AVS2001 Session BI-MoM: Molecular Recognition

Monday, October 29, 2001 9:40 AM in Room 102

Monday Morning

Start	Invited?	Item
9:40 AM	Invited	BI-MoM-1 Chemical Recognition on Lipid Membrane Surfaces D.Y. Sasaki (Sandia National Laboratories) Cell communication and sensing are processes mediated by chemical recognition events that occur on the lipid membrane surface. Chemical signals in bulk solution are recognized by membrane receptors, which subsequently organize into specific structures that activate signal cascades. Through a biomimetic approach, we have examined chemically induced molecular reorganization events in lipid membranes in an effort to learn how to control this process for sensor applications and nanoscale architecture. By using a synthetic approach we have simplified interactions between molecular species to evaluate their effects on molecular reorganization. Simple two-component lipid bilayer assemblies were prepared with receptors for heavy metal ions, proteins, and polypeptides. The aggregational state of these fluorophore-labeled receptor molecules, as they responded to chemical recognition events, were monitored globally by spectroscopic means (e.g., excimer formation of pyrene labels) and locally via in situ atomic force microscopy (AFM). We found that the dispersion and aggregation of receptors in a bilayer can be directed through multiple levels of interactions, such as electrostatic charge from metal ion chelation, multiple-point binding interaction with polyfunctional guests, and phase separation. In situ AFM studies observed that nanoscale structures composed of aggregated receptors but could be made to disappear or reappear upon the addition or removal of specific chemical ligands. At a slightly larger scale, the functionalized bilayers displayed a unique ability to self-assemble into hierarchical structures of stacked bilayers through a process mediated by chemical recognition. These lipid bilayers with their unique optical response, biocompatibility, and self-organizational properties demonstrate a versatile array of possibilities for sensing and nanoarchitecture.
10:20 AM		BI-MoM-3 Surface Docking Sites for Macromolecules: Interface Architecture based on PLL-g-PEG/PEGbiotin-(Strept)avidin N.-P. Huang, J. Vörös, S.M. De Paul, M. Textor, N.D. Spencer (ETH Zürich, Switzerland) Surface docking sites (nanoscale islands) are desirable for the specific adhesion of macromolecules, such as proteins and oligonucleotides, onto surfaces. The surrounding areas of such docking sites should be non-adhesive so that the adsorbed macromolecules are prevented from denaturing after adsorption. We have mixed poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) and a variant of PLL-g-PEG in which some of the PEG chains are biotinylated (PLL-g-PEG/PEGbiotin) to form a novel polymeric interface, in order to tailor chip surfaces in terms of non-specific and specific analyte-surface interactions. By means of optical waveguide lightmode spectroscopy (OWLS), streptavidin and avidin are shown to bind specifically to the biotin-functionalized PEG, while the resistance of the remaining PEG chains to protein adsorption yields a high specific-binding-to-non-specific-binding ratio. Subsequent binding of biotinylated goat-anti-rabbit immunoglobulin (αRIgG-biotin) to (strept)avidin as a capture molecule allows the system to be used as an immunoassay for the target molecule, rabbit immunoglobulin (RIgG). Changing the ratios of PLL-g-PEG and PLL-g-PEG/PEGbiotin in the mixture changes the distribution of docking sites (biotin sites) on the interface and, thus, allows optimization of the sensing response. The effects of protein charge and the ionic strength of the buffer are also explored. We expect that such a platform could serve as a powerful tool for the investigation of molecular recognition effects.
10:40 AM		BI-MoM-4 Self-Assembly of Modified Porphyrin Monolayers A.L. Bramblett, M.S. Boeckl, K.D. Hauch, T. Sasaki (University of Washington); J.W. Rogers, Jr. (Pacific Northwest National Laboratory); B.D. Ratner (University of Washington) Assembled monolayers can be used to deliver recognition signals to biological systems. Planar, multi-ring porphyrin structures can provide precision lateral control of chemistry at interfaces. Porphyrin molecules, which have peripheral constituents with the ability to hydrogen bond or coordinate to a metal, are predicted to form ordered molecular monolayers on gold surfaces in a side-by-side orientation. TPyP-Ge-Disulfide (a Ge coordinated pyridyl substituted porphyrin with two disulfide ligands) has been synthesized for use in self-assembling porphyrin monolayers, which form when one of two disulfide ligands binds to the gold surface. Self-assembled monolayers of TPyP-Ge-disulfide have been prepared on gold for characterization. X-ray photoelectron spectroscopy (XPS) of TPyP-Ge-disulfide shows that the atomic composition of the surface is consistent with a porphyrin monolayer, and approximately 50% of the sulfur atoms are bound to gold. Further, ultraviolet-visible spectroscopy (UV/Vis) shows a red shift in the peak of the Soret band, which is indicative of side-by-side porphyrin orientation when self-assembled. ¹ In addition, monolayers formed utilizing hydrogen bonding between the TPyP-Ge-disulfide and TCP (benzoic acid derivatized porphyrin), or utilizing Zn²⁺ coordination with the pyridyl nitrogen in TPyP-Ge-disulfide will be characterized using XPS, UV/Vis, and scanning tunneling microscopy, to examine the porphyrin arrangement on the gold surface. By chemically modifying the remaining free disulfide bond, these monolayers could be used to present biological ligands of interest in a spatially controlled manner. ¹ ¹ Boeckl, M. S.; Bramblett, A. L.; Hauch, K. D., Sasaki, T.;Ratner, B. D.; Rogers, Jr, J. W. Langmuir 2000, 16, 5644-5653
11:00 AM		BI-MoM-5 Efficient New Method of Nucleic Acid Immobilization Y. Wu, P.L. Dolan, L.K. Ista, M.A. Nelson (University of New Mexico); R.L. Metzenberg (Stanford University); G.P. Lopez (University of New Mexico) The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring preferential gene expression in an organism. To that end, a collaborative effort to produce a chemically reactive surface on glass slide substrates to which DNA will covalently bind for use in and advancement of cDNA microarray technology was undertaken. We have developed a chemical process for covalently linking unmodified DNA to an ordinary microscope slide in a manner that preserves the ability of the immobilized nucleic acid to hybridize to complementary sequences. The method of binding DNA to solid surfaces considerably increased the consistency and uniformity of attachment, and reduced DNA loss during the experimental process when compared to other commonly used commercially available methods. Moreover, better hybridization results have been generated compared to commercially available immobilization techniques. In general, this method allows binding of single- and double-stranded nucleic acids onto a solid substrate that can lead to considerable improvements in hybridization of complementary sequences, stability of affixed DNA, and re-usability of microarrays. Following our immobilization process, arrayed slides were reusable for at least 5 times. In addition, the hybridization data has been analyzed quantitatively and successfully correlated with solution concentrations. Although this method is originally designed for forming DNA microarrays, it is likely also suitable for the immobilization of proteins, ribozymes and aptamers onto certain solid substrates..
11:20 AM		BI-MoM-6 Probe Density Effects for Target Hybridization in DNA Monolayer Films Studied By SPR A.W. Peterson, R.J. Heaton, L.K. Wolf, R.M. Georgiadis (Boston University) Understanding probe to target interactions for surface immobilized duplexes is important in the emerging applications of DNA biosensors. We use in-situ surface plasmon resonance spectroscopy to monitor the kinetics of probe immobilization and target/probe hybridization for thiol-modified probes immobilized on gold. We find that both the efficiency and kinetics of probe/target hybridization depend strongly on probe density. Immobilization conditions can be used to control probe density and we investigate the effects of solution ionic strength, electrostatic potential at the interface and whether duplex or single stranded probes are immobilized. Independent of which immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and kinetics for probe/target hybridization. In addition, the hybridization isotherms show a distinct dependence on probe density. Insight into the mechanism of these probe/target interactions are investigated and discussed in the context of the observed kinetics.
11:40 AM		BI-MoM-7 Two-dimensional Arrangements of Streptavidin on Biotin-doped Supported Lipid Bilayars Optimized for DNA-DNA Hybridization C. Larsson, E. Fridell, B. Kasemo, F. Höök (Chalmers University of Technology, Sweden) In this work the quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and ATR-FTIR techniques were used to follow the 2-D arrangement of streptavidin on a biotinylated phospholipid bilayer supported on a SiO2 surface, for optimized capture of single stranded biotin-DNA (15- and 30-mer) and subsequently hybridization-kinetics measurement of their reaction with mixed-sequences of DNA (15- and 30-mer) with various degrees of mismatch. The QCM-D and ATR-FTIR data suggest that streptavidin rearranges into a more rigid and oriented structure at approximately 50 % coverage, interpreted as onset of crystallization. Interestingly, immobilization of biotin-DNA followed by subsequent hybridization prior to this stage results in up to a two-fold more rapid association kinetics compared with immobilization and hybridization at full coverage of streptavidin. Our results demonstrate how real-time control of the immobilization step can be efficiently used to minimize the influence from lateral interactions for surface-based biorecognition detection, and thus optimize hybridization kinetics measurements. The QCM-D data were also used to quantify variations in the viscoelastic properties of the formed layers of DNA and DNA duplexes, which were further correlated to the obtained results onhybridization kinetics.