Two methods for patterning surfaces with supported lipid bilayers and immobilized protein are described. First, proteins are used to fabricate corrals for supported lipid bilayers. Poly(dimethylsiloxane) stamps are used to deposit arbitrarily shaped patterns of thin layers of immobilized protein onto glass surfaces. This is followed by vesicle fusion into the regions that are not coated with proteins. Second, supported bilayer membranes are blotted to remove patterned regions of the membrane, 1 and the blotted regions are filled in or caulked with protein from solution. In both cases, the lipid bilayer regions exhibit lateral fluidity, but each region is confined or corralled by the protein. These two methods can be combined and used iteratively to create arrays with increasing lateral complexity in both the fixed protein and mobilesupported membrane regions for biophysical studies or cell-based assays.

We describe a method for making and erasing barriers to the lateral diffusion of membrane components in fluid lipid bilayers supported on glass substrates. When a bilayer is mechanically partitioned by scratching the membrane-coated surface at basic pH, barriers to lateral diffusion are formed which prevent mixing between the regions separated by the scratches. Upon lowering the pH, the bilayer is observed to spread over the scratch boundary, allowing diffusive mixing between the previously separated regions. This is exploited in combination with electrophoresis within the membrane to separate fluorescently labeled charged lipid probes, partition them with a scratch, and allow remixing to occur when the scratch is healed. This method for membrane manipulation can be used to transform a homogeneous membrane into an array of corrals with different compositions while preserving the ability to allow subsequent remixing. This approach should be useful for examining the kinetics of reactions and the assembly of fluid membrane-associated components in a native setting, and for investigating the dynamics of two-dimensional fluids.

We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357. Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 μm 2 /s; this is 3-5 times smaller than individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this paper, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3 -fold change in viscosity of the bulk medium. Addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own rights, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects which reversibly trap diffusing vesicles, are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior in the timescale of our experiments.

A four‐level grayscale image of Abraham Lincoln on a lipid bilayer membrane(see Figure) is used to demonstrate this novel single‐exposure photolithographic procedure for printing images on fluid surfaces containing partitioned corrals, the very nature of the fluid membrane allowing true grayscale to be achieved. The potential scope and applications—particularly in biotechnology—of the concept are also discussed.