Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores

Lipid bilayers were deposited inside the 0.2 mum pores of anodic aluminum oxide (AAO) filters by extrusion of multilamellar liposomes and their properties studied by H-2, P-31, and H-1 solid-state NMR. Only the first bilayer adhered strongly to the inner surface of the pores. Additional layers were washed out easily by a flow of water as demonstrated by H-1 magic angle spinning NMR experiments with addition of Pr3+ ions to shift accessible lipid headgroup resonances. A 13 mm diameter Anopore filter of 60 mum thickness oriented approximately 2.5 x 10(-7) mol of lipid as a single bilayer, corresponding to a total membrane area of about 500 cm(2). The 2H NMR spectra of chain deuterated POPC are consistent with adsorption of wavy, tubular bilayers to the inner pore surface. By NMR diffusion experiments, we determined the average length of those lipid tubules to be approximately 0.4 mum. There is evidence for a thick water layer between lipid tubules and the pore surface. The ends of tubules are well sealed against the pore such that Pr3+ ions cannot penetrate into the water underneath the bilayers. We successfully trapped poly(ethylene glycol) (PEG) with a molecular weight of 8000 in this water layer. From the quantity of trapped PEG, we calculated an average water layer thickness of 3 nm. Lipid order parameters and motional properties are unperturbed by the solid support, in agreement with existence of a water layer. Such unperturbed, solid supported membranes are ideal for incorporation of membrane-spanning proteins with large intra- and extracellular domains. The experiments suggest the promise of such porous filters as membrane support in biosensors.