AVS2001 Session BI+AS-WeA: Surface Characterization

Wednesday, October 31, 2001 2:00 PM in Room 102

Wednesday Afternoon

Start	Invited?	Item
2:00 PM	Invited	BI+AS-WeA-1 Sum Frequency Generation (SFG) - Vibrational Spectroscopy and Atomic Force Microscopy (AFM) Studies of Biomaterial Liquid and Gas Interfaces. Surface Structures, Compositions and Bonding G.A. Somorjai (University of California, Berkeley) The surface monolayers of polyethylene and polypropylene and its blends, polyurethanes with different hydrophilic and hydrophobic endgroups and their blends and pHEMA have been studied by a combination of SFG and AFM. SFG reveals the different surface structures of polyolefins as a function of molecular weight. Changes of chain orientation occur at the glass transition temperature of polypropylene (-10 ËšC). When polyethylene is stretched, the surface becomes rough as the spherulites align in the stretch direction thereby weakening the polymer normal to the stretching direction. pHEMA with polymer groups that are crosslinked and polymer chains that are not exhibit variations of friction coefficients as measured by AFM. The low friction areas can be associated with polymer chains that are not crosslinked that can also be removed by methanol solution. These studies can also be carried out while the surface is under water. AFM reveals phase separation in blends due to the difference in friction coefficients of the two polymer components.
2:40 PM		BI+AS-WeA-3 Quantitative Analysis of Multicomponent Adsorbed Protein Films by Static Time of Flight Secondary Ion Mass Spectrometry M.S. Wagner, M. Shen, T.A. Horbett, D.G. Castner (University of Washington) Quantitative analysis of multicomponent adsorbed protein films is an integral part in the investigation of biofouling in many marine, food processing, and biomaterial applications. We have previously shown that Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and multivariate analysis (MVA) are ideal techniques for the analysis of single component adsorbed protein films.¹ MVA is also essential for quantitative compositional analysis of multicomponent adsorbed protein films in one parallel ToF-SIMS experiment. Partial Least Squares Regression (PLSR), a multivariate calibration technique, can quantitatively determine the composition of binary protein mixtures adsorbed onto mica using only the single component spectra for calibration. Current research focuses on binary protein mixtures on fluorocarbon and nitrogen-containing plasma polymer surfaces, both providing unique challenges for quantitative analysis by ToF-SIMS. Ternary and quaternary adsorbed protein films adsorbed onto mica were also investigated to determine the limit of complexity for quantitative compositional analysis. Finally, Principal Component Analysis (PCA) and a set of standard spectra were used to obtain semi-quantitative compositional analysis of a time series protein adsorption from dilute plasma solutions. Factor-deficient (fibrinogen and kininogen) plasmas were studied to corroborate this data. Multivariate analysis and ToF-SIMS are useful tools for quantitative compositional analysis of multicomponent adsorbed protein films. ¹ M. S. Wagner and D. G. Castner, Langmuir in press (2001).
3:00 PM		BI+AS-WeA-4 Limits of Detection and Identification for Adsorbed Protein Films using XPS, ToF SIMS and Multivariate Analysis S.L. McArthur, M.S. Wagner, M. Shen, T.A. Horbett, D.G. Castner (University of Washington) In the ongoing development of biomaterial surfaces capable of resisting protein adsorption, surface analytical means of accurately detecting and subsequently identifying sub monolayer amounts of protein are becoming critical. The surface and chemical sensitivity of ultrahigh vacuum techniques such as XPS and ToF-SIMS have been utilized for some time in the detection of adsorbed protein films. More recently, multivariate analysis techniques have enabled the identification of the adsorbed species from both single and binary solutions via ToF-SIMS. In the instances where protein adsorption is low factors such as surface chemistry, surface coverage and roughness can be expected to complicate data interpretation, strongly influencing the both the detection sensitivity and accuracy of the protein identification. In this study a number of different surfaces were incubated in protein solutions of varying concentrations from 0.1ng/ml to 100µg/ml. Protein adsorption was quantified using radiolabelling and each surface analyzed using both XPS and ToF-SIMS. The results of the study illustrated the significant role of surface chemistry on the detection limits for adsorbed proteins. Not surprisingly, the presence of nitrogen in the substrate hindered the detection of protein by XPS, although detection limits remained high (<10ng/cm²) for ToF-SIMS. The most interesting finding was the poor detection limits on PTFE surfaces, where both XPS and ToF-SIMS were unable to detect proteins below 100ng/cm². The detection limit of ToF-SIMS for protein adsorbed onto mica was 0.1 attomole of protein, rivaling the sensitivity of liquid and gas phase MS. Multivariate classification methods were also implemented to identify the adsorbed protein at submonolayer surface coverages. These results were also highly dependent on the substrate chemistry and morphology. Both XPS and ToF-SIMS are useful tools for the characterization of low levels of adsorbed protein.
3:40 PM		BI+AS-WeA-6 High z-resolution Microscopy of Biological Interfaces C.M. Ajo-Franklin, L.C. Kam, S.G. Boxer (Stanford University) Total internal reflection fluorescence microscopy (TIRFM) is widely used to study the structure and dynamics of biological interfaces by confining the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to even further confine the evanescent field, and by varying the angle of incidence, to profile fluorescent objects with high z-resolution. High refractive index materials, such as lithium niobate, sapphire, and zinc sulfide, exhibit different surface chemistries and each presents a unique challenge for defining biomolecular assemblies at the surface, a prerequisite for high resolution TIR techniques. In contrast, many well-developed strategies exist for modifying and tethering biomolecules to SiO₂ surfaces. Furthermore, SiO₂ surfaces are one of the few that can be used as substrates for supported lipid bilayers, a useful model system for studying biological membranes and interactions between membrane components and cells. We report the fabrication of hybrid surfaces consisting of nm layers of SiO₂ on lithium niobate (LiNbO₃, n = 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we show resolution of structures near a dielectric interface displaced by only tens of nanometers. These results demonstrate that it should be possible to profile the vertical location of fluorophores with nm resolution in real time, opening the possibility of many experiments at the interface between supported membranes and living cells.
4:00 PM		BI+AS-WeA-7 Chiral Recognition Observed at the Molecular Level by UHV-STM: Cysteine on Au(110)-(1x2) A. Kühnle, T.R. Linderoth, B. Hammer, F. Besenbacher (University of Aarhus, Denmark) Chirality is a frequently encountered property of organic molecules, leading to the existence of two mirror-image enantiomers. Interestingly, Nature is often homo-chiral in the sense that only one enantiomer participates in biological processes. Molecular recognition with chiral specificity is thus crucial within many fields of chemistry, biology and medicine. It is also essential for strategies to resolve racemic mixtures into enantiopure phases. Here we report on the adsorption of the chiral amino acid cysteine [HS-CH₂-CH(NH₂)-COOH] on the Au(110)-(1x2) surface under Ultra-High Vacuum conditions. Using Scanning Tunneling Microscopy (STM) we have discovered that the cysteine molecules can form isolated molecular pairs that break the mirror symmetry of the gold surface. This provides a model system for a molecular level study of chiral recognition: Deposition of the pure L and D- enantiomers, respectively, leads to identical, but mirror-reflected, cysteine pairs clearly distinguishable by STM. Most interestingly, deposition of the racemic D-L mixture only leads to the known homochiral pairs, showing that heterochiral D-L molecular interaction is avoided. To explore the origins of this novel chiral recognition mechanism, we have performed ab-initio DFT calculations. We find that the cysteine molecules are anchored to the surface via S-Au bonds and interact mutually through hydrogen bonds between the carboxylic groups. Importantly, a favorable interaction between the surface and the amino group is only geometrically feasible for homochiral pairs. The mechanism behind the observed chiral recognition is thus reminiscent of the generic so-called three-point contact model for chiral ligand-receptor interaction.
4:20 PM		BI+AS-WeA-8 Interactions between Calix-6-arene Sulfonates and Poly (Allylamine Hydrochloride) : A Stoichiometric Complexation Able to Release Proteins Bound to the Polyelectrolyte V. Ball, G. Esposito, A.W. Coleman, P. Schaaf (CNRS, France); J.C. Voegel (Unite INSERM 424, France) In the framework of our research, aimed to understand the fundamental mechanisms of interactions between polyelectrolytes and proteins or between polyelectrolytes and ions either in the adsorbed state on surfaces or in solution, we describe here the interaction between calix-6-arene sulfonate (C6S) and poly (allylamin hydrochloride), PAH. At pH = 7.4, the C6S molecules carry 6 negative charges owing to their sulfonate groups whereas the polyelectrolyte is positively charged. Electrophoretic mobility experiments show that neutral particles are formed when the amount of negative charges coming from C6S matches the amount of positive charge from PAH (two chain lengths have been investigated). These particles display sizes in the µm range and are subjected to sedimentation after long term equilibration. Hence, coacervation occurs and this process is irreversible, since addition of either an excess of PAH or C6S does not change the solution turbidity and the particle size distribution. At smaller C6S/PAH stoichiometries, where C6S is totally removed from the unbound state in solution, the particles are positively charged and smaller in size (hydrodynamic radius between 50 and 150 nm). Therefore, the neutral particles obtained at the charge equivalence may result from aggregation of smaller C6S-PAH aggregates. Moreover, H-NMR experiments show that the C6S molecules are tightly bound to the polymer. We show then that one can make use this strong interaction in competition experiments to release quantitatively bovine serum albumin (BSA) that has been bound to PAH molecules in a previous step. These BSA-PAH aggregates are spontaneously dissociated and replaced by C6S-PAH particles which sediment, the supernatant only containing proteins in their native secondary conformation.
5:00 PM		BI+AS-WeA-10 Carbon Based Coatings for Cardiovascular Stents E.A. Evans (University of Akron); U. Hafeli (Cleveland Clinic Foundation) Carbon based films including diamond like carbon have been deposited as a protective layer for coronary stenting applications. Taking advantage of carbon's stability, or resistance to chemical attack, its flexibility, and other properties, our current technical objective is to develop carbon based materials as coatings for radioactive rhenium stents. Radioactive rhenium stents are being investigated to limit smooth muscle cell growth following coronary surgery. The carbon based coating is being investigated to reduce the release of radioactive rhenium (free activity) into the blood following the stenting procedure. Plasma enhanced deposition was used to deposit the carbon based coating onto metallic substrates. Foils, wires, and coils were coated and tested for adhesion, cytotoxicity, and release of radioactive rhenium. A critical requirement for successful stenting devices is a uniform surface over the entire structure. Our initial results indicate a 50% decrease in the release of free activity relative to uncoated rhenium. Relationships between deposition parameters and coating performance will be presented.