Christoph Nowak

Patterning of plasmonic nanoparticles into multiplexed one-dimensional arrays based on spatially modulated electrostatic potential.

We report a new strategy to pattern plasmonic nanoparticles into multiplexed one-dimensional arrays based on the spatially modulated electrostatic potential. The 32 nm Au nanoparticles can be simultaneously deposited on one chip with tunable interparticle distance by solely adjusting the width of the grooves. Furthermore, 32 and 13 nm Au nanoparticles can be selectively deposited in grooves of different widths on one chip. As a result, the surface plasmon absorption bands on the chip can be tuned depending on the interparticle distance or the particle size of multiplex 1D arrays, which could enhance the Raman scattering cross section of the adsorbed molecules and result in multiplex surface-enhanced Raman scattering (SERS) response on the chip. This strategy provides a general method to fabricate 1D multiplex arrays with different particle sizes and interparticle distances on one chip.

Molecular and Functional Characterization of Human Pendrin and its Allelic Variants

Pendrin (SLC26A4, PDS) is an electroneutral anion exchanger transporting I -, Cl -, HCO3-, OH -, SCN - and formate. In the thyroid, pendrin is expressed at the apical membrane of the follicular epithelium and may be involved in mediating apical iodide efflux into the follicle; in the inner ear, it plays a crucial role in the conditioning of the pH and ion composition of the endolymph; in the kidney, it may exert a role in pH homeostasis and regulation of blood pressure. Mutations of the pendrin gene can lead to syndromic and non-syndromic hearing loss with EVA (enlarged vestibular aqueduct). Functional tests of mutated pendrin allelic variants found in patients with Pendred syndrome or non-syndromic EVA (ns-EVA) revealed that the pathological phenotype is due to the reduction or loss of function of the ion transport activity. The diagnosis of Pendred syndrome and ns-EVA can be difficult because of the presence of phenocopies of Pendred syndrome and benign polymorphisms occurring in the general population. As a consequence, defining whether or not an allelic variant is pathogenic is crucial. Recently, we found that the two parameters used so far to assess the pathogenic potential of a mutation, i.e. low incidence in the control population, and substitution of evolutionary conserved amino acids, are not always reliable for predicting the functionality of pendrin allelic variants; actually, we identified mutations occurring with the same frequency in the cohort of hearing impaired patients and in the control group of normal hearing individuals. Moreover, we identified functional polymorphisms affecting highly conserved amino acids. As a general rule however, we observed a complete loss of function for all truncations and amino acid substitutions involving a proline. In this view, clinical and radiological studies should be combined with genetic and molecular studies for a definitive diagnosis. In performing genetic studies, the possibility that the mutation could affect regions other than the pendrin coding region, such as its promoter region and/or the coding regions of functionally related genes (FOXI1, KCNJ10), should be taken into account. The presence of benign polymorphisms in the population suggests that genetic studies should be corroborated by functional studies; in this context, the existence of hypo-functional variants and possible differences between the I -/Cl - and Cl -/HCO3- exchange activities should be carefully evaluated.

beta-1,3-Glucanase from Delftia tsuruhatensis Strain MV01 and Its Potential Application in Vinification

ABSTRACT During vinification microbial activities can spoil wine quality. As the wine-related lactic acid bacterium Pediococcus parvulus is able to produce slimes consisting of a β-1,3-glucan, must and wine filtration can be difficult or impossible. In addition, the metabolic activities of several wild-type yeasts can also negatively affect wine quality. Therefore, there is a need for measures to degrade the exopolysaccharide from Pediococcus parvulus and to inhibit the growth of certain yeasts. We examined an extracellular β-1,3-glucanase from Delftia tsuruhatensis strain MV01 with regard to its ability to hydrolyze both polymers, the β-1,3-glucan from Pediococcus and that from yeast cell walls. The 29-kDa glycolytic enzyme was purified to homogeneity. It exhibited an optimal activity at 50°C and pH 4.0. The sequencing of the N terminus revealed significant similarities to β-1,3-glucanases from different bacteria. In addition, the investigations indicated that this hydrolytic enzyme is still active under wine-relevant parameters such as elevated ethanol, sulfite, and phenol concentrations as well as at low pH values. Therefore, the characterized enzyme seems to be a useful tool to prevent slime production and undesirable yeast growth during vinification.

Two-Dimensional Heterospectral Correlation Analysis of the Redox-Induced Conformational Transition in Cytochrome c Using Surface-Enhanced Raman and Infrared Absorption Spectroscopies on a Two-Layer Gold Surface

The heme protein cytochrome c adsorbed to a two-layer gold surface modified with a self-assembled monolayer of 2-mercaptoethanol was analyzed using a two-dimensional (2D) heterospectral correlation analysis that combined surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced Raman spectroscopy (SERS). Stepwise increasing electric potentials were applied to alter the redox state of the protein and to induce conformational changes within the protein backbone. We demonstrate herein that 2D heterospectral correlation analysis is a particularly suitable and useful technique for the study of heme-containing proteins as the two spectroscopies address different portions of the protein. Thus, by correlating SERS and SEIRAS data in a 2D plot, we can obtain a deeper understanding of the conformational changes occurring at the redox center and in the supporting protein backbone during the electron transfer process. The correlation analyses are complemented by molecular dynamics calculations to explore the intramolecular interactions.

Graphene-based liquid-gated field effect transistor for biosensing: Theory and experiments

We present an experimental and theoretical characterization for reduced Graphene-Oxide (rGO) based FETs used for biosensing applications. The presented approach shows a complete result analysis and theoretically predictable electrical properties. The formulation was tested for the analysis of the device performance in the liquid gate mode of operation with variation of the ionic strength and pH-values of the electrolytes in contact with the FET. The dependence on the Debye length was confirmed experimentally and theoretically, utilizing the Debye length as a working parameter and thus defining the limits of applicability for the presented rGO-FETs. Furthermore, the FETs were tested for the sensing of biomolecules (bovine serum albumin (BSA) as reference) binding to gate-immobilized anti-BSA antibodies and analyzed using the Langmuir binding theory for the description of the equilibrium surface coverage as a function of the bulk (analyte) concentration. The obtained binding coefficients for BSA are found to be same as in results from literature, hence confirming the applicability of the devices. The FETs used in the experiments were fabricated using wet-chemically synthesized graphene, displaying high electron and hole mobility (µ) and provide the strong sensitivity also for low potential changes (by change of pH, ion concentration, or molecule adsorption). The binding coefficient for BSA-anti-BSA interaction shows a behavior corresponding to the Langmuir adsorption theory with a Limit of Detection (LOD) in the picomolar concentration range. The presented approach shows high reproducibility and sensitivity and a good agreement of the experimental results with the calculated data.

The Extended Growth of Graphene Oxide Flakes Using Ethanol CVD .

We report the extended growth of Graphene Oxide (GO) flakes using atmospheric pressure ethanol Chemical Vapor Deposition (CVD). GO was used to catalyze the deposition of carbon on a substrate in the ethanol CVD with Ar and H2 as carrier gases. Raman, SEM, XPS and AFM characterized the growth to be a reduced GO (RGO) of <5 layers. This newly grown RGO possesses lower defect density with larger and increased distribution of sp(2) domains than chemically reduced RGO. Furthermore this method without optimization reduces the relative standard deviation of electrical conductivity between chips, from 80.5% to 16.5%, enabling RGO to be used in practical electronic devices.

2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.

A Two-Layer Gold Surface with Improved Surface Enhancement for Spectro-Electrochemistry Using Surface-Enhanced Infrared Absorption Spectroscopy

A two-layer gold surface is developed for use with surface-enhanced infrared absorption spectroscopy (SEIRAS) consisting of a conducting underlayer onto which Au nanoparticles (AuNPs) are grown by self-catalyzed electroless deposition. AuNPs are grown on protruding substructures of the 25 nm thin underlayer. The enhancement factor of the two-layer gold surface is controlled by the growth conditions. Cytochrome c adsorbed to a self-assembled monolayer of mercaptoethanol is used as a reference system. Under optimum conditions the absorbance of the amide I band is increased by a factor of 5 versus the classical SEIRAS surface. Reversible reduction/oxidation of cytochrome c on the two-layer gold surface is shown to take place by cyclic voltammetry.

Conformational transitions and molecular hysteresis of cytochrome c oxidase: Varying the redox state by electronic wiring

Even though the structures of cytochrome c oxidase (CcO) from different sources have been determined by X-ray crystallography in both the reduced and oxidized redox states, information about redox-induced structure-function relationships is still very limited. In the current work, redox-dependent structural changes are determined for CcO reconstituted in a protein-tethered bilayer lipid membrane by surface-enhanced infrared absorption spectroscopy in the ATR mode. Significantly, the redox changes in the enzyme are attained by direct wiring of CcO to a gold electrode, ensuring that sequential intra-protein electron transfer occurs by a directed pathway that is natural to the system. The characteristics of CcO were observed to be dramatically altered after the reconstituted enzyme was allowed to turn over in the presence of O2. The data suggest that the enzyme is initially in an “inactive” state, but that direct electron transfer in the presence of O2 converts the enzyme to an “activated” form which returns to the inactive conformation when the enzyme remains idle under anaerobic conditions. Potentiometric titrations are performed and reduced-minus-fully oxidized and oxidized-minus-fully reduced absorbance spectra are recorded at decreasing and increasing potentials, respectively, applied to the electrode in a regular succession. The two sets of difference spectra show mirror symmetry, however, they markedly differ from those measured in the presence of redox mediators. Plots of band area of individual bands obtained by Fourier self deconvolution vs. applied potential show a sigmoid dependence as expected for a redox process. However, the sigmoid curves do not coincide but are displaced depending on the direction of the potential change. In other words, these curves show hysteresis, which is an indication of cooperativity and non-equilibrium states for electron transfer and/or conformational changes of the protein. This is discussed in terms of known concepts of molecular hysteresis.

Proteins in biomimetic membranes: promises and facts

Different planar lipid bilayers are considered, which are designed as model systems for membrane proteins in a functionally active form. Tethered bilayer lipid membranes (tBLMs) were investigated in our group based on peptide- or oligo-oxy-ethylene (OEO) tethers. Peptide-tethered BLMs were shown to incorporate large proteins such as cytochrome c oxidase (CcO) isolated from bovine heart and F0F1 ATPase from chloroplasts, whereas OEO-tBLMs were designed to incorporate ion carriers such as valinomycin and small channel proteins such as melittin and gramicidin. The activity of these proteins could be demonstrated. However, a quantitative treatment was not feasible. Therefore, we have concentrated our efforts on a relatively new approach, the protein-tethered bilayer lipid membrane (ptBLM), which uses the protein itself as the essential building block. With the example of cytochrome c oxidase (CcO) from R. sphaeroides with His-tags attached to subunits I and II the protein was immobilized in opposite orientations and reconstituted into the respective ptBLMs. Direct electron transfer from an electrode into the enzyme could be measured by electrochemical and spectro-electrochemical methods when the enzyme was immobilized with the electron entry side directed toward the electrode. The uptake of 4 electrons per molecule CcO measured under anaerobic conditions was treated by rigorous electrochemical theory to follow a sequential, or ECCC mechanism. When the enzyme was exposed to an oxygenated solution an amplified current density indicated catalytic turnover. As far as spectro-electrochemistry was concerned, the most promising results were obtained by electrochemically controlled surface-enhanced infrared absorption spectroscopy (SEIRAS). Potentiometric titrations followed by SEIRAS indicated significant conformational changes of the protein as a function of potential. The enzyme could even be shown to adopt two different conformational states, a non-activated and an activated state before and after it was allowed to undergo catalytic turnover. Differences between the two states could be demonstrated particularly using 2D autocorrelation maps.