| 1. |
- Pace, Hudson, 1982, et al.
(författare)
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Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers
- 2018
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:21, s. 13065-13072
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Tidskriftsartikel (refereegranskat)abstract
- Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.
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| 2. |
- Lubart, Quentin, 1989, et al.
(författare)
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Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO
- 2020
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 22:16, s. 8781-8790
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Tidskriftsartikel (refereegranskat)abstract
- Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.
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| 3. |
- Shaali, Mehrnaz, 1981, et al.
(författare)
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Site-selective immobilization of functionalized DNA origami on nanopatterned Teflon AF
- 2017
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Ingår i: Journal of Materials Chemistry C. - : Royal Society of Chemistry (RSC). - 2050-7534 .- 2050-7526. ; 5:30, s. 7637-7643
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate the use of arrays of Teflon AF nanopillars for directing the assembly of single rectangular DNA origami scaffolds, functionalized with covalently linked fluorophore molecules, in defined positions on patterned surfaces. This is achieved by introducing Teflon AF as a non-amplified negative e-beam resist, which is exposed and chemically developed to generate arrays of hydrophobic nanopillars with a minimum feature size 40 nm. Binding of the DNA origami to the pillars is facilitated by porphyrin moieties that act as hydrophobic molecular anchors, reaching 80% coverage of the available sites. This combination of top-down lithography and bottom-up self assembly is an efficient means of fabricating hierarchically structured bio-nanointerfaces in which the positioning of functional units is precisely controlled on the molecular scale inside the DNA assembly, and on the nanoscale at pre-designed locations on the substrate.
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| 4. |
- Albinsson, Bo, 1963, et al.
(författare)
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Functionalized DNA Nanostructures for Light Harvesting and Charge Separation
- 2012
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Ingår i: Coordination Chemistry Reviews. - : Elsevier BV. - 0010-8545. ; 256:21-22, s. 2399-2413
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Forskningsöversikt (refereegranskat)abstract
- Mimicking natural photosynthesis by covalently arranging antenna and charge separation units is a formidable task. Many such beautiful supramolecular complexes have been designed and synthesized with large efforts, some of which are presented in this special issue. The ability to predict relative position of and electronic coupling between the active components in covalent arrays is quite high but there are two obvious drawbacks with the covalent approach. Firstly, as the size grows the complexity of the organic synthesis increases and secondly, sensitivity to light-induced damage becomes a major issue if covalent bonds are broken. Self-assembly of the photoactive components should, in principle, provide a solution to both these issues but generally the ability to predict position and electronic coupling is too low to have the designed properties needed for a functional artificial photosynthetic complex. Here, we present an approach of using DNA as a template for arranging both charge separation units and antenna molecules that govern long-range energy transfer. Of particular interest is the ability of DNA to function as a scaffold for chromophores, either through covalent attachment, or through non-covalent association by means of intercalation or grove binding. Using controlled positioning of dyes, multichromophoric assemblies can be created, capable of long range communication through multi-step energy transfer. This facilitates creation of DNA-based photonic devices for both light harvesting and directed information transfer. The channeled excitation energy can be transformed site specifically to chemical energy by charge separation of DNA linked porphyrins. A two phase system is discussed, in which the DNA is located in buffered solution whereas the hydrophobic porphyrins, responsible for the charge separation reaction, are located in the lipid bilayer of liposomes or supported lipid bilayers.
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| 5. |
- Albinsson, Bo, 1963, et al.
(författare)
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Multistep FRET and Nanotechnology
- 2013
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Ingår i: FRET - Förster Resonance Energy Transfer: From Theory to Applications. - : Wiley. ; , s. 607-653
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 6. |
- Czolkos, Ilja, 1980, et al.
(författare)
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Platform for Controlled Supramolecular Nano-Assembly
- 2009
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 9:6, s. 2482-2486
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Tidskriftsartikel (refereegranskat)abstract
- We here present a two-dimensional (2D) micro/nano-fluidic technique where reactant-doped liquid−crystal films spread and mix on micro- and nanopatterned substrates. Surface-supported phospholipid monolayers are individually doped with complementary DNA molecules which hybridize when these lipid films mix. Using lipid films to convey reactants reduces the dimensionality of traditional 3D chemistry to 2D, and possibly to 1D by confining the lipid film to nanometer-sized lanes. The hybridization event was observed by FRET using single-molecule-sensitive confocal fluorescence detection. We could successfully detect hybridization in lipid streams on 250 nm wide lanes. Our results show that the number and density of reactants as well as sequence of reactant addition can be controlled within confined liquid crystal films, providing a platform for nanochemistry with potential for kinetic control.
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| 7. |
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| 8. |
- Hannestad, Jonas, 1981
(författare)
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First As Probe, Then As Function - Fluorescence in Bio-inspired Nanotechnology
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, I demonstrate how fluorescence can be used in the context of bio-inspired nanotechnology, both as an indirect probe and as a function in itself. By combining principles and molecules from three different bio-molecular systems, DNA, bacterial light-harvesting complexes and cell membranes, I have constructed nano- and microscale systems for long-range excitation energy transfer, light-harvesting and reaction control.In the first part of the work, DNA is utilized as a scaffold for fluorophores, arranged in a manner that facilitates excitation energy transfer from either one end of a wire to the other, or from a single input to two separate outputs in a nanoscale DNA network. These photonic assemblies use Pacific Blue and Cy3 as input and output fluorophores, respectively. The network also comprises fluorescein as an alternative output. Both systems rely on the intercalator YO-PRO-1 to mediate energy transfer between input and output. With this design, it is possible to construct a 20-mer wire with over 90% end-to-end efficiency and a longer 50-mer wire that enables energy transfer over more than 20 nm. In the network, it is possible to regulate the flow of excitation energy between the two spatially and spectrally separate outputs.In the second part, a DNA-based light-harvesting complex is presented. By loading the DNA scaffold with intercalators it is possible to enhance the excitation of a membrane-anchored porphyrin acceptor through energy transfer from the YO-PRO-1 donors. Using a linear and a hexagonal light-harvesting complex the excitation of the acceptor porphyrin can be enhanced by a factor of 13 and 18, respectively. Finally in the third part, lipid monolayer films with incorporated DNA molecules are created on hydrophobic substrates. DNA moves inside the film and can therefore interact and form duplexes with complementary strands also incorporated in the film. This process is studied in patterned surfaces where the mixing of lipid films is restricted by the shape of the hydrophobic support. The hybridization mechanism is investigated using single molecule fluorescence spectroscopy, showing that the duplex formation rate depends on the length of the DNA strand.
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| 9. |
- Hannestad, Jonas, 1981, et al.
(författare)
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Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy
- 2013
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 7:1, s. 308-315
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Tidskriftsartikel (refereegranskat)abstract
- We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger.
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| 10. |
- Hannestad, Jonas, 1981, et al.
(författare)
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Nanometer-scale molecular organization in lipid membranes studied by time-of-flight secondary ion mass spectrometry
- 2018
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Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- The organization of lipid membranes plays an important role in a wide range of biological processes at different length scales. Herein, the authors present a procedure based on time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize the nanometer-scale ordering of lipids in lipid membrane structures on surfaces. While ToF-SIMS is a powerful tool for label-free analysis of lipid-containing samples, its limited spatial resolution prevents in-depth knowledge of how lipid properties affect the molecular assembly of the membrane. The authors overcome this limitation by measuring the formation of lipid dimers, originating in the same nanometer-sized primary ion impact areas. The lipid dimers reflect the local lipid environment and thus allow us to characterize the membrane miscibility on the nanometer level. Using this technique, the authors show that the chemical properties of the constituting lipids are critical for the structure and organization of the membrane on both the nanometer and micrometer length scales. Our results show that even at lipid surface compositions favoring two-phase systems, lipids are still extracted from solid, gel phase, domains into the surrounding fluid supported lipid bilayer surrounding the gel phase domains. The technique offers a means to obtain detailed knowledge of the chemical composition and organization of lipid membranes with potential application in systems where labeling is not possible, such as cell-derived supported lipid bilayers.
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| 11. |
- Hannestad, Jonas, 1981, et al.
(författare)
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Self-Assembled DNA-Based Fluorescence Waveguide with Selectable Output
- 2011
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 7:22, s. 3178-3185
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Tidskriftsartikel (refereegranskat)abstract
- Using the principle of self-assembly, a fluorescence-based photonic network is constructed with one input and two spatially and spectrally distinct outputs. A hexagonal DNA nanoassembly is used as a scaffold to host both the input and output dyes. The use of DNA to host functional groups enables spatial resolution on the level of single base pairs, well below the wavelength of light. Communication between the input and output dyes is achieved through excitation energy transfer. Output selection is achieved by the addition of a mediator dye intercalating between the DNA base pairs transferring the excitation energy from input to output through energy hopping. This creates a tool for selective excitation energy transfer on the nanometer scale with spectral and spatial control. The ability to direct excitation energy in a controlled way on the nanometer scale is important for the incorporation of photochemical processes in nanotechnology.
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| 12. |
- Hannestad, Jonas, 1981, et al.
(författare)
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Self-assembled DNA photonic wire
- 2008
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Ingår i: Nucleic acids symposium series (2004). - 1746-8272. ; :52, s. 685-685
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Tidskriftsartikel (refereegranskat)abstract
- DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several nanometer regime and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. Our results show that it is possible to create two component DNA-based photonic wires capable of long range energy transfer using a straightforward self-assembly approach.
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| 13. |
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| 14. |
- Hannestad, Jonas, 1981, et al.
(författare)
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Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:25, s. 14178-14186
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Tidskriftsartikel (refereegranskat)abstract
- The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).
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| 15. |
- Huijser, Annemarie, et al.
(författare)
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UV-Dissipation Mechanisms in the Eumelanin Building Block DHICA
- 2010
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Ingår i: ChemPhysChem. - : Wiley. - 1439-7641 .- 1439-4235. ; 11:11, s. 2424-2431
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Tidskriftsartikel (refereegranskat)abstract
- The UV-dissipative mechanisms of the eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and the 4,7-dideutero derivative (DHICA-d(2)) in buffered H2O or D2O have been characterized by using ultrafast time-resolved fluorescence spectroscopy. Excitation of the carboxylate anion form, the dominating state at neutral pH, leads to dual fluorescence. The band peaking at lambda = 378 nm is caused by emission from the excited initial geometry. The second band around lambda = 450 nm is owed to a complex formed between the monoanion and specific buffer components. In the absence of complex formation, the mono-anion solely decays non-radiatively or by emission with a lifetime of about 2.1 ns. Excitation of the neutral carboxylic acid state, which dominates at acidic pH, leads to a weak emission around lambda = 427 nm with a short lifetime of 240 ps. This emission originates from the zwitterionic state, formed upon excitation of the neutral state by sub-ps excited-state intramolecular proton transfer (ESIPT) between the carboxylic acid group and the indole nitrogen. Future studies will unravel whether this also occurs in larger building blocks and ESIPT is a built-in photoprotective mechanism in epidermal eumelanin.
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| 16. |
- Woller, Jakob, 1984, et al.
(författare)
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Self-Assembled Nanoscale DNA-Porphyrin Complex for Artificial Light Harvesting
- 2013
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 135:7, s. 2759-2768
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Tidskriftsartikel (refereegranskat)abstract
- Mimicking green plants' and bacteria's extraordinary ability to absorb a vast number of photons and harness their energy is a longstanding goal in artificial photosynthesis. Resonance energy transfer among donor dyes has been shown to play a crucial role on the overall transfer of energy in the natural systems. Here, we present artificial, self-assembled, light-harvesting complexes consisting of DNA scaffolds, intercalated YO-PRO-1 (YO) donor dyes and a porphyrin acceptor anchored to a lipid bilayer, conceptually mimicking the natural light-harvesting systems. A model system consisting of 39-mer duplex DNA in a linear wire configuration with the porphyrin attached in the middle of the wire is primarily investigated. Utilizing intercalated donor fluorophores to sensitize the excitation of the porphyrin acceptor, we obtain an effective absorption coefficient 12 times larger than for direct excitation of the porphyrin. On the basis of steady-state and time-resolved emission measurements and Markov chain simulations, we show that YO-to-YO resonance energy transfer substantially contributes to the overall flow of energy to the porphyrin. This increase is explained through energy migration along the wire allowing the excited state energy to transfer to positions closer to the porphyrin. The versatility of DNA as a structural material is demonstrated through the construction of a more complex, hexagonal, light-harvesting scaffold yielding further increase in the effective absorption coefficient. Our results show that, by using DNA as a scaffold, we are able to arrange chromophores on a nanometer scale and in this way facilitate the assembly of efficient light-harvesting systems.
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