| 1. |
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| 2. |
- Briand, Elisabeth, 1979, et al.
(författare)
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Chemical Modifications of Au/SiO2 Template Substrates for Patterned Biofunctional Surfaces
- 2011
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 27:2, s. 678-685
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this work was to create patterned surfaces for localized and specific biochemical recognition. For this purpose, we have developed a protocol for orthogonal and material-selective surface modifications of microfabricated patterned surfaces composed of SiO2 areas (100 mu m diameter) surrounded by Au. The SiO2 spots were chemically modified by a sequence of reactions (silanization using an amine-terminated silane (APTES), followed by amine coupling of a biotin analogue and biospecific recognition) to achieve efficient immobilization of streptavidin in a functional form. The surrounding Au was rendered inert to protein adsorption by modification by HS(CH2)(10)CONH-(CH2)(2)(OCH2CH2)(7)OH (thiol-OEG). The surface modification protocol was developed by testing separately homogeneous SiO2 and Au surfaces, to obtain the two following results: (i) SiO2 surfaces which allowed the grafting of streptavidin, and subsequent immobilization of biotinylated antibodies, and (ii) Au surfaces showing almost no affinity for the same streptavidin and antibody solutions. The surface interactions were monitored by quartz crystal microbalance with dissipation monitoring (QCM-D), and chemical analyses were performed by polarization modulation-reflexion absorption infrared spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to assess the validity of the initial orthogonal assembly of APTES and thiol-OEG. Eventually, microscopy imaging of the modified Au/SiO2 patterned substrates validated the specific binding of streptavidin on the SiO2/APTES areas, as well as the subsequent binding of biotinylated anti-rIgG and further detection of fluorescent rIgG on the functionalized SiO2 areas. These results demonstrate a successful protocol for the preparation of patterned biofunctional surfaces, based on microfabricated Au/SiO2 templates and supported by careful surface analysis. The strong immobilization of the biomolecules resulting from the described protocol is advantageous in particular for micropatterned substrates for cell-surface interactions.
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| 3. |
- Claesson, Maria, 1985, et al.
(författare)
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Pore Spanning Lipid Bilayers on Mesoporous Silica Having Varying Pore Size
- 2011
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 27:14, s. 8974-8982
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Tidskriftsartikel (refereegranskat)abstract
- Synthetic lipid bilayers have similar properties as cell membranes and have been shown to be of great use in the development of novel biomimicry devices. In this study, lipid bilayer formation on mesoporous silica of varying pore size, 2, 4, and 6 nm, has been investigated using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescent recovery after photo bleaching (FRAP), and atomic force microscopy (AFM). The results show that pore-spanning lipid bilayers were successfully formed regardless of pore size. However, the mechanism of the bilayer formation was dependent on the pore size, and lower surface coverages of adsorbed lipid vesicles were required on the surface having the smallest pores. A similar trend was observed for the lateral diffusion coefficient (D) of fluorescently labeled lipid molecules in the membrane, which was lowest on the surface having the smallest pores and increased with the pore size. All of the pore size dependent observations are suggested to be due to the hydrophilicity of the surface, which decreases with increased pore size.
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| 4. |
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| 5. |
- Faxälv, Lars, et al.
(författare)
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Imaging of blood plasma coagulation at supported lipid membranes
- 2011
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 1095-7103 .- 0021-9797. ; 364:2, s. 582-587
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Tidskriftsartikel (refereegranskat)abstract
- The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of similar to 6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems.
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| 6. |
- Frost, Rickard, 1979, et al.
(författare)
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Bioreducible insulin-loaded nanoparticles and their interaction with model lipid membranes
- 2011
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 1095-7103 .- 0021-9797. ; 362:2, s. 575-583
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Tidskriftsartikel (refereegranskat)abstract
- To improve design processes in the field of nanomedicine, in vitro characterization of nanoparticles with systematically varied properties is of great importance. In this study, surface sensitive analytical techniques were used to evaluate the responsiveness of nano-sized drug-loaded polyelectrolyte complexes when adsorbed to model lipid membranes. Two bioreducible poly(amidoamine)s (PAAs) containing multiple disulfide linkages in the polymer backbone (SS-PAAs) were synthesized and used to form three types of nanocomplexes by self-assembly with human insulin, used as a negatively charged model protein at neutral pH. The resulting nanoparticles collapsed on top of negatively charged model membranes upon adsorption, without disrupting the membrane integrity. These structural rearrangements may occur at a cell surface which would prevent uptake of intact nanoparticles. By the addition of glutathione, the disulfide linkages in the polymer backbone of the SS-PAAs were reduced, resulting in fragmentation of the polymer and dissociation of the adsorbed nanoparticles from the membrane. A decrease in ambient pH also resulted in destabilization of the nanoparticles and desorption from the membrane. These mimics of intracellular environments suggest dissociation of the drug formulation, a process that releases the protein drug load, when the nanocomplexes reaches the interior of a cell. (C) 2011 Elsevier Inc. All rights reserved.
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| 7. |
- Frost, Rickard, 1979, et al.
(författare)
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Structural Rearrangements of Polymeric Insulin-loaded Nanoparticles Interacting with Surface-Supported Model Lipid Membranes
- 2011
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Ingår i: Journal of Biomaterials and Nanobiotechnology. - 2158-7027. ; 2:2, s. 181-193
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Tidskriftsartikel (refereegranskat)abstract
- The design and screening of nanoparticles for therapeutic applications (nanodrugs) belong to an emerging research area, where surface based analytical techniques are promising tools. This study reports on the interaction of electro- statically assembled nanoparticles, developed for non-invasive administration of human insulin, with cell membrane mimics. Interactions between the nanoparticles and differently charged surface-supported model membranes were studied in real-time with the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, in some ex- periments combined with optical reflectometry. Based on the experimental observations, we conclude that structural rearrangements of the nanoparticles occur upon adsorption to negatively charged lipid membranes.␣ The degree of structural changes in the nanoparticles will have important implications for the induced release of the protein drug load. The presented results provide an example of how a surface-based experimental platform can be used to charac- terize the physico-chemical properties of nanosized drug carriers with respect to their interactions at different surfaces.
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| 8. |
- 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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| 9. |
- 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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| 10. |
- 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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| 11. |
- Nilebäck, Erik, 1984, et al.
(författare)
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Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules
- 2011
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Ingår i: Biosensors and Bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 28:1, s. 407-413
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Tidskriftsartikel (refereegranskat)abstract
- The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10 h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.
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| 12. |
- Sundh, M., et al.
(författare)
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Formation of Supported Lipid Bilayers at Surfaces with Controlled Curvatures: Influence of Lipid Charge
- 2011
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 115:24, s. 7838-7848
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Tidskriftsartikel (refereegranskat)abstract
- We have developed and characterized novel biomimetic membranes, formed at nanostructured sensor substrates with controlled curvatures, motivated by the many biological processes that involve membrane curvature. Model systems with convex nanostructures, with radii of curvatures (ROCs) of 70, 75, and 95 nm, were fabricated utilizing colloidal assembly and used as substrates for supported lipid bilayers (SLBs). The SLBs were formed via vesicle adsorption and rupture, and the vesicle deposition pathway was studied by means of quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. SLBs conforming to the underlying nanostructured surfaces, which exhibit increased surface area with decreased ROC, were confirmed from excess mass, monitored by QCM-D, and excess total fluorescence intensities. The formation of SLBs at the nanostructured surfaces was possible, however, depending on the ROC of the structures and the lipid vesicle charge the quality varied. The presence of nanostructures was shown to impair vesicle rupture and SLB formation was progressively hindered at surfaces with structures of decreasing ROCs. The introduction of a fraction of the positively charged lipid POEPC in the lipid vesicle membrane allowed for good quality and conformal bilayers at all surfaces. Alternatively, for vesicles formed from lipid mixtures with a fraction of the negatively charged lipid POPS, SLB formation was not at all possible at surfaces with the lowest ROC. Interestingly, the vesicle adsorption rate and the SLB formation were faster at surfaces with nanostructures of progressively smaller ROCs at high ratios of POPS in the vesicles. Development of templated SLBs with controlled curvatures provides a new experimental platform, especially at the nanoscale, at which membrane events such as lipid sorting, phase separation, and protein binding can be studied.
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| 13. |
- Sundh, M., et al.
(författare)
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Supported Lipid Bilayers With Controlled Curvature via Colloidal Lithography
- 2011
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Ingår i: IEEE Transactions on Nanobioscience. - 1536-1241. ; 10:3, s. 187-193
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Tidskriftsartikel (refereegranskat)abstract
- Supported lipid bilayers (SLBs) at surfaces provide a route to quantitatively study molecular interactions with and at lipid membranes via different surface-based analytical techniques. Here, a method to fabricate SLBs with controlled curvatures, in the nanometer regime over large areas, is presented, utilizing lipid vesicle rupture onto nanostructured sensor substrates. Heat treated colloidal particle masks were used as templates to produce silicon dioxide films with systematically varied radius of curvature (ROC, 70 to 170 nm are demonstrated) and quartz crystal microbalance with dissipation monitoring (QCM-D) was used to confirm vesicle rupture onto such structured surfaces. Fluorescence microscopy was used to show fluidity of the supported membranes. The formation of confluent SLBs is demonstrated at the nanostructured surfaces from vesicles composed of POPC lipids. However, at surfaces with decreasing ROCs, vesicle rupture was hindered but with an increasing fraction of the positively charged lipid POEPC in the vesicles, it was possible to form good quality supported bilayers on all curvatures studied. Curved SLBs open up the possibility to systematically study the influence of curvature on molecular interactions at lipid membranes.
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