Altered Expression of Antimicrobial Peptides in the Upper Gastrointestinal Tract of Patients with Diabetes Mellitus.

Antimicrobial peptides (AMP) are essential components of innate immunity with a broad range of antimicrobial activities against bacteria, viruses, and fungi. The aim of this study was to investigate AMP expression in the upper gastrointestinal tract in normal and pathological metabolic states in humans. Furthermore, we examined the correlation between vitamin D levels and AMP expression in the same cohort. Serum concentrations of 25-hydroxyvitamin D3 were measured, and mRNA expression of ß-defensins HBD-1, -2, -3, -4, α-defensins HD-5 and -6 and cathelicidin in the upper gastrointestinal tract epithelia were determined by quantitative RT-PCR in 31 individuals (10 with type 2 diabetes, 10 with insulin resistance, and 11 healthy controls). The majority of the cohort showed low vitamin D concentrations, which were negatively correlated with mRNA expression levels of HBD-3 in corpus mucosa. HBD-1 and HBD-3 mRNA were expressed in corpus mucosa, with the former significantly decreased in patients with diabetes. Hence, we conclude that type 2 diabetes is associated with reduced AMP expression in the upper gastrointestinal tract, which might contribute towards epithelial barrier dysfunction and increased bacterial translocation in these patients.

Shedding Light on the Dark Corners of Metal-Organic Framework Thin Films: Growth and Structural Stability of ZIF-8 Layers Probed by Optical Waveguide Spectroscopy.

Metal-organic framework (MOF) thin films are promising materials for multiple technological applications, such as chemical sensing. However, one potential limitation for their widespread use in different settings is their stability in aqueous environments. In the case of ZIF-8 (zeolitic imidazolate framework) thin films, their stability in aqueous media is currently a matter of debate. Here, we show that optical waveguide spectroscopy (OWS), in combination with surface plasmon resonance (SPR) spectroscopy, offers a convenient way for answering intriguing questions related to the stability of MOF thin films in aqueous solutions and, eventually provide a tool for assessing changes in MOF layers under different environmental conditions. Our experiments relied on the use of ZIF-8 thin films grown on surface-modified gold substrates, as optical waveguides. We have found a linear thickness increase after each growing cycle and observed that the growing characteristics are strongly influenced by the nature of the primer layer. One of our findings is that substrate surface modification with a 3-mercapto-1-propanesulfonate (MPSA) primer layer is critical to achieve ZIF-8 layers that can effectively act as optical waveguides. We observed that ZIF-8 films are structurally stable upon exposure to pure water and 50 mM NaCl solutions but they exhibit a slight swelling and an increase in porosity probably due to the permeation of the solvent in the intergrain mesoporous cavities. However, OWS revealed that exposure of ZIF-8 thin films to phosphate-buffered saline solutions (pH 8) promotes significant film degradation. This poses an important question as to the prospective use of ZIF-8 materials in biologically relevant applications. In addition, it was demonstrated that postsynthetic polyelectrolyte modification of ZIF-8 films has no detrimental effects on the structural stability of the films.

Inhomogeneous Growth of Micrometer Thick Plasma Polymerized Films.

Plasma polymerization is traditionally recognized as a homogeneous film-forming technique. It is nevertheless reasonable to ask whether micrometer thick plasma polymerized structures are really homogeneous across the film thickness. Studying the properties of the interfacial, near-the-substrate (NTS) region in plasma polymer films represents particular experimental challenges due to the inaccessibility of the buried layers. In this investigation, a novel non-destructive approach has been utilized to evaluate the homogeneity of plasma polymerized acrylic acid (PPAc) and 1,7-octadiene (PPOD) films in a single measurement. Studying the variations of refractive index throughout the depth of the films was facilitated by a home-built surface plasmon resonance (SPR)/optical waveguide (OWG) spectroscopy setup. It has been shown that the NTS layer of both PPAc and PPOD films exhibits a significantly lower refractive index than the bulk of the film that is believed to indicate a higher concentration of internal voids. Our results provide new insights into the growth mechanisms of plasma polymer films and challenge the traditional view that considers plasma polymers as homogeneous and continuous structures.

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.

A mixed alkanethiol based immunosensor for surface plasmon field-enhanced fluorescence spectroscopy in serum.

This paper describes a simple and sensitive immuno-based biosensor for interference-reduced detection of C-reactive protein (CRP) in serum. The detection was performed by using a non-competitive sandwich immunoassay in combination with surface plasmon field-enhanced fluorescence spectroscopy (SPFS). CRP is an important marker for the diagnosis of inflammatory processes and cardiovascular diseases (CVD). It is nowadays detected by high-sensitivity enzyme-linked immunosorbent assays (ELISA) in blood serum. CRP was used as a model analyte in this work because it is well-characterized. However, interfering effects of matrix components affect the limit of detection (LOD) and quantification (LOQ) in general. Therefore, the availability of fast, sensitive and robust analytical methods is of major interest. A number of biosensor approaches have been described already, but only a few have demonstrated their usefulness in authentic samples such as serum. Thus our aim was to develop a simple and sensitive immunoassay-based biosensor for an interference-reduced detection of CRP in serum with surface plasmon enhanced fluorescence spectroscopy (SPFS). LODs and LOQs were experimentally determined both for CRP spiked buffer and serum. SPFS in combination with our biosensor allows sensitive analysis of CRP, achieving in buffer a LOD of 0.016 µg mL(-1) and a LOQ of 0.049 µg mL(-1). In serum the accomplished LOD was 0.026 µg mL(-1) and the LOQ was found to be 0.08 µg mL(-1). These low LODs and LOQs demonstrate the applicability of the designed biosensor for qualitative and semi-quantitative analysis of trace amounts of substances in very small sample volumes of body fluids.

Resonance enhanced dynamic light scattering.

We present a novel light scattering setup that enables probing of dynamics near solid surfaces. An evanescent wave generated by a surface plasmon resonance in a metal layer is the incident light field in the dynamic light scattering experiment. The combination of surface plasmon resonance spectroscopy and dynamic light scattering leads to a spatiotemporal resolution extending a few hundred nanometers from the surface and from microseconds to seconds. The comparison with evanescent wave dynamic light scattering identifies the advantages of the presented technique, e.g., surface monitoring, use of metal surfaces, and biorelevant systems. For both evanescent wave geometries, we define the scattering wave vector necessary for the analysis of the experimental relaxation functions.

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.

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.

Probing dynamics at interfaces: resonance enhanced dynamic light scattering.

Experiments addressing supramolecular dynamics at interfaces are of paramount importance for the understanding of the dynamic behaviour of polymers, particles, or cells at interfaces, transport phenomena to and from surfaces, thin films or membranes. However, there are only few reports in the literature due to the paucity of experimental methods that offer the required spatial and time resolution. Evanescent wave dynamic light scattering originally developed to meet these needs has limited sensitivity and is restricted to glass substrates. Here we report the first experimental realization of a dynamic light scattering experiment close to an interface using surface plasmon polaritons as light source offering a strong increase in the signal to noise ratio and allowing for the use of metallic interfaces. As a proof of concept, we consider the diffusion of particles with radii down to 10nm in dilute dispersions close to a gold surface.

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.

Responsive thin hydrogel layers from photo-cross-linkable poly(N-isopropylacrylamide) terpolymers.

The structural features and swelling properties of responsive hydrogel films based on poly(N-isopropylacrylamide) copolymers with a photo-cross-linkable benzophenone unit were investigated by surface plasmon resonance, optical waveguide mode spectroscopy, and atomic force microscopy. The temperature-dependent swelling behavior was studied with respect to the chemical composition of the hydrogel polymers containing either sodium methacrylate or methacrylic acid moieties. In the sodium methacrylate system, a refractive index gradient was found that was not present in the free acid gel. This refractive index gradient, perpendicular to the swollen hydrogel film surface, could be analyzed in detail by application of the reversed Wentzel-Kramers-Brillouin (WKB) approximation to the optical data. This novel approach to analyzing thin-film gradients with the WKB method presents a powerful tool for the characterization of inhomogeneous hydrogels, which would otherwise be very difficult to capture experimentally. In AFM images of the hydrogel layers, a macroscopic pore structure was observed that depended on the polymer composition as well as on the swelling history. This pore structure apparently prevents the often-observed skin barrier effect and leads to a quickly responding hydrogel.

Azobenzene-containing polyamic acid with excellent Langmuir-Blodgett-Kuhn film formation behavior suitable for all-optical switching.

To generate command surfaces for all-optical switching, highly ordered polymeric Langmuir-Blodgett-Kuhn (LBK) multilayers were fabricated onto silicon substrates and gold-coated optical glass slides from novel azobenzene-bearing polyamic acid systems. Pronounced Bragg peaks and well-defined Kiessig fringes observed in the X-ray reflectivity measurement for samples on silicon substrates indicate that these films possess a regularly repeated Y-type LBK multilayer structure and ultrasmooth surfaces. Fourier transform infrared (FT-IR) spectra taken by grazing incidence reflection suggest specific orientations of the functional groups in the layers. The excellent film-forming properties of the polyamic acid allow for a smooth buildup of several hundreds of layers of the LBK films onto gold-coated glass slides, which in turn allows for determining the geometrical thickness and the anisotropic refractive indices of the films by using optical waveguide spectroscopy. Interestingly, the probe laser beam induced a distinct fluorescence signal from the films, which remained even after the film underwent a thermal imidization process at 160 degrees C for 8 h in vacuo. LBK films fabricated from these compounds can be successfully applied for all-optically switching the alignment of liquid crystals by irradiation with light of different wavelengths.

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.