Langmuir 1994,10, 1877-1881
1877
Deposition and Structural Properties of Langmuir-Blodgett Films of a Fluorescently Labeled Phospholipid P. J. Lukes,tJ G. R. Ivanov,g M. C. Petty,? J. Yarwood,*JJ M. H. Greenhall,$ and Y. Lvovll Molecular Electronics Research Group, School of Engineering and Computer Science, University of Durham, Durham DHl 3LE, U.K., Department of Chemistry, University of Durham, Durham DH1 3LE, U.K., Department of Biomolecular Layers, Institute of Solid State Physics, Bulgarian Academy of Sciences, 72 Tzarigradsov Chaussee Boulevard, 1784 Sofia, Bulgaria, and Institut fiir Physikalische Chemie, Johannes Gutenburg Universitat, Mainz, Postfach, Jakob Welder-Weg 11,D-6500 Mainz, Germany Received August 13, 1993. In Final Form: March 16, 1994' Films of the fluorescently labeled phospholipid molecule DPPE-NBD (dipalmitoylphosphatidylethanolamine-nitrobenzoxadiazole) have been deposited and characterized by FTIR spectroscopy,ellipsometry, and low-angle X-ray diffraction. A combination of these techniques is required to give a reliable picture of the molecular structure of the film since there are a number of possible conformations which could account for the measured d spacing. We have used FTIR spectroscopy to distinguish these different conformationsand have shown that the data are consistent with an angle of 150° between the head group and the hydrocarbon tail. The P=O bonds of the head group are parallel to the surface, and the benzene ring is arranged in a perpendicular manner. The molecular arrangement implied by our measurements is consistent with data derived from isotherms and from molecular modeling.
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1. Introduction
The possibility of depositing Langmuir-Blodgett (LB) films of phospholipids onto solid supports has attracted the interest of many researchers, particularly those involved in membrane and biomimetic work. While many lipids, including phosphatidylcholine and phosphatidylethanolamine,' form compact stable monolayers, few deposit well as LB films. We have previously reported deposition parameters2 for the lipid DPPA (dipalmitoylphosphatidic acid) and have characterized the film by the tilt angle3 of the chains using infrared spectroscopy. The LB films of DPPA were found to have well-ordered chains oriented at an angle of 20' to the surface normal. The fluorescently labeled phospholipid DPPE-NBD (dipalmitoylphosphatidylethanolamine-nitrobenzoxadiazole) (Figure 1) is also an exceptional compound in its ability to form Langmuir-Blodgett films. This lipid is of special interest because the fluorescent characteristicsare quite sensitive to the dielectric permittivity of the solvent, and therefore the molecule may be used as a probe of membrane structure. A novel effect of fluorescence selfquenching in Langmuir films composed of DPPE-NBD upon transition from the liquid-expanded to the liquidcondensed state has been dis~overed.~ In order to explain this effect a detailed knowledge of the structure of the condensed phase is essential. We have used low-angle X-ray diffraction and ellipsometry to determine the thickness of DPPE-NBD films
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t
School of Engineering and Computer Science, University of
Durham.
Department of Chemistry, University of Durham. Bulgarian Academy of Sciences. A Present address: MaterialsResearchInetitute,SheffieldHallam University, Pond St., Sheffield S1 lWB,U.K. IJohannes Gutenburg Universitiit, Mainz. t
Abstract published in Advance ACS Abstracts, April 15,1994. (1)Roberta, G . G.,Ed.Langmuir-BlodgettFilns;Plenum: NewYork, 1990. (2) Cui, D.F.; Howarth,V. A.; Petty, M. C.; Ancelin, H.; Yarwood, J. Thin Solid Films 1990, 192, 391. (3) Lukes, P. J.; Petty, M. C.; Yarwood, J. Langmuir 1992, 8, 3043. (4)Ivanov, G.R. Chem. Phys. Lett. 1992,193, 323.
0743-7463/94/2410-1877$04.50/0
and have combined these results with an infrared analysis in order to provide structural information. DPPE-NBD is a more complex molecule than DPPA in that the possibility of a tilt of the head group relative to the chains arises (see Figure 1). Therefore, the X-ray results alone are not able to give an unambiguous determination of film structure from d spacing measurements. However, a combination of IR and X-ray data gives a clearer picture of the molecular organization. 2. Experimental Section L-a-Dipalmitoylphosphatidylethanolamine-nitrobenzoxadia-
zole (DPPE-NBD) was obtained from the Sigma Chemical Co. as a 2 g dm4 solution under inert gas in chloroform. It was used without further purification. Monolayer films were spread from the undiluted solution, and the solvent was allowed to evaporate on the water surface for a period of 30-60 min prior to the compression of the monolayer at a rate of approximately 1o-S nm2molecule-' s-l. The isotherm of this material has appeared el~ewhere.~ Monolayers were compressed to a pressure of 35 mN m-' and were allowed to stabilizefor a period of at least 1 h before the initiation of the dipping process. The substrates used for grazing incidenceIR measurements were gold-coated microscope slides prepared in the normal way.2 A 12-reflection ZnSe attenuated total reflection (ATR) crystal was used to measure ATR spectra. The first layer of the lipid was deposited on the upstroke in the case of both the gold slide and the ZnSe ATR crystal, the substrates having been immersed into the subphaseprior to the spreading of the film. The dipping speed was 2 mm mi+, and the transfer ratio (Y-type deposition)was found to be 1.0 f 0.1. Details of the troughs, water purification, and pressure measurements have appeared previously.2 IR spectra were recorded on a Mattson Sirius 100 FTIR instrument, equipped with a MCT detector at a resolution of 4 cm-'. Ellipsometricthickness measurements were made on a Rudolf Auto EL IV ellipsometer using a wavelength of 633 nm. For these measurements, a film comprising a series of stepped layers was deposited onto a polished silicon substrate. Thus, a series of measurements could be made on the same substrate. The thickness of the Si02layer and the refractive index of the silicon were measured,and the values recorded were used subsequently in the calculation of the thickness of the organic layer. Low-angle X-ray diffraction measurements were performed on two separate, but supposedly identical, samples. The 0 1994 American Chemical Society
1878 Langmuir, Vol. 10, No. 6, 1994
Lukes et al.
0
II
c15H31-
"1
ea2
Slope (Layer Thickness) = 33.12
I
No of Layers DPPE-NBD
Figure 2. Ellipsometric film thickness determination for a stepped film of DPPENBD. 4 10'
B
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310'
'.j 210'
g $
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LI
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1.0
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Figure 3. Low-angle X-ray reflection curves for two samples of DPPE-NBD Langmuir-Blodgett films. Table 1. Thickness Determination of DPPE-NBD (nm) d1/2 ( n m j d2/2 (nm) ellipsometry (nm) ~~
,R
3.10 f 0.02
Figure 1. (A, top) Chemical structure of DPPENBD. (B, bottom)Spatial arrangement of the atoms of DPPE-NBD. measurements were made with a Siemens D-500 powder diffractometer equipped with a graphite monochromator on the detector side, and using Cu Ka radiation with a wavelength of X = 0.154 nm. Data were acquired via a DACO-MP interface connected to a personal computer using a step width of O.0lo (in 28) and counting intervals of 5 s. The intensity curves were I = I d 4 ,where q = 4.rr sin multiplied by a Fresnel corre~tion,~~
eix.
3. Results and Discussion 3.1. Molecular Orientation. (a) X-ray and Ellipsometric Data. A plot of film thickness versus number of layers for a stepped film is shown in Figure 2. A value of 33 f 0.3 nm was obtained for the thickness per layer from the slope of the graph. Modeling experiments using standard molecular models (Corey-Pauling-Koltun (CPK)) show that the molecule has a maximum length of 3.8 nm.
3.07 i 0.02
2.94 f 0.02
~~
____
3.3 f 0.3
This is composed of 2.0 nm for the alkyl chain in the alltrans conformation, 0.4 nm for the phosphate linkage, and 1.0-1.1 nm for the NBD head group. Figure 3 shows the intensity curves for the two samples for which X-ray data were recorded. The curves contain one clear maximum (Bragg reflection) which is due to the well-ordered multilayer composition of the films. The angular position of the peak permits the calculation of the spacings in the films using the Bragg equation. The width of the reflection (Aq) at half-height is related to a correlation length in the packing of layers;&' R,, = 274 Aq. In the case of ideal (nondefective) packing, the correlation length is equal to the film thickness. The correlation length of the multilayer samples was the same in both of the test samples and corresponds to the total film thickness. This means that the molecules in the LB layers are well ordered with respect to each other. A summary of the X-ray results (R" and the d spacings of the two samples) is given in Table 1,which also includes the ellipsometrically determined film thickness. (5) Ebashi,S.,Koch, M.,Robenstein, E., Eda Handbook o f S y m h t r o n Radiation; Elsevier: Amsterdam, 1991; Vol. 4, pp 1-53. (6)Tippman-Krayer,P.; MGhwald, H.; Lvov, Y. Langmuir 1991, 7,
2298. (7) Feigin, L.; Lvov, Y.; Troitsky, V. Sou. Sei. Reu. 1989,11,283. (8) Bradshaw,A. M.;Schweizer,E. In Spectroscopy of Surfaces;Clark, Hester, Eds.; Wiley New York, 1988, Vol. 16, Chapter 8. (9) Lukes, P. J.; Vaughan, M. H.; Y a r w d , J.; Petty, M.C.; Lvov, Y.
Unpublished results.
LB Films of a Fluorescently Labeled Phospholipid ATR spectrum of cast DPPE (cm-1)
2955 2916
Langmuir, Vol. 10, No. 6, 1994 1879
Table 2. Infrared Band Aseipments for Thin Films of DPPE-NBD ATR spectrum of an LB film of RAIRS spectrum of cast DPPENBD (cm-l) DPPENBD (cm-9 DPPENBD (cm-l) ATR spectrum of
2955 2918
2850 1726
2955 2917 2872 2849 1738
1465
1467
1467 1444
~. ..
assignment
2961 2922 2873 2852 1738 1719
2850 1738
1265 1245 1222 1199 1180 746 721
739 722
The presence of only one sharp reflection and absence of the higher order Bragg peaks may be due to a sinusoidal electron density distribution across the layers. Thus, the layer packing may be similar to smectic-type multilayer ordering without sharp borders between the neighboring bilayers. The differencein monolayer thicknessesbetween samples 1 and 2 (2.94 A and 3.07 nm) may represent the natural variation of film thickness due to aging effects in the monolayer prior to dipping. Using slow dipping speeds (2 mm/min), this type of aging effect may be more pronounced than is usually the case because of the prolonged dipping time. The absence of Kiessig fringes in the X-ray curves shows that the films are not flat at a molecular level (Kiessigfringes are due to an interference of the X-ray beams from the substrate/film and film/air interfaces). On the basis of the X-ray results, it is also evident that there must be some tilt in the chains or in the head group (or both) in order to arrive a t a film thickness of 3.1 nm (assuming no interdigitation). From the X-ray analysis alone it is not possible to distinguish the various possibilities. However, by the use of IR spectroscopy, the orientation of the lipid chains can be estimated, and hence the various models of packing may be distinguished. (b) Infrared Data. The assignment of alkyl chain vibrations of the DPPE-NBD is shown in Table 2. These consist of the C-C stretching, C-H symmetric and antisymmetric stretching vibrations, and the CH2 bending, scissoring, wagging, and rocking vibrations. The CH3 and CH2 antisymmetric and symmetric stretching bands are observed to exhibit significant dichroism between the grazing incidence angle reflection (RAIRS) and ATR data (Figure 4). Since RAIRS couples only transition dipoles perpendicular to the (metal) substrate, it might be expected that the strong band due to us (CHs) at 2870 cm-l may indicate molecules in an all trans configuration oriented at a small angle to the surface n0rmal.~J5However, the position of the band a t 2922 cm-l a and a width of -30 cm-1 in RAIRS considerable population of gauche conformations. A more quantitative approach to the problem of chain tilting for these molecules is to attempt to calculate an order parameter,lO f , using two different polarization directions (TE and TM) in the ATR experiment. This is givenlo in the “thick film” approximation1’ by (10)Okamura, E.;Umemura, J.; Takenaka,T. Biochem. Biophys. Acta 1985,812,139. (11)Vaughan, M. H.; Froggatt, E. S.; Swart,R. M.;Yanvood, J. Thin Solid Films 1992,210/211,512.
738 723
6;iCH;j
Figure 4. Dichroism in the v(CH) bands of the alkyl chains: (A) grazing incidence angle reflection (RAIRS) and (B)ATR spectra of DPPE-NBD (31 layers). f
+
= - 2 . l ) g - 2-W)) 1.20)
where R = (Abs)TE/(Abs)mis the dichroic ratio. fvaries from +1.0 to -0.5 on going from orientation of the CH2 transition dipoles parallel to the substrate to perpendicular orientation of such transition dipoles. For randomized orientations f = 0 and R = 2. In our case, the dichroic ratio R was found to be 0.81, quite close to the value expected if the lipid molecule chains are aligned nearly parallel to the z axis (givingCH2 transition dipoles parallel to the substrate). The expression for f will be only approximately correct here since the LB films are thin compared with the wavelength, but nevertheless, we deduce that the DPPE-NBD is very similar in behavior18 to DPPA where the average tilt angle has been determined more accurately. In that case18 a combination of X-ray and infrared dichroism have shown that this tilt angle is in the range 17-24’ (from the surface normal). Assuming that the angle is similar here, say 20 f 4O, and assuming that interdigitation is not an issue (it seems not to be for other lipids’s), then a simple model allows us to estimate the range of (rigid) structures which may be adopted. For a 20° chain tilting angle the head group makes an angle of 5 6 O to the normal; for 24O tilt this angle is 49”. The angle between the alkyl chain and the head group is thus 150 f 7O. This could only be a provisional value, however, given the indication that the structure may not be rigid. 3.2. Molecular Packing. The 6(CHz)rocking band at 721 cm-1 is the strongest of two bands in this region which form the rocking progression. Interaction of the individual rocking vibration with other CH2 groups in the hydro-
Lukes et al.
1880 Langmuir, Vol. 10, No. 6, 1994 I A ~ ATR
DPPE-NBD
o f
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DPPE-NBD
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---
,1593
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'
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1200
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"
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1600
1400
1200
Wavenumber
1000
800
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Figure 5. ATR and RAIRS spectraof DPPE-NBD. Dichroism can be observed in the C=O stretching band at 1720 cm-l and in the 1180-1340-~m-~ region.
1400
1000
1200
Wavenumber
(cm-I)
a00
(cm-1)
Figure 6. RAIRS spectra of DPPE-NBD (A) and DPPA (B) showing the similarity in appearance of the wagging progression (1180-1340-cm-'1. The v ( C - 0 ) band underlying the progression shows up more strongly in the DPPE-NBD spectrum and accounts for the difference between the two spectra in this region.
carbon chain gives rise to a series of bands though in this case only two are observed (the second being at 746 cm-', Table 2). Crystal field splitting of the rocking and scissoring bands12-14is not evident. The CH2 scissoring vibration is, however, accompanied by a shoulder at 1444 cm-1, but it is too well separated from the main band a t 1474 cm-l to have arisen from crystal field effects (the separation due to crystal field splitting being of the order of 10 cm-' for both rocking and scissoring modes). The surface selection rule results in a weakening in the rocking and scissoring bands in the RAIRS spectrum (Figure 6) when the chains are oriented at a small angle to the substrate normal, since these transition dipoles are parallel to the surfaceas Thus, the absorptions at 720 and 1474 cm-' in the RAIRS spectrum are considerably attenuated and arise only due to the small deviation of the lipid chains from the surface normal. The antisymmetric v(CH2) and symmetric u(CH2) band positions at 2918 and 2850 cm-l in ATR (seen in Figure 4) are of the scale of values reported by Takenaka,lo but we have found this also to be the case with other lipids we have studied.3 If we extrapolate Takenaka's data, it would appear that the area per molecule for DPPE-NBD would approach or perhaps be lower than 40 A2, indicating a very tightly packed and "rigid" lipid array. Significant differences exist between the RAIRS and ATR data in the 1180-1340-~m-~ region (Figures 5 and 6).
Bands in this region are usually assigned to the CH2 wagging vibrations, and for fatty acids the number of bands is correlated with the chain length.3J6 Underlying the CH2 wagging progression are the C-0 and P=O stretching vibrations a t 1309 and 1242 cm-', respectively. Comparison of the IR data in this region with the spectrum of DPPA3,9shows that the wagging progression is much more evident in the ATR spectrum of DPPA and is masked by the C-0 and P=O vibrations in the spectrum of DPPE-NBD (Figures 5 and 6). Figure 5 shows the ATR and RAIRS spectra of DPPENBD. The wagging progression is evident in the RAIRS spectrum (1180-1265 cm-I), but the ATR spectrum is dominated by the C-0 and P=O vibrations at 1309 and 1242 cm-l. The C=O vibration shows a significant reduction in intensity in going from ATR to RAIRS, consistent with the chains being aligned nearly perpen-
(12) Chapman, D., Ed. Biomembrane Structure andFunction;Verlag Chimie: Weinheim, Germany, 1984; Chapter 4. (13) Cameron,D. G.; Gudgin, E. F.;Mantach,H. H.Biochemistry 1981, 20, 4496. (14) Coulthrop, N. B.; Daly, L. H.; Wilberly, S. E. Introduction to Infrared and Raman Spectroscopy, 3rd ed.; Academic Press: London, 1990. (15) Song, Y. P.; Petty, M. C.; Yarwood, J. Langmuir 1993,9, 543.
(16) Meiklejohn, R. A.; Meyer, R. J.; Aronovic, S. M.; Schuette, H. A.; Meloch, V. W. Anal. Chem. 1957,29, 329. (17) Mirabella, F. M. Appl. Spectrosc. Reo. 1985, 21, 45. (18) Greenhall, M. H.; Lukes, P. J.; Lvov, Y.; Petty, M. C.; Yarwood, J. Thin Solid Films, Eds.; in press. (19) Allara, D. L.; Parikh, A. N. J . Chem. Phys. 1992, 96, 927. (20) Laibbienis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y.-T.; Parikh, A. W.; Nuzzo, R. G. J . Am. Chem. SOC.1991, 113, 7152.
Table 3. Normalized Relative Intensity of Chain Stretching Vibrations
RAIRS (DPPE-NBD) ATR (DPPE-NBD) RAIRS (DPPA) ATR (DPPA)
0.68 0.35 0.58 0.22
1 1 1 1
0.39 verysmall 0.48 verysmall
0.42 0.64 0.58 0.72
LB Films of a Fluorescently Labeled Phospholipid ATR spectrum of cast DPPE (cm-1)
Langmuir, Vol. 10, No. 6,1994 1881
Table 4. Assignment of Head Group of Ring Vibrations of DPPE-Nl3D ATR spectrum of ATR spectrum of an LB f i of R A W spectrum of cast DPPE-NBD (cm-') DPPE-NBD (cm-1) DPPE-NBD (cm-1)
assignment of head group vibrations
1655 1649 1621 1588
1618 1586
1627 1593
ring Breathing
1305 1265
1309 1242
1318
v(C-0)
v(C=N)
1554 1244 1222
dicular to the substrate. Coupling of the CHz wagging and C-0 vibrations usually gives rise to a strong progression when the lipid chain is in the all-trans conformation. The progression of bands can be clearly seen for DPPA though it only shows up clearly in the RAIRS spectrum of DPPE-NBD (due to the enhancement of absorption by perpendicularly oriented dipoles). The appearance of only one band in the carbonyl region (1740 cm-9 of the ATR spectrum indicates the dominance of one conformation of the chains about the glycerol linkage in the films of DPPE-NBD. The carbonyl band in LB films of phospholipids often appears accompanied by a shoulder a t 1720 cm-l due to the gauche conformation being adopted about the glycerol linkage. The absence of this band in the ATR spectrum is probably due to the relatively high surface pressure (35 mN m-9 used for the deposition, forcing the lipid chains into a single, all-trans configuration. The head group vibrations of DPPE-NBD are shown in Table 4. f'heiinclude the ring breathing mode of the benzene ring, the C-N ring stretching, the NO2 group vibrations, and the phosphate group vibrations. We assign the ring breathing vibration to the band a t 1622 cm-l, and the band at 1585 cm-1 to C=N stretching in the ring. The ring breathing mode is enhanced in the RAIRS spectrum due to a twist of the benzene ring making the ring plane perpendicular to the plane of the surface. However, this observation does not in itself imply a tilt between the head group and the hydrocarbon chains. The aromatic v(NOz) bands are normally observed between 1530 and 1500 cm-l (va(NOz))and 1370 and 1330 cm-l (v,(NOa)). Thus, we assign the band at 1510 cm-l to the antisymmetric va(NO~) vibration and the band at 1369 cm-' to the corresponding symmetric vibration. We note, however, that there is a degree of overlap of a number of vibrations in this region, including, for instance, the CH3 umbrella mode which is normally assigned to a band near 1379 cm-l. Since aromatic nitro groups absorb strongly between 760 and 705 cm-l, it is possible that the band at 738 cm-1 is an aromatic mode, and not part of the rocking progression.
mixed rw(CHz)and v(P==O)
Finally, the phosphate vibration a t 1265 cm-l is drastically attenuated in the RAIRS spectrum, indicating alignment of the P=O bonds parallel to the substrate. This is further direct evidence for a tilt about the phosphate group in the lipid (see Figure 1). 4. Conclusions
The fluorescently labeled lipid DPPE-NBD has been shown by low-angle X-ray diffraction, ellipsometry, and FTIR spectroscopy to form well-ordered films. A comparison of the ATR and RAIRS data for the hydrophobic chain obtained for DPPE-NBD and that previously recorded for DPPA shows similarities in the dichroism observed for the C-H stretching vibrations, indicating a similar tilt angle for DPPE-NBD. Assuming that the molecules are not interdigitated, we find that the tilting angle of the alkyl chains is within 20 f 4O of the surface normal. This implies that the head group is arranged a t an average angle of about 150 f 7O to the alkyl chain. The packing density and fluidity of DPPE-NBD dipped at 35 mN m-1 are reflected in the position of the C-H stretching bands and in the absence of any shoulder on the carbonyl band in the ATR spectrum. We have observed a shift in the strongest band of the wagging progression which we believe is due to a change in the electronic structure of the head group of the lipid. The head group of the lipid is tilted with respect to the chain about the phosphate group, causing the P=O bonds to lie approximately parallel to the substrate. The benzene ring of the head group is twisted so that the plane of the ring is nearly perpendicular to the substrate. Thus, a combination of X-ray and FTIR data have been used successfully to provide detailed structural information not available from X-ray data alone.
Acknowledgment. We would like to thank the SERC and the Bulgarian National Fund "Scientific Studies" (Contract MU-NM-12) for their support of this work.
