Two-Step Formation of Streptavidin-Supported Lipid Bilayers by PEG-Triggered Vesicle Fusion. Fluorescence and Atomic Force Microscopy Characterization

Abstract

We have used fluorescence microscopy, fluorescence photobleaching recovery (FPR), and atomic force microscopy (AFM) to investigate the formation of tethered lipid bilayers on plane aluminum oxide or glass surfaces. The bilayers were assembled with the help of a two-step methodology recently proposed for microporous templates (Proux-Delrouyre et al. J. Am. Chem. Soc. 2001, 123, 8313). The first step consists of the accumulation of intact biotinylated vesicles (PC + DOPE) on a streptavidin sublayer itself immobilized on the substrate. The second step, clearly time separated, is the deliberate triggering of bilayer formation with the help of poly(ethylene glycol) (PEG), a fusion agent of lipidic vesicles. AFM and FPR measurements confirm that the vesicles do not spontaneously fuse during the first step provided that the streptavidin sublayer is present on the substrate. On the contrary, the treatment with PEG provokes the fast formation of a continuous lipid bilayer, as attested at the hundred nanometer scale by the AFM images and at the hundred micrometer scale by the lateral diffusion of a fluorescent probe (D ) 2.2 × 10 -8 cm 2 s -1 for NBD-DMPE at 22 °C). † Part of the Langmuir special issue entitled The Biomolecular Interface.

Sensing Immobilized Molecules of Streptavidin on a Silicon Surface by Maldi-Tof Mass Spectrometry and Fluorescence Microscopy

“…The immobilization was carried out following previously reported procedures 11,37 . The stability of the biotinstreptavidin complex during intensive washing permits removal of nonspecifically bound and non-biotinylated material 10 .…”

“…One group of streptavidin applications involves solid phase assays, in which streptavidin is immobilized on a solid surface 9,11 . The extremely high biotin-binding affinity of streptavidin allows tight, efficient immobilization or capture of biotinylated materials on streptavidin-coated solid surfaces.…”

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

“…Introducing 10 mM Mg 2+ often helps solving this issue. Other agents can be introduced to help bursting the SUVs, such as other divalent cations 51 , a polymer solution 52 , or osmotic shock 25 .…”

Formation and fluidity measurement of supported lipid bilayer on polyvinyl chloride membrane

“…The photobleaching was performed within 2 ms with 1778 time stronger laser power than that for observation, and the size of the photobleached area (S) was 26.7×26.7 µm 2 . On the condition of a pure two-dimensional diffusion of the dye-labeled lipid from an infinite reservoir, the recovery ratio of the fluorescence intensity as a function of time t is given by 22,23 …”