Article pubs.acs.org/Langmuir
Mechanism of Lipid Vesicles Spreading and Bilayer Formation on a Au(111) Surface Jan Pawłowski, Joanna Juhaniewicz, Alişan Güzeloğlu, and Sławomir Sęk* Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Ż wirki i Wigury 101, 02-089 Warsaw, Poland S Supporting Information *
ABSTRACT: Spreading of small unilamellar vesicles on solid surfaces is one of the most common ways to obtain supported lipid bilayers. Although the method has been used successfully for many years, the details of this process are still the subject of intense debate. Particularly controversial is the mechanism of bilayer formation on metallic surfaces like gold. In this work, we have employed scanning probe microscopy techniques to evaluate the details of lipid vesicles spreading and formation of the lipid bilayer on a Au(111) surface in a phosphate-buffered saline solution. Nanoscale imaging revealed that the mechanism of this process differs significantly from that usually assumed for hydrophilic surfaces such as mica, glass, and silicon oxide. Formation of the bilayer on gold involves several steps. Initially, the vesicles accumulate on a gold surface and release lipid molecules that adsorb on a Au(111) surface, giving rise to the appearance of highly ordered stripelike domains. The latter serve as a template for the buildup of a hemimicellar film, which contributes to the increased hydrophilicity of the external surface and facilitates further adsorption and rupture of the vesicles. As a result, the bilayer is spread over a hemimicellar film, and then it is followed by slow fusion between coupled layers leading to formation of a single bilayer supported on a gold surface. We believe that the results presented in this work may provide some new insights into the area of research related to supported lipid bilayers.
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INTRODUCTION Supported lipid bilayers are often considered as simplified models mimicking the architecture of biological membranes.1,2 Like those of the natural systems, their properties are determined by lipid composition, the size and shape of the molecules, the chemical nature of polar headgroups, and the presence of unsaturated bonds in hydrocarbon chains. However, when supported lipid bilayers are considered, there is one more crucial factor affecting their structure and the properties, which is the surface of the substrate. Most commonly used supports are hydrophilic surfaces such as glass, quartz, silicon, and mica.3−6 However, metal surfaces offer certain benefits, because the structure of the supported film as well as its properties can be examined under electrochemical control, i.e., in the presence of an external electric field.7,8 This enables investigation of voltage-dependent membrane processes. Moreover, immobilization of lipid films at a metal surface is advantageous when considering their potential application for the construction of biomimetic interfaces and the development of biosensors with electrochemical signal transduction.2,9,10 A noble metal, in particular gold, is also required for surface plasmon resonance (SPR) studies. Usually, supported lipid bilayers are prepared either by the transfer of the monolayers from the air−water interface or by spreading of small unilamellar vesicles (SUVs).1,2,11,12 The second method is based on a self-assembly concept. According to a widely accepted model, the mechanism of bilayer © XXXX American Chemical Society
formation by SUVs spreading on hydrophilic substrates involves adsorption of intact vesicles and their spontaneous rupture on a solid surface.13,14 The rupture events can be driven by several different factors. These include substrate-induced deformation of vesicles, fusion between neighboring vesicles, the high surface density of vesicles, and the presence of active edges of already formed bilayer patches. Numerous experimental observations confirmed the mechanism of bilayer formation on mica, silicon, and glass.15−17 However, in the presence of metallic surfaces, significant differences can be expected. Successful formation of lipid bilayers supported on gold using vesicle spreading was demonstrated by the Lipkowski group.18,19 They used electrochemical methods and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) to study the spreading of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles on a Au(111) electrode surface. It was demonstrated that at charge densities between −15 and 0 μC/cm2, lipid molecules form a condensed film. At more negative charges, the film was desorbed from the electrode surface but lipid molecules remained close to the surface in an ad-vesicle state, while at more positive charge densities, reorientation of the molecules was observed followed by a desorption. Later, these Received: April 12, 2015 Revised: May 20, 2015
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DOI: 10.1021/acs.langmuir.5b01331 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir results were complemented by atomic force microscopy (AFM) measurements, which showed that indeed an ∼4.3 nm thick DMPC bilayer is formed on gold at the open circuit potential.20 Moreover, potential-induced changes in film thickness were examined, demonstrating that the tilt angles of the molecules vary in response to the potential, which was in line with previous PMIRRAS results. Quite similar conclusions were drawn for a two-component system consisting of DMPC and cholesterol in a 7:3 molar ratio.21 Interestingly, the studies of DMPC/cholesterol vesicles spreading on Au(111) with use of electrochemical scanning tunneling microscopy (EC-STM) revealed that at the early stages of film formation the molecules adsorb on the surface with a flat-lying orientation and separate into domains enriched with either DMPC or cholesterol.22 Such an observation suggests that the mechanism of lipid vesicles spreading on Au(111) has to be stepwise and distinctly different from that observed on mica or other hydrophilic substrates. Although several other groups reported successful formation of a lipid bilayer on gold by fusion and spreading of vesicles, the process is still poorly understood and the literature lacks the details concerning the intermediate stages occurring between adsorption of flat-lying molecules and formation of the compact bilayer. The aim of this work is to provide a detailed description of the mechanism of lipid vesicles spreading on a Au(111) surface. We have employed STM and AFM techniques to follow the dynamics of the formation of a mixed bilayer composed of DMPC and cholesterol in a 7:3 molar ratio. This particular model system has been chosen because its structure and electric field-induced transformations are well-characterized using numerous techniques such as PMIRRAS, AFM, and neutron reflectivity. To follow the changes in thickness during the formation of the lipid film, we have also utilized force spectroscopy measurements. Thus, the results presented in this work demonstrate how the topography and thickness of the lipid film change as a function of time. On this basis, we have reached conclusions about the structure of the lipid film at various stages of bilayer formation and proposed a detailed mechanism of SUV spreading on a Au(111) surface.
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Scheme 1. Structures of DMPC and Cholesterol
with a mean effective size in the range of 30−50 nm. An exemplary distribution of SUVs size is shown in Figure 3SI of the Supporting Information. For imaging experiments, the suspension of the lipid vesicles was added to the electrochemical cell in such an amount that the total concentration of lipids was 3.0 × 10−5 M. A low concentration of lipids was preferable to slow the kinetics of film formation and allowed imaging of intermediate structures. STM and AFM images were obtained with 5500AFM (Keysight Technology) and Dimension Icon (Bruker) instruments. The measurements were taken at 21 °C. All images were recorded in a buffer solution [0.01 M PBS (pH 7.4)] under electrochemical control. A single-crystal Au(111) electrode (MaTeck) was used as a working electrode, and a miniaturized Ag/AgCl (saturated KCl) electrode and a platinum wire served as reference and counter electrodes, respectively. The exemplary image of bare gold is shown in the Supporting Information. The electrochemical cell as well as gold and platinum electrodes was cleaned in piranha solution [3:1 (v/v) concentrated H2SO4/30% H2O2] for at least 1 h and rinsed with thoroughly Milli-Q ultrapure water. A single-crystal Au(111) electrode was flame annealed prior to assembly of the electrochemical cell. STM imaging was conducted with electrochemically etched tungsten tips coated with polyethylene to minimize leakage currents. The AFM images were taken either in MAC mode using type VII MAC cantilevers (Keysight Technology, nominal spring constant of 0.14 N/m) or in soft tapping mode using PPP-BSI cantilevers (Nanosensors, nominal spring constant of 0.1 N/ m).24 The imaging was performed on five independently prepared samples. The AFM images shown in this work represent a time-lapse sequence recorded in a single experiment, but they are representative for the data collected from all samples being studied. The thickness of the lipid films was determined by force spectroscopy measurements taken at different spots on each sample, and ∼20 force−distance curves were collected from a single sample. Quartz crystal microbalance measurements were taken using a model 400B time-resolved electrochemical quartz crystal microbalance (CH Instruments). The working resonators were commercially available 8 MHz gold-coated AT-cut quartz crystals with an area of 0.196 cm2. The frequency change of 1 Hz corresponded to a mass increase of 6.90 ng on the gold electrode. Prior to the measurements, the QCM cell was sonicated in acetone for 10 min and rinsed with Mili-Q water. The gold QCM surface was first cleaned in piranha solution for 2 min. Afterward, the crystals were rinsed with an abundant amount of water and absolute ethanol, then sonicated in acetone for 10 min, and rinsed with ethanol and Mili-Q water. The crystals were mounted in a QCM cell and stabilized in PBS buffer (3 mL, pH 7.4) until the frequency shift was
