Papers

AVS1997 Session BI-TuM: Surface Characterization of Biological Materials

Tuesday, October 21, 1997 8:20 AM in Room F

Tuesday Morning

Start	Invited?	Item
8:20 AM		BI-TuM-1 Direct Measurements of Microenvironmental Influences on Biomolecular Recognition at Interfaces D.E. Leckband (University of Illinois, Urbana) Molecular recognition at the surfaces of biomaterials or biosensors is a complex function of both the receptor-ligand interactions and the influence of the substrate/biomaterial properties on the net forces between immobilized cognate receptors and soluble analytes. The work presented describes direct measurements with the surface force apparatus (SFA) of the influence of both long and short-range substrate forces on the ability of soluble analytes to recognize immobilized species. In particular, we quantified the influence of the electrostatic properties of the supporting materials on site-selective binding. We also studied the effectiveness of strategies intended to mask or suppress these influences. Near the surface, short-range steric forces due to surface hydration or thermal roughening of soft materials presents an additional energy barrier to selective surface adsorption. We demonstrated the relevance of measured short-range repulsive forces to experimentally determined surface binding kinetics. By incorporating measured, and therefore realistic, repulsive potentials into a hydrodynamic model for site-selective surface binding by proteins, the rates of adsorption were predicted quantitatively. These findings show the importance of considering not only the properties of receptor-ligand bond, but also the influence of the microenvironment on biomolecular recognition at surfaces.
9:00 AM		BI-TuM-3 Orientation of "G-wires" on Mica J. Vesenka, I. Kumar, C. West (California State University, Fresno); J. Bolonick (Science Consultant); T. Marsh (University of Wisconsin, Greenbay); E. Henderson (Iowa State University) Quadruplex DNA, in the form of "G-wires", allowed to incubate in buffer on the surface of freshly cleaved mica preferentially orient themselves over a period of time, as determined by atomic force and low current scanning tunneling microscopy investigations. Furthermore, the G-wires orient parallel to the next nearest neighbor of mica's atomic surface, and in greater abundance in one of these orientations than others. The mechanism for the preferential adsorption mechanism between mica and purported structure of G-wires may be a response to the octahedrally coordinated layer of magnesiuim ions between the silicate tetrahedral layer in muscovite mica. Thus it is possible the observation of G-wire alignment is an experimental measure of the muscovite's "stagger vector". In addition, the G-wires provide reproducible "networked patterns" and might be manipulated into suitable structures for "nano-circuitry" purposes using scanning probe microscopes.
9:20 AM		BI-TuM-4 Atomic Force Microscope Measurements of Enzyme Interactions with Various Cotton Fibers I. Lee, B. Evans, J. Woodward (Oak Ridge National Laboratory) Tapping-Mode Atomic Force Microscope (AFM) has been used to study the interaction between catalytically active and inactive cellobiohydrolase I (CBH I) and cotton fibers. Images of both enzyme and microfibril molecules can be resolved from the AFM. CBH I was catalytically inactivated by treating with ammonium hexachloropalladate while still possess the ability to bind to the cotton fiber. We have observed inactivated CBH I on cotton fiber under the AFM. The images from AFM suggesting that the catalytic activity of CBH I was required for fiber disruption. However, the cottons containing brown pigments, the natural colored varieties Buffalo (registered trademarks of Natural Cotton Colours, Inc., Wickenburg, Arizona), was quite resistant to CBH I and EG II. The AFM images of colored cotton fibers treated with CBH I shows peeling of the outer surface. The surface treated with EG II appeared to smoothed or polished.
9:40 AM		BI-TuM-5 Water Interfaces and Tunnel Channels in STM Studies J. Halbritter (Forschungszentrum Karlsruhe, Germany) Water is the base not only of electrochemistry or biology but acts also as substrate in STM measurements in air or even in UHV. Such water-hydroxide coatings are difficult to identify or to quantify. STM depends sensitively on the state of those waterous coatings enhancing charge transfer, which allows a quantification of such coating. So "dead" interfaces with fast decaying direct tunneling given by exp(-2d√2mΦ₀/h) with the gap width d and the tunnel barrier height Φ₀ > 1 eV are no longer the appropriate description. Instead, via water tunnel currents decay very slowly with gap width described by d_eff=d/(n+1) by n intermediate states efectively bridging the tunnel gap, which translates into a low tunnel barrier Φ_eff = Φ₀/(n+1)². Thus, the reported small Φ_eff-values, i.e. large barrier transparencies for d > 0.3 nm, are not "energetic" barrier heights but resemble intermediate state tunneling. By using the distance dependence of the tunnel current several intermediate states in oxide-, hydraxide-, and polarized water layers have been identified by small Φ_eff-values. For polarized HOH-HO^--layers we propose dipole resonances as intermediate states performing the STM imaging. Dipole resonances, like other electron attachment states, strengthen tunneling by small Coulomb charging energies. Such attachment states not only enhance barrier tunneling across, but also yield incoherent tunnel exchange along layers of attachment states. For example, water layers allow charge transfer along surfaces important, e.g. for biological systems or the STM imaging on mica. If insulated against tip and substrate such layers act as intermediate electrodes causing a Coulomb barrier in tunneling.
10:00 AM		BI-TuM-6 Solid State NMR Methods for Determination of Peptide Structure on Crystalline Surfaces G. Drobny, J. Dindot, J. Long, P. Stayton (University of Washington) The Materials Science community has focused a great deal of attention recently on the materials processing strategies by which biological composites and ceramics are synthesized and processed. The molecular control necessary to produce these materials is directly provided by proteins in most known examples. Despite their well established importance, however, little is known of the direct molecular mechanisms by which protein surfaces control the nucleation and growth of biological composites such as calcium oxalate and hydroxyapatite. Our research program is aimed at developing and applying novel solid state Nuclear Magnetic Resonance techniques to elucidating the structural nature of peptide-surface interactions. In this talk, we will describe: 1) the principles undelying structure determination in biological solids by solid state NMR; 2) the application of these techniques to determining the structure of a peptide derived from salivary statherin and adsorbed onto hydroxyapaptite crystals. 3) future plans and prospects for determining the structure and orientation of larger protein domains on crystal surfaces will be discussed.
10:40 AM		BI-TuM-8 Biological Material Analysis by Matrix-Enhanced SIMS K.J. Wu, R.W. Odom (Charles Evans & Associates) Matrix-enhanced secondary ion mass spectrometry (ME-SIMS) combines the matrix enhanced ionization of MALDI (matrix-assisted laser desroption/ionization) with ion beam sputtering processes characteristic of secondary ion mass spectrometry¹. The studies were performed with a PHI-EVANS high resolution triple focusing time-of-flight mass spectrometer (TRIFT) equipped with a nitrogen laser for MALDI analysis and ion sources (Ga, In and Cs) for TOF-SIMS applications. Molecular ions are observed in this ion beam sputtering of organic mixtures for peptides and oligonucleotides up to masses on the order of 10,000 daltons (Da). This matrix enhanced SIMS exhibits substantial increases in the ionization efficiency of selected analyte molecules compared to conventional static SIMS processes. Thus, higher mass peptides, proteins and nucleic acids become accessible to near surface analysis by ion beam techniques, and subpicomole sensitivity has been demonstrated. A number of matrices were examined for their efficiency in ME-SIMS applications, and these initial matrix studies focused on common MALDI matrices and their isomers. The survey indicate that 2,5-dihydroxybenzoic acid provides the best general enhancement of molecular secondary ions emitted from analyte/matrix mixtures. The results demonstrate the utility of ME-SIMS for near-surface (top 10 nanometer) molecular analysis of organic thin films. The feasibility of ME-SIMS analysis of bimolecules directly on tissues will also be discussed. ¹1. K. J. Wu and R. W. Odom, Anal. Chem. 68, 847 (1996).
11:00 AM		BI-TuM-9 Two Laser Mass Spectrometry for the Surface Analysis of Biological Materials L. Hanley (University of Chicago, Illinois); J.T. Trevor, O. Kornienko (University of Illinois, Chicago); M.J. Pellin, K.R. Lykke (Argonne National Laboratory) Various surface chemical preparations have been developed to enhance the biocompatibility of medical implants, induce biomimetic growth of ceramics, and direct cellular growth in tissue engineering. These surface chemical systems include self-assembled monolayers, Langmuir-Blodgett films, covalently bound polypeptide monolayers, and functionalized polymers. Time-of-flight secondary ion mass spectrometry has become a standard method for the analysis of these systems, but is limited by low ion yields and matrix effects. As an alternate method of surface mass spectrometry, we have been applying two laser desorption/photoionization to the analysis of straight chain and biotinylated alkanethiolate self-assembled monolayers, amino acid thin films, and polymer surfaces modified by ion beams. Laser desorption/single photon ionization with vacuum ultraviolet radiation is performed in a time-of-flight instrument which allows micron scale chemical imaging. Laser desorption/multiphoton ionization is performed in an ion trap instrument which permits tandem mass spectrometry for the selective analysis of components of a surface mixture. The general application of these techniques to biological materials will be discussed. The submitted manuscript has been created by the University of Chicago as Operator of Argonne National Laboratory ("Argonne") under Contract No. W-31-109-ENG-38 with the U.S. Department of Energy. The U.S. Government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government.
11:20 AM		BI-TuM-10 TOF-SIMS Analysis of Streptococcus Salivarius R.E. Peterson, B.J. Tyler (Montana State University) The outermost surface of bacterial cells plays a crucial role in bacterial adhesion phenomena but chemical characterization of the cell surface is a difficult and time consuming process. The objective of this work, is to determine the utility of SIMS for studying cell surfaces. Four strains of Streptococcus salivarius, a parent strains and three mutants that show an increasing loss of the proteinaceous fibrillar layer have been studied with TOF-SIMS. The surfaces of the four strains have been extensively characterized for antigenic composition, presence or absence of surface appendages, the relative amounts of lipo(teichoic acid)/teichoic acid exposed at the surface, the surface free energy, adhesive behavior, infrared spectrum, XPS spectrum and zeta potentials¹. SIMS spectra of the freeze dried cells were collected on an PHI/Evans TRIFT Time of Flight Secondary Ion Mass Spectrometer with a Cs Ion source. The spectra indicate the presence of proteins, hydrocarbons, and carbohydrates. Teichoic acid on the cell surfaces was clearly identifiable in the TOF-SIMS spectra and correlated strongly with previous studies of the cell surface. Distinctive protein fragments could be seen for each of the cell strains. The SIMS PO3- / CNO- peak increases as the number of proteinaceous surface appendages decrease. This ratio shows an excellent correlation with the XPS N/P. Multivariate models are being used to find correlations between the TOF-SIMS results and the other physical/chemical measurement previously made on the cells. ¹Van der Mei H.C, J Noordmans, and HJ Busscher, Biochim. Biophys. Acta, 991: 395-398, 1989
11:40 AM		BI-TuM-11 Diffusion at the Adhesive/Dentin Interface D.M. Wieliczka, M.B. Kruger, P. Spencer (University of Missouri, Kansas City) The dentin/adhesive interface continues to be the subject of investigation using a number of different techniques. The goal of these studies is to obtain detailed information of the morphologic and chemical composition of this interface. In actuality, the system is composed of two interfaces, an interface exists between the adhesive and what has been termed the "hybrid layer" and a second interface is present between the "hybrid" layer and the unmodified dentin. The characteristics of each of these interfaces as well as the separation distance and the composition of the "hybrid" layer are crucial to the understanding of the performance of the adhesive/dentin system. The purpose of this project was to study the dentin/adhesive interface using micro-Raman spectroscopy in conjunction with laser light that is optimized to reduce the background fluorescence. The commercial dentin adhesives Scotchbond Multi-Purpose Plus(3M) and Superbond (Sun Medical) were placed according to manufacturers' instructions on coronal dentin substrates that were cut from extracted, unerupted third molars. The Raman spectra were obtained using a Dilor spectrometer with a resolution of 4 cm^-1 over the spectral range of 100 to 2000 cm^-1 as measured from the laser line. The excitation source was a Kr⁺ laser operating at 647 nm. All data were obtained using a microscope with a 100x objective and with the sample mounted on a precision linear stage allowing for 0.5 µm positioning. Data were obtained from successive positions on the sample providing a spectral record of the interface from the pure adhesive to the pure dentin. Adhesive penetration into the dentin was determined by comparing the relative intensities of spectral bands attributable to the dentin versus the adhesive. With a spot size of 1 µm it was possible to obtain spectra from pure adhesive, pure dentin and to evaluate the hybrid zone from a single sample.