Papers

AVS1996 Session BI+AS-WeM: Surface Characterization of Biological Materials

Wednesday, October 16, 1996 8:20 AM in Room 203A

Wednesday Morning

Start	Invited?	Item
8:20 AM		BI+AS-WeM-1 Surface Compatibility of Micromachined Devices for PCR E. Picozza, E. Winn-Deen (Applied Biosystems Division of Perkin Elmer); K. Bateman, D. Jung, J. Hickman (Science Applications International Corporation); M. Albin (Applied Biosystems Division of Perkin Elmer) The polymerase chain reaction (PCR), an //ital in vitro// DNA amplification technique, has become ubiquitous throughout the scientific community. However, as the technique comes on line for various applications such as in diagnostics, the demands for sample throughput and reduction of cost per sample increase. An approach taken here addresses these demands by applying microlithography to generate dense arrays of sample compartments on small ~1 cm\super 2\ silicon chips. Typical volumes of these compartments are 1-5 microliters. A consequence of reducing these volumes in sealed compartments is that the surface to volume ratio is increased, resulting in a negative effect on the product yield. The micromachined vessels for PCR also differ in surface chemistry and composition from the polypropylene, PP, tubes traditionally used in PCR. We have used Xray photoelectron spectroscopy, XPS, analysis combined with contact angle wettability to characterize the PCR vessel surfaces before and after PCR. We are designing a surface interface, which in combination with traditional chemical PCR optimization, will enhance the miniaturized PCR technique.
9:00 AM		BI+AS-WeM-3 Multiplexed DNA Sequencing and Diagnostics on a Genosensor Chip H. Arlinghaus, M. Kwoka, X. Guo (Atom Sciences, Inc.); K. Jacobson (Oak Ridge National Laboratory) Sequencing by hybridization (SBH) enables DNA sequencing faster, more economically, and more comprehensively than is possible with other techniques. SBH employs known probe DNA to analyze target DNA for sequence homology. The target DNA hybridizes to complementary sequences of the probe DNA only if the base pairing rules apply. Multiple enriched stable tin-labeled DNAs were used for SBH to demonstrate the basic advantages of such labels, to show the multiplexing feasibility, and to provide a very rapid analytical process. After hybridization, the bound tin-labeled DNA was detected by laser atomization resonance ionization microprobe (LARIMP) or sputter-initiated RIMP (SIRIMP), which are element selective, isotope specific, rapid mass spectrometric analytical methods. Hybridization and detection were performed on DNA immobilized on nylon, platinum, and glass surfaces. The data demonstrated (a) successful attachment of Sn isotopes to specific sequences of DNA and (b) selective hybridization between multiple free Sn-labeled DNAs and DNAs immobilized on a surfaces. Discrimination between complementary and non-complementary sites was 10-100 fold. The multiplex method was demonstrated by first selectively hybridizing \super 118\Sn- and \super 120\Sn-labeled DNA simultaneously to a DNA genosensor matrix and then simultaneously detecting the tin isotopes with LARIMP/SIRIMP. It can be concluded that this new technique has the potential to analyze several million separate hybridization spots per day and thus offers a means to rapidly obtain data from hybridization matrices. This work was partly supported by NIH.
9:20 AM		BI+AS-WeM-4 Self Assembly and Hybridization of DNA Functionalized Alkanethiols on Gold: An In Situ Two Color Surface Plasmon Resonance Study K. Peterlinz, R. Georgiadis (George Washington University) Self assembled alkanethiol films have received much attention for use as biologically active surfaces. For example, in many biosensor applications, biochemically active groups are incorporated into stable self assembled organic films via covalent attachment. The process of self assembly determines the structure and therefore the activity of these biologically active surfaces. Despite the propensity for simple aliphatic alkanethiols to self assemble into stable, well ordered monolayers, little is known about the effects of these biochemically active groups on the self assembly process. Surface plasmon resonance (SPR) spectroscopy is a relatively simple, all optical technique which is extremely sensitive to chemical and physical changes at the metal/dielectric interface. Using both multi-solvent SPR and two color SPR, we have monitored the kinetics of self assembly for single stranded DNA terminated alkanethiols. From the kinetics, we determined the effects of pH and ionic strength on the self assembly process. We compare the adsorption kinetics of the single stranded DNA terminated thiol with the adsorption kinetics of the unfunctionalized DNA. In addition, we monitored the hybridization of the DNA functionalized film using SPR.
9:40 AM		BI+AS-WeM-5 Distinguishing between 6-Carbon Sugars using Time-of-Flight SIMS B. Tyler, Y. He, J. Heys, A. Willse (Montana State University) Researchers have suggested the possibility of using TOF-SIMS to probe complex biological surfaces. In order to use static SIMS to probe biological surfaces, protocols for the interpretation of the resulting spectra must be developed. These surfaces consist primarily, of proteins, lipids, glyco-proteins and saccharides. Although work has been published on interpretation of protein and lipid spectra, saccharides have largely been ignored. It is well established that saccharides and glyco-proteins are involved in anti-body recognition, receptor ligand binding, and biological adhesion processes. The stereo chemistry of the saccharides are critical for biological recognition. The objective of this study has been to determine if SIMS is sensitive to these stereo-chemical differences. We have studied TOF-SIMS spectra of eight different aldopyranoses: allose, galactose, mannose, glucose, gulose, idose, and talose. These eight sugars have a 6 membered ring and differ only in stereo chemistry. The conformational stability of the ring and the crystal structure of the sugars are affected by the stereochemistry of the sugars. Positive and negative TOF-SIMS spectra of the eight pyranoses have been collected from multiple samples. The effect of sample preparation and sodium chloride concentration on the spectra has been investigated. Clear differences can be seen in the spectra of the eight sugars. Based on multivariate statistical tests, each of the eight sugars can be uniquely identified. Multivariate models are currently being developed to identify the sugars from TOF-SIMS spectral data. Tests are being done to determine if these pyranoses can be accurately identified in mixtures.
10:00 AM		BI+AS-WeM-6 Lattice Resolution Observations and Defect Structure of Protein and Virus Crystals Studied by In Situ Atomic Force Microscopy Y. Kuznetsov, A. Malkin (University of California, Riverside); J. Konnert (Naval Research Laboratory); A. McPherson (University of California, Riverside) In situ atomic force microscopy was utilized for the visualization of surface morphology and defect structure in protein (thaumatin, canavalin, lipase) and virus (satellite tobacco mosaic virus) crystals. High resolution images of crystal lattices were observed and the measured lattice spacings shown to agree with X-ray crystallographic data for these structures. These images provided additional information on molecular packing useful for structure determination. High resolution images of the molecular structure of the step edge on the surfaces of growing thaumatin crystals were observed and compared with computer simulations. Stacking faults and their combinations with screw dislocations, single dislocations and point defect were observed. Defect densities in several different macromolecular crystals were calculated (for example for canavalin, both dislocation and stacking faults densities were calculated to be 10\super-5\ to 10\super-6\cm\super-2\). Formation of various defects and changes in surface morphology due to the incorporation of microcrystals and debris particles were observed. Etching on various kinds of defects will be described.
10:20 AM		BI+AS-WeM-7 An Approach to Quantitative Analysis of Surface Carboxyl Groups of Biodegradable Polyanhydrides using XPS J. Chen (State University of New York, Buffalo); J. Gardella, Jr. (AVS) Polyanhydrides have shown advantages for controlled drug release devices and tissue regeneration on the adjustability of its hydrolysis rate and favorable biocompatibility. The hydrolysis could undergo either surface etching and/or bulk degradation mechanisms that affect the drug release rate directly. The change of surface carboxyl group concentration during the hydrolysis is the key point in investigating the dynamics of reaction rates of polyanhydride hydrolysis. In this study, the surfaces of polyanhydrides were modified with different reagents to label the surface carboxyl groups and the quantitative analysis was conducted using XPS. Both nucleophilic substitution and acid/base reactions were used for the labeling of surface carboxyl groups. Our results show that acid/base reactions must be used in the surface labeling which takes the carboxylic acid hydrogen as the tagging target. Nucleophilic substitution reactions, which were traditionally used for labeling hydroxyl groups, would not work for this purpose since the reactive nature of the polyanhydride backbone linkage toward nucleophilic reactions causes further degradation. Semiquantitative analysis results have been obtained by XPS measurements with acid/base reaction labeled polyanhydride surfaces.
10:40 AM		BI+AS-WeM-8 Surface Properties of Chemically-Defined Substrates used to Enhance Long Term Embryonic Neuronal Survival in Defined Medium K. Bateman, D. Jung, J. Hickman (Science Applications International Corporation); A. Schaffner, J. Barker (National Institutes of Health) Embryonic rat ventral spinal cord neurons were cultured on artificial surfaces (self assembled silane monolayers, or SAMs) in defined medium. Cells were mechanically or enzymatically (papain) dissociated from the developing hippocampus of 19-day old rat embryos and cultured in serum-free MEM-N3. The cells were grown on poly-D-lysine, a standard culture substratum, and glass coverslips modified with various amino-, alkyl-, phenyl-, thiol-containing and polymeric organosilanes that differed in their chemical structure, degree of wettability and net charge. Cultures were assayed qualitatively for their growth promoting properties including initial cell adhesion, degree of cellular reaggregation, neurite outgrowth and long-term survival. Surfaces were analyzed prior to and following cell culture by X-ray photoelectron spectroscopy (XPS) in order to measure film thickness and elemental composition of the amount of protein deposited by the cells. Interestingly, growth promoting potential of a substrate was not positively correlated with wettability alone. Several hydrophobic and hydrophilic surfaces better enhanced neuron survival than the conventional biological surface, poly-D-lysine. Some surfaces appear to accelerate the extension of neurites. Some of the alkyl surfaces were found to promote the survival of mature oligodendrocytes (myelinating glial cells) as well as neurons. We have correlated several surface properties with the substrates ability to promote cellular survival and differentiation over the long term.
11:00 AM		BI+AS-WeM-9 Structure and Growth of the Surfactant/Silica Films L. Zhou, P. Fenter, M. Trau, S. Manne, I. Aksay, P. Eisenberger (Princeton University) In-situ and ex-situ X-ray study, coupled with AFM characterization, has been performed on the self-assembled surfactant/silica thin films which are synthesized with C\sub 19\H\sub 42\ClN (CTAC)/(C\sub 2\H\sub 5\O)Si (TEOS) under aqueous conditions at pH=1 on freshly cleaved mica substrates. Under these conditions a film spontaneously adsorbs to the mica substrate. Our results show that the film is strained with respect to the bulk hexagonal structure, highly ordered and consists of a lattice of hexagonally packed tubes which are oriented parallel to the substrate surface. A striking feature of our data is that despite the quasi-hexagonal structure of the mica surface the tubes are only aligned along two of the equivalent next-nearest-neighbor directions of the mica substrate. The absence of the alignment in the third direction, which is unique due to the distorted atomic arrangment of the mica, provides direct evidence that the alignment of the tubes is due to an epitaxial arrangement in spite of the large lattice mismatch between the film and the substrate spacings.