Bernhard Menges

Sensitivity studies for specific binding reactions using the biotin/streptavidin system by evanescent optical methods

The combination of various evanescent optical methods such as surface plasmon spectroscopy, waveguide mode spectroscopy and an integrated optical Mach-Zehnder-interferometer are used to characterize biotinylated self-assembled monolayers as well as the binding of streptavidin to these labels. The aim of designing a highly specific and sensitive, re-usable affinity sensor for antigens on the basis of an integrated optical Mach-Zehnder interferometer is based on a proper understanding of the characteristics of the entire binding matrix architecture. Therefore, a variety of biotin-derivatives immobilized in a monolayer are investigated with respect to their affinity to streptavidin and the possibility to remove the steptavidin layer specifically. The density of the streptavidin layer as well as the optical constants of the involved molecules are measured. Finally the integrated optical Mach-Zehnder interferometer is tested with respect to the sensitivity to an antigen-antibody binding reaction. An attempt to further increase the sensitivity by simultaneous detection of a fluorescence signal failed due to bleaching effects.

Simultaneous determination of optical and acoustic thicknesses of protein layers using surface plasmon resonance spectroscopy and quartz crystal microweighing

The optical and the acoustic thicknesses of protein layers during adsorption were simultaneously determined by a combination of surface plasmon resonance spectroscopy and quartz crystal microweighing. Coupling of the surface plasmon was achieved by etching a diffraction grating into the quartz surface prior to the deposition of the metal electrode. The evolution of the acoustic and the optical thickness was markedly different, which is attributed to surface roughness and partial coverage.

Refractive Index and Thickness Determination of Monolayers by Multi Mode Waveguide Coupled Surface Plasmons

Abstract The coupling of p-polarized waveguide modes into waveguide-surface plasmon coupled modes is a promising concept to combine the features of propagating waveguide modes with the resonant field enhancement of surface plasmons. We demonstrate a waveguide device which allows to couple two TM modes to the surface plasmon. For a given waveguide device and a fixed laser wavelength the development of the imaginary part, κ, of the propagation constant, Neff, of the waveguide-surface plasmon coupled modes are simulated to optimize a monolayer sensitive device. The waveguide attenuation is very sensitive to the refractive index architecture within the evanescent field of these combined modes. This resonant behavior was used to measure the refractive index and the geometrical thickness of self-assembled monolayers independently from each other. In addition the binding of uracil to a binary monolayer containing adenin was investigated.

Immobilization of biomolecules to plasma polymerized pentafluorophenyl methacrylate.

Thin films of plasma polymerized pentafluorophenyl methacrylate (pp-PFM) offer highly reactive ester groups throughout the structure of the film that allow for subsequent reactions with different aminated reagents and biological molecules. The present paper follows on from previous work on the plasma deposition of pentafluorophenyl methacrylate (PFM) for optimum functional group retention (Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927) and reactivity in aqueous solution (Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Förch, R. Plasma Process. Polym. 2010, accepted for publication) to investigate the binding of a biologically active peptide known to induce cellular adhesion (IKVAV) and of biochemically active proteins such as BSA and fibrinogen. Analyses of the films and of the immobilization of the biomolecules were carried out using infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The attachment of the biomolecules on pulsed plasma polymerized pentafluorophenyl methacrylate was monitored using surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the presence of immobilized biomolecules on the plasma polymer and was used to determine the mass coverage of the peptide and proteins adsorbed onto the films. The combined analysis of the surfaces suggests the covalent binding of the peptide and proteins to the surface of the pp-PFM.

An anthracene-containing PMMA derivative for photoresist and channel waveguide applications

Abstract We study the possibilities for photopatterning a polymethylmethacrylate derivative carrying an anthracene group in the side chain. This material allows the preparation of high quality optical films by spin coating. Upon irradiation of these films with UV light changes in the optical constants, refractive index and absorption, and the degree of solubility can be induced by photocrosslinking and endoperoxide formation. These features qualify this polymer as a promising material for both integrated optics and photolithography or a combination of both.

Probing mobility and structural inhomogeneities in grafted hydrogel films by fluorescence correlation spectroscopy

We employed fluorescence correlation spectroscopy to investigate the effect of crosslinking density, annealing in the dry state, temperature, and solvent quality on the one-dimensional swelling, permeability, and mobility of tracer molecules in thermoresponsive hydrogel films. These consist of a carboxylated poly(N-isopropylacrylamide) derivative (PNIPAAm) covalently anchored to glass substrates. Upon increasing the temperature beyond the collapse transition at about 32 °C, the gels shrunk from the swollen to a collapsed state. A molecular dye (Alexa 647) and green fluorescent protein were chosen as tracers as they display only weak interaction with the carboxylated PNIPAAm. At large swelling ratios (low temperatures) the hydrogel layers are spatially homogeneous and both tracers show single Fickian diffusion. Diffusion coefficients scale with the PNIPAAm volume fraction. Upon temperature increase a qualitatively different behavior is observed already in the pretransition region (25–32 °C) concurrently with moderate swelling ratios (<4). This is manifested by an additional, faster Fickian diffusion and structural inhomogeneities, which are also found by optical waveguide mode spectroscopy. Above the collapse transition all diffusants are expelled from the hydrogels at a limiting swelling ratio ∼1.5. Subtle differences in the solvent quality influence the diffusion of tracers in the PNIPAAm hydrogel films. In the transition temperature range structural inhomogeneities at the nanoscale appeared.

The swelling behaviour of thermoresponsive hydrogel/silica nanoparticle composites

A new responsive nanocomposite material consisting of a poly-(N-isopropylacrylamide) hydrogel and “super-crosslinking” silica nanoparticles was prepared by mixing both components in solution, spincoating a thin film, and photocrosslinking by UV irradiation. Detailed analysis of the thermal response of these water-swollen films by means of surface plasmon resonance and optical waveguide spectroscopy revealed that the composite is very stable and has excellent responsive properties; it is of high optical homogeneity; admixture of the nanoparticles (up to 50%-wt) does not affect the critical volume collapse temperature; and swell-collapse cycles are highly reproducible and display only limited hysteresis. Thus, the composite is promising as a scaffold for further functionalisation and incorporation in sensors or actuators.

Analysis of optical gradient profiles during temperature- and salt-dependent swelling of thin responsive hydrogel films

Surface-attached, cross-linked hydrogel films based on thermoresponsive N-isopropylacrylamide with a dry thickness >1 microm were studied with surface plasmon resonance/optical waveguide mode spectroscopy (SPR/OWS) to monitor temperature-dependent and salt-induced changes of their swelling state. In combination with the reversed Wentzel-Kramers-Brillouin and Bruggeman effective medium approximation and by modeling the hydrogel film as a composite of sublayers with individual complex refractive indices, refractive index/volume fraction gradient profiles perpendicular to the surface are accessible simultaneously with information about local inhomogeneities. Specifically, the imaginary refractive index kappa of each sublayer can be interpreted as a measure for static and dynamic inhomogeneities, which were found to be highest at the volume transition collapse temperature in the layer center. These results indicate that the hydrogel collapse originates rather from the film center than from its boundaries. Upon addition of NaCl to a swollen hydrogel below its transition temperature, comparable optical loss characteristics as for the thermal gel collapse are observed with respect to inhomogeneities. Interestingly, in contrast to the thermally induced layer shrinkage and collapse, swelling increases at intermediate salt concentrations.

Hydrogel-supported protein-tethered bilayer lipid membranes: a new approach toward polymer-supported lipid membranes

Polymer-supported bilayer lipid membranes offer great opportunities for the investigation of functional membrane proteins. Here we present a new approach in this direction by introducing a thin hydrogel layer as a soft ‘cushion’ on indium–tin oxide (ITO), providing a smooth, functional surface to form the protein-tethered BLM (ptBLM). ITO was used as a transparent electrode, enabling simultaneous implementation of electrochemical and optical waveguide techniques. The hydrogel poly(N-(2-hydroxyethyl)acrylamide-co-5-acrylamido-1-carboxypentyl-iminodiacetate-co-4-benzoylphenyl methacrylate) (P(HEAAm-co-NTAAAm-co-MABP)) was functionalized with the nickel chelating nitrilotriacetic acid (NTA) groups, to which cytochrome c oxidase (CcO) from Paracoccus denitrificans was bound in a well defined orientation via a his-tag attached to its subunit I. Given that the mesh size of P(HEAAm-co-NTAAAm-co-MABP) was smaller than the protein size, binding to the hydrogel occurred only on the top of the layer. The lipid bilayer was formed around the protein by in situdialysis. Electrochemical impedance spectroscopy showed good electrical sealing properties with a resistance of ∼1 MΩ cm2. Furthermore, surface plasmon resonance optical waveguide spectroscopy (SPR/OWS) indicated an increased anisotropy of the system after formation of the lipid bilayer. Cyclic voltammetry in the presence of reduced cytochrome c demonstrated that CcO was incorporated into the gel-supported ptBLM in a functionally active form.

Photoinduced C–C Reactions on Insulators toward Photolithography of Graphene Nanoarchitectures

On-surface chemistry for atomically precise sp(2) macromolecules requires top-down lithographic methods on insulating surfaces in order to pattern the long-range complex architectures needed by the semiconductor industry. Here, we fabricate sp(2)-carbon nanometer-thin films on insulators and under ultrahigh vacuum (UHV) conditions from photocoupled brominated precursors. We reveal that covalent coupling is initiated by C-Br bond cleavage through photon energies exceeding 4.4 eV, as monitored by laser desorption ionization (LDI) mass spectrometry (MS) and X-ray photoelectron spectroscopy (XPS). Density functional theory (DFT) gives insight into the mechanisms of C-Br scission and C-C coupling processes. Further, unreacted material can be sublimed and the coupled sp(2)-carbon precursors can be graphitized by e-beam treatment at 500 °C, demonstrating promising applications in photolithography of graphene nanoarchitectures. Our results present UV-induced reactions on insulators for the formation of all sp(2)-carbon architectures, thereby converging top-down lithography and bottom-up on-surface chemistry into technology.