Fabrication of Au-Nanoparticle-Embedded Lipid Bilayer Membranes

Apr 17, 2017 - embedded supported lipid bilayers (SLBs) by two methods. ... Key techniques for the successful vesicle-membrane fusion are hydrophobiza...
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Fabrication of Au-Nanoparticle-Embedded Lipid Bilayer Membranes Supported on Solid Substrates Naotoshi Sakaguchi,† Yasuo Kimura,‡ Ayumi Hirano-Iwata,§ and Toshio Ogino*,† †

Yokohama National University, 79-1, Tokiwadai, Hodogaya, Yokohama 240-8501, Japan Tokyo University of Technology, 1404-1, Katakura, Hachioji, Tokyo 192-0982, Japan § Tohoku University, 6-6, Aramaki, Aoba, Sendai, Miyagi 980-8578, Japan ‡

S Supporting Information *

ABSTRACT: We fabricated gold nanoparticle (Au-NP)embedded supported lipid bilayers (SLBs) by two methods. In the vesicle−vesicle fusion method, vesicles with hydrophobized Au-NPs are ruptured and fused on SiO2/Si substrates. In the vesicle-membrane fusion method, SLBs without Au-NPs were preformed on the substrate and then vesicles with Au-NPs were fused into the preformed membranes. In the former method, Au-NP incorporation into the SLBs was observed as an increase in the membrane thickness in atomic force microscopy (AFM) images and directly observed by transmission electron microscopy. In the latter method, fusion of vesicles into the preformed membranes was confirmed by the fluorescent color change in the preformed membranes, and Au-NP incorporation was also confirmed by an increase in the membrane thickness in the AFM images. Key techniques for the successful vesicle-membrane fusion are hydrophobization of Au-NPs, approach control of vesicles by mixing the charged lipids, and destabilization of the lipid bilayers by adding lipids with a small polar headgroup. bilayer, lipid molecules are mobile and SLBs are fluidic on the solid substrates.18 SLBs are usually fabricated on the substrates using vesicle fusion,14,19 self-spreading,20 and Langmuir− Blodgett methods.21 Recently, new biosensors22,23 consisting of SLBs and membrane proteins buried into the SLBs have been reported to investigate drug side effects. For device applications of the lipid bilayers with embedded metalnanoparticle, SLBs are much more useful because the conventional device fabrication process developed in the semiconductor technology can be combined with these metal-lipid biosystems. Therefore, the SLBs with the embedded metal nanoparticles will be a novel platform for flexible devices that are operated in aqueous environment. The applications of these platforms are, for example, single-electron transistors (SETs) that has circuit-reconstruction ability through fluidity of the SLBs, plasmonic devices that has fluidic metal nanoparticle arrays, and biotransistors that has biosolid hybridized functions realized by the combination of artificial cell membranes as electrically insulating layers and conductive metal islands. In this article, we report on fabrication of SLBs with embedded Au-NPs. Utilizing the spontaneous incorporation mechanism of Au-NPs into lipid bilayers, we fabricated the SLBs with Au-NPs by two processes: one is rupture and fusion of the Au-incorporated vesicles, and the other is fusion of the

1. INTRODUCTION Lipid bilayers are fluidic biomembranes that package cells, organelle, and exosomes, and their main roles are separation of their interior from the outside and support of membrane proteins,1,2 which transport materials and signals via cell membranes. Recently, applications of artificial cell membranes to biosensors have attracted much interest because the artificial cell membranes are indispensable components as a basic platform for in vitro biosensing and screening devices. Recently, nanocomposites consisting of lipid bilayers and inorganic nanomaterials, such as metal nanoparticles3−5 and nanocarbon,6−8 have been proposed as new functional materials toward electronic devices where lipid bilayers are used as electrically insulating substrates that confine the nanomaterials. In the previous studies, gold nanoparticles (Au-NPs) were modified with self-assembled alkanethiol monolayers to exhibit hydrophobicity and spontaneously incorporated into lipid bilayer vesicles.3,9−13 It was demonstrated that dense, uniformly distributed Au-NPs are embedded between the opposing hydrophobic tails of lipid molecule layers. These results suggest that metal-nanoparticle arrays can be self-assembled in the lipid bilayer vesicles and that the individual nanoparticles can be separated with a uniform gap distance. These nanocomposites may potentially be applied to single-electron devices and plasmonic devices. Supported lipid bilayers (SLBs) are lipid membranes formed on solid substrates with ordinarily planar surfaces, and widely used as biocompatible membranes for biodevices.14−17 Because there is a thin water layer between the substrate and the lipid © XXXX American Chemical Society

Received: January 17, 2017 Revised: April 1, 2017 Published: April 17, 2017 A

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2. MATERIALS AND METHODS 2.1. Materials. Molecular structures of lipids used in this study are shown in Figure S1. DPhPC (1,2-diphytanoyl-snglycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3phosphoethanolamine), DOEPC (1,2-dioleoyl-sn-glycero-3ethylphosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), NBD-PC (1-Myristoyl-2-[12-[(7-nitro-2−1,3benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine), and Rhod-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[lissamine rhodamine B sulfonyl]) were purchased from Avanti Polar Lipids (Alabastar, AL). Chol (Cholesterol) was purchased from Sigma-Aldrich. Aqueous suspensions of Au-NPs of 10 mM (in particle number) dispersed by modification of their surfaces with polyethylenimine were purchased from Wako Pure Chemical Industries, Ltd., Japan. The Au-NP size distribution data supplied from the supplier are shown in Figure S2. The ligand density on the Au-NP surfaces is unknown. 1-Dodecanethiol was purchased from Nacalai Tesque, Inc. In the vesicle−vesicle fusion experiments described later, a buffer solution was a mixture of N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid solution (HEPES, 10 mM) and CaCl2 (20 mM), and its pH was regulated to be 7.4 by adding an appropriate amount of NaOH. In the vesicle−membrane fusion described later, pH of the buffer solution was regulated to be 7.0 by adding an appropriate amount of NaOH. The chemicals for the buffer solutions were purchased from Nacalai Tesque, Inc. For fabrication of SLBs, SiO2/Si substrates with SiO2 thickness of 300 nm were used. 2.2. Preparation of Lipid Bilayers with Embedded AuNPs. The whole processes to prepare SLBs with embedded AuNPs are shown in Figure 1. Hydrophobic Au-NPs were prepared by the following procedure. One milliliter of chloroform and 2 μL 1-dodecanethiol were mixed in a glass vial, and the as-purchased polyethylenimine-modified Au-NP suspension of 1 mL was added to the solution. The mixture was shaken by hand for 2 min, and vortexed at least for 12 h. After the vortexing, we observed that Au-NPs were precipitated at the bottom of the glass vial, 24 indicating that the polyethylenimine molecules on the Au-NP surfaces were substituted for hydrophobic dodecanethiol ones. The prepared hydrophobic Au-NPs were resuspended in chloroform, which we refer to as hydrophobic Au-NP suspension. To prepare SLBs with embedded Au-NPs, we used two methods, as shown in Figure 1. In the first method, vesicles with embedded Au-NPs are fabricated and raptured on the substrate surfaces, and the formed small lipid bilayer islands are fused to form large membranes. We refer to this method as the vesicle−vesicle fusion. In the second method, Au-NP-free lipid bilayers were preformed by the normal vesicle fusion method and then the vesicles with embedded Au-NPs were fused onto the preformed lipid bilayers. We refer to the second method as the vesicle-membrane fusion. In both methods, SiO2/Si substrates were immersed in a mixture of H2SO4 (98%) and H2O2 (33%) in a volume ratio of 3:1 at 90 °C for 10 min and sonicated in deionized water for 5 min21 to obtain clean surfaces. In the vesicle−vesicle fusion, a mixture of DPhPC and RhodPE (1% in mole ratio) was dissolved in chloroform, where the lipid amount was regulated to be 10 mg/mL. When incorporating Au-NPs, the hydrophobic Au-NP suspension

Figure 1. Experimental procedure of this work: (a) goal of this study, (b) preparation procedure of vesicles with incorporated Au-NPs, (c) subsequent SLB formation by the vesicle−vesicle fusion, and (d) the vesicle-membrane fusion.

was added to the lipid. Lipid vesicles were prepared from lipid thin films that were fabricated by dissolving the lipids in 20 μL chloroform in a glass vial and evaporating the chloroform in a vacuum for 12 h. Amount of the Au-NPs was regulated by the following procedure. Concentration of the hydrophobic Au-NP suspension was decreased to 2.5 mM from that of the aspurchased aqueous suspension, 10 mM, after the abovementioned hydrophobization process. We mixed 1 μL of the Au-NP suspension (2.5 mM) and 20 μL of the lipid one (10 mg/mL). Considering the lipid molecular weight (DPhPC: 734), we can roughly estimate that one gold nanoparticle occupies 100 lipid molecules if the whole Au-NPs are uniformly incorporated into the bilayers. To prepare multilamellar vesicles, a lipid thin film of 20 mg was mixed with 2 mL buffer solution, heated in a water-bath above 50 °C, shaken for 5 min, and finally vortexed for 1 h. To obtain large unilamellar vesicles from the multilamellar vesicles, the multilamellar vesicle suspension was frozen in liquid-nitrogen and thawed in a 50 °C water bath, and this cycle was repeated 3 times.17 The obtained large unilamellar vesicle suspension was sonicated in a water bath above 50 °C to produce small unilamellar vesicles.18 To form SLBs, the vesicle suspension and a HEPES/CaCl2/ B

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Figure 2. AFM images of SLBs fabricated using DPhPC lipid when (a) no Au-NPs, (b) polyethylenimine-modified (hydrophilic) Au-NPs, and (c) dodecanethiol-modified (hydrophobic) Au-NPs were added, respectively.

observed by TEM with 200 kV. Incorporation of Au-NPs into SLBs was confirmed by electron probe microanalysis (EPMA) by detecting Au-Lα specific X-ray.

NaOH buffer solution of pH 7.4 were mixed, and dropped onto a SiO2/Si substrate at room temperature (24 ± 1 °C). After 1 h, the excess vesicles were removed by replacing the vesicle suspension with the fresh buffer solutions. In the vesicle-membrane fusion, a mixture of DPhPC, DOPE, DOEPC (positively charged), and Chol in a mole ratio of 5:2:1:2 was dissolved in chloroform, and 1%-DOPC modified with NBD-fluorescent dye was added. For preformation of large area SLBs without Au-NPs, giant unilamellar vesicles (GUVs) were prepared by the electroformation method in a 1 mM sorbitol buffer solution. The GUV suspension was mixed with the HEPES/NaOH buffer solution of pH 7.0 and dropped onto a SiO2/Si substrate at room temperature (24 ± 1 °C) to preform the Au-NP-free membrane on the substrate. After 1 h, the excess vesicles were removed by replacing the vesicle suspension with the fresh buffer solutions. To incorporate AuNPs onto the preformed Au-NP-free lipid bilayers, we used a mixture of DPhPC, DOPE, DOPS (negatively charged), and Chol in a mole ratio of 5:2:1:2 or 4:2:2:2. The lipids were mixed with hydrophobic Au-NPs and 1%-DOPE modified with Rhod-fluorescent dye, and dropped onto the SiO2/Si substrate. After 1 h, the excess vesicles were removed by replacing it with the fresh buffer solutions. 2.3. Characterization. We investigated Au-NP incorporation into the SLBs by atomic force microscopy (AFM, Hitachi-High-Tech Science, E-sweep) at room temperature using the cyclic-contact mode. The samples were immersed in a HEPES/CaCl2/NaOH buffer solution with ionic strength of about 2.5 × 10−2 (mol/L), in a standard fluid cell and Si cantilevers with a spring constant of 1.6 (N/m) were used. The scan rate was 0.5 Hz and the images shown in this study were processed by first order flattening. We also used transmission electron microscopy (TEM) to confirm the incorporation of Au-NPs into the lipid bilayers. The samples for TEM observation were prepared by the freeze-dry method,25 which is often used to characterize nanoscaled biological samples.26,27 An Au-NP-embedded vesicle suspension was dropped onto a hydrophilic TEM grid covered with Formvar films, and a 2% osmium tetroxide solution of 100 μL was dropped to the TEM grid at room temperature. After 1.5 h, the osmium tetroxide solution was removed by the pure water and ethanol. Then, the ethanol solution was completely replaced with t-butyl alcohol. The freeze-dried SLBs were vacuumed, and the samples were

3. RESULTS 3.1. Vesicle−Vesicle Fusion. Figure 2a shows an AFM image of SLBs on a SiO2/Si substrate fabricated by the vesicle− vesicle fusion from DPhPC lipid without Au-NPs. The observed membrane islands have planar surfaces and their height is about 3 nm, indicating that normal SLBs formed. Figure 2b shows an AFM image of SLBs fabricated by the same method as those in panel a when polyethylenimine-modified (hydrophilic) Au-NPs were added to the lipid. Height of the membranes in Figure 2b is 2.3 nm, but the SiO2 surface is rougher than that in Figure 2a. Figure 2c shows AFM image of SLBs fabricated by the same lipid as that used in panel a when dodecanethiol-modified (hydrophobic) Au-NPs were added. Height of the membranes in Figure 2a is about 3 nm, whereas that of the membranes in the lower area of Figure 2c is about 5 nm. From the other samples, we have confirmed that the heights of the SLBs are always divided into two groups, about 3 nm and about 5 nm, when Au-NPs are added to the lipid. We also show a bar graph of the heights measured for the Figure 2c image in Figure S3. When the polyethylenimine-modified AuNPs were added, the SLB thickness is almost the same as that without addition of the Au-NPs, indicating that the Au-NPs were not incorporated into the SLBs. The slightly smaller height in Figure 2b than that in Figure 2a may occur probably because the SLBs in Figure 2b coexist with the Au-NPs excluded from the lipid bilayer and because dense bilayer formation was inhibited. In the top area in Figure 2c, we can observed SLBs with height of about 3 nm, but we did not observe any SLB with both heights in one SLB island nor that with an intermediate height. When the dodecanethiol-modified Au-NPs were added, the formed SLBs were divided into two groups according to their height. It was reported that lipid vesicles with embedded Au-NPs form domains with dense AuNPs and those without any Au-NPs. We can conclude that the SLBs in the top area in Figure 2c and those in the bottom area in Figure 2c correspond to SLBs without and with the Au-NPs. Because the stable position of the hydrophobic Au-NPs in lipid bilayers is the interface between the hydrophobic tails opposing C

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Figure 3. Agglomeration of gold nanoparticles: (a) AFM image of partially double-layered SLBs and (b) illustration of their cross-section.

each other, it is natural that the Au-NPs are embedded in the SLBs, as shown in Figure 1a. The flat surfaces of Au-embedded SLBs indicate that Au-NPs are densely, uniformly distributed in the Au-NP-embedded domains in the SLBs. Occasionally, we observed multilayered SLBs and specific morphology when the hydrophobic Au-NPs were incorporated. Figure 3a shows an AFM image of SLBs that include doublelayered SLBs in the center area, where heights of the areas A and B with respect to the substrate surface are 5 and 8 nm, respectively. Therefore, the area A corresponds to an Au-NPembedded SLB, and the area B to a double lipid bilayers consisting of an Au-NP-embedded SLB and a stacked Au-NPfree SLB. On the area B, spherical and elongated islands marked by C are observed, and their heights are 5−10 nm with respect to the surface of the area A. Because these islands are as high as that of the Au-NP-embedded SLBs, they are attributed to agglomeration of the Au nanoparticles, as shown in Figure 3b. The agglomerated Au-NPs are also one of the stable states of the hydrophobic Au-NPs. The agglomerated Au-NPs formed only in the upper layers of double-layered SLBs, in which the substrate for the upper lipid bilayer is the lower SLBs if the upper bilayer is considered as an independent layer. This suggests that Au-NP incorporation fashion, two-dimensional sheets or three-dimensional clusters, depends on whether the substrate for the SLBs has solid surfaces like SiO2 or soft ones like a lipid bilayer. When double bilayers were observed, the surfaces of the membranes were always very rough, as shown in Figure 3a, whereas surfaces of the lipid bilayer islands observed in Figure 2c are flat. The flatness of the bilayer islands could support that the height difference in the bilayer islands in Figure 2c is not attributed to double bilayer formation. Figure 4a,b shows TEM images of SLBs embedding Au-NPs with diameters of (a) about 2 nm and (b) 3−4 nm, the latter of which happened to be mixed in the nominally 2 nm Au-NPs. These values were estimated from the TEM images. In Figure 4a, we can observe two-dimensionally dispersed nanodots with uniform distribution, as already suggested by the AFM image shown in Figure 2c. In Figure 4b, nanodots with 3−4 nm diameter are agglomerated. Figure 4c shows a specific X-ray spectrum from the SLBs embedding Au-NPs with diameters of about 2 nm in the squared area displayed in the inset of Figure 4c. Because an Au-Lα X-ray emission is clearly observed from the nanodot area, we can confirm that Au-NPs are incorporated into SLBs. These results show that Au-NPs as small as about 2 nm can be embedded in SLBs, and Au-NPs as large as about 3 nm cannot be uniformly embedded within the lipid bilayers. In the previous studies using vesicles, Au-NPs larger than 4 nm in diameter, for example, cannot be loaded into lipid bilayers.28,29

Figure 4. TEM images of dodecanethiol-modified (hydrophobic) AuNPs embedded in SLBs: Au-NPs with diameters of (a) about 2 nm and (b) 3−4 nm. (c) An EPMA spectrum from the area indicated by the square in the inset.

3.2. Vesicle-Membrane Fusion. Figure 5 shows AFM images after the vesicle-membrane fusion. In this sample, composition of the preformed SLBs was DPhPC (50%), DOPE, (20%), DOEPC (10%, positively charged), and Chol (20%) in a mole ratio. The vesicles to be fused had a composition of DPhPC (50% or 40%), DOPE, (20%), DOPS (10% or 20% respectively, negatively charged), and Chol (20%), and dodecanethiol-modified (hydrophobic) Au-NPs were added to the mixed lipid with a ratio of one gold nanoparticle for 100 lipid molecules. In Figure 5a, thickness of the preformed SLBs was 2.6 nm, which is slightly smaller than that in Figure 2a owing to electrostatic attractive force between the positively charged membrane and the negatively charged SiO2/Si surface.30 After the vesicle-membrane fusion, the height increased to 5.3 nm in the lower area in Figure 5b. As indicated by the results of the vesicle−vesicle fusion, the Au-NPembedded domains and the Au-NP-free domains are separated, and the Au-NPs are uniformly embedded into the SLBs in the D

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observed, indicating that more vesicles were incorporated into the preformed membrane. The increase in the preformed membrane thickness and the larger color change caused by the increase in the electrostatic attractive force support occurrence of the vesicle-membrane fusion.

4. DISCUSSION 4.1. Vesicle−Vesicle Fusion. From the experimental results of AFM and TEM observations, we can conclude that Au-NPs as small as 2 nm in diameter are uniformly incorporated into lipid. Hydrophobic Au-NPs in contact with water are energetically unstable, but when embedded between the hydrophobic tails of the lipid bilayers, the excess energy should dramatically decrease. Additionally, when the incorporated Au-NPs form a two-dimensional sheet inside the lipid bilayers, the lipid bilayer surfaces formed by the hydrophilic head of lipids tend to be flat, as shown in Figure 7a. The surface roughness on the SLBs increases the contact areas between the hydrophobic tails near the heads and the water molecules at the lipid-solution interface, as shown in Figure 7b. Therefore, total free energy decreases when the Au-NPs form two-dimensional sheet, and the Au-NP density is determined by a balance between energy gain owing to the surface flatness and energy loss owing to repulsive force between the Au-NPs. These results are basically the same as the previous reports that observed arrays of metal nanoparticles in the suspended lipid vesicles.9 In the TEM images, we also observed two modes of Au-NP incorporation that depend on nanoparticles diameter, as shown in Figure 4. When the diameter of Au-NPs are approximately smaller than the thickness of the SLBs, they are uniformly incorporated into the SLBs and two-dimensional Au-NP sheets are constructed. When the diameter of Au-NPs is approximately larger than the thickness of the SLBs, Au-NPs incorporated into SLBs are agglomerated to form Au-NP clusters encapsulated with lipid molecules, which are also stable states in the systems of Au-NPs and lipid-molecules. M. R. Rashch reported that morphology of the metal nanoparticle sheets incorporated into lipid vesicles depends on the nanoparticle diameters,10 which are also the same as the present results. Therefore, lipid bilayers with embedded AuNPs can be fabricated on solid substrate surfaces, which are more widely applicable to electronic and biological devices. In Figure 2, we observed both Au-NP-free SLBs and Au-NPembedded SLBs, but few SLBs include both domains inside them. This is similar to the vesicles that were clearly separated into Au-NP-incorporated and Au-NP-free vesicles.9 Therefore, the separation into Au-NP-incorporated and Au-NP-free SLB islands in Figure 2c occurred in the vesicle preparation stage before the vesicle fusion stage. If the phase-separation is perfect as the same manner as the vesicle cases,9 the fraction of surface areas between the Au-NP-incorporated and Au-NP-free SLBs should be determined by the initial Au-NP concentration. From the comparison between AFM and TEM observations and the above discussion, we can have another suggestion that occurrence of the Au-NP incorporation can be confirmed only by the AFM characterization when the Au-NP diameter is smaller than the thickness of lipid bilayers. This suggestion is used in the discussion in the Vesicle-Membrane Fusion Section. 4.2. Vesicle-Membrane Fusion. In the vesicle-membrane fusion experiments, we observed membrane color change, as shown in Figure 6, and increase in the preformed SLB thickness, as shown in Figure 5. The color change demonstrates

Figure 5. AFM images of (a) preformed SLBs and (b) the SLBs after fusion of the Au-NP-embedded vesicles into the preformed membrane.

thicker domains. This means that Au-NP-embedded regions can be clearly distinguished only by the thickness of the SLBs. Therefore, we can conclude that Au-NPs were successfully embedded in the preformed SLBs. The larger increase in the height, from 2.6 to 5.3 nm in Figure 5, compared with Figure 2a,c, is owing to neutralization of the positive charge in the preformed membrane after fusion of the negatively charged vesicles. Figure 6 shows the vesicle-membrane fusion process observed by color change in the membrane fluorescence that

Figure 6. Color change in fluorescent images observed by optical microscope (a,c) before and (b,d) after the vesicle-membrane fusion. (a,b) Vesicles with relatively small negative charge (10% DOPE) and (c,d) vesicles with relatively large negative charge (20% DOPE) were fused into the preformed, positively charged (10% DOEPC) membrane.

is accompanied by the fusion. In Figure 6a,b, lipid composition of negatively charged DOPS in the fused vesicles was 10%. After the vesicle-membrane fusion, the observed color change was small, indicating that only small amount of the vesicles were fused into the preformed membrane. When composition of negatively charged DOPS was 20%, a larger color change was E

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Figure 7. Model for the stability of SLBs embedding hydrophobic gold nanoparticles. (a) A SLB with a closely packed Au-NP sheet and (b) a SLB with randomly distributed Au-NPs.

Figure 8. Illustration of the vesicle-membrane fusion and Au-NP incorporation into the preformed SLBs: (a) preferential approach of the vesicles to the preformed SLBs and repelling them from the bare substrate, (b) interaction between lipid-bilayers without DOPE lipid, (c) interaction between lipid-bilayers with DOPE lipid, (d) fusion of the vesicles with Au-NPs into the preformed SLB, and (e) formation of planar SLBs with embedded AuNPs owing to fluidity of the membranes.

constructed to spontaneously hide their hydrocarbon groups inside them.1 Moreover, DPhPC is a stable lipid among phospholipids because its lateral membrane tension is, in many cases, larger than the other phospholipids.39 When using only DPhPC lipid, membrane fusion is less likely to occur because hydrophobic interaction between the lipid bilayer of the preformed SLBs and that of the vesicles to be fused is weak. When a part of DPhPC lipids of the preformed membrane and the vesicles is replaced with DOPE lipids, their lateral membrane tension decreases and hydrophobic interaction between the preformed SLBs and the Au-NP-embedded vesicles increases. Therefore, increased instability of the preformed SLBs should promote the vesicle-membrane fusion. Another important factor for the successful vesiclemembrane fusion is electrostatic attractive force between the vesicles to be fused and the preformed SLBs because electrostatic interaction is one of the factors that control approach of the vesicles to the preformed membrane.40,41 In addition to this charge effect, electrostatic force between the vesicles and the SiO2/Si substrate should be considered when the preformed membranes are supported on the solid substrate. The isoelectric point of SiO2 is generally from 2 to 3 because its surface is terminated with OH group, and the surfaces of the SiO2/Si substrates are negatively charged around pH of 7. Therefore, an electrostatic repulsive force is generated between the negatively charged vesicles and the SiO2/Si substrate, and

that membrane fusion occurred, as discussed in a previous report.31 The increase in the SLB thickness indicates that AuNPs were incorporated into the preformed SLBs, as discussed in Section 4.1. Therefore, we can conclude that Au-NPs were successfully incorporated into the preexisting SLBs by the vesicle-membrane fusion. Next, we discuss factors that promote the vesicle-membrane fusion. Previously, a couple of methods were reported: (a) addition of acidic phospholipid as the lipids to be fused and calcium ion,32 (b) addition of polyethylene glycol for control of vesicle aggregation and fusion,33−35 and (c) use of lipid with a small polar headgroup to lower the threshold for the vesicle fusion.36,37 Among them, we focused on the threshold lowering for the vesicle-membrane fusion by use of the lipids that has a small polar headgroup. When the vesicles contain lipids with a small polar headgroup, the hydrophobic tails of the lipids are not fully hidden and a part of them are exposed to the solution. Then, hydrophobic interaction appears between hydrocarbon groups of the lipids and water, resulting in the threshold lowering for the fusion, as shown in Figure 8. This mechanism was applied to the present vesicle-membrane fusion to incorporate Au-NPs into preformed lipid bilayers without any heterogeneous materials as a promotor. In this study, we replaced part of DPhPC with DOPE as the lipid with a small polar headgroup37,38 both in the preformed SLBs and the vesicles to be fused. In general, lipid bilayers are F

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The Journal of Physical Chemistry B the approach of the vesicles to the bare SiO2/Si areas without the preformed SLBs should be blocked. To realize the abovementioned charge conditions, DOEPC, one of the positively charged lipids, was mixed to the preformed SLBs, and DOPS, one of the negatively charged lipids, to the Au-NP-incorporated vesicles. Figure 8 shows the model of the vesicle-membrane fusion that includes the selective approach of the vesicles owing to the electrostatic interactions and the threshold lowering owing to the enhanced instability of the membranes. In Figure 8a, the negatively charged vesicles preferentially approach the positively charged, preformed SLBs, and their adsorption to the substrate surface without the SLBs is blocked. When DOPE is not mixed to the preformed SLBs and the vesicles to be fused, hydrophobic attractive interaction is relatively weak and steric-hydration repulsive interaction is relatively large,42 as shown in Figure 8b. Therefore, membrane fusion is difficult to occur. When the hydrophobic interaction is larger than the steric-hydration repulsive interaction, the vesicles and the preformed SLBs fuse each other through the hydrophobic interaction, as shown in Figure 8c. In the whole process, the most stable sites of the Au-NPs are always between opposing hydrophobic tails of the lipid bilayers, as shown in Figure 8d. Therefore, the Au-NPs in the vesicles are spontaneously transferred to the membrane after the fusion, as shown in Figure 8e. The key techniques to fabricate Au-NP-embedded SLBs by the vesicle-membrane fusion are hydrophobizing AuNPs, vesicle approach control by addition of the charged lipids, and destabilization of the lipid bilayers by mixing lipids with a small polar headgroup.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Toshio Ogino: 0000-0002-3241-5652 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by CREST-JST and a Grant-inAid for Scientific Research (15K13361) from the Ministry of Education, Culture, Sports, Science and Technology.

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5. CONCLUSIONS We have fabricated Au-NP-embedded lipid bilayers supported on solid substrates (SLBs), which can be used in novel devices that have features of flexibility in device structures and operation in aqueous environment. Au-NPs were incorporated into lipid vesicles by hydrophobization of the Au-NP surfaces, and Au-NP incorporation into SLBs were achieved by the vesicle−vesicle fusion, where adsorption, rapture, and fusion of the vesicles are involved, or by the vesicle-membrane fusion, where vesicles with Au-NPs are fused into preformed Au-NPfree lipid bilayers. The Au-NP embedded SLBs were characterized by AFM and TEM, and it was found that AuNPs form a two-dimensional particle sheet with uniform distribution. Fabrication of Au-NP-embedded SLBs by the vesicle-membrane fusion has been achieved by combination of hydrophobization of the Au-NPs, vesicle approach control by addition of the oppositely charged lipids to the preformed lipid bilayers and the vesicles to be fused, and destabilization of the lipid bilayers by mixing lipids with a small polar headgroup. Using the vesicle-membrane fusion method, vesicles with the Au-NPs can be selectively fused into only oppositely charged bilayer areas. It would be also possible that vesicles with AuNPs is fused with partially suspended areas of the SLBs owing to the relative instability in the suspended areas. Therefore, it is versatile for fabrication of any device structure consisting of lipid bilayers and metal nanoparticles.

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Structural formula of lipids used in this study (Figure S1), Au-NP size distribution used in this experiment (Figures 2, S2), and bar graphs of the lipid bilayer islands without and with Au-NPs (Figures S2, S3) (PDF)

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DOI: 10.1021/acs.jpcb.7b00500 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jpcb.7b00500 J. Phys. Chem. B XXXX, XXX, XXX−XXX