| 1. |
- Lundgren, Anders, 1978, et al.
(författare)
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Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations
- 2018
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 18:1, s. 381-385
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Tidskriftsartikel (refereegranskat)abstract
- The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of β-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (∼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.
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| 2. |
- 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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| 3. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Low-temperature fabrication and characterization of a symmetric hybrid organic–inorganic slab waveguide for evanescent light microscopy
- 2018
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Ingår i: Nano Futures. - : IOP Publishing. - 2399-1984. ; 2:2
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Tidskriftsartikel (refereegranskat)abstract
- Organic and inorganic solid materials form the building blocks for most of today's high-technological instruments and devices. However, challenges related to dissimilar material properties have hampered the synthesis of thin-film devices comprised of both organic and inorganic films. We here give a detailed description of a carefully optimized processing protocol used for the construction of a three-layered hybrid organic–inorganic waveguide-chip intended for combined scattering and fluorescence evanescent-wave microscopy in aqueous environments using conventional upright microscopes. An inorganic core layer (SiO2 or Si3N4), embedded symmetrically in an organic cladding layer (CYTOP), aids simple, yet efficient in-coupling of light, and since the organic cladding layer is refractive index matched to water, low stray-light (background) scattering of the propagating light is ensured. Another major advantage is that the inorganic core layer makes the chip compatible with multiple well-established surface functionalization schemes that allows for a broad range of applications, including detection of single lipid vesicles, metallic nanoparticles or cells in complex environments, either label-free—by direct detection of scattered light—or by use of fluorescence excitation and emission. Herein, focus is put on a detailed description of the fabrication of the waveguide-chip, together with a fundamental characterization of its optical properties and performance, particularly in comparison with conventional epi illumination. Quantitative analysis of images obtained from both fluorescence and scattering intensities from surface-immobilized polystyrene nanoparticles in suspensions of different concentrations, revealed enhanced signal-to-noise and signal-to-background ratios for the waveguide illumination compared to the epi-illumination.
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| 4. |
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| 5. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Optical waveguide-based single-molecule studies for medical diagnostics and drug screening applications
- 2014
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Ingår i: Latin America Optics and Photonics Conference, LAOP 2014, Cancun, Mexico, 16-21 November 2014. - Washington, D.C. : OSA. - 9781557528254
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Konferensbidrag (refereegranskat)abstract
- Measurements of single binding events between membrane-protein receptors and their ligands in near-natural environments are presented, and the advantage with single-molecule sensitivity is discussed in the context of biophysics, medical diagnostics and drug discovery.
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| 6. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Rutile TiO 2 thin films grown by reactive high power impulse magnetron sputtering
- 2013
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Ingår i: Thin Solid Films. - : Elsevier BV. - 0040-6090 .- 1879-2731. ; 545, s. 445-450
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Tidskriftsartikel (refereegranskat)abstract
- Thin TiO 2 films were grown on Si(001) substrates by reactive dc magnetron sputtering (dcMS) and high power impulse magnetron sputtering (HiPIMS) at temperatures ranging from 300 to 700 C.Optical and structural properties of films were compared both before and after post-annealing using scanning electron microscopy, low angle X-ray reflection (XRR), grazing inc idence X-ray diffractometry and spectroscopic ellipsometry.Both dcMS- and HiPIMS-grown films reveal polycrystalline rutile TiO 2 , even prior to post-annealing.The HiPIMS-grown films exhibit significantly larger grains compared to that of dcMC-grown films, approaching 100% of the film thickness for films grown at 700 C.In addition, the XRR surface roughness of HiPIMS-grown films was significantly lower than that of dcMS-grown films over the whole temperature range 300-700 C.Dispersion curves could only be obtained for the HiPIMS-grown films, which were shown to have a refractive index in the range of 2.7-2.85 at 500 nm.The results show that thin, rutile TiO 2 films, with high refractive index, can be obtained by HiPIMS at relatively low growth temperatures, without post-annealing.Furthermore, these films are smoother and show better optical characteristics than their dcMS-grown counterparts.© 2013 Elsevier B.V.All rights reserved.
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| 7. |
- Agnarsson, Björn, 1977-
(författare)
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Surface studies on α–sapphire for potential use in GaN epitaxial growth
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This Licentiate thesis summarizes the work carried out by the author the years 2004 to 2008 at the University of Iceland and the Royal Institute of Technology (KTH) in Sweden. The aim of the project was to investigate the structure of sapphire (alpha-Al2O3) surfaces, both for pure scientific reasons and also for potential use as substrate for GaN-growth by molecular beam epitaxy. More generally the thesis describes some surface science methods used for investigating the substrates; the general physical back ground, the experi- mental implementation and what information they can give. The described techniques are used for surface analysis on sapphire substrates which have been treated variously in order to optimize them for use as templates for epi- taxial growth of GaN or related III-V compounds. The thesis is based on three published papers. The first paper focuses on the formation a thin AlN layer on sapphire, which may act as a buffer layer for potential epitaxial growth of GaN or any related III-V materials. Two types of sapphire substrates (reconstructed and non- reconstructed) were exposed to ammonia resulting in the formation of AlN on the surface. The efficiency of the AlN formation (nitridation efficiency) for the two surfaces was then compared as a function of substrate temperature through photoelectron spectroscopy and low electron energy diffraction. The reconstructed surface showed a much higher nitridation efficiency than the non-reconstructed surface. In the second paper, the affect of different annealing processes on the sapphire morphology, and thus its capability to act as a template for GaN growth, was studied. Atomic force microscopy, X-ray diffraction analysis together with ellipsometry measurements showed that annealing in H2 ambient and subse- quent annealing at 1300 °C in O2 for 11 hours resulted in high quality and atomically flat sapphire surface suitable for III-V epitaxial growth. The third paper describes the effect of argon sputtering on cleaning GaN surfaces and the possibility of using indium as surfactant for establishing a clean and stoichiometric GaN surface, after such sputtering. Soft sputtering, followed by deposition of 2 ML of indium and subsequent annealing at around 500 °C resulted in a well ordered and clean GaN surface while hard sputtering introduced defects and incorporated both metallic gallium and indium in the surface.
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| 8. |
- Agnarsson, Björn, 1977, et al.
(författare)
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Waveguide structure
- 2018
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Patent (övrigt vetenskapligt/konstnärligt)abstract
- A waveguide structure for evanescent wave microscopy and/or spectroscopy, comprising an optically transparent core layer, a lower dielectric cladding layer and an upper dielectric cladding layer arranged on opposite sides of the core layer. The core layer has a refractive index higher than the refractive indices of the cladding layers. The upper cladding layer is made of an organic material. A sample well is arranged on an upper surface of the core layer formed by a cavity in the upper cladding layer, the sample well being adapted to contain a sample medium with one or more sample objects. The core layer is made of a first dielectric inorganic material, and the upper cladding layer has a refractive index which closely matches the refractive index of the sample medium. A method for manufacturing such waveguide structure, and a measurement system comprising the waveguide structure are also disclosed.
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| 9. |
- Ahmadi, Sareh, et al.
(författare)
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Site-dependent charge transfer at the Pt(111)-ZnPc interface and the effect of iodine
- 2014
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 140:17, s. 174702-
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Tidskriftsartikel (refereegranskat)abstract
- The electronic structure of ZnPc, from sub-monolayers to thick films, on bare and iodated Pt(111) is studied by means of X-ray photoelectron spectroscopy, X-ray absorption spectroscopy, and scanning tunneling microscopy. Our results suggest that at low coverage ZnPc lies almost parallel to the Pt(111) substrate, in a non-planar configuration induced by Zn-Pt attraction, leading to an inhomogeneous charge distribution within the molecule and an inhomogeneous charge transfer to the molecule. ZnPc does not form a complete monolayer on the Pt surface, due to a surface-mediated intermolecular repulsion. At higher coverage ZnPc adopts a tilted geometry, due to a reduced molecule-substrate interaction. Our photoemission results illustrate that ZnPc is practically decoupled from Pt, already from the second layer. Pre-deposition of iodine on Pt hinders the Zn-Pt attraction, leading to a non-distorted first layer ZnPc in contact with Pt(111)-I(root 3x root 3) or Pt(111)-I(root 7x root 7), and a more homogeneous charge distribution and charge transfer at the interface. On increased ZnPc thickness iodine is dissolved in the organic film where it acts as an electron acceptor dopant.
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| 10. |
- Aliakbarinodehi, Nima, 1986, et al.
(författare)
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Time-Resolved Inspection of Ionizable Lipid-Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early Endosomal Membrane Mimic
- 2024
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Ingår i: ACS Nano. - 1936-086X .- 1936-0851. ; In Press
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Tidskriftsartikel (refereegranskat)abstract
- Advances in lipid nanoparticle (LNP) design have contributed notably to the emergence of the current clinically approved mRNA-based vaccines and are of high relevance for delivering mRNA to combat diseases where therapeutic alternatives are sparse. LNP-assisted mRNA delivery utilizes ionizable lipid-mediated cargo translocation across the endosomal membrane driven by the acidification of the endosomal environment. However, this process occurs at a low efficiency, a few percent at the best. Utilizing surface-sensitive fluorescence microscopy with a single LNP and mRNA resolution, we have investigated pH-controlled interactions between individual LNPs and a planar anionic supported lipid bilayer (SLB) formed on nanoporous silica, mimicking the electrostatic conditions of the early endosomal membrane. For LNPs with an average diameter of 140 nm, fusion with the anionic SLB preferentially occurred when the pH was reduced from 6.6 to 6.0. Furthermore, there was a delay in the onset of LNP fusion after the pH drop, and upon fusion, a significant fraction (>70%) of mRNA was released into the acidic solution representing the endosomal lumen, while a fraction of mRNA remained bound to the SLB even after reversing the pH to neutral cytosolic conditions. Finally, a comparison of the fusion efficiency of two LNP formulations with different surface concentrations of gel-forming lipids correlated with differences in the protein translation efficiency previously observed in human primary cell transfection studies. Together, these findings emphasize the relevance of biophysical investigations of ionizable lipid-containing LNP-assisted mRNA delivery mechanisms while potentially also offering means to optimize the design of LNPs with enhanced endosomal escape capabilities.
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