Papers

AVS1997 Session BI-TuA: Biomolecule Solid-Surface Interactions

Tuesday, October 21, 1997 2:00 PM in Room F

Tuesday Afternoon

Start	Invited?	Item
2:00 PM		BI-TuA-1 Discrimination of Surface-bound DNA via AFM L.T. Mazzola, C.W. Frank (Stanford University); S.P.A. Fodor (Affymetrix, Inc.); E. Henderson (Bioforce Laboratory, Inc.) The specificity of biomolecular recognition can be probed with an atomic force microscope (AFM) using a chemically-modified probe. This technique is possible by derivatizing the microscope tip with a ligand or receptor that shows affinity for an immobilized substrate. Often referred to as "chemical force" microscopy, the adhesive interaction between the tip and substrate is controlled through molecular affinity. We show this technique to be useful in probing the recognition and discrimination of complementary strands of DNA. Photolithography has been used to direct the synthesis of a two-dimensional matrix of oligonucleotides of the sequence 3'-CAGTTCTACGATGGCAAGTC, alternating with regions containing a different base sequence. These arrays were then probed with AFM tips derivatized with the complement to the above oligonucleotide. These modified tips display sequence-specific discrimination of the multi-component substrate DNA.
2:40 PM		BI-TuA-3 Electronic Properties of "G-wire" DNA Investigated by Low Current Scanning Tunneling Microscopy T.C. Marsh (University of Wisconsin, Green Bay); J. Root (California State University, Fresno); W. Han, S.M. Lindsay (Arizona State University); E. Henderson (Iowa State University); J. Vesenka (California State University, Fresno) Low current scanning tunneling microscopy (LCSTM) of quadruplex DNA on mica was carried out under ambient conditions at high relative humidity (>75%), large bias voltages (about -5V) and currents in the neighborhood of 3 pA. LCSTM images of these molecules showed remarkable similarity with atomic force microscope images of DNA in dry air, i.e. increased lateral width but with diameters (heights) consistent with x-ray determined dimensions. We believe that much of the lateral broadening is the result of residual buffer salts surrounding the DNA that also act as a bridge to the mica substrate. A model predicting DNA high as a function of humidity is compared with previous AFM investigations ¹. Since LCSTM measures the electronic tunneling of surface currents over electronic insulators such as mica, these buffer salts play an important role in the observed electron conduction pathway ². ¹Vesenka, J et al., Scanning Microscopy 7, 781 (1993) ²Yuan, J-Y et al., Phys. Rev. Letts. 67, 863 (1991)
3:00 PM		BI-TuA-4 Monitoring Protein Surface Interactions by MALDI Mass Spectrometry. G.R. Kinsel, A.K. Walker, R.B. Timmons, Y. Wu (University of Texas, Arlington) We have initiated a program where matrix assisted laser desorption / ionization (MALDI) mass spectrometry (MS) is used to characterize peptide and protein binding to unmodified and RF pulsed plasma modified polymer surfaces. Our initial studies show an inverse relationship exists between surface protein binding affinity and observed MALDI ion signal. These studies are correlated with protein binding studies performed using radiolabeled proteins. A second group of studies employed the MALDI MS technique to explore the effect of various surface chemistries, produced by pulsed RF plasma modification of the polymer materials, on protein binding affinity. The interactions were examined as a function of protein pI and molecular weight. In these studies protein pI was clearly identified as a relevant parameter when considering protein binding to amine modified surfaces. Studies of surface roughness by atomic force microscopy further suggest a complex relationship exists between surface morphology-surface chemistry-protein pI and binding affinity. The detailed insights obtained were clearly advanced by the sensitive and efficient MALDI MS technique. A third group of studies demonstrates that MALDI MS can be used to quantitate surface protein binding. This feature is exploited to monitor the time dependence of protein adsorption to unmodified and modified surfaces. In addition, the ability to selectively detect the adosrption of specific biomolecules by MALDI MS has allowed us to explore the time dependent competitive binding of proteins from a complex mixture of biomolecules.
3:20 PM		BI-TuA-5 Molecular Nanotechnology with 2D-Protein Crystals (S-layers) at Surfaces and Interfaces U.B. Sleytr, D. Pum (Universitat f. Bodenkultur, Austria) Crystalline bacterial cell surface layers (S-layers) have shown to be ideal patterning structures for supramolecular engineering. S-layers are composed of monomolecular arrays of identical (glyco)proteins showing high molecular order, defined mass distribution and isoporosity, and high binding capacity for functional macromolecules (e.g.enzymes, antibodies). The possibility for recrystallizing isolated S-layer subunits into large scale coherent protein lattices at solid supports (e.g. silicon, gallium arsenide, gold, glass), at the air/water interface or on lipid films and for handling such layers by standard Langmuir-Blodgett techniques opens a broad spectrum of applications in molecular nanotechnology and biomimetics. S-protein recrystallized on solid supports may be patterned by Deep Ultraviolet excimer laser radiation in the 200nm range. A further application of S-protein monolayers is their use as templates in the fabrication of nanometric metallic point patterns. The concept of an S-layer supported functional lipid membrane seems promising since scientific and practical applications of supported lipid films require nanometer thick biocompatible, water containing interfaces as seperators between lipid membrane and substrate in order to maintain the thermodynamic and structural properties of the free lipid film. The mechanical stability of composite S-layer/lipid films at interfaces can be increased by inter- and intramolecular crosslinking of the subunits from the subphase. The proportion of lipid molecules in the monolayer that can be covalently linked to the porous S-layer lattice will significantly modulate the lateral diffusion of the free lipid molecules and consequently the fluidity of the whole membrane ("semifluid membrane"). S-layer supported functional phospholipid bilayers or tetraether lipid films mimic the molecular architecture of those archaebacterial cell envelopes that are exclusively composed of an S-layer and a plasma membrane. This novel concept could lead to new techniques for exploiting large scale structural and functional principles of membrane associated and integrated molecules (e.g. ion channels, proton pumps, receptors).
4:00 PM		BI-TuA-7 Order and Orientation of Proteins Bound to Self Assembeled Monolayers as Determined by NEXAFS L. Gamble, D.G. Castner, K. Nelson, P. Stayton (University of Washington); D.A. Fischer (National Institute of Standards & Technology) The ordering and orientation of streptavidin (SA) adsorbed onto biotinylated alkane thiol (BAT) monolayers (SAMs) self assembled onto gold surfaces has been studied by near edge x-ray absorption fine structure (NEXAFS) and x-ray photoelectron spectroscopy (XPS). The biotinylated self assembled monolayers (SAMs) were prepared from mixtures of BAT and C16 alkane thiols. NEXAFS spectra indicates that SAMs made from mixed BAT-alkane thiol solutions are better ordered than those made from 100% BAT solutions. NEXAFS spectra (taken in the total electron yield mode) of SAMs prepared from a 60% BAT solution show polarization dependence in the CH* and CC* resonance similar to those seen for Langmuir-Blodgett monolayers with alkane chains. The CN* peak, which is emphasized when data is collected in partial electron yield mode, also shows polarization dependence indicating preferential orientation of the biotin head group. Angle dependent XPS is used to support the NEXAFS data. SA was adsorbed onto the mixed SAMs and a pure alkane thiol monolayer. Radiolabeling was used to determine the amount of SA adsorbed on these surfaces. NEXAFS data collected in partial electron yield mode indicates a small degree of SA ordering on the methyl terminated surface. A higher degree of ordering is observed for SA adsorbed on SAMs with biotin terminated surfaces created from mixtures of BAT and the alkane thiol.
4:20 PM		BI-TuA-8 Ordered Protein Assembly at Biomaterial Surfaces. K. Nelson, D.G. Castner, L. Gamble, L. Jung, C.T. Campbell, P. Stayton (University of Washington) The successful assembly of ordered protein and peptide arrays that mediate desired cell responses to biomaterials requires the complementary design of surfaces and proteins. It is also crucial that the surface background minimize nonspecific binding and cell activation. We have used alkyl thiol assembly on gold surfaces to immobilize oriented and highly ordered streptavidin monolayers. ESCA and surface plasmon resonance studies have been conducted and demonstrate that the quality of the protein monolayers depends on the chemical nature of the mixed alkyl thiol components and their ratio on the surface. When a biotinylated alkyl thiol is mixed with a methyl-terminated alkyl thiol, nonspecific protein binding is observed. The nonspecific binding can be minimized with the use of a PEO-terminated alkyl thiol. The streptavidin molecules have been redesigned to contain RGDS peptides to promote cell adhesion and to trigger receptor-mediated biological pathways. These peptide sequences have been engineered into the streptavidin surface that is oriented away from the self-assembled monolayer, and initial studies of endothelial cell adhesion to these oriented peptides will be reported.
4:40 PM		BI-TuA-9 Adsorption and Electron Transfer of Cytochrome C at Graphite-Water Interface Studied by In Situ Tapping Mode AFM. S. Boussaad, N.J. Tao, J. Arechabaleta (Florida International University) We have studied the adsorption and electron transfer of cytochrome C at the graphite-water interface with an in situ tapping mode atomic force microscope (TMAFM). At a concentration of 0.1 mM in phosphate buffer (20 mM) individual proteins appear in a globular form with an average size of about 10 nm (tip broadening not corrected). The distribution of the proteins in the adsorbed film is uniform. Increasing the substrate potential above 0.8 V (v.s. Ag/AgCl), a pair of peaks, corresponding to the reversible electron transfer of Fe(II) <-> Fe(III) in cytochrome C, appears at about 0.3 V. Our TMAFM study of this phenomenon suggests that the protein reorients itself at ~0.8 V so that the electron transfer center becomes closer to the substrate electrode. ¹Work supported by NIH (GM-08205) and Research Corporation (#CC3608).
5:00 PM		BI-TuA-10 Adsorption and Electron Induced Dissociation of Glycine on Graphite P. Löfgren, A. Krozer, D.V. Chakarov, B. Kasemo (Chalmers University of Techn. & Göteborg Univ., Sweden) To model the material-biosystem interface we are studying the adsorption and desorption of the in-situ deposited amino acids and simple peptides on single crystal surfaces in UHV using surface physics techniques. Previously we have reported results for glycine, alanine, cysteine and the peptide glycylglycine adsorbed on Pt(111) obtained using mainly TDS, but also XPS. Here we report on adsorbtion of glycine (Gl) on a HOPG (0001) surface studied by TDS and HREELS. The TDS experiments reveal a single desorption peak at 300 K for (sub)monolayer and multilayer coverages due to desorption of intact Gl molecules which indicates that Gl-HOPG and Gl-Gl bindings are of similar magnitude.The observed desorption kinetics and the lateraly variable e^--reflectivity suggest glycine clustering with the onset < 0.1 ML. The adsorbed Gl layer subject to electron irradiation decomposes down to electron energies as low as 5 eV. TDS from adsorbates exposed to 70 eV electrons (generated by the MS filament) reveal two new desorption peaks at lower temperatures than the peak from non-irradiated Gl. The cracking pattern from these peaks differs from that of the intact Gl molecules which suggests electron induced dissociation of glycine. At a given e^--energy the dissociation depends on the initial Gl coverage, and on the electron dose. Gl adsorbed on Pt(111) and exposed to X-rays during XPS data collection shows also beam induced damage, although to a much lesser degree than electron irradiated Gl on graphite. It is thus clear that adsorbed Gl is sensitive to electron irraddiation and one has to be carefull when applying electron spectroscopies to study these organic molecules.