Formation of individual protein channels in lipid bilayers suspended in nanopores.

Free-standing lipid bilayers are formed in regularly arranged nanopores of 200, 400 and 800 nm in a 300 nm thin hydrophobic silicon nitride membrane separating two fluid compartments. The extraordinary stability of the lipid bilayers allows us to monitor channel formation of the model peptide melittin and alpha-hemolysin from Staphylococcus aureus using electrochemical impedance spectroscopy and chronoamperometry. We observed that melittin channel formation is voltage-dependent and transient, whereas transmembrane heptameric alpha-hemolysin channels in nano-BLMs persist for hours. The onset of alpha-hemolysin-mediated conduction depends on the applied protein concentration and strongly on the diameter of the nanopores. Heptameric channel formation from adsorbed alpha-hemolysin monomers needs more time in bilayers suspended in 200 nm pores compared to bilayers in pores of 400 and 800 nm diameters. Diffusion of sodium ions across alpha-hemolysin channels present in a sufficiently high number in the bilayers was quantitatively and specifically determined using ion selective electrodes. The results demonstrate that relatively small variations of nano-dimensions have a tremendous effect on observable dynamic biomolecular processes. Such nanopore chips are potentially useful as supports for stable lipid bilayers to establish functional assays of membrane proteins needed in basic research and drug discovery.

Nano for bio: nanopore arrays for stable and functional lipid bilayer membranes (Mini Review).

The usefulness of nanotechnology for biotechnological applications is frequently emphasized. The recent development for using nanostructured materials as supports for free-standing lipid bilayers is briefly reviewed. The authors then demonstrate that the stability of fragile free-standing lipid bilayers in nanopores is enhanced up to days depending on the surface chemistry, the lipid composition, and the diameter of the pores. The insertion of a pore forming protein into bilayers can be monitored over time as a stepwise decrease of membrane resistance. Since membrane proteins are major drug targets, such stable and functional proteo-bilayers integrated in microfluidics are the key components of in vitro devices for drug screening. This conference paper reviews the recent literature and provides preliminary results from own research.