Papers

AVS2004 Session BI-MoP: Poster Session

Monday, November 15, 2004 5:00 PM in Room Exhibit Hall B

Monday Afternoon

Time Period MoP Sessions | Topic BI Sessions | Time Periods | Topics | AVS2004 Schedule

BI-MoP-1 Immobilization of Avidin on COOH-modified SiO₂/Si(100) Surface and Characterization by AFM and BML-IRRAS N. Misawa, S. Yamamura (The Graduate University for Advanced Studies, Japan); T. Urisu (Institute for Molecular Science, Japan) Nowadays bio-mimetic sensing techniques, using immobilization of intact biomolecules on solid surfaces, attract significant attentions. For solid substrates, silicon is a suitable material since precise micro-fabrication has been established. New biosensors can be combined with electronics devices on the same chip. Characterizations by IR spectroscopy and AFM observation are useful tools to investigate biomolecules immobilized on silicon surface. It is known that BML-IRRAS (Infrared Reflection Absorption Spectroscopy using Buried Metal Layer substrate [1]) is a high-resolution surface vibration spectroscopy on the semiconductor or insulator materials, which has sub-monolayer sensitivity for the wide frequency range including fingerprint regions. In this study we have immobilized avidin, which has high versatility for conjugation of biomolecules with solid surface, on the SiO₂/Si(100) surface modified with carboxyl group, and characterized the surfaces by AFM and BML-IRRAS for the first time. The -COOH modification was produced by the deposition of 2-(carbomethoxy) ethyltrichlorosilane and sequential hydrolysis by HCl. Immobilization of avidin was performed after condensation reaction by N-hydroxysucciniimide and EDC, which enhanced the reactivity of carboxyl group with amino group of avidin. AFM images showed that the roughness of the -COOH modified surface was less than 0.5 nm, and protrusions with about 15 nm diameter and 2 nm height appeared after the avidin immobilization. The BML-IRRAS measurements showed clear peaks at 1650 cm^-1 and 1550 cm^-1, which were assigned to Amide I and Amide II bands of avidin. These bands also consisted of several fine structures which might be assigned to secondary structures such as α-helix and β-sheet etc. The detailed shape analysis of these bands could give the information with orientations of these immobilized proteins. [1] S.Yamamura. et al. Jpn. J. Appl. Phys. 42 3942 (2003).
BI-MoP-2 An Application of Microcantilever for Biosensor using Piezoresistivity K.H. Na, C.J. Kang, Y.S. Kim (Myongji University, Korea) A microcantilever-based biosensor with piezoresistor has been fabricated using surface micromachining technique that allows a simple fabrication procedure and a low cost sensor. A microcantilever integrated with piezoresistive readout enables sensing even for non-transparent liquids such as blood and miniaturizing the size of biosensor compared with optical readout. Adsorptions of bio-chemical species on a functionalised surface of a microcantilever will cause surface stress. This makes microcantilever bending and results in the change of the resistance of piezoresistor in the microcantilever. The structural layer of the microcantilever is fabricated with LPCVD silicon nitride film and the sacrificial layer is LPCVD polysilicon film. The cystamine terminated with thiol was covalently immobilized on the gold-coated side of the microcantilever. The immobilization process was characterized by measurement of the microcantilever deflection in real time monitoring. We are going to present a cantilever deflection after a formation of cystamine/glutaraldehyde/protein-A/anti-protein-A composite layers and to measure a limited resolution of the cantilever-based biosensor for a concentration of biomolecule.
BI-MoP-3 Architectured Surfaces for On-Probe Affinity Capture MALDI Mass Spectrometry G.R. Kinsel, Z. Segu, R.B. Timmons (University of Texas at Arlington) MALDI MS is a powerful analytical tool for the characterization of proteins and peptides and is presently used extensively for the analysis of biomolecules extracted from biological media. In these applications one or more purification / fractionation steps are typically required prior to MALDI MS analysis. The development of affinity fractionation approaches performed directly on the MALDI probe is considered attractive because of shortened analysis time, inherent simplicity and reduced sample loss. However, a significant challenge for these on-probe affinity fractionation approaches is the limited capacity of the MALDI probe surface. The significance of protein solution concentration, surface-protein binding constant, and total protein surface capacity, with respect to the MALDI limit of detection for a given protein in a mixture, is revealed in theoretical studies utilizing the systematic treatment of equilibria. Additional experimental studies confirm the predictions of the theoretical model and reveal the importance of the MALDI probe capacity on protein detection using modified probes. In addition, a novel method to increase the capacity of modified MALDI probes is presented. In this approach gold particles are attached to allyl amine RF plasma polymer modified MALDI probes and subsequently modified to incorporate affinity capture ligands through the attachment of biotinylated alkane thiols. Preliminary data demonstrates that these modified gold bead attached MALDI probes allow the selective capture of targeted biomolecules and offer significant increases in the biomolecular binding capacity of the MALDI probe surface.
BI-MoP-4 Single Nanoparticle Detection of Biological Molecules via Darkfield Microscopy G. Nusz, A. Chilkoti (Duke University) Noble metal nanoparticles have been used as sensors for biological molecules because of their unique interactions with light due to the resonant collective oscillations of the conduction electrons known as surface plasmon resonance (SPR). The frequency at which this resonance occurs is strongly dependant upon the dielectric constant of the medium surrounding the particles. We have previously shown that gold nanoparticles chemisorbed on to glass and subsequently functionalized with a biological receptor can optically transduce analyte binding at the surface of the nanoparticles. This is because when the target analyte binds to the receptor functionalized nanoparticles, the dielectric constant of the surrounding media increases, resulting in a measurable shift of the SPR frequency that can be measured as a color change. In this study, we report the extension of this label free optical biosensor to single nanoparticles that are chemisorbed onto glass. Gold nanoparticles in the size range of 13-40 nm and gold nanorods (30 nm diameter, aspect ratio 2.7) are synthesized in solution. The nanoparticles are chemisorbed on to the surface of an amino-terminated silane monolayer under conditions that result in sparse coverage of the nanoparticles on the substrate. Darkfield microscopy is used to detect the SPR shift of a single nanoparticle by analyzing its scattering spectrum as a function of the change in the dielectric constant in the vicinity of a single nanoparticle. Applying this technique to a single nanoparticle offers the advantage of effective detection of a target analyte with detection limits on the order of a few hundred molecules.
BI-MoP-5 Development of Organic Semiconductors Using Metal Doped Fish Protein T. Arockiadoss (Central Leather Research Institute, India); F.P. Xavier (LIFE, Loyola College, India); B.K. Prabhu, M. Babu (Central Leather Research Institute, India) Bioelectronics is an emerging field, which extensively uses the supra-molecular structure of proteins, DNA etc to evolve products applicable in biosensors, switchable membranes, organic thin film transistors and fuel cells. This study shows that a partial purified metal-doped muscle protein from the fish, Clarius battracus, was fabricated with poly vinyl alcohol (PVA) to form a biopolymer thin film. The initial protein characterisation was done by gel electrophoresis, followed by analysing the thin film, using circular dichorism, fourier transformed infrared spectrum, scanning electron microscopy, electrical conductivity with and without temperature dependency and finally cyclic voltameter was used to study the architectures of donor and acceptor molecules. The study concludes that the metal-doped fish muscle protein and PVA gives rise to a conducting biopolymer, leading to a versatile molecular electronic material having a unique electrical and optical property, which could be used as a semiconductor in the arena of biochip, fuel cell and nanotechnology. KEY WORDS: FISH, CONDUCTIVITY, METAL DOPING, PROTEIN.
BI-MoP-6 Biocompatibility of Microelectronic Materials H.D. Wanzenboeck, C. Almeder, E. Bertagnolli (Vienna University of Technology, Austria); E. Bogner, M. Wirth, F. Gabor (University Vienna, Austria) Cell-based biosensors endeavor to use microelectronic data acquisition and processing to evaluate specific signals from living cells. The potential of these bioelectronic sensors has been recognized for numerous applications in medicine, pharmaceutical research, environmental diagnostics and the food and processing industries. The interaction between living tissue and microelectronic materials is the critical issue for all those systems, as the inorganic material must neither interfer with nor harm the cells. The effects of different microelectronic materials on the growth of human colon carcinoma cells have been investigated. A systematic study of the survivability and the growth of an exemplary cell culture on various materials used in microelectronics was performed. The viability and the adhesion of colon carcinoma cells (Caco-2) was tested on 15 different materials - metals, dielectrics and semiconductors - commonly used in microelectronics fabrication. Growth inhibiting materials such as copper and blank gallium arsenide have been identified as well as highly biocompatible materials such as silicon, silicon nitride, chromium and gold. Cells have also been cultured on a microelectrode array consisting of metal and dielectric materials on the same substrate. Neither the sub200 nm height step nor the change of the material showed to effect the cell growth. Several materials have been successfully tested to facilitate the growth of cell structures. The results allow a versatile application for microelectrode arrays and demonstrates the wide compatibility of semiconductor technology for fabrication of cell-based biosensors.
BI-MoP-7 The Use of Novel Self-Assembled Monolayers for Enhancing Biosensor Performance W. Laureyn, F. Frederix, K. Bonroy, T. Ghoos, R. De Palma, K. Jans, C. Zhou, G. Reekmans (IMEC, Belgium); P. Declerck, W. Dehaen, G. Maes (KULeuven, Belgium); C. Van Hoof (IMEC, Belgium) The increasing miniaturisation of biochips and the demand for higher sensor detection sensitivities put severe demands on the process and methodology of coupling biomolecules to surfaces. More specifically, controlled thin film structures have to be created which allow the bio-affinity elements to be arranged and addressed in a reproducible and controlled manner. Addressing these issues, IMEC has developed promising methodologies for the construction of novel, well-defined biosensor interfaces, based on the deposition of Self-Assembled Monolayers (SAMs) of alkane thiols or disulfides on metal (e.g. gold) and alkyltrichlorosilanes on oxide (e.g. tantalum pentoxide) surfaces. In addition, polymeric biosensor interfaces have been created on gold using grafted polysiloxane-g-poly(ethylene glycol) polymers. In order to retain biological activity and to allow for the necessary accessibility, the biomolecular functional units have been immobilised onto gold and oxide surfaces derivatised with mixed SAMs. In the mixed SAM approach, the first molecule carries a functional group to firmly attach the bioreceptor molecule and the second molecule resists the non-specific adsorption of undesired biological entities. Different types of mixed SAMs have been optimised, containing e.g. molecules with a molecular backbone comprised of protein-resistant functionalities and molecules with highly reactive functional moieties. For protein detection, mixed SAMs were optimised in order to increase the amount of receptor molecules (antibodies and fragments) on the surface, while still mitigating non-specific adsorption, allowing for highly sensitive immunosensing in non-specific matrices. For the detection of small molecules, highly reproducible and tuneable immobilization protocols were developed, based on mixed SAMs. The optimisation of these (mixed) SAMs was conducted using various surface characterisation tools and using SPR and QCM-D for immunosensing experiments.
BI-MoP-8 Surface Modified MALDI Probes for Affinity Fractionation of Protein Mixtures G.R. Kinsel, M. Li, G. Fernando, L. van Waasbergen, R.B. Timmons (University of Texas at Arlington) Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a powerful analytical tool for the characterization of proteins. As the effectiveness of the MALDI method has advanced, the need for high-speed separation and purification of peptides/proteins in complex mixtures (e.g. culture media, serum or urine) has increased. The approach described in this presentation focuses on the use of RF plasma polymer coated MALDI probes as platforms for peptide and protein separation based on their hydrophobicity. Pulsed RF plasma deposition of allyl alcohol directly on the MALDI probe surface is used to produce surfaces with various hydrophobicities. Control of the degree of the hydrophobicity is achieved through changes in the duty cycle of the pulsed RF plasma. Testing of the surfaces for peptide/protein separation based on their hydrophobicity is performed using various laboratory prepared control mixtures and mixtures obtained from biological sources. In all cases fractionation of the protein/peptide mixture was evaluated through the acquisition MALDI mass spectra using a Bruker AutoFLEX MALDI TOFMS or a laboratory-constructed linear MALDI TOFMS. Data has been obtained from surfaces with different hydrophobicities that demonstrate the efficacy of these modified MALDI probe surfaces for achieving on-probe fractionation of peptide/protein mixtures.
BI-MoP-9 Micropatterned Substrate Screening under Shear Flow (MiSSUS): Direct Comparison of Receptor-ligand Binding K.A. Burridge, M.A. Figa, J.Y. Wong (Boston University) Microfluidic patterning has been combined with a parallel plate flow chamber to enable screening of combinatorial variations in targeted drug delivery carrier surface properties under tunable physiologically-relevant shear conditions. Carriers containing either drugs or imaging agents must have surface properties that promote binding to targets yet at the same time block rapid immune system clearance. In addition, ligand-receptor mediated attachment must overcome shear flow in the vasculature which decreases contact times and applies forces on bonds. Patterned bilayers which mimic the surface of liposomal delivery vehicles are created by injecting pre-mixed vesicle solutions into lanes formed by a polydimethylsiloxane stamp reversibly sealed to a glass slide. After removing the stamp in an albumin solution to form protein barriers that prevent bilayer expansion, the slide is assembled into a flow chamber for binding studies. MiSSUS provides direct quantitative comparison of the effects of variations in ligand architecture such as relative molecular weights of liganded and unliganded polyethylene glycol. Experiments using MiSSUS revealed that ligand spacer length is an important factor in maintaining adhesion under flow, i.e. that longer spacers confer higher detachment resistance.
BI-MoP-10 Microfluidic Circuit Fabrication and Packaging for Surface-Controlled Bioprocesses in BioMEMS T.M. Valentine, J.J. Park, G.W. Rubloff, R. Ghodssi (University of Maryland) We have previously demonstrated that biopolymers (specifically, the polysaccharide chitosan) can be deposited in vitro at patterned, voltage-programmed electrodes, and that subsequent coupling and conjugation of biomolecules (proteins, nucleic acids) opens the door to applications such as bioassays and enzymatic catalysis. To extend these capabilities beyond simple devices to more complex bioMEMS systems, robust means for microfluidic circuit fabrication and packaging are required. We have designed and fabricated microfluidic networks to support combinatorial test site libraries for surface-controlled bioprocesses, along with packaging approaches to control fluidics and electrical inputs/outputs to the bioMEMS systems, with emphasis on robust technologies for leak-free microfluidics, use of polymer-based MEMS, incorporation of electrode structures and optical access to them, and reliable exchange of bioMEMS systems through reusable packaging which allows rapid connection of fluid and electrical inputs/outputs to external control systems. Three different approaches to microfluidic design, based on both sealing and bonding, are promising in concert with the packaging strategy. The efficacy of these fabrication and packaging approaches is demonstrated through results on fluidic sealing and on the electrically programmable deposition of NHS-fluorescein labeled chitosan at internal sites in the bioMEMS system. The impact of flow rates and residence times on biopolymer deposition and biomolecular conjugation reflect key chemical engineering questions associated with surface bioreactions in microfluidic systems, laying the groundwork for future applications in miniaturized bio-reactors and chemical and biological sensors.
BI-MoP-11 Near-Field Interaction of Infrared Radiation with an Atomic Force Cantilever E.S. Gillman (NanoSpec Corporation) Infrared vibrational spectroscopy is a powerful tool for chemical identification, however most infrared spectroscopic techniques usually do not obtain spatially resolved chemical information at a nanoscopic level due to the fact that they are diffraction limited. On the other hand, a scanning near-field optical microscope (SNOM) can reveal features with spatial resolution less than the diffraction limit because it relies on near-field probing instead of optical focusing. Using an apertureless approach that is based on atomic force microscope (AFM) cantilever tip ineracting with a infrared source of sufficient intensity, spatially resolved chemical information as well as conventional topographic information can be obtained. Critical to this is the near-field interaction of the AFM cantilever tip and the surface in the presence of an electromagnetic field. Modeling of this interaction will show that this approach can result in parctical device for nanoscale chemical imaging.
BI-MoP-12 Nanoscale Chemomechanical Patterning of Silicon and Germanium Surfaces Using an Atomic Force Microscope R.C. Davis, M. Tonks, K. Barnett, M. Lee, M.R. Linford (Brigham Young University) In nanoscale chemomechanical patterning, an atomic force microscope (AFM) probe is used to scribe a flat silicon surface under a solution of reactive molecules, attaching a layer of the molecules to the scribed line. This technique was previously used to functionalize silicon surfaces using a moderate range of scribing forces (5 µN to 80 µN). Here we will present a study focusing on the smaller forces (1µN to 5 µN) on both silicon and germanium surfaces resulting in significantly reduced tip wear and yielding lines down to 20 nm wide. We will also present the extension of chemomechanical patterning to include the attachment of proteins to the functionalized lines.
BI-MoP-13 Force Spectroscopy of Mechanically Stretched Fibronectin K.L. De Jong, P.R. Norton, N.O. Petersen (The University of Western Ontario, Canada) Cell motility is a delicate balance between adhesion and detachment. Identifying key proteins involved (for example, fibronectin, Fn) and understanding the mechanisms employed to find this balance, will provide new insight into the means by which motility can be controlled. Mechanical forces play a key regulatory role in biological cells, and therefore to understand how cells move and adhere, ultimately relies on knowledge of how forces are generated and propagated or in essence, how the cell interacts with the surface. The stress on Fn fibres may be the deciding factor in determining the attachment of the cell to the matrix, and the adhesion of the matrix to the substrate. Studies geared toward understanding fibronectin structure, organization, and binding affinity under mechanical stretching, are providing information crucial to the understanding of the effect of mechanical forces on cell function. To test the prediction that stretching Fn reduces integrin-binding activity, an AFM compatible device is designed to apply a mechanical force to fibronectin while the change in intermolecular interactions that result is monitored. The force required to rupture the interaction between an integrin mimic and fibronectin is determined to be approximately 100 pN. After stretching fibronectin, a trend towards fewer rupture events characterized by smaller pull-off forces in each force curve is observed; this implies a decrease in potential binding sites available to the integrin mimic and concomitant weaker interactions.
BI-MoP-15 Plasma Polymerization of Tetraglyme for PEO-like Surfaces and Plasma Immobilization of PEO Surfactants for Improved Blood Compatibility J.L. Lauer, J.L. Shohet, R. Muguresan, R.M. Albrecht (University of Wisconsin-Madison); U.H. von Andrian (Harvard Medical School); S. Esnault, J.S. Malter (University of Wisconsin-Madison); S.B. Shohet (University of California, San Francisco) The realization of small scale biomedical devices will be closely related to the non-fouling/biocompatibility properties of the exposed surfaces and the uniformity of the surface treatment throughout the device. PEO and PEO-like surfaces are significantly advantageous in preparing medical devices that require good blood compatibility. In this work, we explore two plasma process techniques, plasma polymerization (PP) and plasma immobilization (PI), to improve the blood compatibility of various polymer and non-polymer surfaces. Thrombus formation and embolization are significant problems for blood-contacting biomedical devices. To minimize these affects, plasma polymerized tetraethylene glycol dimethyl ether (tetraglyme) was deposited on flat Si3N4, and SiO2 samples to produce a PEO-like surface coating. In addition, a microplasma was used to immobilize a Poly(ethylene oxide) (PEO) surfactant to the lumenal surface of PE and PTFE tubing (ID 1.14mm). A microwave-cavity diagnostic was used to measure the plasma density of the microplasma inside of the polymer tube. Emitted light from the plasma during the PP and the PI processes was fed into a monochromator. Coating thickness and chemical composition of the flat surfaces was measured using ellipsometry and XPS, respectively. Contact-angle measurements were made for both the flat PP surfaces and the PI polymer tubes. To test blood compatibility, both the flat PP surfaces and the PI polymer tubes were exposed to heparinized human blood. After blood exposure, the tubes were examined with a scanning electron microscope to assess the density of adhering platelets on the flat PP surfaces and along the length of the PI polymer tubes. The plasma-treated surfaces showed fewer blood adherents than the untreated surfaces. By suitably modifying the plasma parameters, the treatment for both plasma processes can be optimized.
BI-MoP-17 Nonfouling Microstructures on Hydroxylated Substrates via Chemical Vapor Deposition and Surface Initiated Atom Transfer Radical Polymerization H. Ma, A. Chilkoti (Duke University) The ability to covalently modify hydroxylated substrates such as glass and metal oxides with a non-fouling polymer coating is an important goal, in view of their wide application as biomaterials and in biotechnology. Most current approaches rely upon physisorption of PEG-containing polymers or grafting of the polymers from solution to the surface (â?ografting toâ? approach). We report here a â?ografting fromâ? strategy in which an oligo(ethylene glycol) functionalized monomer is polymerized in situ from the surface of glass to provide high-density polymer brushes that overcomes the intrinsic limitation of low surface density of PEG chains realized by â?ografting toâ? strategies. A silane initiator presenting a terminal bromoisobutyrate moiety was used to form a SAM on hydroxylated substrates via chemical vapor deposition (CVD). This SAM was used as substrate for surface initiated atom transfer radical polymerization (SI-ATRP) of oligo(ethylene glycol) methyl methacrylate (OEGMA). The SI-ATRP was carried out in an oxygen free environment with CuBr/bipyridine as catalysts in a water /methanol mixture. Poly(OEGMA) brushes with a tunable thickness between 2 and 10 nm can be synthesized in situ, and these brushes are exceptionally resistant to protein adsorption, even from 100% fetal bovine serum. We also report a new masking strategy to pattern the surface with the initiator silane SAM, which enabled facile patterning of the surface with the poly(OEGMA) brushes. When NIH 3T3 fibroblasts were seeded onto those surfaces, cells were confined within the regions demarcated by the polymer and were maintained within the pattern for over a week.
BI-MoP-18 Plasma Grafted Anti-Fouling Films on Ethylene Oxide Base for Biosensors and Biotechnologies M. Kormunda, G. Ceccone, A. Papadopoulou, M. Hasiwa, F. Rossi (EU-JRC-IHCP, Italy) The generation of anti-fouling surface is a key element in the design of biosensors, medical devices and implants. Protein adsorption resistant surfaces have to avoid or reduce non-specific protein adsorption, platelet adhesion and thrombus formation, to prevent undesirable responses of the living systems to the device. The highly cross-linked anti-fouling coatings on ethylene oxide base were plasma grafted from Dimethoxydiethylene glycol vapour in mixture with reactive (N₂) or inert (Ar) gas at low-pressure 40mTorr. The plasma discharge in capacitive configuration of reactor was powered by continuous (from 10 to 100W) or pulsed RF (13.56MHz) voltage. The coatings were plasma grafted on glass, Si and PTFE substrates placed on grounded substrate holder. The film chemical composition has been analysed by XPS and FTIR as well as the surface morphology (AFM) and the protein adsorption (QCM-D). The coatings toxicity and an adhesion of Fibroblast L929 have been investigated on selected samples. The lower power plasma grafted coatings have very good antifouling properties together with low receding contact angles although the methyl function groups in the films are more populated than ethylene oxides groups. The coatings are stable with low contact angles over period about 6 months at normal conditions. No significant changes were observed on functional groups in coatings washed for 40 days in ethanol bath. The oxygen plasma etching during a nano-patterning process for biosensors fabrication has not significant influence on chemical composition, surface morphology and contact angles.}
BI-MoP-19 Local Property of Linear Plasmid DNA on the Metal Wires with Different Potential S.H. Jin, J.M. Son, N.J. Lee, C.J. Kang (Myongji University, Korea) Linear plasmid DNA crossed on the patterned nano metal wire is characterized by scanning capacitance microscopy and electrostatic force microscopy. The metal wires processed on the silicon wafer followed by a capping thin dielectric film were used as a local potential source for the DNA molecule. By varying the voltage bias applied to the metal wire, initial stage of DNA deposition on the substrate is observed and local electrical properties, such as capacitance variation and change of surface potential are measured. With images and spectroscopy, we investigate DNA moleculeâ?"substrate interaction.
BI-MoP-20 Analysis of Contaminants in Commercial Thiolated Single-stranded DNA Oligomers by XPS and ToF-SIMS C.-Y. Lee, L.J. Gamble, D.G. Castner (University of Washington) Commercially produced thiolated single-stranded DNA (SH-DNA) molecules are used in a variety of biotechnology applications including biosensors and DNA microarrays. The diversity of techniques used by different vendors in the synthesis and treatment lead to a significant variation in the quality of SH-DNA. In this work, we used x-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to perform a comparative study of the relative purity of commercially-available SH-DNA from several vendors. We find that thiol-terminated 16-base pair oligomers from two of the vendors, which were self-assembled as thin layers onto gold surfaces, contain excess carbon and sulfur. By using ToF-SIMS, several contaminants including poly(dimethylsiloxane) (PDMS), lipid molecules, and sulfur-containing molecules were identified by their molecular fragments. Preliminary ToF-SIMS data indicates that the excess sulfur arises from the reductant (dithiothreitol) used to purify the SH-DNA by some of the vendors. Time dependent studies of purified versus contaminated SH-DNA were performed to determine the effect of contamination on DNA surface assembly over time. XPS results of purified SH-DNA show increased P, N, O and C atomic percentages over a 24-hour time period, confirming increasing DNA surface coverage on the gold. In contrast, XPS results of contaminated SH-DNA show that C and O atomic percentages increased over time, but no increase was observed in the P and N. This indicates that, after the initial SH-DNA adsorption during the first five minutes, the excess material adsorbed for the rest of the 24 hours was due to contamination.
BI-MoP-21 Empirical Force Field Evaluation for the Molecular Simulation of Protein Adsorption V. Raut, S.J. Stuart, R.A. Latour (Clemson University) Molecular simulation provides a direct method to theoretically investigate the molecular mechanisms governing the adsorption behavior of proteins to biomaterials surfaces. Because of their size, empirical force field based methods must be used for these types of simulations. Force fields (ffs), however, must be parameterized for specific molecular systems. While ffs have been designed to accurately represent the behavior of proteins in solution, none have considered peptide-surface adsorption behavior in their parameterization. Therefore, there is currently no basis to support the accuracy of simulation results. The objective of this research was to develop computational methods to complement our previous experimental studies that measured the adsorption free energy (G) for a host-guest peptide on Au-alkanethiol self assembled monolayer surfaces (SAMs), and to use these methods for ff evaluation. Host-guest peptides were modeled in the form of SGSG-X-GSGS, where G=glycine, S=serine, and X=any selected peptide type. Molecular dynamics simulations were conducted using the GROMACS ff to calculate G for these peptides over functionalized SAMs (CH3, OH, NH2, COOH, PEG) in a 40Å x 40Å x 60Å simulation cell with explicit solvation (water with Na+ and Cl- ions) contained within periodic boundary conditions. While simulation results for certain peptide-SAM systems are generally in agreement with experiments, others show substantial deviations from expected adsorption behavior. Parameter modifications of this ff are thus required for this application. Further work is planned for the eventual development of a validated ff for protein adsorption simulations.
BI-MoP-22 Investigation of the Binding Mechanism of the Bacterial Adhesin Scp to Fibronectin J.R. Hull (The University of Washington); D.G. Castner (University of Washington); G. Tamura (Children's Hospital and Regional Medical Center) Several pathogens bind specifically to immobilized fibronectin (Fn), and not to soluble Fn, including Group B Streptococci (GBS), S. pneumoniae, S. sanguis, and p-fimbriated strains of E. coli. However, the structural basis for the specific adherence of Scp to immobilized Fn has not been resolved. There are two possible mechanisms for Scp binding to Fn. First, specific conformational determinants of Fn that allow Scp binding may be unmasked when Fn is immobilized. Second, Scp may bind to a combinatorial determinant formed by the clustering of multiple immobilized Fn molecules. The Scp-Fn binding interaction is being studied with two complementary analysis methods. Surface Plasmon Resonance (SPR) is being used to study the kinetics of the binding interactions and Atomic Force Microscopy (AFM) is being used to investigate single molecule interactions. Supporting experiments to determine surface compositions was done using X-ray Photoelectron Spectroscopy. SPR has shown that the binding affinity of soluble Scp to adsorbed Fn is approximately nano-molar and that Scp does not bind soluble Fn. A model system was set up to develop AFM methods for examining the Scp-Fn interactions. Collagen related peptides (CRPs) on the AFM tip were used to interact with adsorbed Fn. The CRPs were attached to the AFM tip via homo-functional N-hydroxysuccinimide (NHS) poly(ethylene glycol) (PEG) cross-linker. The jump heights of the force distance curves were graded based on Student´s t-test and only events with a nominal grade were further analyzed. Analysis of unbinding events shows that there are multiple interactions centered at 75 pN and there are multiple length scales over which these interactions occur. There are at three length scales over which these interactions occur: 5 nm which is attributable to nonspecific adhesion, 15 nm which is the NHS-PEG spacer length, and 30 nm and greater which is attributable to CRP-Fn interactions.
BI-MoP-23 L-Cysteine Adsorption on Cu(100) Studied by Sulfur K-edge NEXAFS and XPS S. Yagi, Y. Matsumura, T. Nomoto (Nagoya University, Japan); J.A. Syed, S.A. Sardar (Hiroshima University, Japan); K. Soda (Nagoya University, Japan); E. Hashimoto, M. Taniguchi (Hiroshima University, Japan) For the application to medical materials it is important to study the mechanism of reaction at interface between amino acid, which is the component of protein, and metal, and the influence of water on the mechanism. We have paid attention to L-Cysteine [HSCH₂CH(NH₂)COOH] and investigated the adsorption behavior of L-Cysteine/Cu(100) by Sulfur K-edge Near Edge X-ray Absorption Fine Structure (NEXAFS) and X-ray Photoelectron Spectroscopy (XPS) techniques. Sulfur K-edge NEXAFS spectrum for L-Cysteine/Cu(100) shows that the main peak of σ^*(S-C) is decreased in comparison with the spectrum for L-Cysteine powder. This means that L-Cysteine molecule obtains some electrons from the Cu(100) surface.
BI-MoP-24 Orientation of a Y-shaped Biomolecule Adsorbed on a Charged Surface Y.-J. Sheng (National Taiwan University, Taiwan); H.-K. Tsao (National Central University, Taiwan); S. Jiang (University of Washington) The adsorption and orientation properties of a Y-shaped biomolecule, which models an immunoglobulin (Ig), on a charged surface are analyzed mesoscopically by Monte Carlo simulations. The orientation is a consequence of the interplay between van der Waals interactions and electrostatic interactions. For adsorption dominated by van der Waals attraction, the molecule prefers lying flat on the surface. For weak attraction, we observe a depletion zone in the concentration profile, which can result in a negative surface excess. A secondary peak is found for strong adsorption. For electrostatically dominated adsorption, the orientation is mainly determined by electric dipole and a vertically adsorbed molecule can be attained as it possesses strong electric dipole. Our study provides an explanation for experimental observations of preferential orientation.
BI-MoP-25 Interfacial Dynamics of a Gelatin Solution with Surfactant H.-K. Tsao (National Central University, Taiwan); S.-Y. Lin (National Taiwan University of Technology, Taiwan) In terms of dynamic surface tension, the interfacial dynamics of gelation solutions with various surfactants are investigated by pedant bubble tensiometry. On the basis of local equilibrium assumption, the thermodynamics of dynamic surface tension is analyzed. The adsorption efficiency of gelatin is low compared to that of small surfactant. However, the surface activity of gelatin may be enhanced due to intrachain and interchain rearrangement at the interface, which results in long relaxation time signature. The interplay between adsorption efficiency and surface activity categorizes our experimental results into two types of dynamic behavior. For type I dynamics, the gelatin molecule is completely displaced from the interface and the interfacial dynamics is dominated by that associated with surfactant. Nevertheless, the interaction of gelatin with surfactant in the bulk solution can alter the surfactant surface excess and hence the surface tension. For type II dynamics, the gelatin molecule is partially displaced from the surface and the dynamics displays a long relaxation characteristic. The extent of surface tension decrement due to gelatin conformational change manifests the degree of gelatin displacement from the interface. Our conclusion is able to elucidate the peculiar dynamic surface tension of a solution of gelatin and PEG.
BI-MoP-26 Detergency Effectiveness with Respect to Proteins M. Richard, T. Le Mogne (Ecole Centrale de Lyon, France); J. Criquelion (Laboratoires Anios, France); A. Perret-Liaudet (Hopital Neurologique de Lyon, France); J.M. Martin (Ecole Centrale de Lyon, France) In the detergency field of re-usable medical devices, a special attention is focused on the non-conventional transmissible agent called prions which is a proteinaceous infectious agent. Few cleaning procedures are effective against prions and few techniques are available to study cleaning effectiveness with respect to proteins in general. The first part of our study shows that X-ray Photoelectron Spectroscopy (XPS) is a useful and reliable technique to evaluate detergent formulations effectiveness to remove proteins from stainless steel surface soiled with a human brain homogenate. A semi-quantitative evaluation of the detergency effectiveness could also be performed. XPS makes it possible to study chemical species remaining on surface, substrate properties after cleaning procedures and also the water quality effect on detergency effectiveness. In the second part of our study and in the light of the complexity of the previous system, a simplification of each part of the system was carried out. XPS analysis was used to study the effectiveness of some simple chemical functions to remove proteins from a native oxide layer of a pure chromium surface. The results will be presented and discussed in this paper.
BI-MoP-27 Reflex-Arc on a Chip: an in Silico Cell Culture Analogue K.A. Wilson, M. Das, L.C. Riedel, C.A. Gregory, M. Poeta, D. Damjanovich, P. Molnar, J.J. Hickman (Clemson University) To date understanding of and development of therapies for traumatic spinal cord injury (SCI) and neurodegenerative diseases have been problematic due in part to difficulties associated with the various models used to test new drug therapies. Animal studies are expensive, time consuming, and raise ethical issues. In vitro studies are less expensive and avoid many of the ethical issues associated with animal studies, but are often poor predictors of human response. To overcome the shortcomings of existing models we are developing a microscopic cell culture analogue (microCCA) of the spinal reflex-arc. This system should retain the cost effectiveness of in vitro systems while allowing complex tissue interactions and environmental dynamics that more realistically reflect the in vivo state. The present work draws on advances in a wide variety of technical fields including cell culture, surface chemistry, and microfabrication. These advances have allowed us to begin development of a microCCA device comprised of the basic components of the reflex-arc: a muscle fiber, a dorsal root ganglion (DRG) cell, and a motoneuron. Silicon microstructures serve as the foundation of the device. Surface modification with alkyl-silane SAMs followed by patterning with deep UV photolithography was performed to selectively control cell adhesion and growth. XPS analysis indicated monolayer formation. Furthermore, we have demonstrated the control of neuronal growth and myotube differentiation on the microstructures. Electrophysiology results have confirmed that the neurons and myotubes have physiological properties consistent with previous findings. With this system it will ultimately be possible to report on a variety of properties of the reflex-arc, thereby creating a cost-effective, predictive test bed for the development of novel drug therapies for traumatic SCI and a wide variety of neurodegenerative diseases.
BI-MoP-28 Compartmentalized Bioreactor for Long-Term Culture of Bone Cells R. Dhurjati, E.A. Vogler, P.W. Brown, H.J. Donahue (Pennsylvania State University) A compartmentalized bioreactor designed around the concept of continuous growth and dialysis, was used to study long-term (15-30 days) phenotypic behavior of human fetal osteoblastic cells (hFOB). This specific design separates the growth and feeding functions and permits long term culture sustaining the pericellular environment, otherwise disturbed by continuous or punctuated growth medium replacement. The attachment and growth behavior of model human osteoblasts cultured in the bioreactor was evaluated using scanning and transmission electron microscopy (SEM/TEM) and were compared to those obtained from conventional tissue culture methods. Results suggest that stable culture conditions afforded by the reactor has enhanced utility in the long-term culture of osteoblasts in terms of growth, proliferation and mineralized matrix deposition characteristics and would serve as an ideal in-vitro test system for the study of cell/protein mediated interactions with synthetic bone analog materials.
BI-MoP-29 Cell-Surface Interactions between Marine Diatoms and Fouling-Release Coatings Studied with Atomic Force Microscopy P.F.M. Terán Arce, R. Avci (Montana State University); I.B. Beech (University of Portsmouth, UK); K.E. Cooksey, B. Wigglesworth-Cooksey (Montana State University) Interactions between marine microorganisms, and fouling release coatings are of major significance to the Navy and maritime industry. This interaction usually occurs by means of adhesive exopolymers (EPS) secreted by the microorganisms, which allow them to attach and settle on different surfaces. In the present study, viable marine diatom cells were immobilized on tipless AFM cantilevers and used as bioprobes to investigate the adhesion forces between exopolymers, produced by the immobilized diatoms, and surfaces of Intersleek (^TM International Paints) elastomers. These forces, as well as the work exerted against them, were statistically compared with the forces between the same diatoms and mica surfaces. In spite of the dissimilar character of Intersleek (hydrophobic) and mica (hydrophilic) surfaces, comparable results were obtained on both. Force vs distance curves on both surfaces presented several adhesion peaks with force magnitudes that ranged from hundreds of pNs to tens of nNs and polymer elongations up to several microns. These results demonstrate the ability of diatoms to produce hydrophobic and hydrophilic exopolymers.
BI-MoP-30 Development and Characterization of RGD Peptide Coatings for Cell Adhesion R. Canteri, C. Pederzolli, L. Lunelli, P. Villani, L. Pasquardini, M. Vinante, G. Speranza, S. Forti, M. Anderle (ITC-irst, Italy); J.J. Park, G.W. Rubloff (University of Maryland) The development of biomaterials able to modulate the interaction of mammalian cells with solid substrates is important for many applications, e.g., tissue replacement/regeneration and substrates for cell culture. A common mechanism that mediates cell adhesion involves the interactions between integrin receptors on the surface of mammalian cells and ligands of adhesive proteins present in extracellular matrices (ECM) and bloodstream. These proteins include fibrinogen, fibronectin, vitronectin, collagen, laminin, Von Willebrand factor. It has been demonstrated that the adhesive domains comprise a short peptide sequence, Arg-Gly-Asp (RGD), the most important recognition site for about half of all known integrins. This work describes a three-step reaction procedure for coupling a six-amino-acid (GRGDSY) fibronectin fragment synthesized with an additional cysteine (C) at the C-terminus, to solid substrates. The first step is the activation of the substrate with an amine layer, introduced by chemical modification (silanization) or by using an amino-containing biopolymer (chitosan). In a second reaction step, N-hydroxysuccinimidyl (NHS) ester polyethylene glycol (PEG) is grafted to the aminated surfaces. Fluorescence quantitative showed 1-5 x10¹³ PEG molecules/cm² immobilized on the surface. The distal end of these PEG molecules carry two possible chemical groups: a maleimide or a vinylsulfone group, both selectives for reaction with sulfhydryl groups. The final step is the covalent attachment of RGD-containing peptides on the resulting terminal PEG derivatives. XPS, ToF-SIMS, AFM, SEM, fluorescence spectroscopy and microscopy were applied to characterise the surface. The RGD modified surfaces were tested using different cell lines. The results obtained on the functionalized surface showed an higher extent of cell adhesion, with mainly round-shaped cells at the initial stage of the spreading, compared to the non-modified surface.
BI-MoP-31 Characterization of Nuclear Impalement by Vertically Aligned Carbon Nanofibers for Gene Delivery A.V. Melechko (University of Tennessee, Knoxville); T.E. McKnight, G.D. Griffin, D.K. Hensley, M.J. Doktycz, D.H. Lowndes (Oak Ridge National Laboratory); M.L. Simpson (Oak Ridge National Laboratory, University of Tennessee) Penetration of DNA-modified vertically aligned carbon nanofibers (VACNF) into live cells provides efficient delivery and expression of exogenous genes, similar to â?~microinjectionâ?T-styled methods, but on a massively parallel basis. The efficiency of this method however depends on many factors including plasmid coverage on each nanofiber, maintaining transcriptional activity of these plasmids following immobilization, and retention or release of plasmid from the VACNF scaffold during and after insertion into cell. For DNA that remains tethered, it is believed that gene expression occurs if plasmid is delivered not only into intracellular domain but moreover into nuclear domain. In this work we report on a study of insertion and residence of VACNFs into the nuclei of mammalian cells (Chinese hamster ovary) using Laser Scanning Confocal Microscopy and Scanning Electron Microscopy.
BI-MoP-32 Deposition of Lipid Bilayers on the Silicon Dioxide Surfaces Patterned by Focused Ion Beam and Synchrotron Radiation Etching R. Tero (Institute for Molecular Science, Japan); M. Rahman (The Graduate University for Advanced Studies, Japan); Z.-H. Wang (Nagoya University, Japan); M. Sugawara (Nihon University, Japan); K. Nagayama (National Institute for Physiological Sciences, Japan); T. Urisu (Institute for Molecular Science, Japan) Nano-bioelectronics is one of the most attractive research fields in these days. Microfabrication and modification with biomaterials on solid surfaces have fascinated enormous attentions as important techniques for the development of new biosensors and new devices. We have fabricated fine structures on SiO₂/Si surfaces by combination of the focused ion beam (FIB) and the synchrotron radiation (SR) etching, and deposited lipid bilayers on the SiO₂ surface by the vesicle fusion method. FIB patterning was performed using JEOL-JFIB2300 with the 30 keV of Ga ion. SR etching was performed in the BL4A2 in UVSOR in IMS using Co photomask with the thickness of 400 nm under the mixture gas of SF₆ and O₂ (P_SF6=5.0x10^-2 Torr, P_O2=2.0x10^-3 Torr). AFM images were obtained by Picoscan (Molecular Imaging). In the deposition of lipid bilayers, the sample was incubated above the gel-liquid crystal transition temperatures in the suspension of the pure dipalmitoylphosphatidylcholine (DPPC) or the mixture of egg PC, dioleoylphosphatidylethanolamine (DOPE) and cholesterol (3:1:1), which were prepared by agitating the vacuum-dried lipid films in a buffer solution. We have patterned the Co photomask on the SiO₂/Si substrate by FIB in micrometer order. Three-dimensional micrometer-order structures were successfully obtained by one time SR irradiation (2.0x10⁴ mA min). After deposition of DPPC vesicles on the SiO₂ surface, flat membranes with the height of 5 nm were observed by atomic force microscopy (AFM). The thickness of the membranes well corresponded to that of the single lipid bilayer. The morphology, electronic resistance and additional effect of protein (gramicidin A) will be discussed.
BI-MoP-33 Interfacing Natural and Synthetic Biomaterials: Development of a Multilayered Vascular Scaffold E.J. Taschner, J.B. Leach, J.Y. Wong (Boston University) One of the greatest challenges in designing functional small diameter vascular grafts is to mimic key arterial mechanical properties (e.g., strength and compliance). Our hypothesis is that the underlying scaffold organization is a crucial factor in cellular remodeling, and ultimately, the mechanical properties of biologic vascular grafts. Thus, the overall goal of our research is to develop a multilayered or lamellar vascular scaffold biomaterial that more closely mimics the organization of native artery extracellular matrix. Our approach is to seed vascular smooth muscle cells between layers of poly(lactic-co-glycolic) (PLGA) thin films and naturally derived hydrogels (e.g., collagen, fibrin). However, the major challenge to the creation of such a multilayered scaffold is to promote stability and adhesion between the hydrogels and the relatively hydrophobic PLGA films. Therefore, to promote adhesion between the composite layers, the PLGA films were surface modified to contain specific highly reactive groups. First, the PLGA films were treated in NaOH to expose surface carboxylic acid and alcohol groups. Then, carbodiimide-mediated reactions were used to covalently bind photoreactive moieties to the PLGA films as well as the hydrogel precursor monomers. The film-cell-hydrogel composite constructs were assembled and then exposed to ultraviolet light to initiate the photopolymerization. We tested the adhesion between the layers using a modified peel/creep test that applied a constant "peel" force over time. The modified PLGA composites were associated with a significant amount of resistance to the peeling force while the unmodified PLGA controls failed instantaneously. We therefore demonstrate a promising method of creating stable, multilayered tissue scaffolds from composites of natural and synthetic biomaterials.
BI-MoP-34 Immobilization of the Enamel Matrix Derivate Protein Emdogain onto Polypeptide Multilayers as studied by in situ Ellipsometry and QCM-D T.J. Halthur (Royal Institute of Technology, Sweden); I. Slaby, A. Lindeheim (Biora AB, Sweden); P. Claesson (Royal Institute of Technology, Sweden); U. Elofsson (YKI AB, Institute for Surface Chemistry, Sweden) The build-up of the biodegradable poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers on silica and titanium surfaces, with and without an initial layer of polyethyleneimine (PEI), was investigated and characterized by means of in situ ellipsometry and Quartz Crystal Microbalance with Dissipation (QCM-D). A two-regime build-up was found in all systems, where the length of the first slow growing regime is dependent on the structure of the initial layers. In the second fast growing regime, the film thickness grows linearly while the mass increases more than linearly (close to exponentially) with the number of deposited layers. The film refractive indices as well as the water contents, indicate that the film density changes as the multilayer film builds up. The change in film density was proposed to be due to polypeptides diffusing into the multilayer film as they attach. Furthermore, the use of PEI as initial layer was found to induce a difference in the thickness increments for PGA and PLL. Comparisons between ellipsometry and QCM measurements revealed that the multilayer film was highly hydrated (as much as 70-75% water) and might therefore serve as a good template for proteins and cells. The hydrophobic aggregating Enamel Matrix Derivatives (EMD) protein Emdogain was successfully immobilized both on top of as well as within the multilayer structures while measured in situ with ellipsometry and QCM and in vitro with ELISA. These polypeptidemultilayer/EMD films are thought to be able to trigger cell response and induce biomineralization and might therefore be used as bioactive and biodegradable coatings for future dental implants.
BI-MoP-35 In-situ Formation of Bioactive-Titanium Coating using Reactive Plasma Spraying M. Inagaki, Y. Yokogawa, T. Kameyama (National Institute of Advanced Industrial Science and Technology (AIST), Japan) A surface modification technique using reactive plasma spraying (RPS) was studied to form bioactive-titanium (Ti) coating. An in-situ surface-modification of Ti particles is conducted by making use of plasma-enhanced reactions between the Ti particles and the reactive gaseous species in the plasma flame during plasma spraying. Surface-modified Ti coatings were deposited on Ti substrates by radio-frequency (rf)-RPS using a thermal plasma of Ar gas containing 1-6% N₂ and/or 1-6% O₂ at an input power of 16 kW. As a means of surface modification, Ti powders impregnated with 0.05-0.2 mol% Ca were also sprayed. Compositional changes in the coatings' surface after soaking in simulated body fluid (SBF) were examined by Fourier transform infrared spectroscopy (FT-IR) and thin film X-ray diffraction (TF-XRD). The Ti coatings prepared with Ar-O₂ and Ar-N₂-O₂ plasma formed apatite after 3 days of soaking in SBF. This indicated that such coatings have the ability to form a biologically active bone-like apatite layer on their surface. In the TF-XRD patterns for the Ti coatings sprayed with oxygen-containing plasma, minute peaks ascribable to TiO₂ (anatase and rutile phase) were commonly observed. On the other hand, no compositional change was observed in the surface of the Ti coatings sprayed with Ar-N₂ plasma, even after 7 days of soaking in SBF. In SBF tests, we observed a retardation of apatite deposition for the Ca-added Ti coatings prepared with Ar-O₂ and Ar-N₂-O₂ plasmas. Analyses by X-ray photoelectron spectroscopy indicated that the Ca impregnated in the RPS-Ti coatings formed a Ca-O compound.
BI-MoP-36 Effects of He, Ar Ion Implantation on the Surface Chemistry and Structure of Biomedical Polymers M. Manso Silvan (Institute for Health and Consumer Protection, European Commission, Italy); A. Valsesia, M. Lejeune, D. Gilliland, G. Ceccone, F. Rossi (Joint Research Centre, European Commission, Italy) Ion beams have become during the last years an outstanding tool for the processing of biomedical devices due to their ability to modify the structural and chemical properties of polymers. The surface chemistry, determinant factor in the performances of biosensor and tissue engineering devices, can be tailored by exposure to ion beams in different ranges of energies and ion doses. Regarding the case of noble gas implantation (Ar, He) at energies from 25 up to 100 KeV, we have found that the surfaces of biomedical polymers such as Polymethylmethacrylate (PMMA), Polystyrene (PS), Polycaprolactone (PCL) and Polyethyleneglycol (PEG) can be notably modified by exposure to doses below 10¹⁴ cm^-2. These transformations were applied to adapt the polymer stability in aqueous media or the surface activity towards protein attachment. A series of physico-chemical characterization tools were used in order to follow the surface and structural changes related to the implantation conditions. Fourier transformed infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS) were used to monitor compositional changes of the implanted samples before and after interaction in biomolecular assays at protein and peptide level. Atomic force microscopy (AFM) and Ellipsometry revealed topographic and structural changes while contact angle and Zeta potential measurements evidenced changes in the molecular interaction of the polymers surface. In particular, it is shown that relevant modifications can be observed in polymer samples exposed to identical ion doses but with beams characterized by different ion density.
BI-MoP-37 Plasma Sterilisation of Thermalabile Materials H. Halfmann, M. Schulze, M. Czichy, P. Awakowicz (Ruhr-Universitaet Bochum, Germany) In recent years plasma sterilisation has been developed to a certain degree that makes the corresponding results reliable. Due to the advantages of plasma sterilisation great efforts in world wide investigations are made. The dry and cold process without toxic ingredients is the large benefit of the plasma sterilisation. At the institute of Electrical Engineering and Plasma Technology (AEPT) investigations on plasma sterilisation of medical implants and PET are performed. The experiments are focused on spores and germs which are important in medical, pharmaceutical and food branches. Additionally the influence of the plasma treatment on pyrogens is examined. With the sterilisation and surface modification our attention is on medical implants made of titan, UHMWPE and degradable polylactide. Conventional procedures have several disadvantages besides the long total treatment time. The plasma process reduces germs by 6 decades in a total treatment time of less than 2 minutes. Unlike common sterilisation processes the procedure is also able to reduce pyrogens. In addition to the sterilisation the surface of UHMWPE is hardened by the plasma process. Gel content measurements indicate the improvement of abrasion resistance while the bulk material is not modified. An increasing part of non-carbonated and non-acidic beverages have been bottled in PET. For sensitive products aseptic filling must be guaranteed. A plasma process prepares bottles for aseptic filling within seconds without toxic residua. To improve the shelf-live of oxygen-sensitive soft drinks a diffusion barrier made of a SiO_x layer can be deposited on the inner side of a PET bottle in a second process step. The whole process may be performed on a plasma line microwave reactor and is done in less than 10 seconds. The future work is aimed at unterstanding the mechanisms of sterilisation in the plasma with regard to ions, neutrals and radicals to optimise the procedure.
BI-MoP-38 A Scanning Small Angle X-Ray Scattering (SAXS) Study of the Nanometer Length Scale Bone Structure in Connection with Implants M. Foss, M.H. Bunger, K. Erlacher (University of Aarhus, Denmark); L. Haisheng, Z. Xuenong, B.L. Langdahl (Aarhus University Hospital, Denmark); F. Besenbacher, J.S. Pedersen (University of Aarhus, Denmark) The understanding of the interaction between bone and orthopaedic implants is important for the development of biomaterials with improved biocompatibility. The SAXS technique has previously been applied to offer structural information on mean crystal thickness, predominant orientation and degree of orientation of mineral particles in bone. Therefore, one possible application of SAXS is to investigate the nanostructure of bone in connection with ingrowth on implants, which is not possible with conventional optical techniques. Three sections of pig vertebrae with a thickness of 190µm were examined. One sample included the neurocentral growth zone, while the other two were sections with bone and pieces of either titanium or tantalum implants. Bone provided a strong SAXS signal and relatively low transmission intensity, whereas regions within the neurocentral growth zones showed a high transmission as well as a high SAXS signal. Combining the transmission and the SAXS data, it was possible to differentiate between areas of fibrous tissue and bone. This was supported by elemental analysis performed by SEM-EDAX. The mineral particles in the cancellous bone were aligned along the trabeculae, with less orientation close to the growth zone. Also, the mineral particles tended to be aligned along the implant surfaces. Within the individual bone samples, a large variation in all SAXS parameters were observed depending on the bone position relative to the implant. Furthermore, larger particle thicknesses were found in areas of bone formation, which matches our growth zone data. The data suggests that the parameters obtained by SAXS can be used to assay the local mineral particle growth. This indicates that SAXS is a powerful tool for the characterization of the detailed mineral structure of bone in the vicinity of implants.
BI-MoP-39 Photoacoustic Analysis of Bone Osteogenesis to Different Doses of Irradiation Laser P. Lomelí Mejia (IPN SEPI-ESIME, Mexico); J.L. Jiménez Pérez (CICATA-IPN, Unidad Legaria, Mexico); A. Cruz Orea (CINVESTAV-IPN, Mexico); G. Urriolagoitía Calderón, L.H. Hernández Gomez (IPN SEPI-ESIME, Mexico); H. Lecona Butron (Centro Nacional de Rehabilitación y Ortopedia, Mexico) The photoacoustic analysis of fractured bone callus to different consolidation times in presence of the irradiation laser, was performed. In this study we take into account the fractured tibias of sacrified Wistar rats. By using photoacoustic spectroscopy (PAS) technique it was possible to determine, for different doses of laser irradiation (doses from 5 to 10 Jcm-2) the presence of characteristic absorption peaks of p-Nitrophenylphosphatase (p.Npp) in the fractured bone callus. The evolution of bone consolidation was accelerated by laser radiation when compared with non irradiated fractures bones.

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