Sofia Svedhem

Patterns of DNA-Labeled and scFv-Antibody-Carrying Lipid Vesicles Directed by Material-Specific Immobilization of DNA and Supported Lipid Bilayer Formation on an Au/SiO2 Template

Much effort is currently concentrated on research devoted to biofunctional patterned surfaces, which constitute the fundament for the development of microarrays for high-throughput gene and protein analyses. DNA microarrays have proved very successful, and the concept is in the process of being applied to protein arrays. However, in contrast to DNA fragments, proteins are easily denatured in contact with solid supports, and robotic printing of proteins onto chemically reactive glass slides will not necessarily be applicable as a generic protocol for the preparation of protein arrays. Supported phosphatidylcholine lipid bilayers have emerged as interesting candidate substrates for protein chips, since they efficiently reduce nonspecific protein adsorption 5] and, at the same time, allow different strategies for protein immobilization with biospecific water, desalted with a NAP5 column (Amersham Pharmacia, USA) according to manufacturers protocols, and stored as working stock solutions at 20 C until use. Epoxy-derivatized slides were prepared from plain glass slides (Sigma, USA) as previously described. Nhydroxysuccinimide slides were also used to spot the proteins but consistently gave inferior results. Proteins were prepared in NaHCO3 buffer (0.1M, pH 9) and arrayed on epoxy slides with a spacing of 180 m between the spots by using an statistical microarray analysis arrayer (Engineering Services Inc. , Ontario, Canada). After a 2-hour incubation period the slides were either used immediately, or stored for future use at 4 C. The slides, if stored, were typically used within 48 h of printing.

Cell membrane damage and protein interaction induced by copper containing nanoparticles—Importance of the metal release process

Cu-containing nanoparticles are used in various applications in order to e.g. achieve antimicrobial activities and to increase the conductivity of fluids and polymers. Several studies have reported on toxic effects of such particles but the mechanisms are not completely clear. The aim of this study was to investigate the interactions between cell membranes and well-characterized nanoparticles of CuO, Cu metal, a binary Cu-Zn alloy and micron-sized Cu metal particles. This was conducted via in vitro investigations of the effects of the nanoparticles on (i) cell membrane damage on lung epithelial cells (A549), (ii) membrane rupture of red blood cells (hemolysis), complemented by (iii) nanoparticle interaction studies with a model lipid membrane using quartz crystal microbalance with dissipation monitoring (QCM-D). The results revealed that nanoparticles of the Cu metal and the Cu-Zn alloy were both highly membrane damaging and caused a rapid (within 1h) increase in membrane damage at a particle mass dose of 20 μg/mL, whereas the CuO nanoparticles and the micron-sized Cu metal particles showed no such effect. At similar nanoparticle surface area doses, the nano and micron-sized Cu particles showed more similar effects. The commonly used LDH (lactate dehydrogenase) assay for analysis of membrane damage was found impossible to use due to nanoparticle-assay interactions. None of the particles induced any hemolytic effects on red blood cells when investigated up to high particle concentrations (1mg/mL). However, both Cu and Cu-Zn nanoparticles caused hemoglobin aggregation/precipitation, a process that would conceal a possible hemolytic effect. Studies on interactions between the nanoparticles and a model membrane using QCM-D indicated a small difference between the investigated particles. Results of this study suggest that the observed membrane damage is caused by the metal release process at the cell membrane surface and highlight differences in reactivity between metallic nanoparticles of Cu and Cu-Zn and nanoparticles of CuO.

Graphene Oxide and Lipid Membranes: Interactions and Nanocomposite Structures

We have investigated the interaction between graphene oxide and lipid membranes, using both supported lipid membranes and supported liposomes. Also, the reverse situation, where a surface coated with graphene oxide was exposed to liposomes in solution, was studied. We discovered graphene oxide-induced rupture of preadsorbed liposomes and the formation of a nanocomposite, bio-nonbio multilayer structure, consisting of alternating graphene oxide monolayers and lipid membranes. The assembly process was monitored in real time by two complementary surface analytical techniques (the quartz crystal microbalance with dissipation monitoring technique (QCM-D) and dual polarization interferometry (DPI)), and the formed structures were imaged with atomic force microscopy (AFM). From a basic science point of view, the results point toward the importance of electrostatic interactions between graphene oxide and lipid headgroups. Implications from a more practical point of view concern structure-activity relationship for biological health/safety aspects of graphene oxide and the potential of the nanocomposite, multilayer structure as scaffolds for advanced biomolecular functions and sensing applications.

The Inflammation-associated Protein TSG-6 Cross-links Hyaluronan via Hyaluronan-induced TSG-6 Oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).

QCM-D and Reflectometry Instrument: Applications to Supported Lipid Structures and Their Biomolecular Interactions

A novel setup was recently developed, combining quartz crystal microbalance with dissipation monitoring (QCM-D) and optical reflectometry for measurements on one and the same surface of, for example, biomolecular adlayers and interactions ( Rev. Sci. Instr. 2008 , 79 075107 ). This combination was chosen on the basis of prior experience of using QCM-D and optical techniques in separate instruments, which showed both the advantage of employing multiple techniques and the disadvantage of not working with the same surface and (flow) cell. The new instrument provides, for example, information about associated water and structural changes of the adlayers that would often pass unnoticed or be hard to interpret or quantify, using either technique alone. The triple response instrument (QCM-D frequency and dissipation and reflectometry) is here applied to four model systems: (A) formation of supported lipid bilayers (SLBs), (B) lipid exchange between a SLB and transiently adsorbed vesicles, (C) binding of a hydrated peptide on a functionalized SLB, and (D) streptavidin coupling to a biotinylated SLB, followed by attachment of biotinylated vesicles. The results demonstrate three major advantages of the combination instrument: (i) much faster data collection because the experiments are done on one surface for all signals, (ii) a common time axis and the same relative importance of surface kinetics and mass transport because the same liquid sample and the same transport conditions apply, and (iii) new features are discovered about the studied system that would be difficult to unravel in separate instruments.

Lipid Transfer between Charged Supported Lipid Bilayers and Oppositely Charged Vesicles

The bidirectional transfer of phospholipids between a charged, supported lipid bilayer (SLB) on SiO(2) and oppositely charged, unilamellar vesicles was studied by means of quartz crystal microbalance with dissipation (QCM-D) and optical reflectometry techniques. SLBs and vesicles were prepared from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) mixed with different fractions of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) (negatively charged) or 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) (positively charged). The interaction process consists of an attachment-transfer-detachment (ATD) sequence, where added vesicles first attach to and interact with the SLB, after which they detach, leaving behind a compositionally modified SLB and ditto vesicles. When the process is complete, there is no net addition or reduction of total lipid mass in the SLB, but lipid exchange has occurred. The time scale of the process varies from a few to many tens of minutes depending on the type of charged lipid molecule and the relative concentration of charged lipids in the two membranes. Electrostatically symmetric cases, where only the charge sign (but not the fraction of charged lipid) was reversed between the SLB and the vesicles, produce qualitatively similar but quantitatively different kinetics. The time scale of the interaction varies significantly between the two cases, which is attributed to a combination of the differences in the molecular structure of the lipid headgroup for the positively and the negatively charged lipids used, and to nonsymmetric distribution of charged lipids in the lipid membranes. The maximum amounts of attached vesicles during the ATD process were estimated to be 25-40% of a full monolayer of vesicles, with the precise amount depending on the actual charge fractions in the vesicles and the SLB. Interrupted vesicle exposure experiments, and experiments where the bulk concentration of vesicles was varied, show that vesicles in some cases may be trapped irreversibly on the SLB, when only partial transfer of lipid molecules has occurred. Additional supply of vesicles and further transfer induces detachment, when a sufficient amount of oppositely charged lipids has been transferred to the SLB, so that the latter becomes repulsive to the attached vesicles. Possible mechanistic scenarios, including monomer insertion and hemifusion models, are discussed. The observed phenomena and the actual SLB preparation process form a platform both for studies of various intermembrane molecular transfer processes and for modifying the composition of SLBs in a controlled way, for example, for biosensor and cell culture applications.

Combined QCM-D and EIS study of supported lipid bilayer formation and interaction with pore-forming peptides.

A novel set-up combining the quartz crystal microbalance with dissipation monitoring technique (QCM-D) and electrochemical impedance spectroscopy (EIS) under flow conditions was successfully used to follow supported lipid bilayer (SLB) formation on SiO(2). This study demonstrates the simultaneous detection, in real time, of both the electrical and the structural properties of the SLB. The combination of the two techniques provided novel insights regarding the mechanism of SLB formation: we found indications for an annealing process of the lipid alkyl chains after the mass corresponding to complete bilayer coverage had been deposited. Moreover, the interaction of the SLB with the pore-forming toxin, gramicidin D (grD) was studied for grD concentrations ranging from 0.05 to 40 mg L(-1). Membrane properties were altered depending on the toxin concentration. For low grD concentrations, the electrical properties of the SLB changed upon insertion of active ion channels. For higher concentrations, the QCM-D data showed dramatic changes in the viscoelastic properties of the membrane while the EIS spectra did not change. AFM confirmed significant structural changes of the membrane at higher grD concentrations. Thus, the application of combined QCM-D and EIS detection provides complementary information about the system under study. This information will be particularly important for the continued detailed investigation of interactions at model membrane surfaces.

Reversible Changes in Cell Morphology due to Cytoskeletal Rearrangements Measured in Real-Time by QCM-D

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.

A combined reflectometry and quartz crystal microbalance with dissipation setup for surface interaction studies

We have developed an instrument for surface interaction studies, which combines a newly invented four detector optical reflectometry setup with quartz crystal microbalance with dissipation (QCM-D) monitoring. The design is such that data from both techniques can be obtained simultaneously on the same sensor surface, with the same signal-to-noise ratio and time resolution, as for the individual techniques. In addition, synchronized information about structural transformations, molecular mass, and the hydration of thin films on solid surfaces can be obtained on the same specimen, as validated by monitoring the formation of supported lipid bilayers on a silica-coated QCM sensor surface. We emphasize that the optical (molecular) mass can be separated from the acoustic mass including hydrodynamically coupled solvent, which means, in turn, that the amount of solvent sensed by the QCM-D technique can be dynamically resolved during adsorption processes. In addition, the advantage/necessity to use four, compared to two, detector reflectometry is emphasized.

Tuning Cell Adhesion and Growth on Biomimetic Polyelectrolyte Multilayers by Variation of pH During Layer-by-Layer Assembly

Polyelectrolyte multilayers of chitosan and heparin are assembled on glass where heparin is applied at pH = 4, 9 and 4 during the formation of the first layers followed by pH = 9 at the last steps (denoted pH 4 + 9). Measurements of wetting properties, layer mass, and topography show that multilayers formed at pH = 4 are thicker, contain more water and have a smoother surface compared to those prepared at pH = 9 while the pH = 4 + 9 multilayers expressed intermediate properties. pH = 9 multilayers are more cell adhesive and support growth of C2C12 cells better than pH = 4 ones. However, pH 4 + 9 conditions improve the bioactivity to a similar level of pH = 9 layers. Multilayers prepared using pH 4 + 9 conditions form thick enough layers that may allow efficient loading of bioactive molecules.