AVS2001 Session BI+NS-WeA: Nanobiology

Wednesday, October 31, 2001 2:00 PM in Room 103

Wednesday Afternoon

Start	Invited?	Item
2:00 PM		BI+NS-WeA-1 Light-controlled Molecular Shuttles Based on Motor Proteins H. Hess, J. Clemmens, D. Qin, J. Dennis, J. Howard, V. Vogel (University of Washington) Molecular shuttles, an active transport system to position nanoscale objects, are needed as parts of molecular assembly stations, self-healing materials, or nanoscale actuators. The key problems of such a transport system are finding the motors, guiding the motion, loading cargo, and controlling the speed on the nanoscale. Active transport by single molecules is ubiquitous in biology and the solutions found by nature can serve as inspiration for technology. We demonstrate that molecular shuttles resembling conveyor belts can be constructed utilizing kinesin motor proteins as engines, microtubules as belts, and ATP as fuel. Two different strategies for guiding the microtubules have been explored by us: Arranging the motor proteins in nanometer-wide tracks by selective adsorption or creating micrometer-wide guiding channels by soft-lithography. Selective loading of cargo is accomplished by tagging cargo with streptavidin, and linking it to biotinylated microtubules. User-controlled exposure of caged ATP to UV-light and addition of an ATP-consuming enzyme to the buffer solution can move the microtubules in discrete steps. This forms a tool-set for the assembly of a functional molecular shuttle.
2:20 PM		BI+NS-WeA-2 The Direct Measurement of Drug-enzyme Interactions by Atomic Force Microscopy S.M. Rigby-Singleton, S.J.B.T. Tendler, S. Allen, M.C. Davies, C.J. Roberts, P.M. Williams (University of Nottingham, UK) AFM has been employed to directly probe the rupture forces upon the mechanical dissociation of the drug-enzyme complex formed between the anticancer compound methotrexate and the protein dihydrofolate reductase (DHFR). AFM probes were functionalized with methotrexate immobilized beads and rupture forces recorded between the probe and a DHFR monolayer attached via Lys residues. Three variables were studied, AFM retraction rates, the presence of the enzyme cofactor NADPH and the protonation of the key enzyme Asp26 residue. Rupture forces of 91 pN were recorded at a retract velocity of 1 micrometer per second, a ten fold decrease in velocity resulted in an observed decrease in rupture force. The influence of the enzyme cofactor was negligible suggesting little effect on the dissociation pathway, this is in marked contrast to literature fluorescent binding assays. Notably a decrease in rupture force of approximately 25pN was observed when the pH was decreased below the pKa of the key Asp26 residue which is situated deep within the methotrexate binding site. These studies indicate that the AFM will be a valuable tool in the drug discovery process.
2:40 PM		BI+NS-WeA-3 Single and Multiple Molecule Binding Forces Measured Using Modified Atomic Force Microscope Cantilevers R.G. Rudnitsky, F. Drees, K.S.H. Wu, T.D. Perez, W.J. Nelson, T.W. Kenny (Stanford University) Although the energies and forces controlling protein interactions are frequently inferred from traditional equilibrium and kinetic measurements, recent developments in chemical force microscopy allow for the direct quantification of the ranges and magnitudes of binding forces between individual protein pairs and between groups of proteins. We report here on the use of specially modified Atomic Force Microscope cantilevers to measure bond strength down the single-molecule level, with pico-Newton force resolution, using the cellular binding protein E-cadherin as our model system. Previous E-cadherin studies focused on the energetics of large systems of molecules, typically in-vivo, to demonstrate their role in cellular adhesion. Our novel AFM force spectroscopy method tracks the unbinding process of single and multiple E-cadherin molecules under force loads, to quantitatively differentiate specific from non-specific binding, and single and multiple binding events, in surface bound protein. The measurements isolate the extracellular domain of the molecule, thought to be essential for stable cell adhesion, and demonstrate the dependence of binding forces at a molecular level on Ca++ concentrations. The data correlates the relationship of homophilic E-cadherin adhesion to surface protein density in a way not previously demonstrated in cellular studies.
3:00 PM		BI+NS-WeA-4 Selective Molecular Assembly Patterning - A New Approach to Micro- and Nanochemical Patterning of Surfaces for Biological Applications R. Michel (Laboratory for Surface Science and Technology, Switzerland); J.W. Lussi (Laboratory for Biomedical Engineering, Swiss Federal Institute of Technology, Zurich, Switzerland); I. Reviakine, M. Textor, N.D. Spencer (Laboratory for Surface Science and Technology, Switzerland) A novel method for producing chemically patterned surfaces based on selective self-assembly of alkane phosphates on metal oxide surfaces is presented. Standard photolithography is used to create patterns of titanium oxide within a matrix of silicon oxide by successively depositing 40 nm of TiO₂, 10 nm of SiO₂ onto a silicon wafer, followed by photoresist application and anisotropic etching. Ordered SAMs of alkane phosphates form on the TiO₂, but not on the SiO₂ surfaces by self-assembly. Poly-L-lysine-g-poly(ethylene glycol) (PLL-g-PEG) is used to render the exposed SiO₂ protein-resistant. X-ray photoelectron spectroscopy and imaging time-of-flight secondary ion mass spectrometry were used to characterize the surfaces. Protein adsorption studies conclusively established that the resulting surfaces presented protein adhesive (the TiO₂/alkane phosphate SAM region) and non-adhesive (the PLL-g-PEG-coated SiO₂) areas. This novel Selective Molecular Assembly Patterning (SMAP) technique was used to grow fibroblasts in the presence of serum on 55 µm TiO₂ spots. Cytoskeletal organization in the fibroblasts was induced above the 55 µm TiO₂ patches, while no interaction with the PLL-g-PEG background was evident. The SMAP technique is considered to be highly suitable for reproducible and cost-effective fabrication of biologically-relevant patterns over large areas, by combining state-of-the-art photolithography and simple self-assembly dip-and-wash processes. Its applicability to sub-micrometer patterns is currently being evaluated.
3:20 PM		BI+NS-WeA-5 Studies of 20 nm Gold Particle Systems for Biosensing Applications & Optical Properties of Gold Nanostructures L. Olofsson, F. Höök, P. Delsing, D.S. Sutherland, J. Gold, B. Kasemo (Chalmers University of Technology, Sweden) xxx
3:40 PM		BI+NS-WeA-6 Nanofabricated Lipid Bilayers Patterned on Metal Electrodes R.N. Orth, I. Hafez, J. Kameoka, M. Lindau, H.G. Craighead (Cornell University) Lipid molecules were immobilized on the surface of photolithographically patterned chromium and titanium. Large unilamellar lipid vesicles were found to bind on the native oxide surface of patterned support metals. Metal evaporation and resist liftoff techniques were used to pattern metal on a hydrophobic polymer surface. Lipids bound on solid substrates provide a biological interface for impedance measuring electrodes to detect bound cells or biomaterial. This patterning technique provides means to specifically bind lipids and conjugated biomaterials (polyethelene glycol (PEG), biotin, fluorescence dyes, and DNA oligimers) to the electrode surface. This technique may be applied to patterning biomaterial on metal inside thermally bonded microfluidic channels, to form titanium coated biomedical implants, and to create robust lipid-conjugated electrodes for biosensor applications.
4:00 PM		BI+NS-WeA-7 The Micropatterning of Mixed SAM Surfaces Using Inkjet Printing Technology: A Comparison Study to Microcontact Printing L.F. Pardo, T. Boland (Clemson University) Micropatterning is a powerful method for controlling surface properties, with a myriad of applications ranging from cell biology to electronics. Self-assembled monolayers (SAMs) of alkanethiolates on gold, the structures most widely used for preparing organic films with specific surface properties, are usually patterned by partitioning the surface into regions formed from different thiols. In microcontact printing for example, patterned self-assembled monolayers (SAMs) are printed onto a surface using a polydimethilsiloxane (PDMS), made using a microfabricated mold. Although this technique is suitable, the distortion of patterns, pattern limitation to binary mixtures and expensive mold design are limiting the efficient use of stamps. In this study, a new method utilizing inkjet printing technology for patterning mixed thiols is introduced. Methyl and carboxyl-terminated hexadecanethiols were patterned onto clean gold surfaces using a modified inkjet printer. The topography of the micropatterned samples was visualized and measured by atomic force microscopy. The chemico-physical properties investigated by Fourier Transform infrared spectroscopy and dynamic contact angle measurements suggest that inkjet printer yielded high throughput patterning on surfaces. This new inkjet printing technique provides a quick and inexpensive method for micropatterning alkanethiols of surfaces.
4:20 PM		BI+NS-WeA-8 Fabrication of High Aspect Ratio Vertically Aligned Carbon Nanofiber-based Electrochemical Probes for the Probing of Intact Whole Cells T. McKnight (Oak Ridge National Laboratory); M.A. Guillorn (Oak Ridge National Laboratory & University of Tennessee); A.V. Melechko, D.W. Austin (University of Tennessee); V.I. Merkulov, M. Doktycz (Oak Ridge National Laboratory); D.H. Lowndes, M.L. Simpson (Oak Ridge National Laboratory & University of Tennessee) Molecular biology and genomics are providing great insight into gene sequence, regulation and function. At the same time, imaging technology is elucidating cellular structure. Unfortunately, we have limited ability to monitor processes within and around living cells in real time and with high spatial resolution. This limitation is largely technological - our current research instruments are simply not on the same size scale as the functional components of cells. Here we present the fabrication and operation of high aspect ratio vertically aligned carbon nanofiber (VACNF)-based electrochemical probes for the probing of intact whole cells. Electron beam lithography was used to define the catalytic growth sites of the VACNFs. Following catalyst deposition, VACNF were grown using a novel plasma enhanced chemical vapor deposition (PECVD) process. Photolithography was performed to realize interconnect structures. These probes were passivated with a thin layer of SiO2, which was then removed from the tips of the VACNF, rendering them electrochemically active. We have demonstrated their functionality by selectively electrodepositing Au clusters onto the tips of the probes. We believe that these probes are ideally suited for characterizing intracellular phenomena in real time with an unprecedented degree of spatial resolution.