M.L. Walker, J.T. Woodward (831), C.W. Meuse (831), and A.L. Plant (831)

Objective: To develop an approach for topographical and phase contrast imaging of self-assembled monolayers and hybrid bilayer membranes, with minimal perturbation to the studied system.

Problem: Self-assembled alkane thiol monolayers are a powerful means of surface modification, resulting in a surface with properties that can be far different than those of the supporting substrate. Lipid layers have numerous potential uses as biomimetic surfaces, allowing the study of structure-function relationships of transmembrane proteins in environments biologically similar to those found in cells. Lipid layers assembled on self-assembled monolayers offer an extremely versatile system with far-reaching implications for biological studies, biocatalysis, and sensor development, but the relationship between surface structure and bioactivity must be well-characterized to exploit the usefulness of this hybrid construct.

Approach: Various diagnostic tools, such as surface plasmon resonance, neutron reflectivity, and infrared spectroscopy have been used to understand the interactions between the layers of the hybrid bilayer membrane system, but little is known about the surface of the membrane and how specific features or properties will affect surface chemistry. Non-contact atomic force microscopy (AFM) is being used to explore the surfaces of the membrane. This scanned-probe technique exerts much less force on the sample than the more commonly used contact AFM, resulting in less structural perturbation to the sample. In addition, employing the non-contact mode simultaneously allows the use of phase contrast imaging, which can detect regions of varying composition, adhesion, and other properties.

Results and Future Plans: We have shown that non-contact AFM can successfully image delicate structures easily altered by the force exerted in contact AFM. This capability was demonstrated when one method of substrate preparation led to the formation of unexpected, non-uniform assemblies of the alkane thiol used. These assemblies (or multilayers), clearly and easily detectable with non-contact AFM, were formed regardless of whether octadecanethiol, hexadecanethiol, or dodecanethiol were used, and proved to be insoluble in ethanol, hexane, and even hexadecane after their formation. The images of these complexes, using non-contact AFM, are in sharp contrast to the images (suggestive of a uniform monolayer), seen with contact AFM. The demonstrated capability of imaging fragile samples using non-contact AFM is a clear advance in the scanned-probe program of the Division.

Imaging of hybrid bilayers with non-contact AFM has been accomplished in air, with some determinations made about their structure. The main research effort now is devoted to generating a longer-chain SAM in a grid-shape pattern on the substrate surface by microcontact printing, then backfilling with a shorter-chained SAM. Topographical and phase contrast imaging will be used to confirm the generation of this pattern. A lipid layer will then be overlaid on this pattern of taller and shorter SAMs, in an attempt to create a lipid bilayer over the shorter SAM, resulting in "wells" in which proteins can be immobilized. The covering of the gold substrate by the organic self-assembled monolayer, combined with the lipid bilayer, should simulate a more natural "cell-like" environment and thus promote the study of membrane proteins in their native configurations. These experiments will be done in an AFM fluid cell, providing straightforward characterization of the lipid bilayer and any species situated in the wells under hydrated conditions.