| 1. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells
- 2015
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 9:12, s. 11849-11862
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Tidskriftsartikel (refereegranskat)abstract
- 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 rim 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.
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| 2. |
- Rydberg, Hanna, 1982, et al.
(författare)
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Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring
- 2014
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 1432-1017 .- 0175-7571. ; 43:6-7, s. 241-253
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
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| 4. |
- Jing, Yujia, 1985, et al.
(författare)
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Phase Transition-Controlled Flip-Flop in Asymmetric Lipid Membranes
- 2014
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 118:9, s. 2389-2395
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Tidskriftsartikel (refereegranskat)abstract
- Lipid membrane asymmetry is of fundamental importance for biological systems and also provides an attractive means for molecular control over biomaterial surface properties (including drug carriers). In particular, temperature-dependent changes of surface properties can be achieved by taking advantage of distinct phase transitions in lipid membrane coatings where lipids exchange (flip-flop) between leaflets. In this study, temperature is used to control flip-flop of lipids in asymmetric lipid membranes on planar solid supports, where the two leaflets of the lipid membrane are in different phase states. More specifically, the lower leaflet is prepared from a supported lipid membrane composed of a high T-m lipid mixture of phosphocholine (PC), phosphatidylserine (PS), and a bioactive lipid on TiO2, followed by selective removal of the top leaflet by detergent. Next, at a lower temperature, where the remaining leaflet is in the gel state, a top leaflet of a different lipid composition and in the fluid phase is formed. Phase transition-induced changes in membrane surface properties following upon temperature-activation of the prepared asymmetric membrane are demonstrated by the detection of biotinylated lipids, which were initially located (thus "hidden") in the lower-gel phase leaflet, at the surface of the top leaflet. These processes were monitored in real-time by the quartz crystal microbalance with dissipation (QCM-D) and the dual polarization interferometry (DPI) techniques, allowing modeling of the mass and the anisotropic property of the lipid structures in different phase states.
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| 5. |
- Kunze, Angelika, 1978, et al.
(författare)
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Electrodeless QCM-D for lipid bilayer applications
- 2011
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Ingår i: Biosensors and Bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 26:5, s. 1833-1838
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Tidskriftsartikel (refereegranskat)abstract
- An electrodeless quartz crystal microbalance with dissipation monitoring (QCM-D) setup is used to monitor the formation of supported lipid bilayers (SLBs) on bare quartz crystal sensor surfaces. The kinetic behavior of the formation of a 1-palmitoyl-2-oleoyl-sn-glycero-3-Phosphocholine (POPC) SLB on SiO2 surfaces is discussed and compared for three cases: (i) a standard SiO2 film deposited onto the gold electrode of a quartz crystal, (ii) an electrodeless quartz crystal with a sputter-coated SiO2 film, and (iii) an uncoated electrodeless quartz crystal sensor surface. We demonstrate, supported by imaging the SLB on an uncoated electrodeless surface using atomic force microscopy (AFM), that a defect-free, completely covering bilayer is formed in all three cases. Differences in the kinetics of the SLB formation on the different sensor surfaces are attributed to differences in surface roughness. The latter assumption is supported by imaging the different surfaces using AFM. We show furthermore that electrodeless quartz crystal sensors can be used not only for the formation of neutral SLBs but also for positively and negatively charged SLBs. Based on our results we propose electrodeless QCM-D to be a valuable technique for lipid bilayer and related applications providing several advantages compared to electrode-coated surfaces like optical transparency, longer lifetime, and reduced costs.
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| 6. |
- Kunze, Angelika, 1978, et al.
(författare)
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Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions
- 2013
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- 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 Le(x) glycosphingolipids (Gal beta 4( Fuc alpha 3) GlcNAc beta 3Gal beta 4Glc beta 1Cer), which were employed to mimic cell-cell contacts. The kinetic parameters of the self-interaction between Le(x)-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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| 7. |
- Kunze, Angelika, 1978, et al.
(författare)
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In situ preparation and modification of supported lipid layers by lipid transfer from vesicles studied by QCM-D and TOF-SIMS
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:7, s. 2450-2451
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Tidskriftsartikel (refereegranskat)abstract
- 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 50% in the SLB after D31-POPS exchange, significantly higher in the monolayer prepared in situ by SDS rinse, and 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.
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| 8. |
- Kunze, Angelika, 1978, et al.
(författare)
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Ion-mediated changes of supported lipid bilayers and their coupling to the substrate. A case of bilayer slip?
- 2011
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 7:18, s. 8582-8591
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Tidskriftsartikel (refereegranskat)abstract
- 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-3-phosphocholine (POPC, neutral) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-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.
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| 9. |
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| 10. |
- Kunze, Angelika, 1978, et al.
(författare)
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Non-Invasive Acoustical sensing of Drug-Induced Effects on the Contractile Machinery of Human Cardiomyocyte Clusters
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 10:5, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- There is an urgent need for improved models for cardiotoxicity testing. Here we propose acoustic sensing applied to beating human cardiomyocyte clusters for non-invasive, surrogate measuring of the QT interval and other characteristics of the contractile machinery. In experiments with the acoustic method quartz crystal microbalance with dissipation monitoring (QCM-D), the shape of the recorded signals was very similar to the extracellular field potential detected in electrochemical experiments, and the expected changes of the QT interval in response to addition of conventional drugs (E-4031 or nifedipine) were observed. Additionally, changes in the dissipation signal upon addition of cytochalasin D were in good agreement with the known, corresponding shortening of the contraction-relaxation time. These findings suggest that QCM-D has great potential as a tool for cardiotoxicological screening, where effects of compounds on the cardiomyocyte contractile machinery can be detected independently of whether the extracellular field potential is altered or not.
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