Electronic Olfactory Sensor Based on A.†mellifera Odorant-Binding Protein†14 on a Reduced Graphene Oxide Field-Effect Transistor.

An olfactory biosensor based on a reduced graphene oxide (rGO) field-effect transistor (FET), functionalized by the odorant-binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real-time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand-receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K(d)=4 µM to K(d)=3.3 mM. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.

Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements.

In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular ß-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule.

Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany.

Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no distinct region specificity of O. oeni populations.

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.

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.