EC-STM observation on electrochemical response of fluidic phospholipid monolayer on Au(1 1 1) modified with 1-octanethiol

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Abstract

Electrochemical scanning tunneling microscopy (EC-STM) was applied to observe phospholipid layers over thiol-modified gold substrates as a model biological cell membrane. On a monolayer of 1-octanethiol on Au (1 1 1), a synthetic lipid, 1,2-dihexanoyl-sn-glycero-3-phosphocholine, was introduced in a neutral 0.05 M NH4ClO4 buffer solution. The lipid molecules formed a fluidic layer at 0.0 V vs. RHE of the substrate electrode potential. By cycling the electrode potential between +0.2 V and −0.2 V, the lipid layer reversibly changed over between the fluidic phase and a striped/grainy structure. This structural change might involve partial decomposition and oligomerization of phospholipids. This method will contribute for molecular biology by revealing the nanometer-scale structure of cell membrane.

Introduction

Cell membranes are biological interfaces of prime importance, consisting of lipid bilayers. The lipid molecules move within the membrane, forming a 2-dimensional fluid, providing important biological functions. For example, “lipid raft structure”, which is popular nanostructure on the cell membrane [1], [2], [3], is composed by the 2D phase separation on the cell membrane [4]. Fluidic motion is a prerequisite for the phase separation. It is a challenging task to apply scanning tunneling microscopy (STM) to observe such biological objects in the nanometer-scale. Electrochemical scanning tunneling microscopy (EC-STM) is a tool suitable for observing organic thin films on metal surfaces immersed in a solution in general [5]. Application of EC-STM will widen the scope of nanometer-scale phenomena on lipid membranes in specifying the physical phases and detecting phase separation [6], [7].

We composed a model cell membrane to realize STM observation. STM specimens need to be physically flat and rigid, and the 2D fluidity of lipid bilayer must be introduced into the model. Our model cell membrane consists of two layers. The lower leaflet is a hydrophobic 1-octanethiol (1-C8H17SH) self-assembled monolayer (SAM) on Au (1 1 1) surface. The upper leaflet is a DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) lipid layer on the lower layer. This layer was formed on the SAM by hydrophobic interaction. This sort of model has been adopted for spectroscopic [8], [9] and voltammetric research [10], [11]. So far no STM observation has been reported on a moving lipid layer in the real space. In this work, we prepared a lipid layer in aqueous electrolytic solution and observed it in the real space. We also tried to reveal the effects of the electrode potential applied on the structure of lipid layer.

Section snippets

Experimental

The materials and chemicals used in this work were as follows: Au wire (1 mm ∅, 99.999%: Furuya Metals, Japan), Wires of W (0.25 mm ∅), Pt wire (0.25 mm ∅) and Pt plate (0.1 mm t) from Nilaco (Japan), aqueous ultrapure NH3 and HClO4, 1-octanethiol, hexane and ethanol from Kanto Chemicals (Japan). As for the phospholipid, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) was purchased from Avanti Polar Lipids, Inc. (USA).

All the in situ EC-STM images were obtained using Nanoscope E (Veeco Instruments

Results and discussion

In situ STM image of 1-octanethiol SAM on gold recorded in the aqueous solution is shown in Fig. 1. As have been noted frequently on SAM on gold [14], [15], a number of holes are observed. These holes are called gold vacancy islands (VI’s). The depth of holes are always 0.25 ± 0.01 nm, which corresponds to Au(1 1 1) monatomic height. VI’s are considered to be formed by ejection of excessive Au atoms condensed in the Au(1 1 1) herringbone structure during the relaxation process by adsorption of thiol.

Conclusion

A flat model cell membrane suitable for STM observation was composed of a hydrophobic 1-octanethiol SAM on Au (1 1 1) immersed in an aqueous solution containing DHPC molecules. We observed an ultra-thin fluidic lipid layer forming a 2-dimensional liquid on the SAM substrate under electrode potential control. The 1-C8H17S groups anchored on Au participated as one hydrophobic side of lipid bilayer, and the DHPC molecules acted as the other side of bilayer largely inclined towards the substrate,

Acknowledgements

This work was supported in part by RIKEN President Discretionary Fund (2004–2006) and Grants-in-Aid for Scientific Research on Promotion of Novel Interdisciplinary Fields Based on Nanotechnology and Materials from Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Prof. Mischa Bonn (FOM and Linden University, the Netherlands) for valuable discussions.

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