Rudolf Robelek

Label-free and time-resolved measurements of cell volume changes by surface plasmon resonance (SPR) spectroscopy.

Cell volume and its regulation is one of the key players for cellular integrity and a strong indicator for several cell pathologies. But time-resolved volume measurements of adherently grown mammalian cells using established methods, such as extracellular impedance analysis or light microscopy, are complex and time-consuming. In this study, we demonstrate that surface plasmon resonance spectroscopy (SPR) is a powerful transducer device capable of reporting volume changes of cells that are directly grown on the SPR sensor surface. The approach is label-free, non-invasive and provides an outstanding time resolution. In proof-of-principle studies we recorded the volume change of confluent MDCK II cells induced by hypo- or hypertonic stimulation in a time-resolved manner. Comparison of the SPR-based experiments reported here with more recent studies using different approaches suggests a direct correlation between SPR signal shift and cell volume changes.

Label-free monitoring of cell-based assays: combining impedance analysis with SPR for multiparametric cell profiling.

Label-free approaches to monitor cell-based assays provide an unprecedented, time-resolved and non-invasive view on the response of mammalian cells to chemical, biological or physical stimuli. The most widespread techniques are impedance analysis and optical sensing using evanescent waves like SPR. This study describes the combination of both in one experimental setup so that a given cell population can be monitored simultaneously for electrical and optical changes. The device is based on commercial SPR chips that are processed by photolithography to provide electrodes for impedance analysis and gold spots for surface plasmon excitation on the same substrate. Simultaneous recordings do not interfere with each other but provide independent, time-resolved information on cell shape changes (impedance) and dynamic mass redistribution (SPR) as they occur during exposure of the cells to drugs or toxins or along their normal life cycle. This study provides proof-of-concept experiments of the dual biosensor platform in two experimental settings: signals are recorded and analyzed (i) during cell attachment, spreading and differentiation of initially suspended cells and (ii) during the exposure of the mature cells to an actin cytoskeleton disrupting drug. Impedance and SPR recordings provide complementary information that can be used to trace and assign intracellular mechanisms of action.

Real-time label-free monitoring of the cellular response to osmotic stress using conventional and long-range surface plasmons.

Cell volume and its regulation are key factors for cellular integrity and also serve as indicators of various cell pathologies. SPR sensors represent an efficient tool for real-time and label-free observations of changes in cell volume and shape. Here, we extend this concept by employing the use of long-range surface plasmons (LRSP). Due to the enhanced penetration depth of LRSP (~1μm, compared to ~0.4μm of a conventional surface plasmon), the observation of refractive index changes occurring deeper inside the cells is possible. In this work, the responses of a confluent normal rat kidney (NRK) epithelial cell layer to osmotic stress are studied by both conventional and long-range surface plasmons. Experiments are conducted in parallel using cell layers grown and stimulated under the same conditions to enable direct comparison of the results and discrimination of the osmotic stress-induced effects in different parts of the cell.

Studying Cell-Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques

A better understanding of the interactions of animal (or human) cells with in vitro surfaces is the key to the successful development, improvement and optimization of biomaterials for biomedical or biotechnological purposes. State-of-the-art experimental approaches and techniques are a prerequisite for further and deeper insights into the mechanisms and processes involved in cell-surface adhesion. This chapter provides a brief but not complete survey of optical, mechanical, electrochemical and acoustic devices that are currently used to study the structural and functional properties of the cell-surface junction. Each technique is introduced with respect to the underlying principles before example data are discussed. At the end of the chapter all techniques are compared in terms of their strengths, limitations and technical requirements.

Imaging of G protein-coupled receptors in solid-supported planar lipid membranes

This contribution summarizes first some of our efforts in imaging G-protein-coupled receptor (GPCR) functional inserted into planar tethered lipid bilayer membranes (tBLMs) as a novel platform for biophysical studies. The authors introduced recently a novel approach for the functional incorporation of membrane proteins, i.e., by their cell-free expression and in vitro reconstitution in the presence of tBLMs. By the combination of the corresponding coding DNA with the protein synthesis machinery of a cell-extract (in vitro transcription and translation), the authors observe spontaneous and vectorial insertions of an interesting example of the GPCR family into a tethered bimolecular lipid membrane: the olfactory receptor OR5. After synthesis, imaging of the surface area is performed and the resulting signals are analyzed in order to resolve quantity and lateral mobilities. Ligand-independent aggregation behavior of the GPCRs and quantitative analysis of the fluorescent signals are presented in this work.

Surface plasmon resonance spectroscopy and quartz crystal microbalance study of MutS binding with single Thymine-Guanine mismatched DNA

MutS is a DNA mismatch binding protein that recognizes heteroduplex DNA containing mispaired or unpaired bases. In this study, we employ a quartz crystal microbalance (QCM) and a surface plasmon resonance (SPR) device for the study of MutS binding with DNA containing a single Thymine-Guanine (T-G) mismatch at different sites. Multi-step surface binding reactions are involved in the study, including probe DNA immobilization on the sensor surface through biotin-streptavidin-biotin bridge chemistry, target DNA hybridization to form T-G heteroduplexes, and MutS recognition of the mutation sites. The QCM frequency (d f) and motional resistance (d R, an impedance parameter reflective of QCM damping), as well as the SPR angle shift (d q ) are recorded for the binding reactions. The combined SPR and QCM data collection and analysis allow for an assessment of not only the amount of bound biopolymer but provide also information on also the structural properties of the streptavidin, DNA and MutS/DNA complexes. The affinity of the MutS/T-G heteroduplex assembly is determined by both the QCM and SPR methods through titration of the surface bound DNA with increasing MutS concentration. It is found that if the T-G mismatch is in the center of the DNA fragment, the MutS/DNA complex is more stable than if the mismatch is located near the end of the fragment.

Crystal structures of the antitermination factor NusB from Thermotoga maritima and implications for RNA binding

NusB is a prokaryotic transcription factor involved in antitermination processes, during which it interacts with the boxA portion of the mRNA nut site. Previous studies have shown that NusB exhibits an all-helical fold, and that the protein from Escherichia coli forms monomers, while Mycobacterium tuberculosis NusB is a dimer. The functional significance of NusB dimerization is unknown. We have determined five crystal structures of NusB from Thermotoga maritima. In three crystal forms the protein appeared monomeric, whereas the two other crystal forms contained assemblies, which resembled the M. tuberculosis dimers. In solution, T. maritima NusB could be cross-linked as dimers, but it migrated as a monomer in gel-filtration analyses, suggesting a monomer/dimer equilibrium with a preference for the monomer. Binding to boxA-like RNA sequences could be detected by gel-shift analyses and UV-induced cross-linking. An N-terminal arginine-rich sequence is a probable RNA binding site of the protein, exhibiting aromatic residues as potential stacking partners for the RNA bases. Anions located in various structures support the assignment of this RNA binding site. The proposed RNA binding region is hidden in the subunit interface of dimeric NusB proteins, such as NusB from M. tuberculosis, suggesting that such dimers have to undergo a considerable conformational change or dissociate for engagement with RNA. Therefore, in certain organisms, dimerization may be employed to package NusB in an inactive form until recruitment into antitermination complexes.

Surface Plasmon Fluorescence Techniques for Bioaffinity Studies

Among the various sensing principles proposed for bioaffinity studies, optical evanescent wave techniques have gained the lead in popularity. Next to evanescent ellipsometry [1] and the various optical waveguide platforms [2,3], surface plasmon resonance (SPR) spectroscopy [4–6], in particular, has ...

Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions and their mobility within the membrane are of great importance for ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02μm-2min-1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.

NANOSCOPIC BUILDING BLOCKS FROM POLYMERS, METALS, AND SEMICONDUCTORS FOR HYBRID ARCHITECTURES

This paper describes some of our eorts in the area of nanostructured thin lm architectures. The resulting interfacial hybrid assemblies are built from (1) organic/polymeric objects based on dendrimer systems, from (2) surface-functionalized Au nanoparticles, and (3) from a variety of semiconducting quantum dots. Dendrimers as polymeric building blocks with a strictly monodisperse particle size distribution in the nanometer range can be functionalized in the core, the scaold, or at the periphery, thus oering interesting hybrid materials for a wide range of applications. The combination with Au clusters and their local surface plasmon resonances suggests new strategies for optoelectronic devices or unconventional bio-sensor platforms. The possibility of tuning the luminescent properties of semiconducting nanoparticles by size or compositional bandgap engineering complements the assembly kit with building blocks for supramolecular thin lm nanocomposite materials.