AVS2001 Session BI-ThP: Biomolecule and Cell Poster Session
Thursday, November 1, 2001 5:30 PM in Room 134/135
Thursday Afternoon
Time Period ThP Sessions | Topic BI Sessions | Time Periods | Topics | AVS2001 Schedule
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BI-ThP-1 Interactions of Dye Molecules with Poly(Propylene Imine) Dendrimer Monolayers
S. Chen, L. Li, C. Boozer, S. Jiang (University of Washington) Dendrimers have been regarded as interesting candidates for applications in host-guest chemistry, such as complex drug delivery agents. A fundamental understanding of guest-host interactions in dendrimers will facilitate the effort to design new-generation chemical and biological sensors based on dendrimers. In this work, we report a systematic study of interactions between a group of dye molecules and poly(propylene imine) dendrimers (PPI) tethered to carboxyl-terminated self-assembled monolayers (SAMs). Adsorption kinetics is measured by surface plasma resonance (SPR) sensors. The fifth-generation PPI dendrimers are mainly used in this work. Our results show a strong affinity of PPI-5 to Rose Bengal or erythrosine B. The strong affinity results from well matches in charge and structure between the guest and the host molecules. In addition, the effect of various factors (e.g., dendrimer generations, charges and shapes of guest molecules, and pH values and ionic strengths of liquid solutions) on adsorption will be discussed. This is the first attempt to study the adsorption of small molecules using SPR. |
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BI-ThP-2 Protein Adsorption on Mixed Self-assembled Monolayers
L. Li, S. Chen, C. Boozer, S. Jiang (University of Washington) Mixed self-assembled monolayers (SAMs) of alkanethiols on Au(111) can be used to precisely control molecular-scale chemical, structural, and biological surface properties via controlling the abundance, the type, and the spatial (both normal and lateral) distribution of tail group sites. By controlling surface microenvironment, different structures and activities of immobilized proteins are expected. Here, we first report our recent studies on phase behavior of mixed alkanethiols with two compounds having different chain lengths (C8-C18) and terminal groups (-COOH, -OH, -CH3, and -NH2) on Au(111). These mixed SAMs are characterized by scanning tunneling microscopy (STM) and atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), contact angle measurements, and ellipsometry. Results show that closely packed and homogeneously mixed SAMs can be achieved at the molecular level. These controlled surfaces will then be used for the adsorption of various proteins such as albumin, lysozyme, etc. Adsorption behavior is characterized by tapping-mode AFM and surface plasmon resonance (SPR) sensors. Results show that molecular-scale mixed SAMs generally promote protein adsorption. The effect of the abundance, type, and spatial distribution of terminal groups on protein adsorption is explored systematically in this work. Keywords: Mixed SAMs, protein adsorption, AFM, SPR, and XPS. |
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BI-ThP-3 Studies of Phosphopeptides on Metal Impregnated Plasma Polymer Surfaces
J. Zhang, J.D. Whittle, H. Qiu, R.B. Timmons, G.R. Kinsel (The University of Texas at Arlington) Surface-protein interactions play an important role in the fields of biology and medicine. Recent work in our group has focused on the binding affinity of phosphopeptides for metal ions immobilized on vinyl acetic acid modified PET substrates. Our work demonstrates that, under specific solution conditions, phosphopeptides have high binding affinities for copper. This observation has been utilized to purify / clean-up phosphopeptides on-probe before sample analysis by Matrix Assisted Laser Desorption / Ionization (MALDI) Mass Spectrometry (MS). Our initial studies focus on the development of surfaces for extraction / isolation of phosphopeptides via coordination with surface bound metal ions. PET substrates (4.8 mm diameter disks) were modified by pulsed RF plasma deposition of polymerized vinyl acetic acid. Metal ions were incorporated into the vinyl acetic acid modified PET substrates by immersion of the substrates into various metal ion solutions. Specific binding of phosphopeptides to the surface was first demonstrated by exposure of the metal impregnated film to a mixture of the peptide buccalin and the phosphopeptide p60 substrate II. Washing of the surface with the buffer MES (pH = 5.5)/10%acetonitrile led to selective removal of the buccalin peptide from the surface film. (This step was confirmed by MALDI analysis of the wash solution.) Similar methods have also been successfully applied to the extraction of phosphopeptides from alpha-casein tryptic digests. The purification of phophopeptides resulted in an increase in the peptide MALDI ion signal and improved ion signal resolution. Additional studies have focused on the effect of changes in the metal ion used for phosphopeptide binding, changes in the solutions used for washing / peptide release, and metal-ion binding of histidine-rich peptides. |
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BI-ThP-5 Time-of-flight Secondary Ion Mass Spectrometry (ToF-SIMS) Characterization of Adsorbed Proteins with Amino Acid Mutations
N. Xia, R. To, S.L. McArthur, P.S. Stayton, D.G. Castner (University of Washington) Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to detect amino acid sequence mutations in adsorbed streptavidin. A series of mutations were introduced into wild-type streptavidin using site-directed mutagenesis. Both wild-type and mutant strepavidin were adsorbed onto polystyrene. Then positive and negative static ToF-SIMS spectra were acquired. For mutants where one amino acid (either glutamic acid or serine) in each strepavidin monomer was replaced with a cysteine the largest differences were observed in the negative spectra. The appearance of ToF-SIMS peaks at 32 (S-) and 33 (HS -) indicated the incorporation of cysteine into the streptavidin mutants. Only small changes in the positive ion ToF-SIMS spectra were observed upon incorporation of cysteine into streptavidin (e.g., the appearance of a small peak at 45 due to the CHS + fragment from cysteine). Introduction of a larger mutation (linking a histidine tag to the C-terminus of each streptavidin monomer) resulted in larger changes in the positive ion ToF-SIMS spectra. Due to the relative complexity of the positive spectra from proteins, principal component analysis (PCA) was used to analyze the differences in the positive spectra. The largest changes introduced by the presence of the histidine tags were observed in the intensities of the peaks at m/z=81, 82 and 110, which correspond to the primary mass fragments from the amino acid histidine. |
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BI-ThP-7 Dynamics and Chemistry of ELISA Test for Toxins by In-situ and Ex-situ Spectroscopic Ellipsometry
D.W. Thompson, E.M. Berberov, G.L. Pfeiffer, J.A. Woollam (University of Nebraska-Lincoln); T.E. Tiwald, C.M. Herzinger (J.A. Woollam Co., Inc.); T. Datta (University of South Carolina) Fast, in-situ spectroscopic ellipsometry at 225 simultaneous visible wavelengths is used to study dynamics of deposition in liquid solution of each layer in a four-layer enzyme-linked immunosorbent assay (ELISA) for cholera toxin. Time-dependent functional relationships of cholera and Escherichia coli heat-labile enterotoxin attachment to monosialoganglioside (GM1) coated substrates are studied, as well as the attachment of associated antibodies. Ellipsometric selectivity between toxins is demonstrated. In addition, ex-situ spectroscopic ellipsometry from vacuum-ultraviolet (131 nm / 9.5 eV) to mid-infrared (35 microns) on each constituent layer of the ELISA is studied. Several distinct oscillator-like features in the visible to vacuum-UV are found at 1.42 eV, 4.24 eV, 6.32-6.35 eV, and 10.98 eV. Protein resonant chemical oscillators in the individual layers are identified as: C-H in CH2 and CH3, N-H, O-H, and P=O, seen at 3307 cm-1, 2926 cm-1, 2958 cm-1, 1662 cm-1, 1546 cm-1, 1245 cm-1 and 1081 cm-1. In summary, in-situ and ex-situ spectroscopic ellipsometry covering vacuum-UV to mid-infrared is a simple, nondestructive way to study dynamics and chemistry of nanometer dimension ELISA films. Research supported by NSF SBIR contract number NSF II-9901510. |
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BI-ThP-8 Do the Chemical Properties of Polymeric Surfaces Influence the Bacterial Adhesion?
G. Speranza, R. Canteri, C. Pederzolli, G. Gottardi, L. Pasquardini, E. Carli, M. Grosello, A. Lui, M. Anderle (ITC-irst, Centro per la Ricerca Scientifica e Tecnologica, Italy) Understanding the mechanisms underlying the interactions between biological systems and polymeric surfaces is of paramount importance. In fact, for their physico-chemical properties, polymers as biomaterials, are utilized in a even larger variety of applications. However, an increasing significance in the medical routine is assumed by the development of bacterial infections caused by implanted polymeric devices (up to 40% of nosocomial infections). In this respect the investigation of the phenomena which take place at the interface between polymeric surface and bacterial wall, are gaining a great relevance. Due to the high level of complexity, these processes, to the best of our knowledge, are still not well clarified. Aim of this work is to investigate this topic to reach a deeper degree of comprehension. The description of the interfacial interactions at the biomaterial surface normally make use of the van der Waals forces. A new term that involves acid-base interactions is here hypothesized to fully describe the bacterial adhesion to the polymer surface. Two requirements are needed to test this hypothesis: an ideal polymeric surface in terms of chemical and morphological properties and "standard samples" as bacterial strains. Several experiments were worked out using the Escherichia Coli (Gram-) strain and its growth on polymers having an "acid" or "basic" character, was investigated. The first findings seems to indicate that the bacterial adhesion is influenced by the chemical properties of the polymeric surface. This first results may be interpreted taking into account a mechanism in which the acid/base (Lewis) interaction plays an important role. |
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BI-ThP-9 Photoluminescent Study of Bacterial Lipid A Langmuir-Blodgett Monolayers
V.V. Kislyuk, L.D. Varbanets, Z.I. Kazantseva, I.M. Pahuta, V.Z. Lozovski (National Academy of Sciences of Ukraine) The photo luminescent spectra have been studied at room temperature for the Lipid A Langmuir-Blodgett monolayers grown on various substrates of Au, Si and SiO2. The maxima of the spectral profiles are shifted towards long waves as compared to the spectra of 10-2% DMSO solution and water suspension of the lipid. The energy difference of approximately 0.4 eV is, likely, attributed to the molecule-molecule and molecule-surface interaction of the monolayer. The luminescence has been stimulated with pulses of UV laser (337 nm, 100 Hz, 8 ns) or UV lines of Xe lamp and registered with photon counter with the coincidence circuit. The lipid A were extracted from the membrane of Ralstonia Solancearum. |
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BI-ThP-10 Human Foreskin Fibroblast Cell Studies on Micropatterned Substrates Created by Selective Molecular Assembly Patterning
J.W. Lussi, R. Michel, A. Goessl, M. Textor, J.A Hubbell (Swiss Federal Institute of Technology, Switzerland) A novel patterning technique, termed "Selective Molecular Assembly Patterning" (SMAP), was used to produce cell-adhesive patches on a cell-resistant background. The method is based on selective adsorption of alkane phosphates to titanium oxide, but not silicon oxide surfaces. Using standard photolithographic techniques patterns of titanium oxide within a matrix of silicon oxide were created. A self-assembled monolayer of dodecyl phosphate (DDP) formed on the titanium oxide, while poly(-L-lysine)-poly(ethylene glycol) graft copolymer (PLL-PEG) subsequently rendered the silicon oxide matrix resistant to cell adhesion. A combination of phase contrast and fluorescent microscopy was used to examine the spreading of human foreskin fibroblast (HFF) cells seeded on the patterned substrates. Unstained cells, as well as cells stained for cytoskeletal f-actin and the focal adhesion protein vinculin, were examined. HFFs adhered only to the DDP-coated TiO2 patches, clearly recognizing the substrate pattern. The PLL-PEG coated SiO2 matrix remained completely resistant to cell adhesion in fetal bovine serum containing medium for more than 5 days. Cell surface contacts were observed on DDP-coated TiO2 patches and stress fibers traversed patterned features. Cell shape on patterned substrates differed significantly from HFF cultured on homogenously cell-adhesive substrates. Cells exhibited more angular shapes imposed by the square symmetry of the oxide pattern. Cell extensions frequently bridged several features without establishing contacts to the background surface. These cell experiments conclusively demonstrate the usefulness of this patterning method for studying cell adhesion on patterned surfaces. |
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BI-ThP-11 Self-Assembly Approaches to Preparation of Self-Supported Porous Films for Selective Cell Separations and Tissue Engineering Scaffolds
M. Tanaka, M. Miyama, K. Nishikawa, J. Nishida, M. Shimomura (Hokkaido University, Japan) Selective cell separation is one of the most important issues in the field of tissue regenerative medicine at present. We prepared self-supported porous films of poly(βcaprolactone) or poly(lactic acid-co-glicolic acid) with wide range of controlled pore size (0.5-100 µm) and area (diameter 1-20 cm) to develop high-performance filters and tissue engineering scaffolds. The self-supported porous films were fabricated by a simple casting technique and self-assembly proceses1,2 without lithographic methods. The films had highly regular patterns and stable net structure with high mechanical stability and moderate elasticity. Creation of desirable pore size was easy, and the pore size can be controlled simply by changing the evaporation rate of the polymer solutions. The porous film which had been a novel biocompatible poly(2-methoxyethyl acrylate)3,4 coated was attached to a module having an inlet port and outlet port to simulate a leukocyte eliminating. The film provided the 100% selective separation of the leukocyte from human blood. The leukocyte eliminating ratio and platelet recovery were dependent on the pore structures (size and depth) and the chemical properties (hydrophilicity and surface charge). The biocompatible and biodegradable films could be expected as promising materials to produce useful selective cell separations, implants and cell-supported elastic scaffold for various medical devices.
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BI-ThP-13 Analysis of Cell Organization in a Smooth Muscle Culture Grown On High Aspect Ratio Microstructures
J.D. Glawe, D.K. Mills, B.K. Gale (Louisiana Tech University) Tissue engineered organs may someday replace donated organs. The first step towards realizing this goal is to create three dimensional cell cultures that preserve the organization of cellular orientation found in tissue in vivo. One promising method of achieving this goal is to microfabricate scaffolds with microcontainers designed to allow cell growth in only one direction. This is essential for engineering muscle tissue where to remain functional, all cells must contract along the same axis. The tissue culture scaffold was specifically designed to accommodate smooth muscle cells (SMC's). It was molded out of polydimethylsiloxane (PDMS) from a lithographically patterned SU-8 structure. The structure is coated with protein to promote improved cell adhesion. Two methods of seeding the cells into the scaffold were tested, gravitational and centrifugal seeding. The microcontainers are approximately 500 µm in depth and are either square, octagonal, or plus-shaped, with varying diameters. Measurements were made to determine cell density, orientation, actin concentration, and the percent of living cells at four intervals after cell seeding. |
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BI-ThP-14 Ion Implanted Titanium: Relating Surface Chemistry to Cellular Response.
F.H. Jones, L. Shinawi, S. Nayab, I. Olsen, J.A. Hobkirk (University College London, UK); T.J. Tate, D.S. McPhail (Imperial College of Science, Technology and Medicine, UK) Titanium-based materials are used extensively in hard tissue biomedical implants, often with inorganic coatings to promote bone regeneration and integration. The properties of such coatings remain less than ideal; variable composition, dissolution at low pH and fracture failure or delamination continue to present serious problems. Direct modification of the surface using ion implantation has been proposed as a possible alternative, giving a favourable interface for interaction with the host tissue, without affecting bulk properties. The homogeneity and controllability of ion implantation also mean that the technique is ideal for studying the fundamental effects of chemical composition on cellular response to a surface. Ca implantation has been shown to promote osseointegration,1 but little attention has been paid to the effect of the nature of the substrate on the resulting surface chemistry, or the difference in behaviour of surfaces implanted with different ions. The current work examines the effect of ion implantation into native Ti, air oxidised Ti and TiO2 single crystals. Ca, K and Ar ions, selected due to their similar masses, were implanted at doses up to 2x1017 ions cm-2. Preliminary cell culture studies indicate significant differences in cell behaviour depending on the chemical nature of the implanted ion. Of particular interest are adverse effects observed on Ar-implanted surfaces, despite the inert nature of argon. In parallel, XPS and SIMS were used to investigate surface chemistry. The effects of annealing in UHV and immersion in water were found to be element-dependent. The nature of the ion was also found to influence the interaction with model organic species in solution and the rate of calcium phosphate deposition from mineralising solutions. |