Article pubs.acs.org/Langmuir
Mobile Lipid Bilayers on Gold Surfaces through Structure-Induced Lipid Vesicle Rupture Po-Yu Peng, Po-Chieh Chiang, and Ling Chao* Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan S Supporting Information *
ABSTRACT: Forming fluid supported lipid bilayers (SLBs) on a gold surface can enable various lipid-membraneassociated biomolecular interactions to be investigated by several surface sensing techniques, such as surface plasmon resonance and scanning tunneling microscopy. However, forming fluid SLBs on a gold surface through lipid vesicle deposition continues to pose a challenge. In this study, we constructed nanograting structures on a gold surface to induce lipid vesicle rupture for forming a mobile layer of SLBs. Observations based on fluorescence recovery after photobleaching showed that SLBs on the prepared grating supports had some fluidity, while SLBs on the planar support had no fluidity. The anisotropic fluorescence intensity recovery shape changes observed in the SLBs on the grating support suggested that a second layer of SLBs partially formed on top of the first layer in contact with the gold surface and extended along the grating structure. Comparisons of the relative amounts of second bilayer and the fluorescence recovery fractions on supports with various grating edge densities suggested that the second layer formed at the edge regions and that the coverage ratio was directly proportional to the grating edge density. All of these results showed that the grating edges could serve as vesicle-rupture-inducing sites for the formation of a mobile second SLB on a gold surface. The formation of the second layer of SLBs at the edge regions but not in the flat regions enabled us to determine the second layer locations and provided us with an opportunity to pattern mobile lipid bilayers on gold surfaces by controlling the edge locations.
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surface, such as mica and silicon-based materials.9,10,16,17 Developing new methods for enabling lipid vesicles to deposit and form fluid SLBs on a wider variety of supports can facilitate using surface sensors to study cell-membrane-related events. The deposition of SLBs on gold surfaces enables the transduction of biochemical changes on SLBs to electrical or optical signals, which is necessary in many surface sensing techniques such as surface plasma resonance,18,19 infrared reflection-absorption spectroscopy,20 and scanning tunneling microscopy.21 Most current approaches involve either incorporating lipid membranes on a gold surface by modifying the gold surface with thiols and then preparing a monolayer to form a hybrid bilayer membrane22 or using thiols to tether lipid bilayers to the support to form a tethered-lipid bilayer membrane.23,24 However, using these approaches for transmembrane protein incorporation is difficult, and the chemical modification required to the solid support or to the lipids can influence the native environment of the lipid−membraneembedded species. Using the vesicle deposition method to
INTRODUCTION Supported lipid bilayers (SLBs) have been widely used as biomimetic platforms for studying various lipid-membraneassociated cellular processes.1,2 SLBs are planar extended bilayers adsorbed on appropriate solid surfaces,3,4 and their planar geometry is compatible with a wide range of surface analytical tools.5−8 Lipid vesicle deposition9,10 is one of the commonly used SLB preparation methods and has several advantages compared to some other methods such as the Langmuir−Blodgett/Langmuir−Schaefer method11 and lipid spin-coating method.12 The first major advantage is the ease of incorporating native membrane proteins in SLBs. The membrane proteins can be obtained from cells in the forms of embedding in proteoliposomes and cell blebs, which could be directly used for vesicle deposition. Other SLB formation methods usually require the proteins to be exposed to an air− water interface, detergent, or solvent, which may cause the proteins to denature.13 In addition, unlike other methods that can only allow SLB formation on the outer surface of an object, the vesicle deposition method enables SLB formation not only on the outer surface but also inside a flow chamber of a sensor chip.14,15 However, there is a delicate interaction balance between supports and SLBs, and successful vesicle deposition for forming fluid SLBs has been reported on few types of © 2015 American Chemical Society
Received: November 20, 2014 Revised: February 28, 2015 Published: March 6, 2015 3904
DOI: 10.1021/la504532a Langmuir 2015, 31, 3904−3911
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lammonium salt (Texas Red DHPE) was purchased from Life Technologies (Grand Island, NY, USA). All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fused silica slides were purchased from Precision Glass & Optics (Santa Ana, CA, USA). A silicon mode for nanoimprinting lithography was purchased from LightSmyth Technology (Eugene, OR, USA) Preparation of Gold-Coated Nanograting Silica Supports. Silica solid supports with nanograting structures were fabricated using nanoimprinting lithography. Silicon modes with 200, 400, and 800 nm grating structure geometries were used to construct nanograting structures on fused silica supports, and the procedure is detailed in our previous report.35 The final height of the nanograting structures on the fused silica solid supports was controlled to be approximately 150 nm. After the desired nanograting structures were formed, a 2 nm-thick Cr layer and then a 5 nm-thick gold layer were deposited onto the structured surface through electron-beam-induced deposition by using an evaporator (AST PEVA 400E). The thin Cr layer was introduced to enhance the adhesion of gold to the fused silica supports. Support Cleaning and Formation of Supported Lipid Bilayers. The gold-coated supports were cleaned by immersing them in ethanol under sonication for 1 h, and this was followed by immersion in deionized water under sonication for 1 h. The supports were then removed from the immersion bath and dried using nitrogen. The supports were placed on a hot plate at 120 °C for 20 min to remove the ethanol and water residue trapped in the grooves. Before the lipid vesicle deposition step, the support was cleaned using argon plasma for 10 min. Large unilamellar vesicles with a concentration of 0.2 mg/mL in an aqueous solution with 100 mM sodium chloride were used for lipid vesicle deposition to form SLBs. The vesicle solutions were incubated with the cleaned supports for 60 min, and the excess vesicles were removed by washing them with the same solution that was used to reconstitute the lipid vesicles. Fluorescence Photobleaching after Recovery. An intense laser light from a 200 mW diode-pumped solid-state green laser module (Unice, Taiwan) operating at a wavelength of 532 nm was used to bleach a small spot in a membrane doped with a fluorescently labeled lipid, Texas Red DHPE, for 0.5 s. The fluorescence intensity of the bleached spot had a Gaussian profile with an approximate half-maximum width of 10 μm. The recovery images were captured using an inverted microscope (Olympus IX81, Olympus, Tokyo, Japan) equipped with a complementary metal-oxide-semiconductor (CMOS) camera (ORCA-R2, Hamamatsu Photonics, Hamamatsu, Japan). The intensity recovery in the region of interest was processed by MATLAB (Mathworks Natick, MA, USA) to calculate the two-dimensional diffusion coefficients of the SLB (details on pp 11−12 in the Supporting Information (SI), Obtaining Dperpendicular and Dparallel by 2-D FRAP Fitting). Scanning Electron Microscopy (SEM) Measurement of Gold-Coated Supports with Nanograting Structures. The morphology of the gold-coated supports with nanograting structures was observed using a scanning electron microscope (NOVA NanoSEM 230, FEI Company, OR, USA) at an operating voltage of 5.0 kV. Images were recorded in a highvacuum mode.
form SLBs on a gold surface could enable more robust surface sensing of cell membrane related events. An important property for SLBs to be used as biosensing platforms is to have lateral fluidity. Numerous cellular processes are initiated by multivalent ligand receptor interactions, which require membrane associated receptors to move laterally in the lipid bilayer to form clusters for the interactions to occur.25,26 Conventional SLBs on glass supports are shown to preserve the lateral fluidity of their constituents by having a thin water layer between the support and the SLB.9 However, previous studies have suggested that the strong interaction between gold and the lipid bilayer may lead to the absence of the thin water layer between lipid bilayer and the support surface,25−28 and forming fluid SLBs on a gold surface continues to pose a challenge. For example, Reimhult et al. have suggested that the lipid vesicles can adsorb on flat gold surfaces, but they seem not to rupture.29 Groves et al. have shown that lipid vesicles fuse with gold, but the resulting lipid membranes are immobile.30 Recently, some studies show that the bilayer structure of SLBs can form on a gold surface, but forming this structure requires more time than usual because the strong interaction between gold and the lipid bilayer may result in a formation mechanism different from the conventional mechanism on silica and mica supports.21,25,26,28,31 Some studies have observed the appearance of a water layer upon applying an electrical voltage to a gold surface to reduce gold-lipid membrane interaction;25,26,31 however, the electrical voltage may influence the biological analytes or detection signals. Here, we planned to form a mobile bilayer on a gold surface by creating a second layer of SLBs on an immobile first layer of SLBs on the gold surface. The large distance between the second layer and the support may reduce interaction between the second layer and the gold surface and lead to high lateral fluidity. However, as far as we know, the formation of a second SLB on a gold surface by using lipid vesicle deposition has not been observed. The reason might be that vesicle deposition involves the deformation and rupture of vesicles,32−34 and vesicle rupture kinetics might be a crucial factor to determine whether a second bilayer can be formed in a reasonable time scale. In this study, we constructed nanograting structures on gold surfaces to induce vesicle rupture for forming a mobile second layer of SLBs. Observations based on fluorescence recovery after photobleaching showed that SLBs on the prepared grating supports had some fluidity, while the SLB on the flat surface had no fluidity. The anisotropic fluorescence intensity recovery shape changes observed in the SLBs on the grating support suggested that a second SLB partially formed and extended along the grating structure. We hypothesized that the grating edges serve as additional vesicle-rupture-inducing sites. We prepared grating supports with various grating edge densities to examine whether the amount of the mobile SLBs was directly proportional to the density of the possible rupture-inducing site. Comparisons of the relative amounts of second layer and the fluorescence recovery fractions on supports with various grating edge densities supported our proposed bilayer organizations on the gold-coated nanograting supports.
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MATERIALS AND METHODS Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. 1,2Dihexadecanoyl-sn-glycero-3-phosphoethanol amine, triethy-
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RESULTS AND DISCUSSION Theoretical Prediction of Supported Multibilayer Formation on Gold Surfaces. We used the extended 3905
DOI: 10.1021/la504532a Langmuir 2015, 31, 3904−3911
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Langmuir Derjaguin−Landau−Verwey−Overbeek (DLVO) theory36,37 to describe the interactions between SLBs and their solid supports. Figure 1a shows the interaction energy between a
Proposed Mechanism of Using Grating Edges as Rupture Sites to Induce the Formation of a Second Layer of SLBs. Although the theory predicts that the formation of a second bilayer is energetically favorable, no previous study has reported the observation of a second layer on a gold surface by using lipid vesicle deposition. Since the vesicle deposition method requires lipid vesicles to deform and rupture to form SLBs,38 we hypothesized that interaction between the first layer-support composite and lipid vesicles may not be sufficiently strong to deform the vesicles to create rupture sites, as illustrated in Figure 2b. Inspired by the
Figure 1. Interaction energies calculated using the extended DLVO theory: (a) between the support and the first layer of SLBs and (b) between the first layer-support composite and the second layer of SLBs. The overall interaction energy (Eoverall) is the summation of the calculated van der Waals interaction energy (Evdw), electrostatic interaction energy (EEDL), and hydration interaction energy (EH). The subscript 1b indicates the interaction energies associated with the first layer and the support. The subscript 2b indicates the interaction energies associated with the second layer and the first layer-support composite. The SLBs are DOPC membranes doped with 1.35 mol % Texas Red DHPE and deposited on a gold surface with a surface potential of −11.8 mV/m2 at an ionic strength of 100 mM.
Figure 2. Illustration of the proposed situations where (a) the vesicles approach the planar support, (b) the vesicles approach the first layerplanar support composite, (c) the vesicles approach the grating support, and (d) the vesicles approach the first layer- grating support composite. Yellow indicates the gold-coated supports, and blue indicates the lipid bilayer.
observation of a previous studysmall features on supports can facilitate vesicle breakage39we proposed that supports with grating edges can induce local deformation of lipid vesicles and therefore create additional high-tension rupture sites for vesicle rupture. Figure 2 illustrates the proposed situations where lipid vesicles approach the planar and grating supports. In Figure 2a, the vesicle is deformed appreciably because of the strong affinity between the support and the lipid membrane. Previous studies have suggested that vesicles can rupture at the sites where the lipid bilayers bend the most and have the highest tension.32,33 In Figure 2b, the vesicles on the first layer-support composite are deformed less, probably because the affinity between the first layer-support composite and the second layer is lower than that between the first layer and the support, as shown in Figure 1. The reduced affinity may not be sufficiently strong to deform the vesicles enough for the high-tension rupture sites to form, although the formation of the second bilayer above the first layer is energetically favorable. Figure 2c,d shows that the vesicles may bend and deform when the vesicles approach the grating edge. The local curvature of the lipid bilayer can be high at the region in contact with the grating edge, potentially creating additional high-tension rupture sites. Appreciable SLB Fluidity on Gold-Coated Nanograting Support but not on Gold-Coated Flat Support. We formed SLBs on a gold-coated flat support and gold-coated nanograting supports with three geometries and used fluorescence recovery after photobleaching (FRAP) to examine the membrane fluidity. To show the influence of the edge density on the lipid membrane fluidity, we prepared the supports with Geometries I, II, and III with the edge density increasing in this order of geometry. Figure 3a shows the symbols used to describe the geometry of the grating structure, and Figure 3b−d shows the SEM images of the three types of nanograting support. Figure 3e−h shows the fluorescence recovery situations for 1.35 mol % Texas Red DHPE/DOPC on the planar and the three gold-coated nanograting supports at
support and a single-layered SLB as a function of the distance between the support and the SLB (details of how we obtained the interaction energy curves can be found in pp 1−6 in the SI, the interaction energy between the support and a single-layered supported lipid bilayer). According to the extended DLVO theory, the calculated overall interaction energy (Eoverall,1b) is the summation of the contributions from the van der Waals interaction (Evdw,1b), electrostatic interaction (EEDL,1b), and hydration interaction (EH,1b), and the overall interaction (Eoverall,1b) is set as zero when the support and the SLB are separated by an infinite distance. The overall energy between a gold-coated support and a single-layered SLB is always negative and monotonically decreases when the distance decreases; therefore, the energy reward reaches the maximum when the distance is zero. The calculated interaction energy suggests that the first layer of SLBs can fully attach to the support without a water layer in between at equilibrium. To predict whether a second layer of SLBs can form on top of the first layer, we viewed the first layer and the support as a composite and calculated the interaction energy between the composite and the second layer. Figure 1b shows the overall interaction energy (Eoverall,2b) calculated using the extended DLVO theory (calculation details can be found in pp 6−10 in the SI, the interaction energy between the first layer-support composite and the second layer of SLBs). The electrostatic energy contribution was revised to account for solution exclusion by the acyl chain region of the first bilayer. The calculated minimum interaction energy is negative and located a distance of approximately 1−2 nm away from the top surface of the first layer-support composite, suggesting that the formation of the second bilayer was energetically favorable and that the equilibrium location of the second bilayer was approximately 1−2 nm away from the first bilayer surface. The 1−2 nm water layer between the second layer and the first layer-support composite could provide the second layer with lateral fluidity. 3906
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Figure 3. Fluorescence recovery after photobleaching observed in the SLBs on the three types of nanograting support. (a) Symbols used to describe the geometry of the grating structure. Panels b, c, and d show SEM images of the three types of nanograting support: Geometry I, Geometry II, and Geometry III, respectively. Panels e, f, g, and h show the fluorescence recovery situations on the planar support, Geometry I support, Geometry II support, and Geometry III support, respectively. DOPC membranes doped with 1.35 mol % Texas Red DHPE at an ionic strength of 100 mM were used. The images were taken at 0, 95, and 250 s after photobleaching and show how the fluorescence intensities at the bleached spot recovered with time.
recover after photobleaching. The increase in the final recovery fraction with the grating edge density suggests that the relative amount of the second layer increased with the support edge density. Anisotropy Fluorescence Recovery on Gold-Coated Nanograting Supports Suggesting Formation of Mobile Second Layers along the Edges. To quantify the anisotropy of the fluorescence recovery, we fitted the intensity recovery data to the 2D classical diffusion equation to obtain the membrane diffusivities in directions parallel to the grating structure and perpendicular to the grating structure (details in pp 11−12 in the SI, Obtaining Dperpendicular and Dparallel by 2D FRAP Fitting). Table 1 shows that all lipid membrane
an ionic strength of 100 mM. The photobleached spot on the planar support rarely recovered in 1 h, indicating that the lipid membrane on the planar gold support had poor fluidity, which is consistent with the observations of many previous studies.30,40 We observed that the photobleached spot partially recovered on the supports with nanograting structures. In addition, the recovery shape change was highly anisotropic, and only the diffusion component parallel to the grating was appreciable. Figure 4 further quantitatively shows the fluorescence intensity recovery with time after photobleaching for the planar support and the three grating supports. The fluorescence intensity rarely recovered on the planar support, and therefore, the recovery fraction was close to zero. Partial recovery observed on the grating supports suggests the formation of a mobile second layer, of which the fluorescence intensity can
Table 1. Diffusivities of the Lipid Membrane in Directions Parallel (Dparallel) and Perpendicular (Dperpendicular) to the Grating Structure geometry
Dparallel (μm2/s)
Dperpendicular (μm2/s)
I II III
1.098 ± 0.78 1.25 ± 0.72 1.27 ± 0.75
0.044 ± 0.088 0.056 ± 0.061 0.063 ± 0.070
diffusivities parallel to the grating structure were similar and in the same order of magnitude as the lipid diffusivities in a conventional SLB (1−10 μm2/s).41,42 By contrast, the diffusivities perpendicular to the grating structure were close to zero, showing the low fluidity in that direction. The anisotropic fluorescence recovery parallel to the grating structure suggested that the mobile second layer partially covered the surface and extended parallel to the grating structure, as illustrated in Figure 5. The analyses of fluorescence-recovery-after-photobleaching results showed that the lipid membrane had a high diffusivity in the parallel
Figure 4. Fluorescence intensity recovery with time after photobleaching for the planar support and the three grating supports. The recovery fraction is the fluorescence intensity at the bleached spot at the specified time normalized by the fluorescence intensity before photobleaching. 3907
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support in order to examine whether the SLBs have the structure we hypothesized in Figure 5. The AFM image in the SI (pp 14−15, Figure S6) shows that the topography close to the grating edge was higher than the topography in the middle of the flat region between the top grating edges. The height difference was around 5.4−5.7 nm, which is similar to the height of a single lipid bilayer plus the water layer between the two bilayers, supporting that an extra second bilayer depositing at the grating edge region. In addition, the control AFM image of the nanograting support without any lipid bilayer showed a flat topography, indicating that the height difference is not due to the support defect or AFM scanning artifact. Using Fluorescence Intensity for Further Examining Membrane Conformations. We further examined the existence of a second bilayer and whether the amount of the second bilayer is proportional to the edge density by comparing the measured fluorescence intensity from the flat support and from the three grating supports with various edge densities. Based on the possible lipid bilayer organizations proposed in Figure 5, we assumed that the experimentally measured fluorescence intensity is from the contribution of both the first bilayer, which fully covers the surface contour, and the second bilayer, which partially covers the surface contour. We estimated the fluorescence intensity per unit area of the first bilayer from the intensity measured from the flat support and calculated the fluorescence intensity contribution of the first bilayer following the contour of the designated nanograting support. Subtracting the first bilayer contribution from the experimentally measured intensity provides the fluorescence intensity contribution from the second bilayer. The fluorescence intensity contribution from the second bilayer allows us to estimate the relative second bilayer amounts in the different nanograting supports and to estimate the possible fluorescence recovery fractions as shown in the later paragraphs. It should be noted that the proportionalities of the bilayer amount to the fluorescence intensity for the first layer and for the second layer on a gold support can be different since gold surface has been reported to enhance or quench the emitted fluorescence intensity.43,44 When the same type of fluorophore and the same incident wavelength are used, the fluorescence enhancement/quenching is a function of the distance between the gold surface and the fluorophore molecules.43,44 Although it is difficult to compare the bilayer amounts of the different bilayers locating at different distances away from the gold surface, it is possible to compare the relative second bilayer amounts on the three different grating supports since their second bilayers should all be the same distance away from the gold support. The sixth column in Table 2 shows the estimated relative second bilayer amounts on the three grating supports based on
Figure 5. Illustration of proposed lipid membrane organizations on the nanograting gold supports with varying grating densities: (a) Geometry I support, (b) Geometry II support, and (c) Geometry III support. Yellow indicates the gold-coated support, lighter blue indicates the first bilayer in contact with the support, and darker blue indicates the second bilayer above the first bilayer.
direction and a diffusivity close to zero in the perpendicular direction. Since the second mobile layer did not appear to form on a flat support, it should not spontaneously form in the flat region on a grating support, and the only other possible region for the vesicles rupture is the grating edge region. The large diffusivities in the parallel direction indicated that lipid vesicles ruptured everywhere along the same grating edge and merged to form a continuous lipid membrane along the same grating edge. The small diffusivity in the perpendicular direction indicated that the bilayer perpendicular to the grating structure may not be continuous. The reason might be that the periodic grating distance is larger than the size of a piece of lipid membrane from the rupture of a 100 nm lipid vesicle used in this study. The amount of bilayer material of a vesicle is insufficient to cover the entire surface contour of the grating supports, rendering the lipid membrane discontinuous in the direction perpendicular to the grating structure. We further used atomic force microscopy (AFM) to obtain the topography of lipid membranes on a nanograting gold
Table 2. Comparison of the Relative Edge Density and the Estimated Relative Second Bilayer Amount and Comparison between the Experimental Final Fluorescence Recovery Fraction and the Estimated Final Recovery Fraction on the Three Grating Supportsa grating geometry
w1 (nm)
w2 (nm)
h (nm)
relative edge density (compared to Geometry I support)
estimated relative 2nd layer amount from measured fluorescence intensity (compared to Geometry I support)
estimated final recovery fraction
experimental final recovery fraction
I II III
881 641 278
676 200 132
179 177 151
1 1.85 3.80
1.00 ± 0.19 2.09 ± 0.19 3.66 ± 0.26
23.2 ± 3.5% 35.5 ± 2.1% 44.0 ± 1.8%
23.0 ± 6.2% 31.8 ± 8.7% 41.1 ± 11.8%
a
The variables w1, w2, and h are the grating widths and the grating height as defined in Figure 3a. Details of how to obtain the relative second bilayer amount and the estimated recovery fraction are provided in pp 10−11 in the SI. 3908
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Compared with other methods used to enhance the rupture kinetics of lipid vesicles, such as osmolality,17 freeze-and-thaw,46 and solvent induction methods,47 this grating edge method can keep the lipids and the associated species in their native environments without changing their conditions during the SLB formation process. In addition, it provides us with an opportunity to cause lipid vesicles to break and form SLBs at the designated locations; in other words, the bilayer location and coverage ratio could be controlled by adjusting the patterned structure geometry.
their relative second layer contributed intensities. Since we assumed that large unilamellar vesicles only break at the grating edge region and form second bilayers, the relative second bilayer amount should be proportional to the edge density. The fifth column in Table 2 lists the relative edge density of the three nanograting supports compared to the geometry I support. The trend and values of the relative grating density and the estimated relative second bilayer amount are close, supporting that the second bilayer forms at the edge region and we could increase the second bilayer amount by increasing the edge density. In addition, according to our hypothesis, the second bilayer has fluidity, while the first bilayer is immobile. Therefore, only the fluorescence intensity from the second bilayer but not the first bilayer could recover. If the fluorophore molecules in the bilayers are bleached from the top view of the support, the estimated final fluorescence recovery fraction could be represented as the ratio of fluorescence intensity from the second bilayer to the overall intensity, as shown in the seventh column in Table 2. The eighth column in Table 2 shows the final recovery fraction experimentally measured from FRAP as shown in Figure 4. The trend and values of the estimated final recovery fraction based on the mobile second layer hypothesis are consistent with the experimentally measured final recovery fraction, supporting that the measured mobility comes from the second bilayer. Role of Surface Structure on the Formation of SLBs through Vesicle Deposition. We observed that a mobile layer, which was possibly the second layer of SLBs, formed on the grating surface but not on the flat surface, and that the mobile layer amount increased with the edge density. These observations suggested that the grating edge can enhance the formation of SLBs by lipid vesicle deposition, probably because the edge can induce substantial local deformation of a lipid vesicle. This situation is similar to that in which a needle is used to induce a substantial local deformation of a balloon surface to burst the balloon. The observation seems to be inconsistent with the previous study suggesting that a high surface roughness may impede the formation of SLBs.45 The differences in observations may be because spreading an SLB on a highly rough surface requires bending the lipid membranes to allow them to follow the contour of the support surface. The bending energy penalty can render the spreading energetically unfavorable. In this study, the bending region of the grating edges is relatively small compared to a highly rough surface with sharp features. The surfaces with controllable bending region and edge angle can help us to ensure that the energy penalty from the lipid bilayer bending does not exceed the energy reward from the SLB formation and therefore allow the formation of continuous and high quality SLBs, while the highly rough surface with uncontrollable sharp features may hinder the formation of SLBs. We opine that the successful formation of SLBs through vesicle deposition has two requirements. The first is that the formation process should be thermodynamically favorable; in other words, the SLB state after vesicle deposition should have a lower energy than the free vesicle state. Whether spreading an SLB on the designated support is thermodynamically favorable could be estimated by using the extended DLVO theory as shown in this and some other studies.35,36 The second is that the lipid vesicles need to rupture in a reasonable time scale. The results in this study suggest that the existence of grating edges on the surface can be used to enhance rupture kinetics.
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CONCLUSION The capability to form mobile SLBs on a gold surface can facilitate the biodetection of cell-membrane-related events. To form a mobile second SLB on the gold surface, we constructed a surface grating structure to create additional rupture sites for lipid vesicles. We showed that the formation of a second SLB was thermodynamically favorable by using the extended DLVO theory to describe the interaction between lipid membranes and their supports. We prepared supports with several grating edge densities to vary the possible rupture-inducing sites. The anisotropic fluorescence intensity recovery parallel to the grating structure suggested that the mobile second layer partially covered the surface and extended parallel to the grating structure. In addition, the consistency between the relative edge density and the relative predicted second bilayer amount and the consistency between the experimentally measured fluorescence recovery fraction and the estimated recovery fraction indicated that the second layer was located in the edge region. All of these results showed that employing a grating geometry can facilitate the formation of a mobile second SLB on a gold surface. The capability to incorporate lipid bilayers on a gold surface without losing their fluidity indicates that the platform has the potential to be incorporated with surface analytical tools for studying biomolecular interactions.
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ASSOCIATED CONTENT
S Supporting Information *
Supporting figures, and data analyses. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank National Taiwan University and the Ministry of Science and Technology, Taiwan, for providing financial support for this study (NTU-CDP-104R7841, and NSC 1022221-E-002-153-MY3).
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ABBREVIATIONS DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; SLB, supported lipid bilayer REFERENCES
(1) Salafsky, J.; Groves, J. T.; Boxer, S. G. Architecture and function of membrane proteins in planar-supported bilayers: A study with photosynthetic reaction centers. Biochemistry 1996, 35 (47), 14773− 14781. 3909
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DOI: 10.1021/la504532a Langmuir 2015, 31, 3904−3911
