Bulletin of the American Physical Society
2005 APS March Meeting
Monday–Friday, March 21–25, 2005; Los Angeles, CA
Session D21: Methods in Nanobiotechnology |
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Sponsoring Units: DBP Chair: Kai Felix Braun, Ohio University Room: LACC 409A |
Monday, March 21, 2005 2:30PM - 2:42PM |
D21.00001: Fluorescence Correlation Spectroscopy of Biomolecules: Kinetic Studies Using Microfluidic Devices Peter Galajda, Jason Puchalla, Robert Riehn, Robert Austin Fluorescence correlation spectroscopy (fcs) uses the autocorrelation of the fluctuation of the fluorescence intensity resulted by single fluorophores traversing a confined excitation volume. Information about motion and interactions can be gathered on the single molecule level. Usually either diffusion or convection is dominant on the timescales a molecule travels through the focal volume of a laser beam. In some conditions both can have a significant contribution to the fluorescence fluctuation, and the diffusion constant and the flow velocity can be determined simultaneously. In most cases an fcs measurement lasts for seconds, making it inadequate for kinetic studies of most biomolecular processes. However, when two or more reactants meet in a microfluidic diffusional mixer a steady-state flow establishes a direct mapping of the temporal evolution of the reaction to the spatial position along the channel. Probing the sample along the mixing channel gives information about the different stages of the reaction. This opens a way to perform kinetic fcs measurements with time resolutions of a fraction of a second. A variety of biological reactions might be studied by the above technique from protein folding to DNA-protein interactions. Examples will be given in the talk. [Preview Abstract] |
Monday, March 21, 2005 2:42PM - 2:54PM |
D21.00002: Identifying the Mechanism for Amyloid Formation Using Single-Molecule Spectroscopy Troy Messina, Jason Giurleo, Jongjin Jung, Hiyun Kim, David Talaga We are investigating the mechanism for the initial stages of protein self-assembly leading to amyloid growth using single molecule spectroscopy (SMS). $\beta $-lactoglobulin ($\beta $-LG) has been shown to form amyloid under denaturing conditions and has been chosen as a model protein for this study. Initial bulk experiments have been performed utilizing dynamic light scattering along with steady state and time-resolved fluorescence of conformationally sensitive fluorophores, and a preliminary mechanism of amyloid growth has been formulated. However, SMS directly identifies critical intermediates that may only be hypothesized by bulk experiments. A single molecule imaging experiment utilizing incubated samples of mono-labeled TMR-($\beta $-LG) has been designed to count number of precursor monomers per aggregate species by counting the number of photobleaching steps required to extinguish a single particle's fluorescence. The time evolution of the particle number distribution is fit to a kinetic model representing a mechanism of amyloid growth. Results of bulk and single-molecule experiments will be discussed. [Preview Abstract] |
Monday, March 21, 2005 2:54PM - 3:06PM |
D21.00003: Fluorescence and confocal microscopy studies of the ice surface - antifreeze protein interactions. N. Pertaya, C.L. Di Prinzio, L. Wilen, E. Thomson, J.S. Wettlaufer, P.L. Davies, I. Braslavsky Biomineralization is a phenomenon in which biological material influences mineral growth on the molecular level. A compelling example involves antifreeze proteins (AFPs) known to prevent fish and insects from freezing. AFPs have many potential applications in agriculture, biomedical science, and can be used as a model platform to understand biomineralization processes for future nanotechnology applications. Here we describe a new approach to study the interaction between AFPs and ice using fluorescence and confocal microscopy combined with a unique ice growth cell. After conjugating green fluorescent protein (GFP) to Type III AFP, we imaged the fluorescence signal around and inside of the ice crystals that emerged from the cooled AFP-GFP solution, and have observed an enhanced fluorescence signal at the edge of the ice crystal. In a second cell we observed a dramatic change in the ice growth morphology when AFPs were introduced into an initially pure system. Further developments of these methods will permit the direct imaging of the location and concentration of the AFPs on ice surfaces and enable a better understanding of their operation. Supported by CIHR, the Bosack and Kruger Foundation, Ohio and Yale Universities. [Preview Abstract] |
Monday, March 21, 2005 3:06PM - 3:18PM |
D21.00004: Multi-scale Imaging of Cellular and Sub-cellular Structures using Scanning Probe Recognition Microscopy. Q. Chen, Y. Fan, V.M. Ayres, L. Udpa, A.F. Rice Scanning Probe Recognition Microscopy is a new scanning probe capability under development within our group to reliably return to and directly interact with a specific nanobiological feature of interest. In previous work, we have successfully recognized and classified tubular versus globular biological objects from experimental atomic force microscope images using a method based on normalized central moments [ref. 1]. In this paper we extend this work to include recognition schemes appropriate for cellular and sub-cellular structures. Globular cells containing tubular actin filaments are under investigation. Thus there are differences in external/internal shapes and scales. Continuous Wavelet Transform with a differential Gaussian mother wavelet is employed for multi- scale analysis. [ref. 1] Q. Chen, V. Ayres and L. Udpa, ``Biological Investigation Using Scanning Probe Recognition Microscopy,'' Proceedings 3rd IEEE Conference on Nanotechnology, vol. 2, p 863-865 (2003). [Preview Abstract] |
Monday, March 21, 2005 3:18PM - 3:30PM |
D21.00005: Scanning Probe Recognition Microscopy Investigation of Cell Elastic Properties Y. Fan, Q. Chen, V.M. Ayres, L. Udpa, A.F. Rice Scanning Probe Recognition Microscopy is a new scanning probe capability under development within our group to reliably return to and directly interact with a specific nanoscale feature of interest [ref. 1]. It is a recognition-driven and learning approach, made possible through combining SPM piezoelectric implementation with on-line image processing and dynamically adaptive learning algorithms. Segmentation plus a recognized pattern is implemented within a scan plan and used to guide the tip in a recognition-driven return to a specific site. In the present work, a preliminary coarse resolution scan and on-line processing is performed to detect a target cell. Force volume imaging is then performed at a finer resolution to determine the elastic properties. Elastic properties of cells are interpreted in terms of healthy and pathological cell functions. [ref. 1]. Y. Fan, L. Udpa, Q. Chen and V. Ayres, ``Scanning Probe Recognition Microscopy Inverstgation of Tissue Scaffolding Properties,'' Proceedings of the 2004 Biomedical Engineering Society Annual Meeting, October 13-16, 2004, Philadelphia, PA [Preview Abstract] |
Monday, March 21, 2005 3:30PM - 3:42PM |
D21.00006: MicroRaman Spectroscopy and Raman Imaging of Basal Cell Carcinoma M.A. Short, X. K. Chen, H. Zeng, H. Lui, D. I. McLean We have measured the Raman spectra of normal and cancerous skin tissues using a confocal microRaman spectrograph with a sub-micron spatial resolution. We found that the Raman spectrum of a cell nucleolus is different from the spectra measured outside the nucleolus and considerably different from those measured outside the nucleus. In addition, we found significant spectroscopic differences between normal and cancer-bearing sites in the dermis region. In order to utilize these differences for non-invasive skin cancer diagnosis, we have developed a Raman imaging system that clearly demonstrates the structure, location and distribution of cells in unstained skin biopsy samples. Our method is expected to be useful for the detection and characterization of skin cancer based on the known distinct cellular differences between normal and malignant skin. [Preview Abstract] |
Monday, March 21, 2005 3:42PM - 3:54PM |
D21.00007: Effects of High Forces on Integrin-Mediated Adhesion Protein Complexes in Fibroblast Cells Monica Tanase, Michael P. Sheetz Magnetic tweezers were designed and used to generate large forces on ferrous beads bound to the surface of adherent fibroblasts. Cells sense and exert forces on the extracellular matrix via integrins, a family of transmembrane receptors. On the cytoplasm end, the signaling from the integrins to the cytoskeleton is mediated by a complex of proteins, which were fluorescently tagged, and tracked in live cells. The magnetic beads were functionalized with a fibronectin construct designed to enhance the spatial efficiency of the integrin binding domain FNIII7-10. Rapid protein reorganization was observed in response to modulated forces applied in the lamella region. This allowed for tracking of the spatial and temporal response of proteins involved in the adhesion pathways. [Preview Abstract] |
Monday, March 21, 2005 3:54PM - 4:06PM |
D21.00008: Exploring Molecular Recognition with Spinning-Disk Laser Interferometry David Nolte, Leilei Peng, Manoj Varma We have developed a new laser heterodyne detection method that is sensitive to 0.1 nm average surface coverage of macromolecules on a solid support(1). Spinning-disk interferometry (SDI) is capable of detecting antibody-based molecular recognition at analyte volume concentrations down to 0.1 ng/ml and surface coverage to less than 500 molecules per square micron. This interferometric technique allows direct detection in the small-N limit through virtual molecular dipole transitions, in contrast to other small-N optical spectroscopy approaches that rely on real optical transitions through absorption or fluorescence detection. Sensitivity to enhanced molecular polarizability is observed in FITC-BSA molecules immoblized on silica. Potential applications of SDI in medical diagnostics and nanostructured materials characterization are being pursued. In these applications we have patterned proteins using a modified soft-lithography approach with polyacrylimide gels that lead to highly uniform and dense patterned BSA protein monolayers with rms surface roughness of 0.3 nm verified using AFM measurements. (1) Varma, M. M., D. D. Nolte, et al. (2004). ``Spinning-disk self-referencing interferometry of antigen-antibody recognition.'' \underline {Optics Letters} \textbf{29}(9): 950-952. [Preview Abstract] |
Monday, March 21, 2005 4:06PM - 4:18PM |
D21.00009: Proteins Crystal Tips for Atomic Force Microscopy Jason Hafner, Nissanka Wickremasinghe Protein crystals are being tested as tip materials for biological atomic force microscopy (AFM) with molecular recognition. A critical limitation of AFM for biological imaging is that it only reveals molecular ``topography'', making it impossible to distinguish similar size proteins or to identify specific proteins in an aggregate. Recognition-AFM combines topographic AFM imaging and biomolecular specificity through the use of antibodies tethered to AFM tips through flexible polymer linkers. [Stroh, et al, PNAS 2004, v101 p12503] The linker is required to allow the antibody to achieve the proper orientation for specific interactions with the sample, and to increase the likelihood of having an active Fab at the tip apex. However, this method of tip functionalization is not highly robust, and the linker can degrade the imaging resolution. The presence of an oriented antibody at the tip apex will be guaranteed with a tip made of a protein crystal. Initial work has been carried out with lysozyme crystals grown directly onto commercial AFM tips. Their mechanical rigidity is sufficient for routine imaging, and the resolution is being optimized. Recognition AFM with these tips is being tested by imaging patterned anti-lysozyme and non-specific serum antibodies. [Preview Abstract] |
Monday, March 21, 2005 4:18PM - 4:30PM |
D21.00010: Orientation of DNA Bases Detected by NEXAFS, XPS, and FTIR D.Y. Petrovykh, V. Perez-Dieste, A. Opdahl, H. Kimura-Suda, M.J. Tarlov, F.J. Himpsel, L.J. Whitman There is increasing evidence that conventional surface characterization methods, including those in ultra-high vacuum (UHV), can be used to understand complex bio-interfaces. We are studying the orientation of DNA bases in self-assembled monolayers of thymine (dT) homo-oligonucleotides on gold in both UHV and ambient environments using a combination of spectroscopic methods. The DNA coverage, chemical composition, and orientation of thymine bases in UHV are determined using near-edge X-ray absorption fine structure spectroscopy (NEXAFS) with fluorescence detection and X-ray photoelectron spectroscopy (XPS). Complementary information about dT orientation in the ambient is obtained with Fourier transform infrared (FTIR) spectroscopy. We observe spectral signatures of oligo(dT) monolayers on Au using all three techniques, including features that can be used to distinguish between different conformations of DNA on the surface. We find that monolayers of thiolated dT 5mers are more ordered than 25mers, and that in both cases the thymine bases are oriented with respect to the substrate. [Preview Abstract] |
Monday, March 21, 2005 4:30PM - 4:42PM |
D21.00011: Dielectrophoretic Tweezers and Micropost Arrays for Cell and Particle Manipulation Tom Hunt, Hakho Lee, Robert Westervelt We describe a micromanipulator system that uses dielectrophoresis to capture and release cells or particles. Dielectrophoretic tweezers are capable of applying hundreds of piconewtons of force to micron scale objects suspended in liquid and precisely positioning objects in three dimensions. Metal electrodes on either side of a sharp pipette tip provide the electric field gradient necessary. This manipulation technique compliments our micropost array (1) for the manipulation of particles in a microfluidic system. We will discuss applications of dielectrophoresis using hybrid integrated circuit/microfluidic devices (2) with applications that include cell sorting and tissue assembly. This work made possible by a gift from Phillip Morris and the NSEC NSF grant PHY-0117795. 1. T. P. Hunt H. Lee and R. M. Westervelt, ``Addressable micropost array for the dielectrophoretic manipulation of particles in fluid," Appl. Phys. Lett. In Press. 2. H. Lee, et Al. ``An IC/ microfluidic hybrid microsystem for 2D magnetic manipulation of individual biological cells," To appear in IEEE ISSCC, Feb. 2005. [Preview Abstract] |
Monday, March 21, 2005 4:42PM - 4:54PM |
D21.00012: Surfactant Activated Dip-Pen Nanolithography C. Patrick Collier Direct nanoscale patterning of maleimide-linked biotin on mercaptosilane-functionalized glass substrates using dip-pen nanolithography (DPN) is facilitated by the addition of a small amount of the biocompatible nonionic surfactant Tween-20. A correlation was found between activated ink transfer from the AFM tip when surfactant was included in the ink and an increase in the wettability of the partially hydrophobic silanized substrate. Surfactant concentration represents a new control variable for DPN that complements relative humidity, tip-substrate contact force, scan speed, and temperature. Using surfactants systematically as ink additives expands the possible ink-substrate combinations that can be used for patterning biotin and other molecules. For example, we are currently exploring the possibility of developing nickel/nitrilotriacetic acid (NTA)-maleimide based inks that will bind to mercaptosilanized glass surfaces for the reversible immobilization of biomolecules containing polyhistidine tags. [Preview Abstract] |
Monday, March 21, 2005 4:54PM - 5:06PM |
D21.00013: Ultrafast Laser Detection of Cancer in a Single Cell Paul Gourley, Judy Hendricks, Anthony McDonald, Guild Copeland, Keith Barrett, Cheryl Gourley, Robert Naviaux Currently, pathologists rely on labor intensive microscopic examination of tumor cells using century-old staining methods that can give false readings. Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of tumor cells without invasive chemical reagents. These techniques are critical to advancing early detection, diagnosis, and treatment of disease. Using our award-winning Hyperspectral Inceptor$^{TM}$ to rapidly assess the properties of cells flown through a micro/nano semiconductor device, we discovered a method to rapidly assess the health of a single mammalian cell. The key discovery was the elucidation of biophotonic differences in normal and cancer cells by using intracellular mitochondria as biomarkers for disease. This technique holds promise for detecting cancer at a very early stage and could nearly eliminate delays in diagnosis and treatment. [Preview Abstract] |
Monday, March 21, 2005 5:06PM - 5:18PM |
D21.00014: Generating function methods for photon counting observables Yujun Zheng, Frank L.H. Brown We recently introduced the Generalized optical Bloch Equation (GBE) framework for simulating photon counting observables from single molecule sources [Y. Zheng and F. L. H. Brown, Phys. Rev. Lett., 90, 238305(2003)]. We will present extensions to the original GBE technique that allow for extension to chromophore models more complicated than the simple model systems originally proposed. In particular, we will discuss cases involving non-Markovian stochastic dephasing of the optical transition and chromophore models that explicitly include vibrational degrees of freedom. [Preview Abstract] |
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D21.00015: Synthesis of DNA monolayers on gold and siliceous supports Rastislav Levicky, Gang Shen, Zhen Liu, M.F. Anand Gaspar, Patrick Johnson DNA monolayers have been prepared on metal and siliceous supports. Chains ranging in size from oligonucleotides to gene- sized polymers have been site-specifically attached without detectable side reactions, ensuring an end-tethered, ``brush'' geometry. On gold, DNA monolayers are anchored to nanometer-thin polythiol films which are irreversibly chemisorbed to the gold support to provide excellent stability with minimal damage to the monolayer even under high temperature ($\sim$ 100 C) conditions. The conductive metal supports allow electrical addressing of the DNA layer, a feature useful for electrochemical sensing as well as for physically probing the layer organization. On silica and glass supports, several surface modification strategies have been tested. On both types of support, optimized approaches lead to close to 100\% hybridization activity toward complementary sequences when surface densities are sufficiently low to mitigate effects of crowding. The dependence of the cross-over between full and suppressed hybridization regimes on chain length has also been investigated. [Preview Abstract] |
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