Contrast Formation in Kelvin Probe Force Microscopy of Single π-Conjugated Molecules

We report the contrast formation in the local contact potential difference (LCPD) measured by Kelvin probe force microscopy (KPFM) on single charge-transfer complexes (CTCs) on a NaCl bilayer on Cu(111). At different tip heights, we found quantitatively different LCPD contrasts that characterize different properties of the molecule. In the small distance regime, the tip penetrates the electron density of the molecule, and the contrast is related to the size and topography of the electron shell of the molecule. For larger distances, the LCPD contrast corresponds to the electrostatic field above the molecule. However, in the medium-distance regime, that is, for tip heights similar to the size of the molecule, the nonspherical distribution of π- and σ-electrons often conceals the effect of the partial charges within the molecule. Only for large distances does the LCPD map converge toward the simple field of a dipole for a polar molecule.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Impact of N2 plasma power and duration on AlGaN/GaN HEMT

urface treatments have been recently shown to play an active role in electrical characteristics in AlGaN/GaN HEMTs, in particular during the passivation processing [1-4]. However, the responsible mechanisms are partially unknown and further studies are demanding. The effects of power and time N2 plasma pre-treatment prior to SiN deposition using PE-CVD (plasma enhanced chemical vapour deposition) on GaN and AlGaN/GaN HEMT have been investigated. The low power (60 W) plasma pre-treatment was found to improve the electronic characteristics in GaN based HEMT devices, independently of the time duration up to 20 min. In contrast, high power (150 and 210 W) plasma pretreatment showed detrimental effects in the electronic properties (Fig. 1), increasing the sheet resistance of the 2DEG, decreasing the 2DEG charge density in AlGaN/GaN HEMTs, transconductance reduction and decreasing the fT and fmax values up to 40% respect to the case using 60 W N2 plasma power. Although AFM (atomic force microscopy) results showed AlGaN and GaN surface roughness is not strongly affected by the N2-plasma, KFM (Kelvin force microscopy) surface analysis shows significant changes in the surface potential, trending to increase its values as the plasma power is higher. The whole results point at energetic ions inducing polarization-charge changes that affect dramatically to the 2-DEG charge density and the final characteristics of the HEMT devices. Therefore, we conclude that AlGaN surface is strongly sensitive to N2 plasma power conditions, which turn to be a key factor to achieve a good surface preparation prior to SiN passivation.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s−1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈104 M−1 and a dissociation rate of ≥6 s−1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Large secretory structures at the cell surface imaged with scanning force microscopy.

Scanning force microscopy was used to image rat basophilic leukemia (RBL-2H3) cell surfaces under different stimulation conditions that either permit or inhibit secretion. Cross-linking the surface IgE receptors with dinitrophenol-conjugated bovine serum albumin initiates secretion in RBL cells with concomitant spreading of the cell body. Structures at the cell surface approximately 1.5 microns in diameter relate to secretion both spatially and temporally. The position of these surface pits and their sizes suggest that they may be related to the dense-core granules positioned along the cytoskeletal filaments in detergent-extracted, unactivated RBL cell processes. Topographic scanning force microscopy images of RBL cell surfaces at 2, 5, and 35 min after activation show that these structures persist and change in cross-sectional profile with time after activation. These structures may be related to the membrane retrieval mechanism of cells after intense stimulation.

Magnetization reversal of ferromagnetic nanowires studied by magnetic force microscopy

The magnetization reversal of two-dimensional arrays of parallel ferromagnetic Fe nanowires embedded in nanoporous alumina templates has been studied. By combining bulk magnetization measurements (superconducting quantum interference device magnetometry) with field-dependent magnetic force microscopy (MFM), we have been able to decompose the macroscopic hysteresis loop in terms of the irreversible magnetic responses of individual nanowires. The latter are found to behave as monodomain ferromagnetic needles, with hysteresis loops displaced (asymmetric) as a consequence of the strong dipolar interactions between them. The application of field-dependent MFM provides a microscopic method to obtain the hysteresis curve of the array, by simply registering the fraction of up and down magnetized wires as a function of applied field. The observed deviations from the rectangular shape of the macroscopic hysteresis loop of the array can be ascribed to the spatial variation of the dipolar field through the inhomogeneously filled membrane. The system studied proves to be an excellent example of the two-dimensional classical Preisach model, well known from the field of hysteresis modeling and micromagnetism.

Experimental investigation of interface states and photovoltaic effects on the scanning capacitance microscopy measurement for p-n junction dopant profiling

Controlled polishing procedures were used to produce both uniformly doped and p-n junction silicon samples with different interface state densities but identical oxide thicknesses. Using these samples, the effects of interface states on scanning capacitance microscopy (SCM) measurements could be singled out. SCM measurements on the junction samples were performed with and without illumination from the atomic force microscopy laser. Both the interface charges and the illumination were seen to affect the SCM signal near p-n junctions significantly. SCM p-n junction dopant profiling can be achieved by avoiding or correctly modeling these two factors in the experiment and in the simulation. (c) 2005 American Institute of Physics.

Atomic composition profile change of SiGe islands during Si capping

The 6% Ge isocomposition profile change of individual SiGe islands during Si capping at 640 degrees C is investigated by atomic force microscopy combined with a selective etching procedure. The island shape transforms from a dome to a {103}-faceted pyramid at a Si capping thickness of 0.32 nm, followed by the decreasing of pyramid facet inclination with increasing Si capping layer thickness. The 6% Ge isocomposition profiles show that the island with more highly Si enriched at its one base corner before Si capping becomes to be more highly Si intermixed along pyramid base diagonals during Si capping. This Si enrichment evolution inside an island during Si capping can be attributed to the exchange of capped Si atoms that aggregated to the island by surface diffusion with Ge atoms from inside the island by both atomic surface segregation and interdiffusion rather than to the atomic interdiffusion at the interface between the island and the Si substrate. In addition, the observed Si enrichment along the island base diagonals is attempted to be explained on the basis of the elastic constant anisotropy of the Si and Ge materials in (001) plane. (c) 2006 American Institute of Physics.

Nanoporous InP: anodic formation and growth mechanism in aqueous electrolytes

Porous layers can be formed electrochemically on (100) oriented n-InP substrates in aqueous KOH. A nanoporous layer is obtained underneath a dense near-surface layer and the pores appear to propagate from holes through the near-surface layer. In the early stages of the anodization transmission electron microscopy (TEM) clearly shows individual porous domains that appear to have a square-based pyramidal shape. Each domain appears to develop from an individual surface pit which forms a channel through this near-surface layer. We suggest that the pyramidal structure arises as a result of preferential pore propagation along the <100> directions. AFM measurements show that the density of surface pits increases with time. Each of these pits acts as a source for a pyramidal porous domain. When the domains grow, the current density increases correspondingly. Eventually the domains meet, forming a continuous porous layer, the interface between the porous and bulk InP becomes relatively flat and its total effective surface area decreases resulting in a decrease in the current density. Current-time curves at constant potential exhibit a peak and porous layers are observed to form beneath the electrode surface. The density of pits formed on the surface increases with time and approaches a plateau value. Porous layers are also observed in highly doped InP but are not observed in wafers with doping densities below ~5 × 1017 cm-3. Numerical models of this process have been developed invoking a mechanism of directional selectivity of pore growth preferentially along the <100> lattice directions. Manipulation of the parameters controlling these curves shows that the fall-off in current is controlled by the rate of diffusion of electrolyte through the pore structure with the final decline in current being caused by the termination of growth at the pore tips through the formation of passivating films or some other irreversible modification of the pore tips.

Atomic vapor deposition of bismuth titanate thin films

c-axis oriented ferroelectric bismuth titanate (Bi4Ti 3O12) thin films were grown on (001) strontium titanate (SrTiO3) substrates by an atomic vapor deposition technique. The ferroelectric properties of the thin films are greatly affected by the presence of various kinds of defects. Detailed x-ray diffraction data and transmission electron microscopy analysis demonstrated the presence of out-of-phase boundaries (OPBs). It is found that the OPB density changes appreciably with the amount of titanium injected during growth of the thin films. Piezo-responses of the thin films were measured by piezo-force microscopy. It is found that the in-plane piezoresponse is stronger than the out-of-plane response, due to the strong c-axis orientation of the films.

The (Un)Folding of Multidomain Proteins Through the Lens of Single-molecule Force-spectroscopy and Computer Simulation

Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.

In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.

The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.

Synthesis and Characterization of Nanostructured Electro-active Materials

Thesis (Ph.D.)--University of Washington, 2016-08

Coupled diusion and mechanics in battery electrodes

Thesis (Ph.D.)--University of Washington, 2016-08