Supported membranes embedded with fixed arrays of gold nanoparticles.

We present a supported membrane platform consisting of a fluid lipid bilayer membrane embedded with a fixed array of gold nanoparticles. The system is realized by preforming a hexagonal array of gold nanoparticles (∼5-7 nm) with controlled spacing (∼50-150 nm) fixed to a silica or glass substrate by block copolymer lithography. Subsequently, a supported membrane is assembled over the intervening bare substrate. Proteins or other ligands can be associated with the fluid lipid component, the fixed nanoparticle component, or both, providing a hybrid interface consisting of mobile and immobile components with controlled geometry. We test different biochemical coupling strategies to bind individual proteins to the particles surrounded by a fluid lipid membrane. The coupling efficiency to nanoparticles and the influence of nanoparticle arrays on the surrounding membrane integrity are characterized by fluorescence imaging, correlation spectroscopy, and super-resolution fluorescence microscopy. Finally, the functionality of this system for live cell experiments is tested using the ephrin-A1-EphA2 juxtacrine signaling interaction in human breast epithelial cells.

Spatial patterning of chemical and physical properties of surfaces has been used to control the behavior of cultured cells for decades.[1−7] Most of these early methods were based on patterning extracellular matrix proteins, either directly or by modulating their deposition by the cells themselves. Subsequently, more refined technologies began to focus more on specific ligand display. Such synthetically designed platforms have already provided substantial insight into how cellular functions such as adhesion,(8) migration,[9,10] proliferation,(11) differentiation,(12) as well as specific receptor activation and the role of spatial organization[13,14] are regulated on the molecular level. For example, micro- and nanopatterned arrays of adhesion molecules have been used to investigate how spatial differences of only a few nanometers can influence cell fate and response.(6) These experiments revealed that fibroblasts can apparently sense even nanoscale gradients of adhesion molecules, and underscore the precision with which cells control and react to the spatial organization of molecules.(15) While useful in many cases, immobile patterning intrinsically defeats any cellular process that naturally involves movement of the ligands, such as is particularly common among juxtacrine signaling in cell–cell junctions where both receptor and ligand reside in the fluid cell membranes.

One material platform technology that has proven particularly useful to address the more fluid nature of intercellular interactions is the supported membrane.(16) Lipid bilayers can be assembled on solid surfaces in such a way that they form a single, continuous, membrane that coats the underlying solid substrate but maintains a high degree of lateral mobility in the membrane.[16−18] Lipid mobilities in supported membranes are typically 3–4 μm2/s, which, while several times slower than that of free bilayer membranes (e.g., in giant unilamellar vesicles(19)), is still faster than lipid mobility (∼1 μm2/s) in the crowded membranes of living cells.(20) Thus supported membranes enable ligand display along with freedom to move and reorganize naturally. Supported membranes have found productive applications in studies of the T cell immunological synapse,[21−26] neuronal interactions,[27,28] and the triggering of EphA2 receptor tyrosine kinase in breast epithelial cancer cells.[29,30] Supported membranes provide the added advantage that materials such as metals can be patterned onto the underlying substrate so as to impose fixed barriers or obstacles to mobility of molecules in the supported membrane.[21,23,24,31] Such patterned supported membranes intrinsically embody a combination of mobile and immobile characteristics, which can be used to glean insights into the function of living cells and especially the role of spatial organization and assembly in cellular processes.[32,33]

In this report, we describe the fabrication and characterization of a hybrid nanoparticle and supported membrane configuration consisting of an immobile array of nanoparticles embedded within a fluid supported membrane (Figure 1A–D). Nanoparticle arrays are formed by block copolymer micelle nanolithography (BCML),(34) in which nucleation sites for nanoparticle growth are first ordered by self-assembly of block copolymer micelle arrays. The organic component is subsequently plasma etched, leaving an ordered array of nanoparticles on the substrate whose spacing is dictated by the original polymer molecular weight. Key features of this system are the extraordinarily small size of the gold nanoparticles (∼5–7 nm), which enables functionalization with individual protein molecules, and the controllable spacing between particles in the array in the important range of 50–150 nm, all of which are under direct synthetic control. Importantly, the BCML method of fabricating nanoparticle arrays is a self-assembly process and does not require complex patterning methods such as electron beam lithography or nanoimprint lithography.(34) Supported membranes can be assembled on these surfaces and orthogonal chemistries can be employed to functionalize the particles themselves. Fluorescence correlation spectroscopy and super-resolution microscopy are used here to examine membrane integrity and ligand coupling efficiency to the nanoparticles. Finally, application of this technology to the ephrin-A1–EphA2 signaling system in breast epithelial cells is examined as a test of its utility in a live cell format.

Figure 1

Schematic overview of the fabrication steps: (A) Gold nanoparticle arrays are formed by block copolymer micelle nanolithography (BCML). (B) Supported lipid bilayer formation by vesicle fusion. (C) Selective labeling of the gold nanoparticles. (D) Live-cell experiments with specific ligands bound to the nanoparticles and the lipid bilayer, respectively. (E) SEM micrographs of gold nanoparticle arrays from five different samples with individual particle spacing varying between 58 and 151 nm. The small particle size of ∼5–7 nm matches the height of supported bilayer. Scale bar: 200 nm.