ARTICLE pubs.acs.org/Langmuir
Determination of the Shear Modulus of Spin-Coated Lipid Multibilayer Films by the Spontaneous Embedment of Submicrometer-Sized Particles Jinhua Wang, Kirthi Deshpande, and Gregory B. McKenna* Department of Chemical Engineering, Texas Tech University, Lubbock, Texas 79409-3121, United States ABSTRACT: A novel submicrometer particle embedment technique has been used to determine the shear modulus of 1,2-dipalmitoyl-sn-glycero-3-phosphotidylcholine (DPPC) lipid multibilayers. The depth of the spontaneous embedment of polystyrene and silica spherical particles of 200 nm nominal size has been determined from atomic force microscopy measurements on colloidal particles dispersed onto the surfaces of the DPPC multibilayers. The standard JKR model was used to relate the shear modulus of the lipid multibilayer films to the depth of embedment of the particles. The thus-determined modulus of the DPPC is within the range of reported literature values. Gold particles are also considered, and it is found that for the smallest particles (13 nm) complete engulfment by the DPPC multibilayer film takes place.
1. INTRODUCTION The cell membrane serves as a permeable barrier for all living organisms, and the lipid bilayer forms the main component of the cell membrane.1 Lipid multibilayers are composed of stacks of lipid bilayers. There are three main kinds of membrane lipids: phospholipids, glycolipids, and cholesterol. Phospholipids are the most commonly found among membrane lipids because they can self-assemble into lipid bilayers (or lipid multibilayer) films with the polar headgroups pointing toward the surface in the presence of water, which is important in biological processes.2,3 A complete understanding of cellular processes and the interactions of cells with nanoscale objects requires an understanding of the mechanical response and surface interactions in these materials.4 Since the pioneering work of Gorter and Grendel,5 who used extracted red blood cells to show that lipids exist as bilayers, there has been much experimental and theoretical interest in the study of the mechanical response of lipids. The mechanical properties of red blood cells were extensively studied by Evans and coworkers,6,7 and many methods68 have been used to examine the mechanical response of cell membranes. Osmotic swelling in hypotonic solution,9 compression between two flat plates,8 and micropipet aspiration6,7,10 have been used to examine the elastic properties of cell membranes. The elastic properties of lipid bilayers have been measured by various methods and for a range of lipid preparations. Brillouin scattering11 and electron energy loss spectroscopy (EELS)12 have also been used to measure the elastic modulus of lipid monolayers prepared in a Langmuir Blodgett trough. Israelachvili and co-workers have used the surface force apparatus (SFA) to determine the elastic properties of lipid bilayers under compression.13 r 2011 American Chemical Society
In the present work, we report the results from the application of a novel particle embedment technique to determine the elastic properties of lipid multibilayer films. The method was initially proposed by Teichroeb and Forrest14 to study the surface properties of polymers on the nanometer size scale and was further developed by Hutcheson and McKenna,15 who provided a viscoelastic contact mechanics solution that explicitly included particlesurface interactions as the driving force for particle embedment, viz., the Johnson, Kendall, and Roberts (JKR) model.16 In the present work, the JKR model was applied to relate the elastic shear modulus of 1,2-dipalmitoyl-sn-glycero-3phosphotidylcholine (DPPC) lipid multibilayers to the embedment depth of polystyrene and silica particles dispersed onto the surfaces of lipid multibilayer films.
2. DATA ANALYSIS The JKR16 model can be used to determine the response of a material surface to an embedding particle subjected to a force P. In our case, we use the relationship determined by Hutcheson and McKenna15 for the force on the particle in terms of the work of adhesion wa. Then the rearranged JKR16 equation gives the shear modulus G in terms of the work of adhesion wa, particle radius R, Poisson ratio ν, and the particle embedment depth δ into the surface pffiffiffi 3wa πR 0:5 ð1 νÞ G¼ ð1Þ 4δ1:5 Received: February 10, 2011 Revised: April 28, 2011 Published: May 10, 2011 6846
dx.doi.org/10.1021/la2005375 | Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Here, the material is assumed to be incompressible (ν = 0.5). Hence, if one knows the work of adhesion (wa) (to get the force), the particle diameter (d), and the particle embedment depth (δ), then one can obtain the shear modulus of the material. The particle embedment depth is defined as the difference between the particle diameter and the height of the particles above the surface of interest, which can be measured by atomic force microscopy (AFM). We remark that the measured particle diameter is an important parameter in the determination of the modulus and subsequently discuss the different measures used for its determination. Then, if the work of adhesion is known, the elastic modulus of the lipid multibilayers can be calculated using the above equation. However, in general the work of adhesion is unknown, and it is difficult to determine by direct experiment. In the present study, we measured the contact angles of different liquids on the lipid multibilayers to determine the surface energy of the lipid multibilayer films. The equation for the work of adhesion17 can be written as wa ¼ γSV þ γLV γSL
ð2Þ
where γ is the surface energy and the subscripts SV, LV, and SL are the solidvapor, liquidvapor, and solidliquid interfaces. In the present experiments, γSV is the surface energy of a particle, γLV is the surface energy of the lipid multibilayers, and γSL represents the interfacial tension between the particle and the lipid multibilayer surface. Here, the surface energy γ is defined as the sum of dispersion γd and polar γp components,1720 and the interfacial energy can be described by eq 3. qffiffiffiffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffi p p ð3Þ γSL γSV þ γLV 2 γdSV γdLV 2 γSV γLV By substituting eq 3 into eq 2 and rearranging terms, we obtain for the work of adhesion qffiffiffiffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffi p p ð4Þ wa ¼ 2 γdSV γdLV þ 2 γSV γLV The modified equation for the work of adhesion relates its value to the dispersive and polar components of the surface energy of the particle and the lipid multibilayers. To determine the surface energy of the DPPC lipid multibilayers, we use a method21 that combines the OwensWendt geometric mean (two-liquids) method with Young’s equation as follows,21 γ1 ð1 þ cos θ1 Þ p p ¼ ðγdLV γd1 Þ1=2 þ ðγLV γ1 Þ1=2 2 γ2 ð1 þ cos θ2 Þ p p ¼ ðγdLV γd2 Þ1=2 þ ðγLV γ2 Þ1=2 2
ð5Þ
A pair of polar and nonpolar liquids was chosen for the contact angle θi measurements, and the data were put into eq 5 to obtain surface energy contributions γdLV and γpLV for the lipid multibilayer films. Equations 15 with the appropriate measurements of the contact angle and embedment depth provide the means to determine the shear moduli of the lipid multibilayers from the particle embedment experiments.
3. EXPERIMENTAL METHODOLOGY 3.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphotidylcholine (DPPC) lipid in chloroform (10 mg/mL) was purchased from Avanti Polar Lipids, Inc. This material has its main phase-transition temperature at 41.5 °C2 for the DPPC in multilamellar aqueous suspensions. Prior to
spin coating, the silicon wafer substrates (Universitywafer.com) were vigorously cleaned ultrasonically (ultrasonic cleaner, Sper Scientific, model 100005) using chloroform (ACS GR, 99.8%), ethanol (ACS GR, 92%), and ultrapurified water (Barnstead/Thermolyne model D8961). Uncoated fused silica windows (Edmund Optics Inc.) were cleaned following the same procedure as that for the silicon wafers. The gold surface was obtained by sputter coating a freshly cleaved mica surface under 0.2 mbar vacuum with a sputter coater (Emitech K550X) using a deposition current of 20 mA with a sputtering time of 90 s. The polystyrene surface was prepared by spin coating a 3% PS toluene solution onto a cleaned silicon substrate. All of these prepared clean samples were used for the determination of the surface tension of the solids. The particles used for the embedment experiments were nominally 235 nm silica microspheres (microParticles GmbH), 200 nm polystyrene microspheres (Polysciences.com), and 150, 80, and 13 nm gold particles (Ted Pella Inc.). Formamide (certified A.C.S., Fisher Scientific), water (HPLC for gradient analysis, Acros Organics), and methylene iodide (Sigma-Aldrich Chemie GmbH) were used for the contact angle measurements. 3.2. Sample Preparation. Following the approach of Mennicke and Salditt,22 25 μL DPPC chloroform solutions with different concentrations were pipetted onto cleaned silicon substrates and spin coated to prepare ultrathin DPPC multibilayer films with thicknesses ranging from 90270 nm (film thickness determined using the AFM by an in-lab-developed thickness-determination method23). The spin-coating steps were set as 100 rpm spin speed for 1 s, followed by 2000 rpm for 4 s and 4000 rpm for 25 s. To remove the residual solvent completely, these ultrathin DPPC lipid films were annealed in vacuum (generated by a laboratory roughing pump) at room temperature for approximately 12 h and then transferred to a refrigerator (5 °C) prior to further use.2 The as-received particle colloidal solutions were diluted to 0.05% (w/w) with ultrapurified water (Barnstead/Thermolyne model D8961). Then, 10 μL of the diluted particle suspension was placed onto the prepared DPPC multibilayers using a micropipet and transferred to an oven maintained at room temperature under N2 purging protection to obtain well-dispersed particles. Optical microscopy indicates that some defects are present in the final multibilayer film samples, but most of the surface seems homogeneous and the surface roughness is determined from the root-mean-square roughness Rq.24,25 The roughness was found to be dependent on the multibilayer thickness.2628 For film thicknesses of less than 200 nm, the surface roughness was found to be around 6 nm from 10 μm 10 μm AFM topographical images. And for a film thickness of around 270 nm, the roughness goes to approximately 12 nm. 3.3. Instruments. A contact angle goniometer (NRL C.A. goniometer, model 100-00 from Rame and Hart, Inc.) was used to make the contact angle measurements of solvent on the surfaces of interest to obtain the surface tension of the investigated lipid multibilayer systems. A Quesant USPM (universal scanning probe microscope) was used to measure the particle heights on the DPPC multibilayers and bare substrates. The images were obtained using silicon nitride tips (NSC 16) in intermittent contact mode. This tapping mode of operation is better for soft biological samples because the tip contacts the sample surface intermittently, hence causing less damage to the sample. The xy scan ranges were 40 40, 10 10, and 6 6 μm2, and the scan (raster) rate was set to 1 Hz. Before the experiment, the AFM instrument was carefully calibrated using grating standards. (The performance of the lateral (xy) direction of the instrument was calibrated by a Multiscan method for more accurate calibration, and the z axis was calibrated by a single measurement with a TZ202 standard.) A Hitachi H-7650 transmission electron microscope (TEM, Hitachi High Technologies America, Inc., Pleasanton, CA) was used to examine the particle diameters and their shapes. All particles were mounted on 200 mesh copper grids with Formvar coating without stain. 6847
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Table 1. Literature Values of Surface Tension and Its Components for Probe Liquids
Table 2. Surface Energy Components of Substrates Calculated from Contact Angle Data by Using the Two-Liquid Method
surface tension at 20 °C, mJ/m2 γ
material formamide18 water18 29
methylene iodide
γd
58
39
19
72.8
21.8
50.0 1.3
50.8
49.5
surface tension at room temperature, mJ/m2
γp
Light scattering was also used to examine the particle sizes and was performed using a Nanotrac particle size analyzer (PSD, Nanotrac Ultra, Microtrac Inc.). The particle diameter was found to be independent of the sample concentration (0.05, 0.2, 2, and 100%, where the concentration was defined as the weight percent of purchased particle colloidal solution with respect to the total weight of the dilute solution, i.e., 100% means the purchased particle solution without further dilution). Ultrapurified water was used to obtain the background information for the measurements because this is the medium in which the submicrometer spheres are obtained. Each sample run was repeated six times with the test time set as 60 s.
4. RESULTS AND DISCUSSION 4.1. Contact Angle Measurements. Formamide (polar) and methylene iodide (nonpolar) liquids were selected for contact angle measurements on DPPC, and water and methylene iodide were used to investigate the surface energies of mica, silicon, gold, and silica bare substrates. The literature values for the surface tension contributions of the probe liquids are shown in Table 1. Adhesion is a complex topic, and different methods have been used to study and describe the real nature of the adhesion force.30 As a result, even for the same material, different surface tension values have been reported on the basis of the measurement methods, material surface structure, and physical state.3036 For example, most studies show a high surface energy for gold (∼1130 mJ/m2),30 whereas some of the literature gives a relatively low value (∼45 mJ/m2) for gold under ambient conditions from contact angle experiments.30 Similar large contact angle value differences have been obtained by different groups for gold.36 The contact angle measurements for a liquid on a solid are influenced by several factors, such as the surface roughness, structure, and contamination.3741 Work conducted on the material’s superhydrophobicity by increasing the surface roughness and changing the surface structure confirms this.40,41 In the present work, we performed contact angle experiments on the solid surfaces of interest and under ambient conditions to obtain the relevant surface tension values. The contact angle experiments were taken immediately on freshly cleaned solid surfaces to avoid surface contamination. The surface roughness is small here, and the measured Rq roughness for mica and silicon wafers is only around 1.6 Å by AFM. Both two-liquid (harmonic mean and geometric mean) and three-liquid (Lifshitzvan der Waals acidbase) methods can give reliable surface energy results when suitable liquids are used.18 In the present work, polar and nonpolar liquids (water and methylene iodide) were selected to perform contact angle experiments on the clean, bare solid substrates, and the geometric mean version of the two-liquid method was used to determine the surface energies. The measured contact angles for water on silicon, mica, polystyrene, gold, and silica are 26.0 ( 1.6, 1.6 ( 0.5, 97.2 ( 1.7, 59.1 ( 2.6, and 24.4 ( 0.7°; the contact
γ
γd
γp
silicon
68.4
34.1
34.3
mica
74.3
32.7
41.6
polystyrene
45.5
44.9
0.6
silica
70.0
36.4
33.6
gold DPPC multibilayers
55.1 73.0
44.6 16.1
11.2 56.9
material
angles for methylene iodide on silicon, mica, polystyrene, gold, and silica are 28.3 ( 1.5, 29.0 ( 0.9, 30.7 ( 0.5, 2.2 ( 0.6, and 21.3 ( 1.6°. From the two-liquid method of analysis, the surface free energy and its components for the bare solid substrates were determined and are listed in Table 2. The determined surface tension values for the solid substrates from our experiments agree well with those literature values3032 from contact angle experiments. To obtain the work of adhesion between the particles of interest and DPPC, we need to determine the surface tension of DPPC. The measured contact angles for formamide and methylene iodide on the DPPC lipid multibilayer systems are 3.8 ( 0.6 and 63.3 ( 0.6°, respectively. From eq 5, we find that the surface tension of DPPC is 73.0 mJ/m2 and that γd and γp for the DPPC multibilayers are 16.1 and 56.9 mJ/m2 (Table 2). The calculated surface energy values of DPPC here are consistent with Jurak and Chibowski’ s work using contact angle measurements to study the surface free energy of DPPC multilayers on different surfaces.32 Then, from eqs 25 and the surface energies of Table 2 we determine the work of adhesion between DPPC and the different particles to be 135.9, 65.5, and 104.1 mJ/m2 for the silica, polystyrene, and gold particles, respectively. These values are used subsequently. 4.2. Particle Shape. In the present work, the standard JKR model16 was used to calculate the shear moduli of the DPPC multibilayers, and the particle shape is assumed to be spherical in the calculation. TEM was used to examine the shapes of the particles. The TEM images in Figure 1 show the clearly spherical shape of the polystyrene and silica particles, and the gold particles were found to be faceted. The nonspherical shape of a particle can change the surface tension and also cause a stress decrease relative to the sphere,42 which may lead to errors in the calculation of the elastic modulus of the lipid multibilayers. In this article, we present modulus values only for DPPC obtained with the polystyrene and silica particles. We do discuss the qualitative features of the gold particle behavior. 4.3. Particle Type and Diameter Determination. One can see from Figure 1c,d that the gold particles are faceted. Therefore, these were not used for the quantitative analysis of the embedment behavior to obtain the elastic properties of the DPPC. Having determined the work of adhesion between the particles and DPPC of the silica and polystyrene particles, the elastic modulus of the DPPC multibilayers can be calculated from the rearranged standard JKR model (eq 1) if we have accurate particle diameters. Several methods can be used to obtain the particle size and its distribution such as dynamic light 6848
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Figure 2. Estimated deformation of polystyrene particles on a silicon substrate as a function of particle radius.
this issue, we assume the work of adhesion is the only force applied to the particle to make it deform. Then from the surface tension interactions between the PS particle and silicon, the work of adhesion wa is ∼135.2 mJ/m2. The JKR theory predicts that the contact radius can be expressed as43 a3 ¼ Figure 1. TEM image of (a) 200 nm PS, (b) 235 nm silica, (c) 150 nm gold, and (d) 80 nm gold particles on copper grids.
Table 3. Measured Particle Average Size and Its Standard Deviation (Both in Nanometers) from Different Methods particle type
AFM
TEM
PSD
Silica
199 ( 11
204 ( 9
227 ( 6
Polystyrene
203 ( 9
204 ( 6
221 ( 6
scattering (referred to here as PSD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). As we show in Table 3, these do not all give the same results. PS particles and silica particles have different properties, for example, PS particles have a lower surface tension and are much softer than silica particles and can therefore deform when adhering to a hard surface (silicon or mica) for AFM measurements of their diameters. Thus, a method such as PSD seems to be potentially better for the determination of the polystyrene particle diameters because the particles are dispersed in a liquid and will not deform unless there is a specific (swelling) interaction with the liquid. Yet, this conclusion is problematic because we have found that the PSD gives larger particle diameter values for silica particles (which are too hard to deform measurably) than either TEM or AFM measurements. The comparison of silica particle size determined from the different methods (Table 3) indicates that PSD may not provide accurate particle sizes for the purposes of the present investigation. Hence, in the present work, we use the AFM to determine the size of the silica particles. Because AFM is also used to measure particle heights above the DPPC surface in the embedment experiments, this provides an internal consistency as well. For the PS particles, the situation is more complicated because these particles may deform as a result of the adhesive forces acting on them when dispersed on a hard substrate. To examine
6πwa 2 R K
ð6Þ
where K = (4/(3π(ks þ kp))) and ks(p) = ((1 vs(p))/(πEs(p))), the subscripts s and p indicate the substrate and the particle. From the literature values44 of Poisson’s ratio and Young’s modulus for PS (ν = 0.38, E = 2.8 GPa) and silicon (ν = 0.27, E = 107 GPa), the ratio of the estimated deformation (relative to particle radius) of a PS particle on a silicon substrate as a function of particle size is shown in Figure 2. Figure 2 shows the estimated percentage of deformation for PS particles on silicon with particle sizes ranging from 50 nm to 1 μm. In the present work, the measured PS height on silicon is 203 ( 9 nm by AFM. As shown in Figure 2, the estimated deformation of these PS particles on silicon is less than 1% and the corresponding diameter change is expected to be 1.3 nm. To verify the PS particle size further, the PS particles were examined by TEM. The consistency of the PS particle size data obtained by TEM and AFM implies that even though the PS is relatively soft, little deformation of the PS particles occurred on the silicon substrate and the estimated errors in the measured height/ embedment depth are on the order of 1.3 nm. This is significantly less than the embedment depths of interest and further shows that direct imaging of the particles seems to provide a better estimation of particle size than does dynamic light scattering. We use the AFM values for the PS particle diameter as well as for the silica particles. 4.4. Particle Embedment Measurements. Particle embedment measurements were performed using AFM. To determine the particle height above the DPPC surface (Figure 3), the height profile of a particle from the AFM image was selected. Figure 3a shows the 2D AFM image of the PS particles dispersed on the DPPC multibilayer surface along with an AFM height profile for a line that cuts across several polystyrene particles on a DPPC multibilayer film. Individual particles can be clearly observed on the DPPC multibilayers from the AFM image. Figure 3b shows how the height of a single particle on the DPPC multibilayers is obtained in this article. The height of each particle can be estimated by subtracting the peak height and the underlying baseline 6849
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Figure 3. (a) AFM image of 200 nm polystyrene particles on a DPPC multibilayer film. (b) Illustration showing how to obtain the apparent height of a particle embedded in a film surface. Base line was chosen at (7.58 μm, 17.52 nm).
Figure 4. (a) Scan area (40 40 μm2) AFM image of 200 nm PS particles partially embedded in a DPPC multibilayer film (90 ( 6 nm thick). (b) Subsections of the whole image (a).
from the particle height profile using the AFM software. Because the height of each particle can also be calculated by averaging the difference between the peak point and two bottoms, hundreds of particles’ apparent heights have been examined by this method and then compared to those obtained by the baseline method that is shown in Figure 3b; it was found that both methods give similar results. The embedment depth δ of each particle on the thin film is the difference between the average particle diameter from the measurement on a bare mica or silicon substrate and the particle height on DPPC. 4.5. DPPC Shear Modulus Determination. 4.5.1. Polystyrene Particle Embedment Results. Figure 4a shows a 40 40 μm2 2D image of the 200 nm PS particles partially embedded into a DPPC multibilayer film. Both individual particles and particle clusters are clearly observed. To reduce the effect of instrument error on the apparent height of particles, the whole image (40 40 μm2) was divided into 16 parts and each subsection was rescanned to get more accurate height values for each particle. The height distribution chart (Figure 5) shows that most of the polystyrene particles are located within the range of 170 to 200 nm. A total of 238 individual particles and 105 aggregated particles were analyzed over the entire region, and the average particle heights were 186 ( 9 and 189 ( 10 nm, respectively. The results show that single particles and clustered particles give almost the same embedment depth, which implies that under the present conditions individual particles and clustered particles provide the same information and will yield similar shear modulus
values for the DPPC multibilayers. The implication is that particle clusters can also be used here to calculate the surface shear moduli of DPPC by the particle embedment technique. Though further work is needed to establish the range for which this is true, it suggests the possibility of using crystal monolayers to determine property (modulus) maps of heterogeneous surfaces because close-packed particles provide the opportunity to assign individual particle sizes from peakpeak distances and hence reduce the uncertainty in the particle embedment depth. Similarly, these 200 nm PS particles were placed onto thicker DPPC multibilayers (184 ( 4 nm), and the average particle height is h = 183 ( 10 nm. It seems that the film thickness has only a weak effect on the particle embedment depth, as discussed subsequently. 4.5.2. Silica Particle Embedment Results. Silica particles (235 nm) were also used for the particle embedment experiments. Figure 6 shows AFM topography images of the SiO2 particles partially embedded in a DPPC multibilayer surface with a thickness of 144 ( 8 nm. The scan areas were chosen randomly for these silica particles, and Figure 6 shows some AFM images of the 235 nm silica particles on this DPPC film. Although both individual particle and particle clusters give similar embedment depths on the surface of interest, as discussed above, in this work only single particles were used to determine the elastic properties of the lipid surface. Similar procedures were applied to the silica particles, as done above for the PS particles, to obtain the particle height and the 6850
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Figure 5. Particle height distributions for (a) 200 nm polystyrene individual particles and (b) particle clusters on a DPPC multibilayer film.
Figure 6. Two-dimensional AFM images of 235 nm silica particles on DPPC multibilayers (144 ( 8 nm).
)
)
embedment depth on DPPC. Histograms of the percent of the number of particles in the relevant particle size range versus particle height are shown in Figure 7. The particle heights are heavily centered between 180 and 200 nm. A total of 220 particles were analyzed, and the average particle height was found to be 192 ( 13 nm. We also examined the film thickness effects on the silica particle embedment result. Then, DPPC films with different thicknesses (77 ( 6, 158 ( 6, and 270 ( 15 nm) were prepared, and the same silica particles were embedded in these films. The results are listed in Table 4. Small differences in the embedment depth values were found for the different DPPC film thicknesses, implying that the particle embedment depth is independent on the film thickness. 4.5.3. Shear Moduli of the DPPC Multibilayers. The shear modulus of DPPC can be calculated from the embedment depth δ (average particle diameter d minus particle height h) based on the JKR model given in eq 1. As discussed above, the AFM measured particle size on a bare surface was selected as the particle diameter for both silica and polystyrene particles. Then, the PS particle size on silicon is 203 ( 9 nm by AFM and the silica particle diameter is 199 ( 11 nm by averaging the obtained silica particle size on the two different surfaces (mica and silicon). Table 4 shows the calculated elastic modulus of DPPC from the embedment of PS and silica particles on the DPPC multibilayers having different film thicknesses. The calculated shear moduli for DPPC from the embedment data of polystyrene particles and silica particles are within the range of reported data for the elastic moduli (E and E10) of bilayer lipid membranes, for which the values are in the range of 20100 MPa for E and 0.250 MPa for E10 depending on the strain, frequency, temperature, and lipid type.3 Because the compression modulus is 3 times the shear modulus (for an isotropic, incompressible material), the obtained shear moduli of
Figure 7. Particle height distribution for 235 nm silica particles on DPPC multibilayers.
DPPC from silica particle embedment depth data also agree well with the compression modulus value by Chen et al. for a supported lipid monolayer.13 The present result shows that for PS particles, all of the embedment depths δ in Table 4 are greater than 10% of the film thickness t, whereas for silica particles, δ < 10%t. Dimitriadis et al.45 showed that the same sample is stiffer when bonded to a rigid substrate and this difference cannot be neglected when the embedment depth δ is greater than 10% of the film thickness. In our results, if we consider the rigid surface effect on the DPPC film properties, then the elastic modulus obtained from the PS particle embedment depth should be higher than those obtained from silica particles. Obviously, we have obtained a different result. One possible explanation is that these commercial PS particles are stabilized by the addition of sodium dodecyl sulfate (SDS) surfactant. The silica particles used are pure silica with 6851
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Table 4. Shear Modulus of DPPC Multibilayers from Particle Embedment Measurementsa probe particle
a
200 PS
235 Si
literature
d (nm)
203 ( 9
t (nm)
90 ( 6
184 ( 4
199 ( 11 77 ( 6
144 ( 8
158 ( 6
270 ( 15
h (nm)
186 ( 9
183 ( 10
192 ( 15
192 ( 13
193 ( 12
189 ( 22
δ (nm)
17 ( 1.1
20 ( 1.4
7 ( 0.7
7 ( 0.6
6 ( 0.5
10 ( 1.3
G (MPa)
6.4 ( 0.7
5.0 ( 0.6
49.8 ( 7.7
49.8 ( 7.1
62.7 ( 8.6
29.2 ( 5.9
0.2100,3 5020013
d, particle diameter; t, DPPC film thickness; h, particle height above DPPC; δ, particle embedment depth; G, shear modulus of DPPC.
Figure 8. (a) AFM image of 150 nm gold on a DPPC multibilayer. (b) Three-dimensional view of image a. (c) AFM image of 80 nm gold on a lipid surface. (d) Three-dimensional view of image c.
only some silanol groups on the particle surfaces. In the experiment, a drop of PS particle colloidal solution placed on the prepared DPPC films is taken from the dispersion that is directly diluted from the purchased solution. It seems that the small amount of surfactant may be absorbed by the DPPC multibilayers to change the surface properties of the DPPC films. Then, this surfactant effect would be larger than the constraining (rigid) silicon substrate effect and could lead to a higher particle embedment depth for the PS particles than for the silica spheres. This phenomenon is consistent with results of Inoue et al., who reported the interactions between DPPC membranes and different types of surfactants with respect to the gel-to-liquid phase transition of lipids.46,47 They reported that the phase-transition temperature of DPPC is depressed by the addition of surfactant, and the transition width was found to increase.46,47 Then, in our case, if SDS incorporates into DPPC it could soften the lipid multibilayers and lead to a larger embedment depth for the PS particles on DPPC. Here, we suggest that the shear moduli of DPPC determined from the silica particle embedment measurements are more reliable than the PS particle embedment results. Future work should explore this in more detail. The results for silica particles on DPPC films with different thicknesses in Table 4 indicate that the shear modulus of DPPC is reasonably independent of the lipid film thickness. However, for the thickest film there seems to be a slight decrease in the determined modulus. Because we found that the film surface roughness increases to 12 nm from 6 nm when the film thickness increases from 158 to 270 nm, this may partially account for the observation of the reduced modulus of the thickest film. The greater surface roughness for the thicker film results in more error in the measurement. In the standard JKR equation, the shear modulus is proportional to δ3/2, so a small change in the particle embedment depth can have a large effect on the value of the DPPC shear modulus. In this case, as long as the particle embedment depth remain less than 10% of the film thickness, it is better to choose an appropriate film thickness to reduce the error caused by the surface roughness. Also, it is worth noting that it is
known that surface roughness and surface asperities can cause a decrease in the apparent modulus,48,49 and it is desirable to have surfaces that are as smooth as possible. 4.5.4. Gold Particle Embedment Results. The nonspherical (faceted) shape of the gold particles implies that the stress applied by each edge of the particle on the lipid multibilayers is different from that applied by a spherical particle.42 The JKR model used in the present work is based on the assumption that the particles are spherical, which means that using the JKR model for nonspherical gold particles may yield errors in determining the shear modulus of the DPPC multibilayers with the submicrometer particle embedment technique. However, the gold particles were also considered, and a qualitative analysis was performed; a very interesting phenomenon was found and discussed. In the gold particle embedment experiments, as shown in Figure 8ad, the 150 and 80 nm gold particles were found to sit on the DPPC surface and the average heights of 150 and 80 nm gold particles on the DPPC surface were found to be 127 ( 15 and 58 ( 12 nm with measured particle heights on silicon of around 138 ( 12 and 71 ( 6 nm. However, for the 13 nm gold particles, it is hard to determine the heights on the lipid film surface. Figure 9 shows AFM height and phase images for a pure lipid surface (Figure 9a,b) and a lipid film with 13 nm gold particles on it (Figure 9c,d). The surface topographies for lipid multibilayers with and without 13 nm gold particles are similar. In Figure 9c, it appears that some gold particles may exist on top of the lipid surface. To investigate this further, AFM phase images were compared to the corresponding AFM height images. The phase images show no differences between the two, which implies that the observed “particles” in Figure 9c are not the gold particles. A series of AFM images for the pure lipid and lipid with particles were taken and compared. The only difference is that both the average surface height and roughness for 13 nm gold particles on the lipid thin film increase somewhat as compared to those of the pure lipid film (e.g., the average surface roughness increases from 3.0 to 6852
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
Figure 9. (a) AFM height image of a pure DPPC multibilayer. (b) Phase image for image a. (c) AFM image for 13 nm gold on a DPPC film. (d) Phase image for image c.
4.1 nm). The most likely reason is that these 13 nm nanogold particles were completely engulfed by the lipid multibilayers, which results in changes in the surface height and roughness whereas individual gold particles were not observed. To provide an explanation of this phenomenon, we consider the “critical radius” concept. If the critical radius equals or is greater than the particle radius, then adhesion-induced particle engulfment may occur.43 This critical radius concept was first proposed by Rimai et al. in their study of the engulfment of glass particles on a plasticized polyurethane substrate.43 In their work, it was reported that when the interfacial energy γ12 is sufficiently high, it may lead to total particle engulfment. To determine whether particles can be totally engulfed by the surface, two equations were proposed by Rimai et al. to calculate the critical contact radius of particles under different circumstances.43 In our case, the total engulfment of the 13 nm gold nanoparticles was observed. By simply assuming that the deformation of the lipid is plastic, the the critical radius Rc is expressed by eq 7.43 Rc
2γ12 3Y
ð7Þ
The interaction energy γ12 is 24.0 mJ/m2 on the basis of the obtained work of adhesion between gold and the DPPC multibilayer film and the surface tension values listed in Table 2. According to the reported relations between the yield stress and Young’s modulus for polymers (σy ≈ 0.025E),50 the yield stress for DPPC would be approximately 2.23.7 MPa on the basis of the values of the shear modulus given in Table 4 from silica particle embedment results. Then, the calculated critical radius Rc is 4.37.3 nm and the examined 13 nm gold particle radius is around 6.5 nm, which falls within this range, so one could anticipate that a 13 nm gold particle would be engulfed by the DPPC multibilayers as observed here. In this work, the probe particles all had similar surface tensions. In future work, it would be of interest to apply a known force to the probe particles or use an AFM tip directly to investigate the DPPC properties systematically. Investigating the properties as a function of temperature would also provide novel information concerning DPPC and other lipid multibilayers. Finally, it is worth remarking that the finding that the clustered particles seem to possess properties similar to those of the isolated particles suggests that a possible benefit of the present particle embedment method is the possibility of mapping the spatial distributions of surface properties with better accuracy than possible with the isolated particles.51
5. CONCLUSIONS A novel submicrometer particle embedment method has been used to obtain the elastic properties of a DPPC multibilayer
system. Results show that single particle and clustered particles give essentially the same values for the particle embedment depths and thus the same values of the shear modulus for DPPC ultrathin films. Although the shear modulus values calculated from silica and PS particle embedment depths are consistent with reported data, the difference in modulus data observed between these two types of particles may be due to the existence of surfactant in the PS particle suspensions. Importantly, 13 nm gold particles were found to be completely engulfed by the lipid multibilayer film, which implies that, for nanoparticles, the interfacial energy at the membrane/particle interface can be sufficient to cause spontaneous full particle engulfment.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT We are grateful to the John R. Bradford Endowment at Texas Tech University and the National Science Foundation under grants CMMI-0555906 and DMR-0804438 for partial support of this work. We also are appreciative of the help in taking the TEM images provided by C. Linch of the Texas Tech University Health Sciences Center. ’ REFERENCES (1) Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry; W. H. Freeman: New York, 2007. (2) Terheiden, A.; Rellinghaus, B.; Stappert, S.; Acet, M.; Mayer, C. J. Chem. Phys. 2004, 121, 510–516. (3) Hianik, T.; Passechnik, V. I. Bilayer Lipid Membranes: Structures and Mechanical Properties; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995. (4) Sackmann, E. Science 1996, 271, 43–48. (5) Gorter, E.; Grendel, F. J. Exp. Med. 1925, 41, 439–443. (6) Evans, E. A.; Hochmuth, R. M. Biophys. J. 1976, 16, 1–11. (7) Waugh, R.; Evans, E. A. Biophys. J. 1979, 26, 115–131. (8) Fung, Y. C. Biomechanics: Mechanical Properties of Living Tissues, 2nd ed.; Springer-Verlag: New York, 1993; p 568. (9) Fung, Y. C. B.; Tong, P. Biophys. J. 1968, 8, 175–198. (10) Evans, E. A.; Waugh, R.; Melnik, L. Biophys. J. 1976, 16, 585–595. (11) Zanoni, R.; Naselli, C.; Bell, J.; Stegeman, G. I.; Seaton, C. T. Phys. Rev. Lett. 1986, 57, 2838. (12) Ibach, H.; Mills, D. L. Electron Energy Loss Spectroscopy and Surface Vibrations; Academic Press: New York, 1982. (13) Chen, Y. L.; Helm, C. A.; Israelachvili, J. N. Langmuir 1991, 7, 2694–2699. (14) Teichroeb, J. H.; Forrest, J. A. Phys. Rev. Lett. 2003, 91, 016104. 6853
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
Langmuir
ARTICLE
(15) Hutcheson, S. A.; McKenna, G. B. Phys. Rev. Lett. 2005, 94, 076103. (16) Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc. R. Soc. London, Ser. A 1971, 324, 301–313. (17) Lim, B. C.; Thomas, N. L.; Sutherland, I. Prog. Org. Coat. 2008, 62, 123–128. (18) Mykhaylyk, T. A.; Evans, S. D.; Fernyhough, C. M.; Hamley, I. W.; Henderson, J. R. J. Colloid Interface Sci. 2003, 260, 234–239. (19) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 1741–1747. (20) Wu, S. J. Polym. Sci., Part C: Polym. Symp. 1971, 34, 19–30. (21) Balkenende, A. R.; van de Boogaard, H. J. A. P.; Scholten, M.; Willard, N. P. Langmuir 1998, 14, 5907–5912. (22) Mennicke, U.; Salditt, T. Langmuir 2002, 18, 8172–8177. (23) O’Connell, P. A.; McKenna, G. B. Rev. Sci. Instrum. 2007, 78, 013901–013912. (24) Tak, Y.-H.; Kim, K.-B.; Park, H.-G.; Lee, K.-H.; Lee, J.-R. Thin Solid Films 2002, 411, 12–16. (25) DeGarmo, E. P.; Black, J. T.; Kohser, R. A. Materials and Processes in Manufacturing, 9 ed.; Wiley: Hoboken, NJ, 2003. (26) Foltyn, S. R.; Jia, Q. X.; Arendt, P. N.; Kinder, L.; Fan, Y.; Smith, J. F. Appl. Phys. Lett. 1999, 75, 3692–3694. (27) Logothetidis, S.; Stergioudis, G. Appl. Phys. Lett. 1997, 71, 2463–2465. (28) Collins, G. W.; Letts, S. A.; Fearon, E. M.; McEachern, R. L.; Bernat, T. P. Phys. Rev. Lett. 1994, 73, 708. (29) Wu, S. Polymer Interface and Adhesion; M. Dekker: New York, 1982; p 630. (30) Cognard, J. Gold Bull. 1984, 17, 131–139. (31) Harnett, E. M.; Alderman, J.; Wood, T. Colloids Surf., B 2007, 55, 90–97. (32) Jurak, M.; Chibowski, E. Langmuir 2007, 23, 10156–10163. (33) Fujii, H.; Matsumoto, T.; Izutani, S.; Kiguchi, S.; Nogi, K. Acta Mater. 2006, 54, 1221–1225. (34) Alexander, G. B. J. Phys. Chem. 1957, 61, 1563–1564. (35) Gaines, G. L.; Tabor, D. Nature 1956, 178, 1304–1305. (36) Schrader, M. E. J. Colloid Interface Sci. 1984, 100, 372–380. (37) Wenzel, R. N. Ind. Eng. Chem. 1936, 28, 988–994. (38) Yoshimitsu, Z.; Nakajima, A.; Watanabe, T.; Hashimoto, K. Langmuir 2002, 18, 5818–5822. (39) Kamusewitz, H.; Possart, W. Appl. Phys. A: Mater. Sci. Process. 2003, 76, 899–902. (40) Bhushan, B.; Jung, Y. C. J. Phys.: Condens. Matter 2008, 20, 225010. (41) Jung, Y. C.; Bhushan, B. Nanotechnology 2006, 17, 4970–4980. (42) Needs, R. J.; Mansfield, M. J. Phys.: Condens. Matter 1989, 1, 7555. (43) Rimai, D. S.; Quesnel, D. J.; Busnaina, A. A. Colloids Surf., A 2000, 165, 3–10. (44) Drelich, J.; Tormoen, G. W.; Beach, E. R. J. Colloid Interface Sci. 2004, 280, 484–497. (45) Dimitriadis, E. K.; Horkay, F.; Maresca, J.; Kachar, B.; Chadwick, R. S. Biophys. J. 2002, 82, 2798–2810. (46) Inoue, T.; Miyakawa, K.; Shimozawa, R. Chem. Phys. Lipids 1986, 42, 261–270. (47) Inoue, T.; Iwanaga, T.; Fukushima, K.; Shimozawa, R. Chem. Phys. Lipids 1988, 46, 25–30. (48) Lima, R. S.; Kucuk, A.; Berndt, C. C. Surf. Coat. Technol. 2001, 135, 166–172. (49) Wai, S. W.; Spinks, G. M.; Brown, H. R.; Swain, M. Polym. Test. 2004, 23, 501–507. (50) Rowe, R. C.; Roberts, R. J. J. Mater. Sci. Lett. 1995, 14, 420–421. (51) Hutcheson, S. A. Evaluation of Viscoelastic Materials: The Study of Nanosphere Embedment into Polymer Surfaces and Rheology of Simple Glass Formers Using a Compliant Rheometer. Ph.D. thesis, Texas Tech University, Lubbock, TX, 2008.
6854
dx.doi.org/10.1021/la2005375 |Langmuir 2011, 27, 6846–6854
