 2 0 ; (f) x = 50 ( t e m p e r a t u r e 296 K).
Correlation of the reflection spectra with reflectionarea isotherms provides information about any changes in the orientation of the optical transition axis of the chromophores during the compression process. The optical transition moment is known [ 12] to be parallel to the long molecular axis. Thus in the absence of any aggregation, the surface density normalized reflection (ARN = AR. A) of the probe molecule should remain constant as a function of area/molecule if the probe molecules do not undergo any reorientation during the compression process. The surface density normalized reflection (at 375 n m ) - a r e a isotherms of S9:DMPC
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
550 0.35~ o
0.30-
.~
0.25-
"5
,,~ 0.20-
n-
"O 0.15O N :'~ 0.10-
E O
z
C
d 0.05-
--
e
f ooo
o:6
i 0'.8 1.o 1.2 Area/Molecule [nm 2 ]
1..
Fig. 5. Normalized reflection ( A R . A ) - a r e a isotherms of the mixed monolayers S9:DMPC, molar ratio l:x, at various compositions: (a) x = l; (b) x = 2; (c) x = 5; (d) x = 10; (e) x = 20; (f) x = 50 (temperature 296 K).
mixed monolayers at various concentrations of $9 are shown in Fig. 5. It is seen that for S9:DMPC molar ratios up to 1:10, the value of A R N remains constant during the whole compression process. This result coupled with the fact that reflection spectra do not change with compression shows that the optical transition axis of $9 does not reorient during the compression process. The earlier conclusion from the n - A isotherms about the change of $9 molecular conformation with the molecular plane of $9 going from parallel to perpendicular to the air/water interface during the compression does not contradict this, as the optical transition axis remains parallel to the interface for both the conformations. For higher concentrations of $9, the ARN isotherms are no longer flat and the values of ARN decrease with decreasing area/molecule. This is mainly because of the aggregation of $9 at higher concentrations, as has already been discussed (see Fig. 4 and section 3.2.). The S9:DMPC mixed monolayer, molar ratio 1:1, shows peculiar behavior in that the value of ARN (at 375 nm) shows first a continuous decrease up to A = 0.63 nm 2 and then a sharp increase with further compression. This is due to the fact that at high surface densities and at such higher $9 concentrations, phase separation of $9 and D M P C in the mixed monolayer occurs (see Fig. 3, curve (a), where a sharp change in isotherm slope is seen at high surface pressure values), leading to the formation of higher aggregates which show red shifted absorption spectra. For example, J-aggregates of $9 show a sharp absorption band at 402 nm. Thus for mixed monolayers of S9:DMPC with molar ratios lower than 1:5, we expect to have mixtures of monomers, dimers and higher aggregates. This is also clearly seen by the asymmetrical broadening of the reflection spectra, as shown in Fig. 4. The dependence of reflection at 375 nm on $9 concentration at different surface pressure values shows that for mixed monolayers with
molar ratio up to 1:5, the reflection increases linearly at all surface pressure values. In addition, at surface pressure values below or equal to 20 m N m - i, the reflection signal increases linearly in the whole concentration range investigated. The deviation from linearity for higher concentrations of $9 and at higher surface pressure values is due to dimer formation. These results show that aggregates are not formed immediately after spreading but show up as a result of compression at high surface densities. 3.2.4. Fluorescence spectra The fluorescence emission and excitation spectra of the mixed monolayers of S9:DMPC at different molar ratios of $9 were measured to check for aggregation effects. The surface pressure was kept constant 40 mN m-~ and the temperature was 296 K. The emission spectrum shows a peak at 410 nm and a shoulder at 390 nm. The excitation spectrum taken at the emission wavelength 415 nm shows a shoulder at 364nm and a peak at 378 nm. The excitation and emission spectra do not change with increasing concentration of $9 up to a molar ratio of 1:10. The excitation spectra resemble the reflection spectra as shown in Fig. 4, which shows the corresponding bands at 363.5 nm and 378 nm respectively. 3.2.5. Fluorescence-area isotherms The surface density normalized fluorescence ( F . A ) area isotherms of the S9:DMPC mixed monolayers at different molar ratios of $9 are shown in Fig. 6. It is seen that independent of the concentration of $9, the fluorescence quantum yield shows a strong dependence on surface pressure in that the sursface density normalized fluorescence intensity (F.A) increases with increasing surface pressure. Thus normalized intensity increases by a factor of 1.5-3 as the area/molecule decreases by a
50 ~ "'-2, 40-
~
30-
o ~ 0
20-
ii 10-
0-
0.4
0.6
0.8
1.0
1.2
1.4
Area/Molecule [nm 2 ] Fig. 6. Normalized fluorescence (F. A ) - a r e a isotherms of the mixed monolayers S9:DMPC, molar ratio l:x, at various compositions: (a) x = 5 ; (b) x = 10; (c) x = 2 0 ; (d) x = 5 0 ; (e) x = 100; (f) x = 200; (g) x = 5 0 0 ; (h) x = 1 0 0 0 (temperature 2 9 6 K , 2 e x = 3 6 0 n m , 2e, , = 415 nm).
R. C. Ahuja, D. M6bius / Association of oxacyanine dyes in matrix monolayers
factor of about 3. The data of Fig. 6 have been replotted (data not shown) to study the dependence of fluorescence intensity on S9:DMPC molar ratio at various surface pressure values. These curves show that the fluorescence intensity varies linearly with increasing $9 concentration up to a molar ratio of 1:50. For higher $9 concentrations, the intensity increase is sublinear, showing fluorescence quenching due to dimer formation [9]. In this context it should be mentioned that the reflection spectra do not show any evidence for dimer formation up to an S9:DMPC molar ratio as high as 1:5. Evidently, reflection/absorption spectroscopy is less sensitive in the detection of dimers. 3.2. 6. Effect of temperature To investigate whether or not the $9 fluorescence quantum yield is sensitive to phase changes of the surrounding matrix monolayer, the temperature dependence of the fluorescence characteristics of S9:DMPC (1:100) mixed monolayers was investigated at various subphase temperatures in the 275-298 K range. The dependence of 7t-A isotherms of DMPC on temperature is shown in Fig. 2. These results show that as the temperature is lowered, the isotherm profile of DMPC changes from that of pure liquid phase (T > 295 K) to that showing liquid, liquid-solid coexistence and solid phases. The appearance of solid phase is also reflected in the fluorescence intensity-area isotherms. The fluorescence intensity increases discontinuously in going from liquid to solid phase. $9 shows a preference for the liquid phase in the liquid/solid coexistance region. The fluorescence quantum yield of $9 in the solid phase is much higher than that in the fluid phase. At low temperatures ( T < 2 7 8 K) and within the solid phase, there is another subsolid transition at 0.42 nm 2 (re = 37 mN m-~), indicated by a further change in the slope of fluorescence intensity-area profile. The temperature dependence of the fluorescence intensity of the S9:DMPC, molar ratio 1:100, mixed monolayer at 40 mN m ~ is shown in Fig. 7. Three regions (marked I, II, III) of fluorescence intensity variation with temperature are observed. The break in intensity at 286 K may be identified by the known subtransition temperature for the DMPC matrix monolayer. Below 286 K, a pronounced solid/liquid coexistence region and solid phase regions (see Fig. 2) within the investigated surface pressure range are observed. The increase of fluorescence intensity with decreasing temperature within region III may be related to increased rigidity of the system. Region II in the fluorescence intensity may be attributed to increasing density of solid domains within this temperature range. The second break point may be attributed to another subtransition temperature which has not yet been described in the literature. In addition to the phase state, interfacial viscosity and the
551
11.5-
.'~--~11.0. ¢-
E lo.5. " 10.0. i
9.52
3.35
a.:~o a.:~5 3.~0
3.~5
3.~0
3.65
1000/T
Fig. 7. Temperature dependence of the fluorescence intensity of S9:DMPC mixed monolayer, molar ratio hl00 (surface pressure 40 mN m -I, 2ex = 360nm, ~'em= 415 rim.
change in location of the chromophore may contribute to the temperature dependence of $9 fluorescence.
4. Conclusions It has been observed that the photophysical properties of the amphiphilic oxacyanine dye depend sensitively on the molecular organization state of the matrix phospholipid monolayer. The reflection spectroscopy data, along with surface pressure-area isotherms, show that the chromophore adopts an edge-on conformation, with the long molecular axis parallel to the air/water interface. The reflection spectra show the presence of dimers and higher aggregates for dye/lipid molar ratios lower than 1:10. The fluorescence characteristics are much more sensitive to the matrix monolayer state. The fluorescence intensity of the mixed monolayers depends linearly on dye/lipid molar ratios higher than 1:50. The fluorescence quantum yield of oxacyanine is highly sensitive to the phase state of the monolayer and is higher in the solid phase. The oxacyanine dye shows preference for the liquid phase in the liquid/solid coexistence region. The temperature dependence of the fluorescence intensity shows two breakpoints, at 286 K and 291 K. The break at 286 K coincides with the known subtransition temperature of DMPC.
Acknowledgments Financial support of the project by the Bundesministerium f/Jr Forschung und Technologie through grant 03M40080D is gratefully acknowledged.
References 1 R. P. Haugland, Handbook of Fluorescent Probes and Chemicals, Molecular Probes Inc., Eugene, OR, 1992. 2 H. M6hwald, Annu. Rev. Phys. Chem., 41 (1990) 441. 3 F. Caruso, F. Grieser, A. Murphy, P. Thistlewaite, R. Urquhart, M. Almgren and E. Wistus, J. Am. Chem. Soc., 113 (1991) 4838.
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4 T. Loughran, M. D. Hatlee, L. K. Patterson and J. J. Kozak, J. Chem. Phys., 72 (1980) 5791. 5 R. Peters and K. Beck, Pro c. Natl. A cad. Sci. U.S.A., 80 (1983) 7183. 6 J. Sondermann, Liebigs Ann. Chem., 749 (1971) 183. 7 H. Grfiniger, D. M6bius and H. Meyer, J. Chem. Phys., 79(1983) 3701. 8 K. Lesch, Ph.D. Thesis, University of Marburg, 1959. 9 R. C. Ahuja and D. M6bius, Thin Solid Films, 179(1989) 457.
10 N. Tamai, H. Matuso, T. Yamazaki and I. Yamazaki, J. Phys.
Chem., 96 (1992) 6550. 11 S. Vaidyanathan, L. K. Patterson, D. M6bius and H. R. Gruniger, J. Phys. Chem., 89(1985)491. 12 H. Kuhn, Pure Appl. Chem., 27(1971) 421. 13. D. M6bius and H. Kuhn, Isr. J. Chem., 18 (1979) 375. 14 I. Yamazaki, N. Tamai and T. Yamazaki, J. Phys. Chem., 94(1990) 516.