In Situ STM and Vibrational Study of Nanometer-Scale Reorganization of a Phospholipid Monolayer Accompanied by Potential-Driven Headgroup Digestion.

In situ dynamic observation of model biological cell membranes, formed on a water/gold substrate interface, has been performed by the combination of electrochemical scanning tunneling microscopy and reflection infrared absorption vibrational spectroscopy. Monolayers of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were formed on alkanethiol-modified gold surfaces in a buffer solution, and the microscopic phase transitions driven by electrochemical potential control were observed more in detail than our previous study on the same system [Electrochem. Commun. 2007, 9, 645-650]. This time the transitions were associated with the chemistry of DHPC by the aid of vibrational spectroscopy and the utilization of deuterium-labeled DHPC molecules. A negative potential shift solidifies the fluidic lipid layers into static striped or grainy features without notable chemical reactions. The first positive potential shift over the virginal DHPC monolayer breaks DHPC into choline and the corresponding phosphatidic acid (DHPA). This is the first case of a phospholipid electrochemical reaction microscopically detected at the solid surface.

Formation of Ordered Phospholipid Monolayer on a Hydrophilically Modified Au(111) Substrate.

The molecular arrangement of phospholipid molecules was investigated on a hydrophilically modified gold surface within an aqueous solution by scanning tunneling microscopy. By suspending phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) nanoparticles in the aqueous electrolyte surrounding a hydrophilically modified gold (111) substrate with 3-mercaptopropionic acid (SH-C2H4-COOH, 3-MPA), well-ordered adlattices of POPC were observed. Traces of particle fusion were visualized before formation of the adlattice. Addition of cholesterol to the suspension seems to facilitate accommodation of POPC on this surface. The observed unit cells of POPC adlattices had dimensions of 0.5 nm × 1.9-2.5 nm. By high-resolution imaging, each unit cell was discerned to be occupied by one upright POPC molecule. The POPC + cholesterol suspension also leads to formation of a flat integrated POPC layer, which may be a lipid bilayer covering the surface.

Scanning tunneling microscope observation of the phosphatidylserine domains in the phosphatidylcholine monolayer.

A mixed monolayer of 1,2-dihexanoyl-sn-glycero-3-phospho-l-serine (DHPS) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on an 1-octanethiol-modified gold substrate was visualized on the nanometer scale using in situ scanning tunneling microscopy (STM) in aqueous solution. DHPS clusters were evident as spotty domains. STM enabled us to distinguish DHPS molecules from DHPC molecules depending on their electronic structures. The signal of the DHPS domains was abolished by neutralization with Ca(2+). The addition of the PS + Ca(2+)-binding protein of annexin V to the Ca(2+)-treated monolayer gave a number of spots corresponding to a single annexin V molecule.

Visualization of phospholipid particle fusion induced by duramycin.

We visualized nanometer-scale phospholipid particle fusion by scanning tunneling microscopy (STM) on an alkanethiol-modified gold substrate, induced by duramycin, a tetracyclic antibiotic peptide with 19 amino residues. Ultrasonic homogenization generated a suspension mainly consisting of minimal lipid particles (MLP) from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in a phosphate buffer solution, confirmed by dynamic light scattering (DLS). In situ STM discerned individual MLP as particles (diameter approximately 8 nm) spread on Au(111), modified with alkanethiol, within the suspension. The MLP became fragile by the presence of duramycin, and the MLP were easily scratched by the scanning tip into multilayers along the surface. This process of particle fusion on the gold surface coincides with the aggregation of MLP in the suspension, observed by DLS. It was demonstrated that STM is capable of discerning and monitoring the nanometer-scale features of phospholipid particles altered by antibiotics with biochemical impact. STM might allow in situ, real-space, nanometer-scale observations of minute particles composed of phospholipids within the real cells with the highest magnification ratio.