| 1. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:15, s. 6538-6545
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Tidskriftsartikel (refereegranskat)abstract
- We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ∼1 Hz with a sensitivity in terms of surface coverage of ∼1 ng/cm2. Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.
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| 2. |
- Bally, Marta, 1981, et al.
(författare)
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Interaction of Single Viruslike Particles with Vesicles Containing Glycosphingolipids
- 2011
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Ingår i: Physical Review Letters. - : American Physical Society. - 0031-9007 .- 1079-7114. ; 107:18
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Tidskriftsartikel (refereegranskat)abstract
- Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.
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| 3. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:38, s. 14852-14855
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Tidskriftsartikel (refereegranskat)abstract
- Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.
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| 4. |
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| 5. |
- Jönsson, Peter, 1981, et al.
(författare)
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Accumulation and Separation of Membrane-Bound Proteins Using Hydrodynamic Forces
- 2011
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 83:2, s. 604-611
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Tidskriftsartikel (refereegranskat)abstract
- The separation of molecules residing in the cell membrane remains a largely unsolved problem in the fields of bioscience and biotechnology. We demonstrate how hydrodynamic forces can be used to both accumulate and separate membrane-bound proteins in their native state. A supported lipid bilayer (SLB) was formed inside a microfluidic channel with the two proteins streptavidin (SA) and cholera toxin (CT) coupled to receptors in the lipid bilayer. The anchored proteins were first driven toward the edge of the lipid bilayer by hydrodynamic forces from a flowing liquid above the SLB, resulting in the accumulation of protein molecules at the edge of the bilayer. After the concentration process, the bulk flow of liquid in the channel was reversed and the accumulated proteins were driven away from the edge of the bilayer. Each type of protein was found to move at a characteristic drift velocity, determined by the frictional coupling between the protein and the lipid bilayer, as well as the size and shape of the protein molecule. Despite having a similar molecular weight, SA and CT could be separated into monomolecular populations using this approach. The method also revealed heterogeneity among the CT-molecules, resulting in three subpopulations with different drift velocities. This was tentatively attributed to multivalent interactions between the protein and the monosialoganglioside G(M1) receptors in the lipid bilayer.
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| 6. |
- Simonsson, Lisa, 1982, et al.
(författare)
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Continuous Lipid Bilayers Derived from Cell Membranes for Spatial Molecular Manipulation
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:35, s. 14027-14032
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Tidskriftsartikel (refereegranskat)abstract
- Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/M3)-CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors.
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| 7. |
- Albet-Torres, Nuria, et al.
(författare)
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Molecular motors on lipid bilayers and silicon dioxide: different driving forces for adsorption
- 2010
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 6:14, s. 3211-3219
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Tidskriftsartikel (refereegranskat)abstract
- Understanding how different types of interactions govern adsorption of the myosin motor fragment heavy meromyosin (HMM) onto different substrates is important in functional studies of actomyosin and for the development of motor powered lab-on-a-chip applications. In this study, we have combined in vitro motility assays and quartz crystal microbalance with dissipation (QCM-D) monitoring to investigate the underlying adsorption mechanisms of HMM onto supported lipid bilayers in comparison with pure and silanized SiO2. The QCM-D results, combined with data showing actin transportation by HMM adsorbed onto positively charged supported lipid bilayers, suggest reversible HMM surface adsorption via the negatively charged coiled-coil tail region. In contrast, the QCM-D data for HMM adsorption onto negatively charged lipids support a model according to which HMM adsorbs onto negatively charged surfaces largely via the positively charged actin binding regions. Adsorption studies at low (30-65 mM) and high (185-245 mM) ionic strengths onto piranha cleaned SiO2 surfaces (contact angle
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| 8. |
- Carlred, Louise M, 1985, et al.
(författare)
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Simultaneous imaging of amyloid-β and lipids in brain tissue using antibody-coupled liposomes and time-of-flight secondary ion mass spectrometry
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:28, s. 9973-9981
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Tidskriftsartikel (refereegranskat)abstract
- The spatial localization of amyloid-β peptide deposits, the major component of senile plaques in Alzheimer's disease (AD), was mapped in transgenic AD mouse brains using time-of-flight secondary ion mass spectrometry (ToF-SIMS), simultaneously with several endogenous molecules that cannot be mapped using conventional immunohistochemistry imaging, including phospholipids, cholesterol and sulfatides. Whereas the endogenous lipids were detected directly, the amyloid-β deposits, which cannot be detected as intact entities with ToF-SIMS because of extensive ion-induced fragmentation, were identified by specific binding of deuterated liposomes to antibodies directed against amyloid-β. Comparative investigation of the amyloid-β deposits using conventional immunohistochemistry and fluorescence microscopy suggests similar sensitivity but a more surface-confined identification due to the shallow penetration depth of the ToF-SIMS signal. The recorded ToF-SIMS images thus display the localization of lipids and amyloid-β in a narrow (∼10 nm) two-dimensional plane at the tissue surface. As compared to a frozen nontreated tissue sample, the liposome preparation protocol generally increased the signal intensity of endogenous lipids, likely caused by matrix effects associated with the removal of salts, but no severe effects on the tissue integrity and the spatial distribution of lipids were observed with ToF-SIMS or scanning electron microscopy (SEM). This method may provide an important extension to conventional tissue imaging techniques to investigate the complex interplay of different kinds of molecules in neurodegenerative diseases, in the same specimen. However, limitations in target accessibility of the liposomes as well as unspecific binding need further consideration. © 2014 American Chemical Society.
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| 9. |
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| 10. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Liposome-Based Chemical Barcodes for Single Molecule DNA Detection Using Imaging Mass Spectrometry
- 2010
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 10:2, s. 732-737
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Tidskriftsartikel (refereegranskat)abstract
- We report on a mass-spectrometry (time-of-flight secondary ion mass spectrometry, TOF-SIMS) based method For multiplexed DNA detection utilizing a random array, where the lipid composition of small unilamellar liposomes act as chemical barcodes to identify unique DNA target sequences down to the single molecule level. In a sandwich format, suspended target-DNA to be detected mediates the binding of capture-DNA modified liposomes to surface-immobilized probe-DNA. With the lipid composition of each liposome encoding a unique target-DNA sequence, TOF-SIMS analysis was used to determine the chemical fingerprint of the bound liposomes. Using high-resolution TOF-SIMS imaging, providing sub-200 nm spatial resolution, single DNA targets could be detected and identified via the chemical fingerprint of individual liposomes. The results also demonstrate the capability of TOF-SIMS to provide multiplexed detection of DNA targets on substrate areas in the micrometer range. Together with a high multiplexing capacity, this makes the concept an interesting alternative to existing barcode concepts based on fluorescence, Raman, or graphical codes for small-scale bioanalysis.
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