Specific Adhesion of Vesicles Monitored by Scanning Force Microscopy and Quartz Crystal Microbalance

Abstract

The specific adhesion of unilamellar vesicles with an average diameter of 100 nm on functionalized surfaces mediated by molecular recognition was investigated in detail. Two complementary techniques, scanning force microscopy (SFM) and quartz crystal microbalance (QCM) were used to study adhesion of liposomes consisting of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine and varying concentrations of N-((6-biotinoyl)amino)hexanoyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (biotin-X-DHPE). Monitoring the adhesion of the receptor-doped vesicles to avidin-coated gold surfaces by QCM (f(0) = 5 MHz) revealed an increased shift in resonance frequency with increasing biotin concentration up to 10 mol% biotin-X-DHPE. To address the question of how the morphology of the liposomes changes upon adhesion and how that contributes to the resonator's frequency response, we performed a detailed analysis of the liposome morphology by SFM. We found that, with increasing biotin-concentration, the height of the liposomes decreases considerably up to the point where vesicle rupture occurs. Thus, we conclude that the unexpected high frequency shifts of the quartz crystal (>500 Hz) can be attributed to a firm attachment of the spread bilayers, in which the number of contacts is responsible for the signal. These findings are compared with one of our recent studies on cell adhesion monitored by QCM.

Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment

“…The low biotin coverages on the TIRFM substrate and the vesicles were chosen to reduce the number of anchoring points and potential vesicle deformation. 54 Taking the sizes of the counterparts of the biotinstreptavidin tether into account, 55 the tether length is estimated to be 15-17 nm. This length is rather large, and accordingly, the effect of the glass/water interface on the dye emission is nearly negligible even if dyes are located at the lower vesicle part.…”

Probing small unilamellar EggPC vesicles on mica surface by atomic force microscopy

“…The latter method determined the free vesicles' diameters while the AFM measured the vesicles after adsorption on the mica. The soft vesicles flatten on the substrate, resulting in larger lateral vesicles size [16,19]. In comparison with tapping mode, the vesicle diameter by contact mode is larger mainly due to tip convolution [36].…”

“…AFM images on liposome are quite challenging since they are soft and dynamic. Thus, definitive visualization of individual vesicles is still one of the most challenging tasks to date, due to vesicle deformation during scanning and possible artifacts induced by the AFM tip in contact mode [16][17][18][19]. Several papers found that both contact and tapping mode were able to capture soft samples with tapping mode showing slightly better resolution [20][21][22].…”

Characterization of Recombinant Amino-terminal NC4 Domain of Human Collagen IX

“…Preparation of Biotinylated Small Unilamellar Vesicles-Biotinylated small unilamellar vesicles (SUV) for the surface plasmon resonance studies were prepared by a method adapted from those described earlier (17,18). A solution of egg yolk L-␣-phosphatidylcholine (Sigma) in chloroform/methanol (9:1) containing 2% biotinylated phosphatidylethanolamine (biotin DHPE; Molecular Probes Europe BV) was dried under a stream of nitrogen on a 37°C water bath and lyophilized in a vacuum for 2 h. Multilamellar vesicles were obtained by quickly hydrating the dried mixture with HBS buffer (150 mM NaCl and 20 mM HEPES buffer, pH 7.4).…”