Anders Lundgren

Immune complement activation is attenuated by surface nanotopography.

The immune complement (IC) is a cell-free protein cascade system, and the first part of the innate immune system to recognize foreign objects that enter the body. Elevated activation of the system from, for example, biomaterials or medical devices can result in both local and systemic adverse effects and eventually loss of function or rejection of the biomaterial. Here, the researchers have studied the effect of surface nanotopography on the activation of the IC system. By a simple nonlithographic process, gold nanoparticles with an average size of 58 nm were immobilized on a smooth gold substrate, creating surfaces where a nanostructure is introduced without changing the surface chemistry. The activation of the IC on smooth and nanostructured surfaces was viewed with fluorescence microscopy and quantified with quartz crystal microbalance with dissipation monitoring in human serum. Additionally, the ability of pre-adsorbed human immunoglobulin G (IgG) (a potent activator of the IC) to activate the IC after a change in surface hydrophobicity was studied. It was found that the activation of the IC was significantly attenuated on nanostructured surfaces with nearly a 50% reduction, even after pre-adsorption with IgG. An increase in surface hydrophobicity blunted this effect. The possible role of the curvature of the nanoparticles for the orientation of adsorbed IgG molecules, and how this can affect the subsequent activation of the IC, are discussed. The present findings are important for further understanding of how surface nanotopography affects complex protein adsorption, and for the future development of biomaterials and blood-contacting devices.

Gradients in surface nanotopography used to study platelet adhesion and activation

Gradients in surface nanotopography were prepared by adsorbing gold nanoparticles on smooth gold substrates using diffusion technique. Following a sintering procedure the particle binding chemistry was removed, and integration of the particles into the underlying gold substrate was achieved, leaving a nanostructured surface with uniform surface chemistry. After pre-adsorption of human fibrinogen, the effect of surface nanotopography on platelets was studied. The use of a gradient in nanotopography allowed for platelet adhesion and activation to be studied as a function of nanoparticle coverage on one single substrate. A peak in platelet adhesion was found at 23% nanoparticle surface coverage. The highest number of activated platelets was found on the smooth control part of the surface, and did not coincide with the number of adhered platelets. Activation correlated inversely with particle coverage, hence the lowest fraction of activated platelets was found at high particle coverage. Hydrophobization of the gradient surface lowered the total number of adhering cells, but not the ratio of activated cells. Little or no effect was seen on gradients with 36nm particles, suggesting the existence of a lower limit for sensing of surface nano-roughness in platelets. These results demonstrate that parameters such as ratio between size and inter-particle distance can be more relevant for cell response than wettability on nanostructured surfaces. The minor effect of hydrophobicity, the generally reduced activation on nanostructured surfaces and the presence of a cut-off in activation of human platelets as a function of nanoparticle size could have implications for the design of future blood-contacting biomaterials.

Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells.

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 nm 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.

Resonance-Mode Electrochemical Impedance Measurements of Silicon Dioxide Supported Lipid Bilayer Formation and Ion Channel Mediated Charge Transport

A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO(2) substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO(2), and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO(2). In addition, by adding the ion channel gramicidin D to bilayers supported on SiO(2), channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA).

Two-dimensional flow nanometry of biological nanoparticles for accurate determination of their size and emission intensity

Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging. Optical microscopy allows precise determination of fluorescence/scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. Here, we show that by attaching BNPs to a supported lipid bilayer, subjecting them to hydrodynamic flows and tracking their motion via surface-sensitive optical imaging enable determination of their diffusion coefficients and flow-induced drifts, from which accurate quantification of both BNP size and emission intensity can be made. For vesicles, the accuracy of this approach is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity for radii down to 15 nm.

Computer generated phase holograms (kinoforms) for millimeter and submillimeter wavelengths

Computer generated Fourier transform phase holograms, known as kinoforms, have been synthesized, manufactured and their performance evaluated at a wavelength of 3 mm (100 GHz). The kinoforms were synthesized to give a prescribed far-field intensity distribution and manufactured by milling the computed kinoform surface relief into a Teflon plate, using a numerically controlled milling machine. The measured diffraction efficiencies exceed 50 percent. Millimeter-wave kinoforms can be used in various quasi-optical applications,e.g. distributing a local oscillator signal to an array of detector elements in heterodyne receivers.

Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations

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.

Membrane Deformation Induces Clustering of Norovirus Bound to Glycosphingolipids in a Supported Cell-Membrane Mimic

Quartz crystal microbalance with dissipation monitoring and total internal reflection fluorescence microscopy have been used to investigate binding of norovirus-like particles (noroVLPs) to a supported (phospho)lipid bilayer (SLB) containing a few percent of H or B type 1 glycosphingolipid (GSL) receptors. Although neither of these GSLs spontaneously form domains, noroVLPs were observed to form micron-sized clusters containing typically up to about 30 VLP copies, especially for B type 1, which is a higher-affinity receptor. This novel finding is explained by proposing a model implying that VLP-induced membrane deformation promotes VLP clustering, a hypothesis that was further supported by observing that functionalized gold nanoparticles were able to locally induce SLB deformation. Because similar effects are likely possible also at cellular membranes, our findings are interesting beyond a pure biophysicochemical perspective as they shed new light on what may happen during receptor-mediated uptake of viruses as well as nanocarriers in drug delivery.