The bidirectional transfer of phospholipids between a charged, supported lipid bilayer (SLB) on SiO(2) and oppositely charged, unilamellar vesicles was studied by means of quartz crystal microbalance with dissipation (QCM-D) and optical reflectometry techniques. SLBs and vesicles were prepared from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) mixed with different fractions of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) (negatively charged) or 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) (positively charged). The interaction process consists of an attachment-transfer-detachment (ATD) sequence, where added vesicles first attach to and interact with the SLB, after which they detach, leaving behind a compositionally modified SLB and ditto vesicles. When the process is complete, there is no net addition or reduction of total lipid mass in the SLB, but lipid exchange has occurred. The time scale of the process varies from a few to many tens of minutes depending on the type of charged lipid molecule and the relative concentration of charged lipids in the two membranes. Electrostatically symmetric cases, where only the charge sign (but not the fraction of charged lipid) was reversed between the SLB and the vesicles, produce qualitatively similar but quantitatively different kinetics. The time scale of the interaction varies significantly between the two cases, which is attributed to a combination of the differences in the molecular structure of the lipid headgroup for the positively and the negatively charged lipids used, and to nonsymmetric distribution of charged lipids in the lipid membranes. The maximum amounts of attached vesicles during the ATD process were estimated to be 25-40% of a full monolayer of vesicles, with the precise amount depending on the actual charge fractions in the vesicles and the SLB. Interrupted vesicle exposure experiments, and experiments where the bulk concentration of vesicles was varied, show that vesicles in some cases may be trapped irreversibly on the SLB, when only partial transfer of lipid molecules has occurred. Additional supply of vesicles and further transfer induces detachment, when a sufficient amount of oppositely charged lipids has been transferred to the SLB, so that the latter becomes repulsive to the attached vesicles. Possible mechanistic scenarios, including monomer insertion and hemifusion models, are discussed. The observed phenomena and the actual SLB preparation process form a platform both for studies of various intermembrane molecular transfer processes and for modifying the composition of SLBs in a controlled way, for example, for biosensor and cell culture applications.

Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 nm gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.

The study of lipid transfer between lipid membranes is of great interest for the fundamental understanding of this complex and important process and, furthermore, for providing a new avenue for the in situ modification of supported lipid bilayers (SLBs). SLBs are conveniently formed by vesicle spreading onto a solid support, but this method is limited to conditions (i.e., combination of vesicle lipid composition, surface chemical properties, and buffer) such that the vesicles break spontaneously upon adsorption to the surface. Many SLB compositions are not accessible by this approach. In the present study, we give an example of how lipid transfer can be made use of to form lipid layers with striking new features, notably with respect to stability. After lipid transfer between negatively charged POPS small unilamellar vesicles and a positively charged POEPC SLB on TiO2, an SLB is obtained, which, upon exposure to SDS, leaves behind a lipid monolayer. It is shown how this monolayer can be used for creating new SLBs. The several step procedure, bilayer formation, lipid transfer, removal of a lipid monolayer and the reassembly of a bilayer, is monitored in real time by the quartz crystal microbalance with a dissipation (QCM-D) technique, and the lipid composition is analyzed for each step in postpreparation spectroscopic analyses using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Comparison of the measured signal ratios with those of the reference samples containing known fractions of D31-POPS directly shows that the relative concentration of D31-POPS is approximately 50% in the SLB after D31-POPS exchange, significantly higher in the monolayer prepared in situ by SDS rinse, and approximately 20-25% after reassembly of the SLB using POEPC vesicles. The results thus provide unambiguous evidence for extensive lipid transfer between the initial POEPC SLB and D31-POPS vesicles in solution. We suggest that the reassembled SLB has a significant asymmetry between the two leaflets, and we propose that the described method is promising for the in situ preparation of asymmetric SLBs.

Ion-mediated (Ca 2+ ) changes in viscoelastic, structural and optical properties of negatively charged solid supported lipid bilayers (SLBs) on SiO 2 surfaces were studied by means of quartz crystal microbalance with dissipation (QCM-D) monitoring and optical reflectometry. Despite the sensitivity of QCM-D to viscoeleastic/structural variations, it has not often been used to probe such changes for SLBs. SLBs were prepared from binary phospholipid mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC, neutral) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1 0 -rac-glycerol) (POPG, negatively charged) on SiO 2 sensor surfaces in a Ca 2+ -containing buffer. Interestingly, for bilayers containing POPG fractions above 35%, large QCM-D dissipation shifts occurred, when Ca 2+ was removed from buffer in contact with the SLB (while maintaining 100 mM NaCl). The accompanying frequency changes were small. These Ca 2+ mediated QCM-D responses are reversible, and a signal for considerable changes in the viscoelastic and structural properties of the SLB. Variation of Ca 2+ -concentration revealed a threshold concentration of around 0.4 mM for the changes in the SLB to occur. Below this value, at >35% POPG concentration in the SLB, the SLB appears to become more weakly attached to the SiO 2 substrate, which is partly attributed to a weakening of the POPG-substrate interaction in the absence of Ca 2+ . A consequence of this is an oscillation-amplitude dependent dissipation, which we attribute to slip of the bilayer at higher oscillation amplitudes. Complementary experiments using a combined QCM-D/reflectometry instrument showed that the Ca 2+ -induced changes in the viscoelastic/structural properties of the SLB are accompanied by changes in the optical properties. We discuss different scenarios to explain the observed reversible effect of Ca 2+ -ions on the dissipative and optical properties of the mixed SLBs. Based on our results we propose the observed phenomenon to be a combination of geometric changes, internal structural changes, changes in the interfacial water layer, and a slip mechanism, i.e. friction between the SLB and the substrate.

Carbohydrate−carbohydrate interactions (CCIs) are of central importance for several biological processes. However, the ultra-weak nature of CCIs generates difficulties in studying this interaction, thus only little is known about CCIs. Here we present a highly sensitive equilibrium-fluctuation-analysis of single liposome binding events to supported lipid bilayers (SLBs) based on total internal reflection fluorescence (TIRF) microscopy that allows us to determine apparent kinetic rate constants of CCIs. The liposomes and SLBs both contained natural Lex glycosphingolipids (Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ1Cer), which were employed to mimic cell−cell contacts. The kinetic parameters of the self-interaction between Lex-containing liposomes and SLBs were measured and found to be modulated by bivalent cations. Even more interestingly, upon addition of cholesterol, the strength of the CCIs increases, suggesting that this interaction is strongly influenced by a cholesterol-dependent presentation and/or spatial organization of glycosphingolipids in cell membranes.

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Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-014-0958-9) contains supplementary material, which is available to authorized users.

Cellular motility is the major driving force of numerous biological phenomena including wound healing, immune response, embryogenesis, cancer formation, and metastasis. We studied the response of epithelial FaDu monolayers cultured on gold electrodes of an acoustic resonator (quartz crystal microbalance, QCM) and impedance sensor (electric cell-substrate impedance sensing, ECIS) to externally applied chemical stimuli interfering with cytoskeleton organization. Epithelial cell motility of confluent monolayers is characterized by subtle cell shape changes and variations in the cell-substrate as well as cell-cell distance without net directionality of individual cells. The impact of small molecules such as cytochalasin D, phalloidin, and blebbistatin as well as paclitaxel, nocodazol, and colchicin on actin and microtubules organization was quantified by conventional sensors' readouts and by comparing the noise pattern of the signals which is attributed to cellular dynamics. The responsiveness of noninvasive and label-free techniques relying on cellular dynamics is compared to classical viability assays and changes of the overall impedance of ultrasmall electrodes or acoustic loads of a thickness shear mode resonator. Depending on the agent used, a distinct sensor response was found, which can be used as a fingerprint of the cellular response. Cytoskeletal rearrangements and nuclear integrity were corroborated by fluorescence microscopy and correlated to the readouts of QCM and ECIS.