Ingo Köper

Reusable Localized Surface Plasmon Sensors Based on Ultrastable Nanostructures

Nanoparticle arrays created by nanosphere lithography are widely used in sensing applications since their localized surface plasmon resonances are extremely sensitive to changes in the local dielectric environment. A major drawback for any biologically oriented sensing application of conventionally produced particle arrays is the lack of stability of the nanoparticles in aqueous media and buffer solutions. Here, a robust and reusable nanoscale sensing platform based on localized surface plasmon resonances of gold nanoparticles embedded in a silicon dioxide matrix is presented. The architecture exhibits extremely high stability in aqueous environments and can be regenerated several times by simple mechanical cleaning of the surface. The platforms surface is ultraflat by design, thus making it an ideal substrate for any bio-oriented sensing application.

Stable insulating tethered bilayer lipid membranes

Tethered bilayer lipid membranes have been shown to be an excellent model system for biological membranes. Coupling of a membrane to a solid supports creates a stable system that is accessible for various surface analytical tools. Good electrical sealing properties also enable the use of the membranes in practical sensing applications. The authors have shown that tethered membranes have extended lifetimes up to several months. Air-stability of the bilayer can be achieved by coating the membrane with a hydrogel. The structure of a monolayer and its stability under applied dc potentials have been investigated by neutron scattering.

Functional ion channels in tethered bilayer membranes - Implications for biosensors

The demand for rapid in situ detection of chemical and biological analytes has increased the interest in the development of biosensors, which combine biological sensing elements with physicochemical transducers. Engineered membrane-bound ion channels are one promising class of biological receptors because they allow for highly sensitive stochastic detection of analytes, and produce a well-defined read-out that is inherently suitable for digitization. However, in order to perform stochastic sensing, it is necessary to measure the ion currents associated with single ion channel opening and closing events. Although, sensors based on supported tethered bilayers that contain various pore forming proteins have been described, there is still great limitations in creating a signal-to-noise ratio that is high enough to allow for single-channel activity detection. An alternative way to design bilayers on a chip, in which the lipid membrane covers an aperture, has been proposed. This technique has proven sensitive enough for detection of single ionchannel activity. However, this approach is fundamentally different to the tethering of bilayers onto a stable solid surface, and is likely to cause problems due to low mechanical stability. Here, we present a biosensor based on modulation of single ion-channel activity, with the ability to detect analytes in the micromolar range. The ion channels were interfaced to a gold surface, where they were reconstituted into tethered bilayer lipid membranes (tBLMs), which were in turn formed at multiple individual pixels of a microelectrode array device. The limited size of the gold sense pad surface (100;100 mm) and the electrical stability of the overlying lipid bilayer membrane made each pixel sensitive enough to measure single ion-channel currents in the picoampere range, and yet the device is convenient for monolithically integrated fabrication schemes. The biosensor is illustrated in Figure 1. Recently we were able to measure, for the first time, single ion-channel activity by using gramicidin A (gA), which was ACHTUNGTRENNUNGdirectly interfaced to a gold device surface. Even though gA can be modified to be used as a sensor there are still limitations in its use due to its relatively simple chemical struc-

Formation of tethered bilayer lipid membranes probed by various surface sensitive techniques

Tethered bilayer lipid membranes are promising biomimetic architectures. Their formation has been investigated using four different surface sensitive techniques, including optical, acoustic, and electrical methods. The lipid bilayers are built in a two-step procedure; the proximal layer is formed by self-assembly and is then completed to a bilayer by fusion with small vesicles. The different technical approaches revealed specific aspects of the layer formation processes, namely, first a fast adsorption process followed by a longer rearrangement period. Similar phenomena have been observed for the vesicle fusion process. The results allow for a more controlled assembly protocol for the preparation of highly insulating lipid membranes.

Structural Analysis of Tethered Bilayer Lipid Membranes

Solid supported membrane systems have been established as biomimetic architectures, which allow for the systematic investigation of various membrane-related processes. Especially tethered bilayer lipid membranes have been a successful concept. They consist of a lipid bilayer that is covalently anchored to a solid substrate through a spacer group. The submembrane part, which is defined by the spacer group, is important especially for the biological activity of incorporated membrane proteins. Anchor lipids with different spacer and anchor groups have been synthesized, and the resulting membrane structures have been investigated by neutron reflectivity. The different molecular architectures had a significant effect on both the amount of water incorporated in the spacer region and the electrical properties of the bilayer. A detailed understanding of the structure-function relationship allows for an optimized design of the molecular architecture with respect to possible applications, for example an optimized protein incorporation.

Vesicle Adsorption and Phospholipid Bilayer Formation on Topographically and Chemically Nanostructured Surfaces

We have investigated the influence of combined nanoscale topography and surface chemistry on lipid vesicle adsorption and supported bilayer formation on well-controlled model surfaces. To this end, we utilized colloidal lithography to nanofabricate pitted Au-SiO(2) surfaces, where the top surface and the walls of the pits consisted of silicon dioxide whereas the bottom of the pits was made of gold. The diameter and height of the pits were fixed at 107 and 25 nm, respectively. Using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique and atomic force microscopy (AFM), we monitored the processes occurring upon exposure of these nanostructured surfaces to a solution of extruded unilamellar 1-palmitolyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles with a nominal diameter of 100 nm. To scrutinize the influence of surface chemistry, we studied two cases: (1) the bare gold surface at the bottom of the pits and (2) the gold passivated by biotinamidocaproyl-labeled bovine serum albumin (BBSA) prior to vesicle exposure. As in our previous work on pitted silicon dioxide surfaces, we found that the pit edges promote bilayer formation on the SiO(2) surface for the vesicle size used here in both cases. Whereas in the first case we observed a slow, continuous adsorption of intact vesicles onto the gold surface at the bottom of the pits, the presence of BBSA in the second case prevented the adsorption of intact vesicles into the pits. Instead, our experimental results, together with free energy calculations for various potential membrane configurations, indicate the formation of a continuous, supported lipid bilayer that spans across the pits. These results are significantly important for various biotechnology applications utilizing patterned lipid bilayers and highlight the power of the combined QCM-D/AFM approach to study the mechanism of lipid bilayer formation on nanostructured surfaces.

Adsorption and Conformation Behavior of Biotinylated Fibronectin on Streptavidin-Modified TiOX Surfaces Studied by SPR and AFM

It is well-known that protein-modified implant surfaces such as TiO(2) show a higher bioconductivity. Fibronectin is a glycoprotein from the extracellular matrix (ECM) with a major role in cell adhesion. It can be applied on titanium oxide surfaces to accelerate implant integration. Not only the surface concentration but also the presentation of the protein plays an important role for the cellular response. We were able to show that TiO(X) surfaces modified with biotinylated fibronectin adsorbed on a streptavidin-silane self-assembly multilayer system are more effective regarding osteoblast adhesion than surfaces modified with nonspecifically bound fibronectin. The adsorption and conformation behavior of biotinylated and nonbiotinylated (native) fibronectin was studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Imaging of the protein modification revealed that fibronectin adopts different conformations on nonmodified compared to streptavidin-modified TiO(X) surfaces. This conformational change of biotinylated fibronectin on the streptavidin monolayer delivers a fibronectin structure similar to the conformation inside the ECM and therefore explains the higher cell affinity for these surfaces.

Photocurrent response from vertically aligned single-walled carbon nanotube arrays

Vertically-aligned arrays of single walled carbon nanotubes were created on an optically transparent electrode (FTO glass) these arrays were found to exhibit a prompt current and voltage when exposed to light. These cells were then investigated by electrochemical impedance spectroscopy and found to exhibit a dampening of the recombination reaction over the first 24 hours. Symmetrical cell modeling was successful in simulating the behavior of normal cell architecture.

Assembly of the M2 Tetramer Is Strongly Modulated by Lipid Chain Length

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.

Functional Tethered Bilayer Lipid Membranes on Aluminum Oxide

Tethered bilayer lipid membranes are established as well-suited model membrane systems adaptable to different surfaces, for example, gold and silicon. These solid supported membranes are highly flexible in their tethering and lipid parts and can thus be optimized for functional incorporation of membrane proteins. The excellent sealing properties of the tethered membranes allow incorporated ion-channel proteins to be investigated. Preparation of ultrasmooth aluminum oxide by sputtering and synthesis of new tethering lipids with phosphonic acid anchor groups enable formation of an electrically sealing membrane on this surface. This process is monitored by electrochemical impedance spectroscopy and by surface plasmon resonance spectroscopy. High sealing performance of the membrane and functional incorporation of the ion carrier valinomycin are demonstrated.