AVS2001 Session BI-TuP: Surface Characterization and Non-Fouling Surfaces Poster Session
Tuesday, October 30, 2001 5:30 PM in Room 134/135
Tuesday Afternoon
Time Period TuP Sessions | Topic BI Sessions | Time Periods | Topics | AVS2001 Schedule
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BI-TuP-2 Short-term Oxidation of Polymer Films Deposited from Pulsed Radiofrequency Allylamine Plasmas
J.D. Whittle, G.R. Kinsel, R.B. Timmons (University of Texas at Arlington) Plasma deposited films are seen as a promising route to the synthesis of novel functional coatings for a large number of potential applications. Allylamine deposited films in particular are of great interest in the biomaterials field as surfaces for protein adsorption. Studies of the long-term aging of these plasma polymers have shown that the oxygen content of the films changes over extended periods of time. Earlier work has shown that the oxygen content of allylamine films deposited from continuous wave plasmas increased from around 2% for a fresh sample, to around 10% after a year of aging in the laboratory, with the greatest change in composition being within the first 48h. In addition, some loss of nitrogen from the films has also been observed. In this study, we concentrate on the changes in chemistry over the first few days, and in particular the first 12 hours following deposition. The surface chemistry is investigated by X-ray photoelectron spectroscopy (XPS) and Matrix Assisted Laser Desorption/Ionization Mass spectrometry (MALDI-MS). Using XPS we investigate the stability of the plasma polymer surfaces in the UHV environment using different substrates for deposition to determine what the source of the oxidative species may be. A small amount of oxygen is always present in these plasma polymers, which may be due to the unavoidable exposure to the atmosphere between completing the deposition, and insertion of the sample into the spectrometer. Further, by analyzing samples exposed to the laboratory atmosphere for specific lengths of time, we show how the surface chemistry evolves in the first few hours following deposition. We also examine the effect of plasma power and pulsing duty cycle on the post-deposition properties of the films. |
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BI-TuP-3 Fast Impedance Spectroscopy Measurements on Supported Lipid Bilayer Membranes with and without Incorporated Ion Channels
G. Wiegand, S. Beyer, N. Arribas-Layton, P. Wagner (Zyomyx Inc.) A substantial part of the mammalian proteome is represented by proteins that are either associated or incorporated into lipid bilayer membranes. Our goal is to provide appropriate platform assays and transducer technologies for the functional analysis of membrane proteins. Our special focus is on ion channels due to their pharmacological relevance. Because ionic flux thru an ion channel generates an electrical signal, electronic transducer technologies are the most direct detection method for ion channel analysis. We developed a method of fast impedance spectroscopy that combines the power of a spectroscopic technique providing high information content with the millisecond time resolution of a fast analytical tool. In biophysical experiments, time dependent quantities such as the membrane resistance and the membrane capacity are obtained from the measured sequences of impedance spectra. Supported lipid bilayers provide membrane matrices for protein incorporation that are coupled to solid surfaces. Supported bilayer applications take advantage of the high membrane stability imparted by the solid support, and of the improved accessibility for analytical tools due to the two-dimensional geometry. As a result of the chip compatibility, supported membrane systems are potentially useful in high-throughput technologies. By application of fast impedance spectroscopy, dynamic properties of supported lipid bilayers with and without incorporated ion channels are studied during formation, relaxation and in various states of conduction. |
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BI-TuP-5 Optical Inverted Microscope with a Scanning Near Field Optical Microscope to Study Biological Material
A. Cricenti, R. Generosi, M. Luce, P. Perfetti (ISM-CNR, Italy) A scanning near field optical microscope (SNOM) has been added to a standard inverted optical microscope with the dedicate aim of characterizing the inner parts of biological molecules. Therefore, in addition to the requirements of reliability and mechanical stability we have carefully looked to analyzing a sample with all available geometries for input/output of photons, in order to get as many information as possible. The SNOM unit consists of a support mounted on the optical microscope arm containing a piezoelectric scanner. The reflectivity of the sample can be measured by applying different methods: the sample can be illuminated on top by an external source, as well as by the optical fiber used for the detection of the reflectivity signal. Absorption experiments can be easily performed by detecting the transmitted signal through the optical apparatus of the inverted microscope.Also fluorescence signal can be simultaneously detected. Reflectivity, transmissivity and fluorescence measurements will be presented on several biological systems, with a resolution well below the diffraction limit. |
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BI-TuP-6 Investigation of Bone Tissues using Infrared Spectroscopic Ellipsometry
G.M.W. Kroesen, J.-C. Cigal, E. Stoffels, B. van Rietbergen, R. Huiskes (Eindhoven University of Technology, The Netherlands) Small fractures on the bone surface, called micro-cracks, are formed throughout the lifetime as a result of e.g. mechanical stress. In individuals of advanced age, these defects are no longer efficiently repaired by the organism. Increasing density of micro-crack is one of the important factors which lead to osteoporosis: the severe loss of bone mass and attendant fragility of the skeleton. The size of micro-cracks is in the order of 10 microns, and they are difficult to detect in vivo. Apart from these fractures, the chemical composition of the bone surface is expected to change in the course of ageing. Spectroscopic ellipsometry is a powerful but non-destructive technique of analysing complex surfaces, and it seems very suitable in a study of bone tissues. We developed a spectroscopic ellipsometer combined with a Fourier transform spectrometer in the middle infrared range (wavelength of 2.5 to 10 microns). This device allows to collect accurate data on the chemical composition of the bone surface. In addition, it can provide information about the surface roughness, which is useful in determining the density of micro-cracks. Ellipsometry is a purely physical method, and this novel application to the complex biological environment poses many technical challenges. We will present preliminary results on ellipsometric analysis of bone surfaces, including infrared spectra of several bone samples. In the subsequent study we will investigate how the ageing of the bone tissue is reflected by its infrared properties. |
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BI-TuP-7 Changes in Bone Surface after Exposure to an Electric Discharge
J.H.R. Feijen, C.Y.M. Maurice, E. Stoffels, G.M.W. Kroesen, B. van Rietbergen, R. Huiskes (Eindhoven University of Technology, The Netherlands) Human bones are subject to a continuous process of regeneration. Due to mechanical stress, cracks on a microscopic scale are generated in bone tissue, but in the healthy situation these cracks are repaired before they can lead to serious damage. In the case of disturbed bone regeneration, however, due to osteoporosis, drugs that inhibit bone resorption or bone cancer, the mechanical integrity of bone is impaired by accumulation of micro cracks or large metastatic defects. Treatment of bone diseases in vivo is nowadays very difficult. We consider an alternative method of bone surface processing, using non-equilibrium (cold) electric discharges. These plasmas combine high reactivity with non-destructive character. In this study we attempt plasma treatment and observe its impacts on the surface of bone tissues. These impacts are change in roughness, etching of some layers, removal of cells, etc. Since the concept of exposing living tissues to electric discharges is new, the presented results are preliminary and the medical implications are not yet resolved. For this experiment we employ a low-pressure inductively coupled plasma (ICP), supplied with diagnostics. A Langmuir probe, an energy-resolved mass spectrometer and a Doppler shifted laser-induced fluorescence (DSLIF) techniques are used to monitor the parameters of the plasma. With an Environmental Scanning Electron Microscopy (ESEM) we record images of the surface before and after exposure to the plasma. Several gases will be investigated, like oxygen, hydrogen and argon, and plasma treatment under various conditions (varying pressure, power and electric bias) will be performed. In the continuation of this work, cold atmospheric discharges will be used for bone treatment. |
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BI-TuP-8 Glass Ionomer Cements: Probing Uptake from Solution using Surface Sensitive Techniques
B.M. Hutton, G. Palmer, P.C. Hadley (University College London, UK); T.A. Steele, A.J. Eccles (Millbrook Instruments Ltd., UK); R.W. Billington, G.J. Pearson (Queen Mary, UK); F.H. Jones (University College London, UK) Glass ionomer cements (GICs) are dental filling materials with the ability to take up ionic species (e.g. F-) from solution and store them within the cement matrix for subsequent re-release. This offers the potential for controlled drug release in-vivo. Previously, ion uptake and release by such materials has been determined primarily by measuring ion concentration in solution.1 Although this approach provides useful data on the concentration of ionic species as a function of time, little is learned about the mechanism of uptake and release. In the current work, X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) have been used to examine ion uptake. Cements were analysed after the introduction of fluoride either by doping or by immersion in KF(aq) of various concentration. Immersion of a GIC based on poly(acrylic acid) and a calcium aluminosilicate glass was found to result in the formation of a calcium and fluoride rich surface layer, while doping by mixing with KF solution during GIC preparation resulted in no such surface layer. The formation of CaF2-like layers on immersion was entirely unexpected on the basis of previous solution-based experiments and may explain differences in measured uptake kinetics. The use of these techniques has been extended to examine the inclusion of molecular active species (amprolium hydrochloride and chlorhexidine acetate) within the GIC matrix. Both species could be detected in GIC samples irrespective of whether they were included by mixing or by immersion in solution. However, relative peak intensities indicated that the binding of the active molecule is dependent on the method of inclusion. Significant applications in the study of the uptake and release mechanisms of active species such as antibacterial and antifungal agents are envisaged. |
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BI-TuP-9 Morphological Analysis of the Collagen Structure of Regenerated Rat Tendons Following Laser Photo-stimulation
V. Baranauskas (Universidade de Campinas - Brazil); N.V. Parizotto (Universidade Federal de Sao Carlos - Brazil) Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were used to comparatively study the structure of regenerated rat tendons submitted to laser photo-stimulation after a surgical injury that extracted around 6 mm of Achilles'' tendon (tenectomy) of the left forefoot. The experiment was performed using male Rattus norvegicus albinus, lineage Wistar, with body weights in the range of 250 to 300 g, and 90 days old. The healing course was monitored at 7, 14, 21 or 28 days after the injury. The animals were divided into four groups. One group was used as a control and the other three were subjected to irradiation of doses of 0.5, 5.0 and 50 Jcm-2, respectively. A He-Ne laser of 6 mW power (L = 632.8 nm) was used. AFM and SEM images, at different magnifications, allowed the identification of the time-dependence of the recovery, measured by the organization of the collagen fibers. Comparison of natural recovery processes with the laser photo-stimulation procedures will be discussed. |
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BI-TuP-10 Evolution of a UHV Compatible Heater for TSG Preparation
M. Hasselblatt, B. Jackson, M. Heidecker, P. Wagner (Zyomyx, Inc.) Template Stripped Gold (TSG) surfaces have been used extensively as a source for ultra-flat substrates. The preparation of these thin gold films relies on accurate temperature control. Heating in UHV / HV is always difficult since an accurate temperature measurement requires excellent thermal contact with the sample due to the lack of convection. Working with layered crystals like mica makes matters worse. Here we present the evolution of an ultra-high vacuum compatible heating plate that is optimized for the preparation of TSG. Initial versions of this heater were successfully built by casting Omegabond 600 High Temperature Chemical Set Cement into a Teflon mold including pre-coiled Omega Nickel Heating Wire and thermocouple wire. The current design features an exceptional degree of thermal homogeneity over approx. 9 square inches with variations of less than 1% and also a modular design to improve serviceability. |
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BI-TuP-11 Imaging Biomolecules for Skin Cancer Demarcation
M.B. Ericson, A. Rosén (Chalmers University of Technology - Göteborg University, Sweden); A.-M. Wennberg, C. Sandberg (Sahlgrenska University Hospital - Göteborg University, Sweden); F. Gudmundsson (Chalmers University of Technology - Göteborg University, Sweden); O. Larkö (Sahlgrenska University Hospital - Göteborg University, Sweden) Protoporphyrin IX, Pp IX, is a photoactive porphyrin molecule formed in the cell heame synthesis. It has been shown that Pp IX is formed to a larger extent in tumor cells due to enzymatic and metabolic differences compared to normal cells. This effect can be enhanced by exposing the cells to an excess of aminolevulinic acid, ALA, a precursor in the heame synthesis. By imaging the fluorescence from Pp IX molecules in the skin, the extension of skin tumor can be visualised with respect to the enhanced Pp IX production. This technique is based on photodynamic therapy, PDT, which is a new clinical treatment for cancer that has developed over the past 25 years. In a clinical study of 40 patients with basal cell carcinoma, a malignant type of skin cancer, the Pp IX fluorescence was recorded by a CCD camera set-up. The lesions were treated with ALA cream and thereafter the fluorescence was visualised by using filtered mercury lamps as excitation light-source. The contrast in the fluorescence images was evaluated as a function of ALA application time in order to optimise the technique. The study showed a correlation between the fluorescence images and histological pattern however the individual variations were large. Further studies are planned in order to further improve the technique. |
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BI-TuP-12 Characterization of the Crotalus Durissus Terrificus Venom by Atomic Force Microscopy
V. Baranauskas, J. Zhao (Faculdade de Engenharia Eletrica e Computacao - UNICAMP, Brazil); D.M. Dourado (UNIDEP - Brazil); M.A. Cruz-Hofling (Instituto de Ciencias Biologicas - UNICAMP - Brazil) Atomic Force Microscopy (AFM) was used to study the morphology of crude venom from the South American rattlesnake Crotalus durissus terrificus. The effects of the crotalic venom on humans are systemic, leading to suffocation in fatal cases due to the neurotoxic, myotoxic and coagulative action of the components of the venom. We used adult snakes from the Pantanal region, Mato Grosso do Sul, Brazil, that remained without food for 30 days before the venom was extracted. The venom was collected manually by a specialist and dried at room temperature. Atomic Force Microscopy images, obtained using low vertical forces, allowed characterization of the surface morphology of the samples at sub-micron resolution. Coiled and porous structures are observed. Characterization of the venom by AFM is potentially of great importance because it may allow the comparison of its natural components. Critical discussion of the experimental results and characterization of the samples by AFM are given. |
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BI-TuP-13 Role of Interfacial Water Structure on the Protein Resistant Properties of Oligo(ethylene glycol) Monolayers
B. Subramanian, J. Yan, G.P. Lopez (The University of New Mexico) Understanding the mechanism of protein adsorption at surfaces is an important issue in the field of biomedical materials, cellular adhesion and clinical diagnostics. Self-assembled monolayers (SAMs) of oligo(ethylene glycol)-terminated alkanethiols on gold are known to be protein resistant and represent a good model system to study the interactions of proteins with organic surfaces. Although these SAMs are resistant to protein adsorption, the mechanism by which these monolayers prevent protein adsorption is not yet established. Recently, it was suggested that protein resistance of these monolayers is a consequence of the formation of a structured interfacial water layer, which prevents direct contact between the surface and the protein. It was further suggested that, this might be a common mechanism for other monolayers, which show resistant to protein adsorption. It has been observed that, interfacial water undergo sharp changes in its properties (e.g., density, surface viscosity) at 15, 30, 45, and 60°C. These changes are attributed to the change in the structure of interfacial water at that temperature. We examine whether the change in the interfacial water structure at these characteristic temperatures affect the protein resistant properties of these monolayers, by carrying out protein adsorption on mixed monolayers of hexa(ethylene oxide)-terminated alkanethiols and methyl terminated alkanethiol (χ EG6 = 0.44) as a function of temperature. The results show that, there is a sharp change in the protein adsorption behavior at 30±1°C. Below this temperature, there is no protein adsorption and above this temperature there is approximately a monolayer of protein adsorbed on the SAM surfaces. These results strongly support the view that interfacial water structure plays an important role in the protein resistant properties of oligo(ethylene glycol) SAMs. |
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BI-TuP-16 Oligo(Ethylene Glycol)-Terminated Self Assembled Monolayers: Protein Resistance and the Effect of Assembly Temperature
C. Boozer, S. Chen, L. Li, S. Jiang (University of Washington) The rational design of protein resistant surfaces is a critical step in the ongoing development of biomaterials and biosensors, yet we lack a fundamental understanding of how such surfaces work. Here, we report a systematic study of the behavior of oligo(ethylene glycol)-terminated self-assembled monolayers (SAMs) prepared at a range of temperatures. The monolayers were formed by self-assembly of (EG)6-terminated thiols, in a heated (or cooled) methanol solution, on both single crystal and polycrystalline gold films. The films were characterized using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and infrared adsorption (FTIR). Protein adsorption on the OEG-terminated SAMs was studied using a home-built surface plasmon resonance (SPR) sensor. It was found that the ability of the OEG-terminated SAMs to resist protein adsorption from a buffer solution correlates with the temperature at which they were prepared. Protein adsorption studies were performed with both bovine serum albumin and fibrinogen, and in both cases we found that protein resistance of the films was greatly diminished by increasing assembly temperature. A possible mechanism will be presented. |
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BI-TuP-17 Determination of Adsorption Thermodynamics for Lysine Residues on Functionalized SAMs Using Surface Plasmon Resonance
V.N. Vernekar, R.A. Latour (Clemson University) Although protein adsorption is key to many bioengineering problems, it is still not well understood. New comprehensive approaches to this problem are needed. In this study we take a systematic approach to address protein-surface adsorption by studying submolecular interactions of peptide residues with model surfaces. We hypothesize that combining the intermolecular thermodynamic contributions for peptide residue-surface adsorption with intramolecular residue-residue interactions will provide an approach to accurately predict overall protein adsorption. Accordingly, the objective of this study was to develop experimental techniques to measure residue-surface adsorption using surface plasmon resonance spectroscopy (SPR). The model residue-surface system selected for this initial study was poly-L-lysine (PL) and OH & COOH terminated Au-alkanethiol self assembled monolayers (SAMs). Preliminary studies were conducted to develop surface preparation and cleaning protocols necessary to obtain a stable SPR signal during the adsorption process. Adsorption studies were then conducted to measure the difference in signal as a function of surface functionality and PL concentration. Results show that the amount of adsorbed PL increases with increasing solution concentration, with the COOH-SAM adsorbing more PL than the OH-SAM for each concentration. These studies provide experimental data that is needed for calculating thermodynamic parameters for adsorption (Gibbs free energy, enthalpy, entropy) for each of these model systems. These values will be compared to results predicted from computational chemistry studies by others in our group for these same residue/surface systems. |