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AFM surface morphology and friction force studies of microscale domain structures of binary phospholipids
Colloids and Surfaces B: Biointerfaces 79 (2010) 205–209
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
AFM surface morphology and friction force studies of microscale domain structures of binary phospholipids Takakuni Oguchi, Kenichi Sakai, Hideki Sakai ∗ , Masahiko Abe Department of Pure and Applied Chemistry in Faculty of Science and Technology and Research Institute of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
a r t i c l e
i n f o
Article history: Received 21 January 2010 Accepted 31 March 2010 Available online 7 April 2010 Keywords: Phospholipid LB film Friction force AFM FFM
a b s t r a c t We have studied friction forces on binary mixtures of phospholipid monolayer films. The phospholipid monolayer films have been prepared via the Langmuir–Blodgett (LB) technique on mica. The twocomponent phospholipids are distearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC). At 25 ◦ C the LB monolayer films give the gel-state DSPC domains surrounded by the liquid crystalline DLPC matrix. The friction forces measured on the DSPC domain region are significantly greater than those on the DLPC matrix region at this temperature. An increased temperature results in a decreased friction of the DSPC domain region, and above the gel-to-liquid crystalline phase transition temperature of DSPC, the difference in the friction forces measured on the two phospholipids becomes negligible. This means that the phase state is a key factor in determining friction forces on the phospholipid monolayer films. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Understanding the factors that influence the formation of phospholipids domains within the lipid bilayer is of significant interest due to their potential participation in transport and signalling in cellular membranes. The domains are expected to be in a liquidordered phase that is characterized by closely packed chains and a high degree of lateral mobility [1,2]. Lipid model membranes have been typically investigated by a wide range of surface analytical techniques such as scanning probe microscopy, ellipsometry, neutron reflectivity, time-of-flight secondary ion mass spectrometry and electrochemical impedance spectroscopy. Solid-supported phospholipid films prepared using the Langmuir–Blodgett or Langmuir–Schaefer technique have been used to investigate the formation and structure of lipid microdomains or rafts [3–6], to probe peptide/lipid interactions [7–9], to generate biologically addressable surface patterns [10,11], and to identify the forces which govern cell adhesion processes [12,13]. The formation of condensed phase structures in the two-phase co-existence region of lipid monolayers at the air/water interface has provided a wealth of information on the 2D growth of micrometer domains [14,15]. This is because the molecular density and phase state of these Langmuir monolayers can be determined via surface pressure–area (–A) isotherms, and readily controlled by
∗ Corresponding author. E-mail address: [email protected] (H. Sakai). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.03.051
varying the area per molecule, subphase temperature and ionic conditions on a Langmuir film balance. Such precise control on the lipid molecular area and phase is not possible when using bilayer vesicles or solid-supported bilayers formed by lipid vesicle fusion. For this reason, much has been learned from previous investigations with use of Langmuir monolayer [4,16,17]. In our current work, we have studied surface morphology and friction forces of binary mixtures of phospholipids as a function of temperature, in order to understand effects of sample temperature on friction forces in the system where lateral phase separation arises from a difference in the hydrocarbon chain length of the phospholipids. Friction force microscopy (FFM) measures simultaneously normal and lateral forces on the scanning tip with the laser beam deflection [18–20]. As far as we are aware, this is the first report focusing on friction forces on such phase-separated phospholipid monolayers from the viewpoint of their phase states. The phospholipid mixtures employed in this study consist of l-␣-distearoylphosphatidylcholine (DSPC) and l-␣-dilauroylphosphatidylcholine (DLPC), which exhibit low miscibility with each other and hence form a phase-separated monolayer film at the air/water interface [21]. Such low miscibility results from the difference in the phase states between the two phospholipids at room temperature: the main gel-to-liquid crystalline phase transition temperatures of DSPC and DLPC are reported to be 80 ◦ C and 0 ◦ C, respectively [22]. The binary phospholipid monolayers with various composition ratios are prepared at the air/water interface under controlled surface pressure (by using a Langmuir trough) and transferred on mica.
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2. Experimental
3. Results and discussion
2.1. Materials
3.1. Surface morphology and –A isotherm data
The phospholipids used in this study (DSPC and DLPC) were kindly supplied from the NOF Corporation and their purities are reported to be >99.6% by the supplier. Chloroform (Spectrochemical Anal., Wako) was used without further purification. Mica plates (Nilaco) were used as an LB film substrate after cleaving. The water used in this work was filtered with a reverse osmosis membrane after deionization and disinfectant with a Barnstead NANO pure Diamond UV System.
A typical topographic AFM image of a mixed DSPC/DLPC monolayer film is presented in Fig. 1a. The DSPC/DLPC mixed molar ratio is set at 0.5/0.5 for this particular case. One can clearly see ellipsoidal phase separation in this image. The diameter of the domain region is measured to be ca. 3–7 m and the step height between the two phases is 0.8 ± 0.1 nm (Fig. 1b). We have confirmed that the area fraction of the domain region increases linearly with increasing DSPC mol fraction (data not shown), and hence, we assume that DSPC forms the domain region whereas DLPC exists in the matrix. Taking the difference in the hydrocarbon chain length into consideration, the step height measured here is deemed to be within an acceptable range [23]. The spherical DSPC domain may result from its greater intermolecular van der Waals interaction as well as the gel structure at this temperature. Sanchez and Badia have studied the surface morphology of binary mixtures of DPPC and DLPC, transferred onto a mica surface, and demonstrated a very similar phase-separated domain structure of DPPC surrounded by DLPC matrix [24]. The lipid monolayer films, prepared by the LB technique, were transferred onto cleaved mica, and friction forces were measured. Before presenting the resultant friction force data, the –A isotherm data are shown in Fig. 2. In the case of the DSPC single system, gas-to-solid film transition occurs at the occupied area of 0.55 nm2 . The limit area per molecule and collapsed pressure of the film was 0.38 nm2 and 52 mN m−1 , respectively. On the other hand, DLPC forms a liquid-expanded film, which is distinguishable from the DSPC monolayer film. The shapes of the isotherms indicate that the DSPC monolayer is characterized as a two-dimensional (2D) solid-like organization, whereas the DLPC monolayer shows a 2D liquid-like behavior. In our current study, the LB monolayer films were transferred on the cleaved mica at 32 mN m−1 , which is close to the biomembrane pressure. The values of the averaged occupied area per molecule, estimated at 32 mN m−1 , are summarized in Fig. 3 as a function of DSPC composition. The occupied area is decreased linearly with increasing DSPC mol fraction, which is predicted by the additive rule [24] as follows. The area of a two-component monolayer at a given surface pressure is comparable to that of the pure components:
2.2. Preparation of DSPC/DLPC mixed films The phospholipids were first dissolved in chloroform, and then the DSPC/DLPC chloroform solution (approximately 100 L) was dropped on water. At this stage the surface pressure was adjusted at 32 mN m−1 , using an HBM700LB trough with a glass Wilhelmy plate (Kyowa Interface Science). The prepared monolayer film was transferred onto a cleaved mica surface according to the horizontal-lifting method, and hence the hydrophobic chains of the lipids face the outer surface of the substrate. –A isotherm data were measured using the same apparatus with a computer controller. 2.3. Measurements DSC measurements were carried out using a Rigaku DSC8230 calorimeter with a stainless vessel. The measurement conditions were set at 1 ◦ C min−1 for the scanning rate, 20–80 ◦ C for the scanning range and 0.42 mJ s−1 for the sensitivity, respectively. An SPI4000 AFM system with an E-Sweep (SII NanoTechnology Inc.) was used for measuring surface morphologies and friction forces on the DSPC/DLPC lipid films. Cantilevers with a silicon tip (SII NanoTechnology Inc. SI–AF01, nominal spring constant = 0.2 N m−1 , nominal torsional spring constant = 81.3 N m−1 ) were used for the contact mode AFM in air at room temperature (approximately 25 ◦ C). Based on this nominal torsion spring constant value, friction forces are evaluated as 1 mV (torsion count) = ca. 0.022 nN. Friction forces on the LB Film were measured by the use of torsion of the cantilever. The sample surface was moved back and forth in a direction perpendicular to the cantilever, and the torsion count against position coordinate was measured. In the current work, we set the scan range and scan speed as 4 m and 4 m s−1 , respectively, and the load of the cantilever was fixed at 0 nN.
A¯ = x1 A1 + x2 A2
(1)
where A¯ is the mean molecular area of the two-component film, x1 and x2 are the mol fraction of components 1 and 2 in the mixed film, and A1 and A2 are the molecular areas of 1 and 2 in pure monolayers.
Fig. 1. (a) An AFM topographic image (20 m × 20 m) of the mixed DSPC/DLPC (molar ratio = 0.5/0.5) monolayer transferred on mica. (b) The section analysis along the solid line in (a).
T. Oguchi et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 205–209
207
Fig. 3. The average occupied are per molecule, estimated at 32 mN m−1 , as a function of DSPC composition. Fig. 2. The surface pressure–area (–A) isotherms of DSPC/DLPC mixed films. The DSPC/DLPC compositions (in mol ratio) are set to 1.0/0.0, 0.5/0.5, 0.0/1.0, respectively.
This gives two possibilities: one is an ideal mixing of the two phospholipids, and the other is a complete phase separation as a result of no chemical or physical interactions between the two components. As is observed in Fig. 1, the latter possibility is a real story in our current case, which is also reported in the previous literature [24]. 3.2. Friction forces on LB monolayer films Friction force measurements were performed immediately after surface morphology measurements. As a typical result, friction force data obtained in the scan line shown in Fig. 4b are presented in Fig. 4a. This scan line is located in the boundary of the phase separation. Gap in the torsion count measured on the domain area (DSPC) is 15 mV, whereas the gap observed in the matrix area (DLPC) is 12 mV. Apparently, friction forces measured in the domain region are greater than those in the matrix. It seems likely that the observed difference in friction force data results from the difference in the phase states (or the degrees of hardness) of the LB film surface.
There are some reports regarding friction forces measured on various film surfaces. Yokoyama et al. have reported friction forces on the LB films consisting of polyamic acid alkylamine salts and polyimide [25] and found that the frictional interaction between the AFM cantilever tip and the sample surface is directly correlated with the chemical nature of the surface functional groups. For example, the single-chain alkylamines tend to form self-aggregated islands (domains) in the LB film, where the greater frictional response is observed compared with the surrounding area. It seems likely that the greater frictional response observed in the domain region results from the well-aggregated alkyl chain conformation, giving a relatively ‘hard’ surface surrounded by the ‘soft’ matrix region. As mentioned earlier, DSPC forms the gel phase at room temperature and the intermolecular van der Waals interaction is assumed to be greater than that of DLPC. This situation of DSPC is similar to the well-aggregated polyamic acid alkylamine salts studied in reference [25], and therefore, the greater friction of DSPC is expected. In order to evaluate effects of phase states on friction forces, we have examined changes in friction as a function of temperature. The friction force data are shown in Fig. 5 for each lipid. As mentioned in the previous paragraph, the friction of DSPC is greater
Fig. 4. (a) Friction forces on the DSPC/DLPC mixed monolayer film (the molar ratio of DSPC/DLPC is set at 0.5/0.5, as shown in Fig. 1), measured along the scan line shown in the AFM topographic image (b).
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T. Oguchi et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 205–209
Fig. 5. Friction forces on the single component films of DSPC or DLPC, measured at (a) 25 ◦ C, (b) 60 ◦ C and (c) 80 ◦ C.
than that of DLPC at 25 ◦ C. The increased temperature results in a smaller difference in friction forces between the two phospholipids, and at 80 ◦ C the difference becomes almost negligible within error. The observed change in the friction force data is clearly seen in Fig. 6, where the torsion count of each phospholipid is plotted against temperature. One can see a decrease in torsion count for the two phospholipids with increasing temperature. Indeed, the decrement is much greater for DSPC than for DLPC. It should be recalled that the two phospholipids exist as different phase states at 25 ◦ C (i.e., gel-state DSPC and liquid crystalline DLPC [26]), whereas at 80 ◦ C both the phospholipids are in the liquid crystalline state. This means that the phase state of the phospholipid monolayer films is a key factor on the friction forces. Here, our DSC measurements demonstrate that the gel-to-liquid crystalline phase transition temperature of DSPC is in the range of 80–90 ◦ C (data not shown). This transition temperature is slightly higher than the temperatures at which we have observed the steep decrease in torsion count for the DSPC monolayer (70–80 ◦ C, see Fig. 6). We assume, for this reason, that FFM is sensitive to any change in film surface states whereas the transition temperature determined by the DSC measurements reflects the overall bulk state. Friction forces have been studied in the literature [27] as a function of molecular weight of polymer samples, to elucidate effects of intermolecular entanglements on friction forces. The presence
of molecular entanglements within the film influences mobility of polymer chains, and hence makes a significant impact on friction forces. For example, when the polymer film is exposed to the UV light, the friction forces are decreased as a result of photo-induced disentanglement. This is similar to the phase transition observed in our current study from the standpoint of molecular packing at the membrane surface. In the gel state, the phospholipid molecules must interact with each other to form a solid-like membrane, and give greater frictional responses. On the other hand, it seems likely that the liquid crystalline phospholipids provide a less opportunity for the intermolecular interaction. This is the reason we have observed smaller friction forces on such liquid crystalline films. 4. Conclusions Friction forces on the phospholipid binary LB films have been studied as a function of temperature from the viewpoint of phase states of the mixed films. The two phospholipids (DSPC and DLPC) form a phase-separated monolayer film at the air/water interface and the film is transferred onto mica to examine the friction forces. At room temperature the friction forces measured on the DSPC gel domain region are significantly greater than those on the DLPC liquid crystalline matrix region. Indeed, the difference in the friction forces measured for the two regions becomes smaller with increasing temperature and it becomes almost negligible within error above the phase transition temperature of DSPC. This means that friction forces on the monolayer films are primarily determined by the phase state of each phospholipid component. References
Fig. 6. Torsion displacements of DSPC and DLPC single films as a function of sample temperature.
[1] D.A. Brown, E. London, Membr. Biol 164 (1998) 103. [2] C.M. McQuaw, A.G. Sostarecz, L. Zheng, A.G. Ewing, N. Winograd, Langmuir 21 (2005) 807. [3] M. Menke, S. Künneke, A. Janshoff, Eur. Biophys. J. 31 (2002) 317. [4] C. Yuan, L.J. Johnston, J. Microsc. 205 (2002) 136. [5] P.E. Milhiet, C. Domec, M.C. Giocondi, N. Van Mau, F. Heitz, C. Le Grimellec, Biophys. J. 81 (2001) 547. [6] V. Vié, N. Mau Van, E. Lesniewska, J.P. Goudonnet, F. Heitz, C. Le Grimellec, Langmuir 14 (1998) 4574. [7] D.Y. Takamoto, M.M. Lipp, A. von Nahmen, K.Y.C. Lee, A.J. Waring, J.A. Zasadzinski, Biophys. J. 81 (2001) 153. [8] B.N. Flanders, S.A. Vickery, R.C. Dunn, J. Microsc. 202 (2001) 379. [9] N. Van Mau, V. Vié, L. Chaloin, E. Lesniewska, F. Heitz, C. Le Grimellec, J. Membr. Biol. 167 (1999) 241. [10] P. Moraille, A. Badia, Angew. Chem. Int. Ed. 41 (2002) 4303. [11] C. Duschl, M. Liley, G. Corradin, H. Vogel, Biophys. J. 67 (1994) 1229. [12] S. Sivasankar, W. Brieher, N. Lavrik, B. Gumbiner, D. Leckband, Proc. Natl. Acad. Sci. U.S.A.× 96 (1999) 11820. [13] Z.W. Yu, T.L. Calvert, D. Leckband, Biochemistry 37 (1998) 1540. [14] H.M. McConnell, Annu. Rev. Phys. Chem. 42 (1991) 171. [15] R.M. Weis, Chem. Phys. Lipids 57 (1991) 227. [16] E. Kalb, S. Frey, L.K. Tamm, Biochim. Biophys. Acta 1103 (1992) 307.
T. Oguchi et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 205–209 [17] J.P. Slotte, Biochim. Biophys. Acta 1238 (1995) 118. [18] C.M. Mate, G.M. McClelland, R. Erlandsson, S. Chiang, Phys. Rev. Lett. 59 (1987) (1942). [19] G. Meyer, N.M. Amer, Appl. Phys. Lett. 57 (1990) 2089. [20] O. Marti, J. Colchero, J. Mlynek, Nanotechnology (1990) 141. [21] H.C. Chang, D.R. Flanagan, J. Pharm. Sci. 84 (1995) 1078.
[22] [23] [24] [25] [26] [27]
D. Marsh, Handbook of Lipid Bilayers, CRC Press, Boca Raton, FL, 1990. S.V. Bennun, R. Faller, M.L. Longo, Langmuir 24 (2008) 10371. J. Sanchez, A. Badia, Thin Solid Films 440 (2003) 223. S. Yokoyama, M. Kakimoto, Y. Imai, Synth. Met. 81 (1996) 265. S.J. Mabrey, J.M. Sturtevant, Proc. Nat. Acad. Sci. U.S.A. 73 (1976) 3862. T.J. Whittle, G.J. Leggett, Langmuir 25 (2009) 2217.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
AFM surface morphology and friction force studies of microscale domain structures of binary phospholipids Takakuni Oguchi, Kenichi Sakai, Hideki Sakai ∗ , Masahiko Abe Department of Pure and Applied Chemistry in Faculty of Science and Technology and Research Institute of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
a r t i c l e
i n f o
Article history: Received 21 January 2010 Accepted 31 March 2010 Available online 7 April 2010 Keywords: Phospholipid LB film Friction force AFM FFM
a b s t r a c t We have studied friction forces on binary mixtures of phospholipid monolayer films. The phospholipid monolayer films have been prepared via the Langmuir–Blodgett (LB) technique on mica. The twocomponent phospholipids are distearoylphosphatidylcholine (DSPC) and dilauroylphosphatidylcholine (DLPC). At 25 ◦ C the LB monolayer films give the gel-state DSPC domains surrounded by the liquid crystalline DLPC matrix. The friction forces measured on the DSPC domain region are significantly greater than those on the DLPC matrix region at this temperature. An increased temperature results in a decreased friction of the DSPC domain region, and above the gel-to-liquid crystalline phase transition temperature of DSPC, the difference in the friction forces measured on the two phospholipids becomes negligible. This means that the phase state is a key factor in determining friction forces on the phospholipid monolayer films. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Understanding the factors that influence the formation of phospholipids domains within the lipid bilayer is of significant interest due to their potential participation in transport and signalling in cellular membranes. The domains are expected to be in a liquidordered phase that is characterized by closely packed chains and a high degree of lateral mobility [1,2]. Lipid model membranes have been typically investigated by a wide range of surface analytical techniques such as scanning probe microscopy, ellipsometry, neutron reflectivity, time-of-flight secondary ion mass spectrometry and electrochemical impedance spectroscopy. Solid-supported phospholipid films prepared using the Langmuir–Blodgett or Langmuir–Schaefer technique have been used to investigate the formation and structure of lipid microdomains or rafts [3–6], to probe peptide/lipid interactions [7–9], to generate biologically addressable surface patterns [10,11], and to identify the forces which govern cell adhesion processes [12,13]. The formation of condensed phase structures in the two-phase co-existence region of lipid monolayers at the air/water interface has provided a wealth of information on the 2D growth of micrometer domains [14,15]. This is because the molecular density and phase state of these Langmuir monolayers can be determined via surface pressure–area (–A) isotherms, and readily controlled by
∗ Corresponding author. E-mail address: [email protected] (H. Sakai). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.03.051
varying the area per molecule, subphase temperature and ionic conditions on a Langmuir film balance. Such precise control on the lipid molecular area and phase is not possible when using bilayer vesicles or solid-supported bilayers formed by lipid vesicle fusion. For this reason, much has been learned from previous investigations with use of Langmuir monolayer [4,16,17]. In our current work, we have studied surface morphology and friction forces of binary mixtures of phospholipids as a function of temperature, in order to understand effects of sample temperature on friction forces in the system where lateral phase separation arises from a difference in the hydrocarbon chain length of the phospholipids. Friction force microscopy (FFM) measures simultaneously normal and lateral forces on the scanning tip with the laser beam deflection [18–20]. As far as we are aware, this is the first report focusing on friction forces on such phase-separated phospholipid monolayers from the viewpoint of their phase states. The phospholipid mixtures employed in this study consist of l-␣-distearoylphosphatidylcholine (DSPC) and l-␣-dilauroylphosphatidylcholine (DLPC), which exhibit low miscibility with each other and hence form a phase-separated monolayer film at the air/water interface [21]. Such low miscibility results from the difference in the phase states between the two phospholipids at room temperature: the main gel-to-liquid crystalline phase transition temperatures of DSPC and DLPC are reported to be 80 ◦ C and 0 ◦ C, respectively [22]. The binary phospholipid monolayers with various composition ratios are prepared at the air/water interface under controlled surface pressure (by using a Langmuir trough) and transferred on mica.
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T. Oguchi et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 205–209
2. Experimental
3. Results and discussion
2.1. Materials
3.1. Surface morphology and –A isotherm data
The phospholipids used in this study (DSPC and DLPC) were kindly supplied from the NOF Corporation and their purities are reported to be >99.6% by the supplier. Chloroform (Spectrochemical Anal., Wako) was used without further purification. Mica plates (Nilaco) were used as an LB film substrate after cleaving. The water used in this work was filtered with a reverse osmosis membrane after deionization and disinfectant with a Barnstead NANO pure Diamond UV System.
A typical topographic AFM image of a mixed DSPC/DLPC monolayer film is presented in Fig. 1a. The DSPC/DLPC mixed molar ratio is set at 0.5/0.5 for this particular case. One can clearly see ellipsoidal phase separation in this image. The diameter of the domain region is measured to be ca. 3–7 m and the step height between the two phases is 0.8 ± 0.1 nm (Fig. 1b). We have confirmed that the area fraction of the domain region increases linearly with increasing DSPC mol fraction (data not shown), and hence, we assume that DSPC forms the domain region whereas DLPC exists in the matrix. Taking the difference in the hydrocarbon chain length into consideration, the step height measured here is deemed to be within an acceptable range [23]. The spherical DSPC domain may result from its greater intermolecular van der Waals interaction as well as the gel structure at this temperature. Sanchez and Badia have studied the surface morphology of binary mixtures of DPPC and DLPC, transferred onto a mica surface, and demonstrated a very similar phase-separated domain structure of DPPC surrounded by DLPC matrix [24]. The lipid monolayer films, prepared by the LB technique, were transferred onto cleaved mica, and friction forces were measured. Before presenting the resultant friction force data, the –A isotherm data are shown in Fig. 2. In the case of the DSPC single system, gas-to-solid film transition occurs at the occupied area of 0.55 nm2 . The limit area per molecule and collapsed pressure of the film was 0.38 nm2 and 52 mN m−1 , respectively. On the other hand, DLPC forms a liquid-expanded film, which is distinguishable from the DSPC monolayer film. The shapes of the isotherms indicate that the DSPC monolayer is characterized as a two-dimensional (2D) solid-like organization, whereas the DLPC monolayer shows a 2D liquid-like behavior. In our current study, the LB monolayer films were transferred on the cleaved mica at 32 mN m−1 , which is close to the biomembrane pressure. The values of the averaged occupied area per molecule, estimated at 32 mN m−1 , are summarized in Fig. 3 as a function of DSPC composition. The occupied area is decreased linearly with increasing DSPC mol fraction, which is predicted by the additive rule [24] as follows. The area of a two-component monolayer at a given surface pressure is comparable to that of the pure components:
2.2. Preparation of DSPC/DLPC mixed films The phospholipids were first dissolved in chloroform, and then the DSPC/DLPC chloroform solution (approximately 100 L) was dropped on water. At this stage the surface pressure was adjusted at 32 mN m−1 , using an HBM700LB trough with a glass Wilhelmy plate (Kyowa Interface Science). The prepared monolayer film was transferred onto a cleaved mica surface according to the horizontal-lifting method, and hence the hydrophobic chains of the lipids face the outer surface of the substrate. –A isotherm data were measured using the same apparatus with a computer controller. 2.3. Measurements DSC measurements were carried out using a Rigaku DSC8230 calorimeter with a stainless vessel. The measurement conditions were set at 1 ◦ C min−1 for the scanning rate, 20–80 ◦ C for the scanning range and 0.42 mJ s−1 for the sensitivity, respectively. An SPI4000 AFM system with an E-Sweep (SII NanoTechnology Inc.) was used for measuring surface morphologies and friction forces on the DSPC/DLPC lipid films. Cantilevers with a silicon tip (SII NanoTechnology Inc. SI–AF01, nominal spring constant = 0.2 N m−1 , nominal torsional spring constant = 81.3 N m−1 ) were used for the contact mode AFM in air at room temperature (approximately 25 ◦ C). Based on this nominal torsion spring constant value, friction forces are evaluated as 1 mV (torsion count) = ca. 0.022 nN. Friction forces on the LB Film were measured by the use of torsion of the cantilever. The sample surface was moved back and forth in a direction perpendicular to the cantilever, and the torsion count against position coordinate was measured. In the current work, we set the scan range and scan speed as 4 m and 4 m s−1 , respectively, and the load of the cantilever was fixed at 0 nN.
A¯ = x1 A1 + x2 A2
(1)
where A¯ is the mean molecular area of the two-component film, x1 and x2 are the mol fraction of components 1 and 2 in the mixed film, and A1 and A2 are the molecular areas of 1 and 2 in pure monolayers.
Fig. 1. (a) An AFM topographic image (20 m × 20 m) of the mixed DSPC/DLPC (molar ratio = 0.5/0.5) monolayer transferred on mica. (b) The section analysis along the solid line in (a).
T. Oguchi et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 205–209
207
Fig. 3. The average occupied are per molecule, estimated at 32 mN m−1 , as a function of DSPC composition. Fig. 2. The surface pressure–area (–A) isotherms of DSPC/DLPC mixed films. The DSPC/DLPC compositions (in mol ratio) are set to 1.0/0.0, 0.5/0.5, 0.0/1.0, respectively.
This gives two possibilities: one is an ideal mixing of the two phospholipids, and the other is a complete phase separation as a result of no chemical or physical interactions between the two components. As is observed in Fig. 1, the latter possibility is a real story in our current case, which is also reported in the previous literature [24]. 3.2. Friction forces on LB monolayer films Friction force measurements were performed immediately after surface morphology measurements. As a typical result, friction force data obtained in the scan line shown in Fig. 4b are presented in Fig. 4a. This scan line is located in the boundary of the phase separation. Gap in the torsion count measured on the domain area (DSPC) is 15 mV, whereas the gap observed in the matrix area (DLPC) is 12 mV. Apparently, friction forces measured in the domain region are greater than those in the matrix. It seems likely that the observed difference in friction force data results from the difference in the phase states (or the degrees of hardness) of the LB film surface.
There are some reports regarding friction forces measured on various film surfaces. Yokoyama et al. have reported friction forces on the LB films consisting of polyamic acid alkylamine salts and polyimide [25] and found that the frictional interaction between the AFM cantilever tip and the sample surface is directly correlated with the chemical nature of the surface functional groups. For example, the single-chain alkylamines tend to form self-aggregated islands (domains) in the LB film, where the greater frictional response is observed compared with the surrounding area. It seems likely that the greater frictional response observed in the domain region results from the well-aggregated alkyl chain conformation, giving a relatively ‘hard’ surface surrounded by the ‘soft’ matrix region. As mentioned earlier, DSPC forms the gel phase at room temperature and the intermolecular van der Waals interaction is assumed to be greater than that of DLPC. This situation of DSPC is similar to the well-aggregated polyamic acid alkylamine salts studied in reference [25], and therefore, the greater friction of DSPC is expected. In order to evaluate effects of phase states on friction forces, we have examined changes in friction as a function of temperature. The friction force data are shown in Fig. 5 for each lipid. As mentioned in the previous paragraph, the friction of DSPC is greater
Fig. 4. (a) Friction forces on the DSPC/DLPC mixed monolayer film (the molar ratio of DSPC/DLPC is set at 0.5/0.5, as shown in Fig. 1), measured along the scan line shown in the AFM topographic image (b).
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Fig. 5. Friction forces on the single component films of DSPC or DLPC, measured at (a) 25 ◦ C, (b) 60 ◦ C and (c) 80 ◦ C.
than that of DLPC at 25 ◦ C. The increased temperature results in a smaller difference in friction forces between the two phospholipids, and at 80 ◦ C the difference becomes almost negligible within error. The observed change in the friction force data is clearly seen in Fig. 6, where the torsion count of each phospholipid is plotted against temperature. One can see a decrease in torsion count for the two phospholipids with increasing temperature. Indeed, the decrement is much greater for DSPC than for DLPC. It should be recalled that the two phospholipids exist as different phase states at 25 ◦ C (i.e., gel-state DSPC and liquid crystalline DLPC [26]), whereas at 80 ◦ C both the phospholipids are in the liquid crystalline state. This means that the phase state of the phospholipid monolayer films is a key factor on the friction forces. Here, our DSC measurements demonstrate that the gel-to-liquid crystalline phase transition temperature of DSPC is in the range of 80–90 ◦ C (data not shown). This transition temperature is slightly higher than the temperatures at which we have observed the steep decrease in torsion count for the DSPC monolayer (70–80 ◦ C, see Fig. 6). We assume, for this reason, that FFM is sensitive to any change in film surface states whereas the transition temperature determined by the DSC measurements reflects the overall bulk state. Friction forces have been studied in the literature [27] as a function of molecular weight of polymer samples, to elucidate effects of intermolecular entanglements on friction forces. The presence
of molecular entanglements within the film influences mobility of polymer chains, and hence makes a significant impact on friction forces. For example, when the polymer film is exposed to the UV light, the friction forces are decreased as a result of photo-induced disentanglement. This is similar to the phase transition observed in our current study from the standpoint of molecular packing at the membrane surface. In the gel state, the phospholipid molecules must interact with each other to form a solid-like membrane, and give greater frictional responses. On the other hand, it seems likely that the liquid crystalline phospholipids provide a less opportunity for the intermolecular interaction. This is the reason we have observed smaller friction forces on such liquid crystalline films. 4. Conclusions Friction forces on the phospholipid binary LB films have been studied as a function of temperature from the viewpoint of phase states of the mixed films. The two phospholipids (DSPC and DLPC) form a phase-separated monolayer film at the air/water interface and the film is transferred onto mica to examine the friction forces. At room temperature the friction forces measured on the DSPC gel domain region are significantly greater than those on the DLPC liquid crystalline matrix region. Indeed, the difference in the friction forces measured for the two regions becomes smaller with increasing temperature and it becomes almost negligible within error above the phase transition temperature of DSPC. This means that friction forces on the monolayer films are primarily determined by the phase state of each phospholipid component. References
Fig. 6. Torsion displacements of DSPC and DLPC single films as a function of sample temperature.
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