Robert F. Roskamp

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.

Dynamics of swollen gel layers anchored to solid surfaces

We employ a dynamic micro light scattering technique to probe the thermal concentration fluctuations in surface-attached poly-N-isopropylacrylamide (PNIPAAm) gel layers swollen in ethanol as a good solvent. At the equilibrium swelling state, the relaxation function exhibits two decays in the time range between microseconds and seconds and the characteristic rates display a pure diffusive behavior. The fast cooperative diffusion increases with crosslinking density as a result of the decrease in the dynamic network mesh size. This increase is significantly stronger than the concentration dependence of the cooperative diffusion in uncrosslinked linear PNIPPAm solutions. Uniaxial swelling due to the surface attachment and structural inhomogeneities intrinsic to photo-crosslinked gels alter the dynamics of the surface anchored networks compared to the solutions. In contrast to the frozen inhomogeneities in conventional gels, the slow diffusion in the present anchored layers was found to be ergodic. It might relate to structural inhomogeneities but its nature is not clarified yet.

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.

Optical Waveguide Spectroscopy for the Investigation of Protein-Functionalized Hydrogel Films

This article reports the implementation of optical waveguide spectroscopy (OWS) for the quantitative time-resolved observation of changes in the swelling behavior and mass density of protein-functionalized hydrogel films. In the experiment, a thin film of an N-isopropylacrylamide (NIPAAm)-based polymer that supported optical waveguide modes is attached to a metallic sensor surface. IgG molecules are in situ immobilized in this gel by using novel coupling chemistry with a charge-attraction scheme based on a tetrafluorophenol sulfonate active ester. The anti-fouling properties of the functionalized hydrogel network and the kinetics of the affinity binding of protein molecules in the gel are investigated.

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.

Probing dynamics near surfaces: waveguide enhanced dynamic light scattering

Interfaces and interphases are a vital part of everyday life—since the influence of their proximity might change considerably the behaviour of systems studied. The ability to study motion on the nanometre scale is essential for the understanding of transport phenomena to and from surfaces, thin films or membranes. It is indispensable to have analytical methods with spatio-temporal resolution adapted to these problems and which are non-invasive to obtain untainted results. The small dimensions lead to weak signals and therefore we need experimental setups which are able to amplify them. Here we describe a dynamic light scattering experiment where the evanescent part of waveguide modes is used as the source of light. Using waveguide modes increased the signal-to-noise ratio by a factor of 8 compared to evanescent waves generated by total internal reflection and it allows adjusting the spatial resolution near the interface in situ. This technique monitors changes of the waveguide surface as e.g. adsorption to it.