AVS2004 Session BI2-WeM: Oligo Nucleotide - Surface Interactions

Wednesday, November 17, 2004 9:00 AM in Room 213C

Wednesday Morning

Start	Invited?	Item
9:00 AM		BI2-WeM-3 Characterization of Surface Order and Structure of Thiolated Single-stranded DNA Oligomers on Gold by XPS and NEXAFS L.J. Gamble, C.-Y. Lee, N.T. Samuel, H.E. Canavan, D.G. Castner (University of Washington) The hybridization efficiency of DNA microarrays and biosensors is determined in part by variables such as the density and orientation of the single stranded DNA oligomers used to build the devices. In this study, we have used x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure spectroscopy (NEXAFS) to characterize the surface order and structure of thiol-terminated ssDNA on gold. While NEXAFS provides an investigation of the order and orientation of the DNA oligomers at a surface, XPS provides a quantitative measure of the amount of DNA at a surface. We find that when thiolated DNA oligonucleotides (T)₁₆, (A)₁₆, and (A)₈(C)₈ are adsorbed to gold surfaces, their surface orientations vary differently with increased adsorption time. Time dependent studies of thiolated (T)₁₆ and (A)₁₆ oligomers by NEXAFS showed opposite polarization dependence between the two sequences possibly due to differences in their interactions with the gold surface. XPS results also indicate variations in the phosphorus to gold ratios among the three thiolated DNA oligomers over time. Furthermore, we explain these contrasting observations by examining the interaction of unmodified DNA strands that are non-specifically adsorbed to gold. Although polyT does not interact with the gold surface, polyA binds to gold possibly through the amine groups of the bases.
9:20 AM		BI2-WeM-4 Detection of DNA Hybridization by Infrared Absorption Spectroscopy K. Miyamoto, Y. Kimura, H. Ishii, M. Niwano (Tohoku University, Japan) We have previously proposed a new, label-free method of in-situ (in-vitro) determining the chemical bonding conformation of DNA in aqueous solution, by infrared absorption spectroscopy in the multiple internal reflection geometry (MIR-IRAS). In our method, a Si prism, through which infrared lights penetrate, internally reflecting a number of times, serves as an electrochemical electrode. By applying a positive or negative potential to the electrode (prism), we can manipulate negatively-charged DNA molecules in aqueous solutions. In this study, we have selected the chemical system of complementary DNA as an appropriate template for testing the possible application of our method to biosensors for detecting DNA hybridization. We first collect IRAS spectra for complementary single-stranded (ss) DNAs that are comprised of 30 bases, and then analyze the hybridization by observing the spectral changes in the IRAS spectra caused by mixture of the two complementary DNAs in D₂O solution. We observed significant spectral changes in the frequency region of 1600-1750 cm^-1, where the bases of DNA have specific vibration modes (C=O stretching and -NH₂ scissoring modes) that are quite sensitive to base-paring. On the other hand, no significant spectral changes were observed for mixture of non-complementary DNAs. This confirms that the observed spectral changes were specifically induced by DNA hybridization. Our method would be capable of detecting and classifying other biomolecules such as proteins and peptides.
9:40 AM		BI2-WeM-5 XPS and SIMS Characterization of Oligonucleotide Immobilisation via Patterned Plasma Polymerized Interlayers P-C. T. Nguyen, R. Metz, S. Kumar (University of South Australia); M. DeNichilo (TGR BioSciences, Australia); N. Voelcker (Flinders University of South Australia); M. Jasieniak (University of South Australia); S. Coultas, S. Hutton (Kratos Analytical Ltd, UK); H.J. Griesser (University of South Australia) Surface immobilised oligonucleotides are an attractive choice as recognition elements in microarrays for parallel, multidimensional, high throughput analysis in many biosensing applications. We investigate the physico-chemical factors affecting efficient oligonucleotide immobilisation and patterning. Furthermore, by using patterned plasma polymer coatings, we implemented a direct, one-step method of fabricating microdot arrays with specific surface chemistries onto which oligonucleotides can be covalently immobilised. Amino terminated oligonucleotides such as 15-T are immobilised via surface aldehyde groups of plasma polymerised polymers. Propanal plasma polymer coatings have been applied to a wide variety of substrates including silicon and Teflon. A mask containing 100 µm diameter holes was used during plasma polymerisation to produce an array of distinct aldehyde surface chemical regions for oligonucleotide immobilisation. XPS analysis of the plasma polymer and the immobilised oligonucleotides gave rise to the expected C, O, N and P peaks. Using atomic concentration, surface coverage is calculated as a measure of immobilisation efficiency. SIMS spectra revealed characteristic thymine containing fragments from the immobilised oligonucleotides. MALDI-MS verified the covalent attachment of the oligonucleotides and their ability to hybridise a complementary strand of 15-A oligonucleotide. Hybridisation kinetics studies are underway. SIMS imaging is used to document the spatial patterns formed by masked plasma deposition and to assess the spatial selectivity of oligonucleotide immobilisation. Small spot XPS will be used to quantify the composition of the microarray dots. Results to date demonstrate that the plasma deposition step, both patterned and unpatterned, is readily transferable to various substrates including Si wafer, Teflon PTFE and Teflon PFA.
10:00 AM		BI2-WeM-6 Molecular Recognition in 2D Binary Mixtures of DNA-Base Molecules Studied by STM M. Schöck, R. Otero, L.M. Molina, E. Laegsgaard, I. Stensgaard, B. Hammer, F. Besenbacher (University of Aarhus, Denmark) Molecular recognition events between complementary nucleic acid bases are fundamental for many biological processes, like DNA replication, and is currently being exploited for self-assembling DNA-based nanostructures. The DNA replication fidelity in living organisms is maintained by a complex molecular machinery of polymerases, exonucleases, etc. On the other hand, in the case of replicating NA molecules in the prebiotic soup, the basic physico-chemical mechanism to steer the replication process is the hydrogen-bonding between DNA bases. The fidelity of this replication process implies that Watson-Crick pairing must be favored over others, like "wobble" or "deviant" pairing. By means of a combination of STM experiments and DFT calculations, in this contribution we compare the 2D molecular networks formed on Au(111) upon deposition of the binary mixtures G-C (purine-pyrimidine pair of complementary bases) and A-C (purine-pyrimidine pair of non-complementary bases). We show that, after a gentle annealing to 80°C the non-complementary bases segregate into islands of pure A and a network of pure C, whereas the complementary bases G and C form a network that cannot be separated by annealing up to the desorption temperature for C. High-resolution STM images allow us to identify the structures for these enhanced thermal stability as structures that contain G-C bonds possibly with the same structure as the Watson-Crick pairs in DNA molecules. This result shows that the hydrogen-bonding interaction alone can steer the molecular recognition process necessary for high-fidelity DNA replication even in the absence of polymerases, exonucleases, etc.,a result that could be relevant to understand the origin and nature of the first self-replicating molecules in the prebiotic soup.
10:20 AM		BI2-WeM-7 Formation of ssDNA Brushes with Controlled Length and Spacing on Gold A. Opdahl (National Institute of Standards and Technology); D.Y. Petrovykh (University of Maryland); H. Kimura-Suda (National Institute of Standards and Technology); L.J. Whitman (Naval Research Laboratory); M.J. Tarlov (National Institute of Standards and Technology) A method is presented that uses block-oligonucleotides to generate single stranded (ss)DNA brushes with controlled length and density on gold surfaces. The method is based on previously reported observations that adenine oligonucleotides (dA) have a higher affinity than thymine oligonucleotides (dT) for adsorbing on gold substrates (Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J.; J. Am. Chem. Soc.; 2003; 125(30); 9014-9015). In this study, adenine/thymine block-oligonucleotides, d(T_m-A_n), with specific (dT) and (dA) sequence lengths, m and n, were adsorbed on gold substrates from aqueous solution and were characterized by FTIR and by XPS. The FTIR and XPS experimental results support a model where the (dA) nucleotides preferentially adsorb on the gold substrate and the (dT) sequences extend away from the substrate. The surface density of the (dA_n) blocks decreases with their length n, such that the overall surface density of (dA) nucleotides adsorbed on the gold is approximately independent of the (dA_n) block length. Therefore d(T_m-A_n) oligonucleotides with long (dA) sequences (larger n) have lower densities of (dT) brush strands in the adsorbed layer. Oligonucleotides with long (dT) sequences, m, are observed to have longer brush strands extending away from the substrate. The n and m dependent adsorption behaviors and the stabilities of the brush layers will be compared to the behavior of alkanethiol derivatized ssDNA monolayers on gold.
10:40 AM		BI2-WeM-8 Coverage and Stability of ssDNA on Gold: Effects of Temperature and Displacement by Alkanethiols D.Y. Petrovykh (University of Maryland and Naval Research Laboratory); A. Opdahl, H. Kimura-Suda, M.J. Tarlov (National Institute of Standards and Technology); L.J. Whitman (Naval Research Laboratory) We characterize self-assembled films of thiolated and unmodified single-stranded DNA (ssDNA) on polycrystalline Au films using Fourier transform infrared (FTIR) and X-ray photoelectron (XPS) spectroscopy. We use homo-oligonucleotides to study the film stability as a function of the DNA-base under conditions used in hybridization experiments. One common method of controlling the ssDNA probe spacing and availability is post-deposition exposure to alkanethiols. Another common post-deposition treatment is exposure to buffer solution at elevated temperature during the hybridization step. In both cases, we find strong base-dependence in agreement with the previous results for film structure and relative adsorption affinities of thiol-modified and unmodified ssDNA [JACS 125, 5219 (2003); 125, 9014 (2003)]. The use of these post-deposition treatments also allows us to compare the relative effects of DNA-DNA vs. DNA-Au interactions for each of the bases. For the three bases that we examined, a wide range of DNA-DNA and DNA-Au interactions is observed: both interactions are weak for oligo(dT); oligo(dA) exhibits a strong affinity for Au surfaces but weak DNA-DNA interactions; and oligo(dC) represents the opposite case, with strong DNA-DNA but weak DNA-surface interactions.
11:00 AM		BI2-WeM-9 Biomolecular Immobilization in a Sugar Polyacrylate Hydrogel M.S. Spector, P.T. Charles, B.D. Martin, C.M. Soto, C.H. Patterson (Naval Research Laboratory) A novel sugar polyacrylate hydrogel has been developed as a substrate for high density microarrays. Copolymerization of chemo-enzymatically synthesized 6-acryloyl-β-O-methyl-galactopyranoside with acrylate monomers containing terminal amine or carboxyl functionalities allows for covalent attachment of the oligonucleotides or proteins. The hydrogels show extremely low non-specific adsorption of biomolecules leading to increased signal-to-noise ratio and enhanced immunoassay sensitivity over two-dimensional surfaces. High density microarrays containing oligonucleotides and protein toxins have been obtained using a non-contact microdispensing system onto thin hydrogel films. The large pore size and solution-like environment of these hydrogels allow for easy penetration of large biomolecules and detection reagents. Confocal microscopy was used for three-dimensional visualization of the gel, immobilized biomolecules, and hybridized DNA. Results indicate that DNA diffuses into the hydrogel as discrete spots with higher concentration near the middle of the gel. Hybridization of 70-mer nucleotides was readily observed in these gels.