Integrating carbon nanotubes and lipid bilayer for biosensing

<p>This thesis presents detailed temperature-dependent Raman and conduction studies on two materials: cupric oxide (CuO) nanostructures and single-walled carbon nanotube (SWNT) networks. SWNT networks are a promising alternative to indium tin oxide as the transparent conducting material in electronic displays. A key factor that complicates fundamental studies on SWNT networks is surfactant residue. Our study utilises SWNTs (HiPCO) dispersed in volatile solvents, which can be removed by annealing to only 100°C. Using this unique solvent system, we have systematically studied charge transport mechanisms in surfactant-free networks using a percolation approach; where sample resistance can be controlled by the amount of deposited material. The chemical environment of these networks was found to be unchanged using Raman spectroscopy; i.e. film fabrication did not cause any significant doping of the network. Around 15 surfactant-free networks, with resistances between 300 kΩ and 8 kΩ, were found to follow a 'universal' charge transport model. Most of the networks could be described by two dimensional variable range hopping (VRH) and thermal activation. The barrier energy or, To parameter, for the VRH mechanism was independent of resistance with a value of 20x 10ᵌ ± 9.4x 10ᵌ K. The activation energy also had a resistance-independent value of 160 ± 20 meV. Four terminal measurements confirm that the activation mechanism is due to processes within the network not, Schottky barriers at the nanotube/metal interface. The effects of extrinsic adsorbants on the network resistance provides evidence for dominant non-metallic conduction pathways within the studied range of resistances. These results strongly suggest a characteristic barrier size in our SWNT networks where non-metallic tubes dominate the resistance. The surfactant-free networks were also used to study the temperature-dependent behaviour of the radial breathing modes (RBM) in bundled nanotubes. Deconvolution of complex RBM spectra was made possible using an interactive routine: based on the higher resolution of second derivatives for fitting spectra with washed-out features. Using this routine, the temperature-dependent characteristics of the RBM lineshape could be identified. We find that RBM modes in our bundled networks soften at the same rate as individual tubes; the linewidth follows a three phonon decay process with a temperature-independent component and the intensity can be modelled from the change in Eii with temperature. The second part of this thesis addresses the unique asymmetric lineshape of the A1g mode in CuO nanowire forests. A symmetric lineshape is recovered at low powers indicating that the underlying mechanism is thermal. To study this effect, the high temperature behaviour of the A1g mode is first analysed in a `bulk' form of CuO. The analytical temperature dependence of the A1g mode frequency, linewidth and intensity were used as the basis of a physical model that connects lineshape asymmetry to laser-induced, spatial temperature gradients in the sample.The peak temperature (under the laser hotspot) was found to be proportional to laser power until it reaches a critical value. We believe that regions with temperature above the critical value cool by radiation rather than convection.</p>

The effect of cleaning the surface of single-walled carbon nanotube (SWNT) networks by thermal and the O2 plasma treatments is presented in terms of NH3 gas sensing characteristics. The goal of this work is to determine the relationship between the physicochemical properties of the cleaned surface (including the chemical composition, crystal structure, hydrophilicity, and impurity content) and the sensitivity of the SWNT network films to NH3 gas. The SWNT networks are spray-deposited on pre-patterned Pt electrodes, and are further functionalized by heating on a programmable hot plate or by O2 plasma treatment in a laboratory-prepared plasma chamber. Cyclic voltammetry was employed to semi-quantitatively evaluate each surface state of various plasma-treated SWNT-based electrodes. The results show that O2 plasma treatment can more effectively modify the SWNT network surface than thermal cleaning, and can provide a better conductive network surface due to the larger number of carbonyl/carboxyl groups, enabling a faster electron transfer rate, even though both the thermal cleaning and the O2 plasma cleaning methods can eliminate the organic solvent residues from the network surface. The NH3 sensors based on the O2 plasma-treated SWNT network exhibit higher sensitivity, shorter response time, and better recovery of the initial resistance than those prepared employing the thermally-cleaned SWNT networks.

<p>Graphene, consisting of a single layer of carbon atoms, is being widely studied for its interesting fundamental physics and potential applications. The presence and extent of disorder play important roles in determining the electronic conduction mechanism of a conducting material. This thesis presents work on data analysis and modelling of electronic transport mechanisms in disordered carbon materials such as graphene. Based on experimental data of conductance of partially disordered graphene as measured by Gómez-Navarro et al., we propose a model of variable-range hopping (VRH) – defined as quantum tunnelling of charge carriers between localized states – consisting of a crossover from the two-dimensional (2D) electric field-assisted, temperature-driven (Pollak-Riess) VRH to 2D electric field-driven (Skhlovskii) VRH. The novelty of our model is that the temperature-dependent and field-dependent regimes of VRH are unified by a smooth crossover where the slopes of the curves equal at a given temperature. We then derive an analytical expression which allows exact numerical calculation of the crossover fields or voltages. We further extend our crossover model to apply to disordered carbon materials of dimensionalities other than two, namely to the 3D self-assembled carbon networks by Govor et al. and quasi-1D highly-doped conducting polymers by Wang et al. Thus we illustrate the wide applicability of our crossover model to disordered carbon materials of various dimensionalities. We further predict, in analogy to the work of Pollak and Riess, a temperature-assisted, field-driven VRH which aims to extend the field-driven expression of Shklovskii to cases wherein the temperatures are increased. We discover that such an expression gives a good fit to the data until certain limits wherein the temperatures are too high or the applied field too low. In such cases the electronic transport mechanism crosses over to Mott VRH, as expected and analogous to our crossover model described in the previous paragraph. The second part of this thesis details a systematic data analysis and modelling of experimental data of conductance of single-wall carbon nanotube (SWNT) networks prepared by several different chemical-vapour deposition (CVD) methods by Ansaldo et al. and Lima et al. Based on our analysis, we identify and categorize the SWNT networks based on their electronic conduction mechanisms, using various theoretical models which are temperature-dependent and field-dependent. The electronic transport mechanisms of the SWNT networks can be classed into either VRH in one- and two-dimensions or fluctuation-assisted tunnelling (FAT, i.e. interrupted metallic conduction), some with additional resistance from scattering by lattice vibrations. Most notably, for a selected network, we find further evidence for our novel VRH crossover model previously described. We further correlate the electronic transport mechanisms with the morphology of each network based on scanning electron microscopy (SEM) images. We find that SWNT networks which consist of very dense tubes show conduction behaviour consistent with the FAT model, in that they retain a finite and significant fraction of room-temperature conductance as temperatures tend toward absolute zero. On the other hand, SWNT networks which are relatively sparser show conduction behaviour consistent with the VRH model, in that conductance tends to zero as temperatures tend toward absolute zero. We complete our analysis by estimating the average hopping distance for SWNT networks exhibiting VRH conduction, and estimate an indication of the strength of barrier energies and quantum tunnelling for SWNT networks exhibiting FAT conduction.</p>

Lipid Bilayer Strengthens the Side Chain Interaction Network of a Membrane Protein

Membrane proteins play crucial roles in numerous biological processes and are important drug targets. However, structural studies of membrane proteins often rely on solubilization with detergents, which may not accurately reflect their native states in a cellular context. Additionally, identifying suitable detergents for individual membrane proteins can be a detailed and time-consuming process. Here, we developed a vesicle-based method that preserves the native lipid environment for subsequent structural and functional studies. Using the bacterial multidrug efflux transporter AcrB as an example, we isolated AcrB-containing vesicles and determined its cryo-EM structure with all protomers in a loose (L) state at 3.88 Å by incorporating our micrograph-based sorting strategy. Notably, compared to the L-state AcrB in liposomes and nanoparticles, the exterior transmembrane helices (TMs) in our map exhibited superior quality, featuring a continuous and clear representation of lα, which is positioned horizontally within the lipid bilayer. We further expanded our method by identifying endogenous membrane proteins, including F-ATPase and respiratory complexes, in vesicles generated using mitochondria from pig hearts. The high-resolution structure of respiratory complex III in vesicles revealed a shared subunit nine between two monomers. Briefly, our method presents a promising and straightforward approach for studying the structure and function of membrane proteins in their native environment, eliminating the need for detergent screening and protein purification.

Statistical analysis of the change in electrical characteristic of single-walled carbon nanotube (swCNT) network after the decoration of Au nanoparticles (NPs) is presented. We have fabricated 100 unitary swCNT network devices on a single chip employing a concentric electrode array by swCNT dip-coating and thermal evaporation of Au. The experimental results show that the decoration of Au-NP on the swCNT network can decrease not only its resistance but also variation in the resistance distribution. Remarkably, we have found that the change in electrical characteristic is correlated with the resistance value of the bare swCNT network.

Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.

Accurate and reliable detection of hypergolic fuels such as hydrazine (N2H4) and its derivatives is vital to missile defense, aviation, homeland security, and the chemical industry. More importantly these sensors need to be capable of operation at low temperatures (below room temperature) as most of the widely used chemical sensors operate at high temperatures (above 300 °C). In this research a simple and highly sensitive single walled carbon nanotube (SWNT) network sensor was developed for real time monitoring of hydrazine leaks to concentrations at parts per million levels. Upon exposure to hydrazine vapor, the resistance of the air exposed nanotubes (p-type) is observed to increase rapidly while that of the vacuum-degassed nanotubes (n-type) is observed to decrease. It was found that the resistance of the sample can be recovered through vacuum pumping and exposure to ultraviolet light. The experimental results support the electrochemical charge transfer mechanism between the oxygen redox couple of the ambient and the Fermi level of the SWNT. Theoretical results of the hydrazine-SWNT interaction are compared with the experimental observations. It was found that a monolayer of water molecules on the SWNT is necessary to induce strong interactions between hydrazine and the SWNT by way of introducing new occupied states near the bottom of the conduction band of the SWNT.

Reconstitution of membrane proteins: a GPCR as an example.

In the field of electronic gas sensing, low-dimensional semiconductors such as single-walled carbon nanotubes (SWCNTs) can offer high detection sensitivity owing to their unprecedentedly large surface-to-volume ratio. The sensitivity and responsivity can further improve by increasing their areal density. Here, an accelerated gas adsorption is demonstrated by exploiting volumetric effects via dispersion of SWCNTs into a percolating three-dimensional (3D) network in a semiconducting polymer. The resultant semiconducting composite film is evaluated as a sensing membrane in field effect transistor (FET) sensors. In order to attain reproducible characteristics of the FET sensors, a pulsed-gate-bias measurement technique is adopted to eliminate current hysteresis and drift of sensing baseline. The rate of gas adsorption follows the Langmuir-type isotherm as a function of gas concentration and scales with film thickness. This rate is up to 5 times higher in the composite than only with an SWCNT network in the transistor channel, which in turn results in a 7-fold shorter time constant of adsorption with the composite. The description of gas adsorption developed in the present work is generic for all semiconductors and the demonstrated composite with 3D percolating SWCNTs dispersed in functional polymer represents a promising new type of material for advanced gas sensors.

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.

Due to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly.

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer

Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.