Ciril Reiner-Rozman

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

Abstract 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.

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.

Enzyme-polyelectrolyte multilayer assemblies on reduced graphene oxide field-effect transistors for biosensing applications.

We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu2+(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition.

Electronic Biosensing with Functionalized rGO FETs

In the following we give a short summary of examples for biosensor concepts in areas in which reduced graphene oxide-based electronic devices can be developed into new classes of biosensors, which are highly sensitive, label-free, disposable and cheap, with electronic signals that are easy to analyze and interpret, suitable for multiplexed operation and for remote control, compatible with NFC technology, etc., and in many cases a clear and promising alternative to optical sensors. The presented areas concern sensing challenges in medical diagnostics with an example for detecting general antibody-antigen interactions, for the monitoring of toxins and pathogens in food and feed stuff, exemplified by the detection of aflatoxins, and the area of smell sensors, which are certainly the most exciting development as there are very few existing examples in which the typically small and hydrophobic odorant molecules can be detected by other means. The example given here concerns the recording of a honey flavor (and a cancer marker for neuroblastoma), homovanillic acid, by the odorant binding protein OBP 14 from the honey bee, immobilized on the reduced graphene oxide gate of an FET sensor.

Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.

An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.

Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS

Time-resolved surface-enhanced IR-absorption spectroscopy (tr-SEIRAS) has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. The enzyme was converted electrochemically into the fully reduced state. Thereafter, in the presence of oxygen, the potential was switched to open circuit potential (OCP). Under these conditions, the enzyme is free to undergo enzymatic oxidation in the absence of an external electric field. Tr-SEIRAS was performed using the step-scan technique, triggered by periodic potential pulses switching between - 800mV and OCP. Single bands were resolved in a broad band in the amide I region using phase sensitive detection. Amplitudes of these bands were analyzed as a function of time. Time constants in the ms time scale were considered in terms of conformational changes of the protein secondary structures associated with the enzymatic turnover of the protein.

pH and Potential Transients of the bc1 Complex Co-Reconstituted in Proteo-Lipobeads with the Reaction Center from Rb. sphaeroides

His-tag technology is employed to bind membrane proteins, such as the bc1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 μm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc1 activated by the RC. To assess the turnover of bc1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.

Proteo-lipobeads to encapsulate cytochrome c oxidase from Paracoccus denitrificans

Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications.