The Nanobiotechnology Center - Microanalysis of Biomolecules

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
404	7010	1060	34%
404	7010	2010	33%
404	7010	2020	33%

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Applied sub-15nm core-shell pH sensor particles to functional tomographic imaging of biofilm microenvironments. Developed sub-7nm diameter core-shell particle synthetic methods which may enable vesicle trafficking visualization. Apical dendrites in brain slices contain uniform polarity microtubules but dendritic microtubule polarity is lost in cultured neurons. First on-chip measurements of single vesicle release events using a fully integrated CMOS chip enabled by improved post-fabrication of Pt electrodes using e-beam deposition. Precise calibration curves of glutamate detection were achieved on glutamate oxidase modified microelectrodes. An antibacterial polymer brush modified surface was used as neuron culture substrate for the first time and patterned to guide neuron outgrowth. Prepared Spongels that are less than 1 micron and absorb model drug. Developed microfluidic devices that can generate droplets in the gel format. Quantified IgE clustered with DNA ligands on cells using SEM and correlation analysis. Imaged carbon nanotube motion in solution using scanned photocurrent microscopy. Attached beads to nanotubes using optical tweezers and measured fluctuations in position. Measured nanotube mechanical response using optical tweezers. Microfabricated Platinum electrodes applied for the first time to measure catecholamines by cyclic voltammetry. C-terminus of SNAP-25 has a role in fusion pore structure indicating a longer fusion pore when this domain is deleted consistent with the zipper model of SNARE mediated fusion. Actin plays a role in fusion pore expansion independent of myosin II but myosin II accelerates release when fusion pore is expanded. Time resolved small angle x-ray scattering detects conformational change of light sensitive protein. Architecture of fusion loop of VSV G protein identified. Baculovirus vectors tested for robust expression system. The Wang lab has used nanofabricated quartz cylinders in an angular optical trap to create a Holliday junction in DNA that may be used as a "nano-sensor" for motor proteins. Working closely with the Kraus lab, the Wang lab has obtained intriguing preliminary data on translocation of the nucleosome remodeling enzyme SWI/SNF against the junction. The Lis and Webb labs have used MPM to monitor histone dynamics in living cells. Specifically, they have created a fly line expressing paGFP tagged H2B in order to directly track how these changes occur. By generating flies coexpressing mCherry tagged LacI with transgenic inserts of LacO sequences flanking the Hsp70 gene loci, they can now identify and specifically activate molecules at these loci before gene activation and track changes in structure over time. The Kraus and Lis labs have used biochemical, genomic, and gene-specific assays to determine the localization and chromatin remodeling effects of the nucleosome binding protein PARP-1. These studies have shown that PARP-1 localizes to promoter regions and, for many promoters, excludes the binding of the linker histone H1 (another nucleosome binding protein). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In the Nanoscale Cell Biology Program of the NBTC interdisciplinary teams develop and apply nano- and microscale tools to approach a mechanistic understanding of cellular function with nanoscale precision. Microfabricated devices are also developed to probe cellular function in highly parallel manner for proteomics and drug testing. The approaches include nanoparticles, molecular force measurements, small angle x-aray diffraction, carbon nanotubes, microelectrode arrays, integrated chip design, nanofabricated devices, AFM, multiphoton (MP), second harmonics generation (SHG) and total internal reflection fluorescence (TIRF) microscopy, and molecular design. All projects involve interdisciplinary teams with PIs, post-docs and students from many different disciplines supplemented by outside collaborations on the national and international level.

Publications

Settlement of Ulva Zoospores on Patterned Fluorinated and PEGylated Monolayer Surfaces; J. A. Finlay, S. Krishnan, Maureen E Callow, J. A. Callow, R. Dong, N. Asgill, K. Wong, E. J. Kramer, C. K. Ober; Langmuir, 2008, 503-510.
Polarized microtubule arrays in apical dendrites and axons; A. C. Kwan, D.A. Dombeck, W.W. Webb; Proc.Natnl.Acad.Sci, 105: 11370-11375 (2008).
Ambiguous polymeric surfaces for marine antifouling applications; S. Krishnan, R. Dong, C.K. Ober, et al.; PMSE Preprints, 2008, 98, 83-84.
Molecular architecture of the bipartite fusion loops of vesicular stomatitis virus glycoprotein G, a class III viral fusion protein. Sun, Xiangjie; Belouzard, Sandrine; Whittaker, Gary; J. Biol. Chem., 283(10): p. 6418-6427, 2008.
Polymer brushes as responsive materials for the biology-material interface; Ober, Christopher K.; Dong, Rong; Rastogi, Abhinav; Weinman, Craig J.; Tanaka, Manabu; Hemmelmann, Mirjam; Chiang, Ethan N.; Park, Daewon; Yi, Yi; Paik, Marvin Y.; Nad, Suddhasattwa; Smith, Norah; Handlin, Dale L.; Willis, Carl L.; Kramer, Edward J.; Bair; PMSE Preprint, 2008, 99, 109-110.
Trivalent ligands with rigid DNA spacers reveal structural requirements for IgE receptor signaling in RBL mast cells; D. Sil, J. Lee, D. Luo, D. Holowka, and B. Baird; ACS Chemical Biology 2: 674-684. (2007).
Hinge stiffness is a barrier to RNA folding; J. Schlatterer, L.W. Kwok, J.S. Lamb, H.Y. Park, K. Andresen, M. Brenowitz and L. Pollack; J. Mol. Biol. 379, 859 (2008).
Mono and tri-valent ions around DNA: a small angle scattering study of competition and interactions; K. Andresen, X. Qiu, S. A. Pabit, J. S. Lamb, H.Y. Park, L.W. Kwok and L. Pollack; Biophys. J., 95, 287 (2008).
Abrupt buckling transition observed during the plectoneme formation of individual DNA molecules; Forth S, Deufel C, Sheinin MY, Daniels B, Sethna JP, Wang MD; Phys Rev Lett. 100:148301 (2008)
Imaging transcription dynamics at endogenous genes in living Drosophila tissues. Yao, J., Zobeck K. L., Lis J. T. and Webb W. W.; Methods. 2008 Jul;45(3):233-41. Epub 2008 Jun 27
Intranuclear Distribution and Local Dynamics of RNA Polymerase II during Transcription Activation. Yao J, Ardehali B, Fecko CJ, Webb WW, and Lis JT; Molecular Cell 28: 978-990 (2007)
Rapid, Transcription-Independent Loss of Nucleosomes over a Large Chromatin Domain at Hsp70 Loci; Petesch SJ and Lis JT; Cell 134: 74-84 (2008)
Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes; Krishnakumar R., Gamble M. J., Frizzell K. M., Berrocal J. G., Kininis M., and Kraus W. L.; Science 319:819-821 (2008)
The DNA binding and catalytic domains of poly(ADP-ribose) polymerase-1 cooperate in the regulation of chromatin structure and transcription; Wacker D. A., Ruhl D. D., Balagamwala E. H., Hope K. M., Zhang T., and Kraus W. L.; Mol. Cell. Biol. 27:7475-7485 (2007) (Featured on the cover)
Transcriptional control by PARP-1: chromatin modulation, enhancer-binding, coregulation, and insulation; Kraus W. L.; Curr. Op. Cell Biol. 20:294-302 (2008) (Review)

Progress 10/01/06 to 09/30/07

Outputs
Accomplishments Synthesized Ratiometric core-shell nanoparticles with hydrodynamic radii of <15 nm, capable of sensing pH or Ca+2 concentration in solution. PEG-coated particles with enhanced particle stability and decreased non-specific adsorption were introduced into cells using single cell electroporation Apical dendrites contain uniform polarity microtubules that can extend for more than 270 um having an estimated polarity of 83%. First electrochemical CMOS chip detection of dopamine. Measurement of cellular exocytosis using our potentiostat circuit. PMAA-b-PHEMA block copolymer brush grown on gold electrode surface and used to immobilize proteins. Prepared rigid Y-shaped DNP-DNA ligands of different lengths that bind with same affinity to IgE and cluster multiple IgE-receptors on cells. Assessed several cellular activities that are stimulated by Y-DNP-DNA ligands and revealed length-based discrimination among signaling pathways in RBL mast cells. Confirmed rigidity and assess shape of Y-shaped DNA templates with fluorescence resonance energy transfer. Created improved carbon nanotube devices for membrane sensing. Used nanotube as electronic sensor to detect binding of streptavidin to supported lipid bilayer. Performed first experiments electrical interaction of suspended nanotube devices with cells. Demonstration of Tyr kinase activities of multiple src isoforms. Development of high throughput immunofluorescence assay for phosphotyrosine. Testing chemical inhibitors of tyrosine kinases. Amperometric detection of exocytic release with transparent 5 nm gold electrodes. Improved SNAP-25 FRET construct using FlAsh as acceptor probes SNAP-25 conformation change during complex formation with syntaxin. Surface patterned poly-D-lysine stimulates mast cell exocytosis detected as amperometric spikes by planar electrode arrays. Purification of HA protein and construction of baculovirus expression system. SAXS profiles and reconstructions of HA and SARS proteins. Successful testing of laser trigger of conformational changes. We performed single molecule experiments using several distinct optical trapping techniques to elucidate the mechanism of nucleosome remodeling by the SWI/SNF and ACF chromatin remodeling enzymes. The DNA unzipping technique, developed by the Wang lab, was used to determine that both ACF and SWI/SNF are capable of displacing single nucleosomes along a DNA template, repositioning them while maintaining their canonical structure. We used AFM to define the structural features of PARP-1 that underlie its ability to promote the localized compaction of nucleosomes. Our results indicate that the DNA binding domain and the catalytic domain function together to form the minimal functional module of PARP-1 required for chromatin compaction. We developed the use of photo-activateable GFP (paGFP) and other photochromic proteins for use with MPM to determine the fate of histone proteins that package a gene that is inactive and which subsequently becomes transcriptionally activated. For example, we have tested fly lines that produce fusions of canonical histone H2B or variant histone H2Av with photoactivatable GFP (paGFP).

Impacts
The Nanoscale Cell Biology Program of the NBTC is aimed at approaching a mechanistic understanding of cellular function with nanoscale precision. To achieve this aim new nano- and microscale tools are developed and applied. Microfabricated devices are also developed to probe cellular function in highly parallel manner for proteomics and drug testing. The approaches include nanoparticles, molecular force measurements, small angle x-aray diffraction, carbon nanotubes, integrated chip design, nanofabricated devices and molecular design. All projects involve interdisciplinary teams with PIs, post-docs and students from many different disciplines supplemented by outside collaborations on the national and international level.

Publications

Lamb, J.S., S. Cornaby, K. Andresen, L.W. Kwok, H.Y. Park, X. Qiu, D.M. Smilgies, D.H. Bilderback and L. Pollack. 2007. Focusing capillary optics for use in solution small-angle x-ray scattering. J. Appl. Cryst., 40, 193.
Yao, J., K. Munson, W.W. Webb and J.T. Lis. 2006. Dynamics of Heat Shock Factor Association with Native Gene Loci in Living Cells. Nature 442(7106),1050-1053.
Deufel, C., S. Forth, C.R. Simmons, S. Dejgosha, and M.D. Wang. 2007. Nanofabricated quartz cylinders for angular optical trapping: torque detection during DNA supercoiling. Nature Methods 4:223-225.
Wacker D.A., Frizzell K.M., Zhang T., Kraus W.L. 2007. Regulation of chromatin structure and transcription by poly(ADP-ribose) polymerase-1: Possible targets for drug based therapies. Subcell. Biochem. 41:45-69.
Choi, J., Burns, A., Williams, R., Zhou, Z., Flesken-Nikitin, A., Zipfel, W., Wiesner, U. and Nikitin, A. 2007. Core-shell silica nanoparticles as fluorescent labels for nanomedicine. Journal of Biomedical Optics.
Burns, A., Ow, H., Wiesner, U. 2007. Fluorescent Core-Shell Silica Nanoparticles: Towards 'Lab on a Particle' Architectures for Nanobiotechnology. Chem. Soc. Rev. 35 (11)1028-1042.
Bandla, S., Gillis, K., Lindau, M. & Minch, B.A. 2007. Design of a CMOS Potentiostat Circuit for Electrochemical Detector Arrays. IEEE Transactions on Circuits & Systems I, 54: 736-744.
Finlay, J.A., Krishnan, S., Callow, M.E., Callow, J.A., Dong, R., Asgill, N., Wong, K., Kramer, E.J. and Ober, C.K. 2007. Settlement of Ulva Zoospores on Patterned Fluorinated and PEGylated Monolayer Surfaces. Langmuir.
Holowka, D., Sil, D., Torigoe, C. and Baird, B. 2007. Insights into IgE receptor signaling from structurally defined ligands. Immunological Reviews 217: 269-279.
Zhou, X., Moran-Mirabal, J.M., Craighead, H.G. and McEuen, P.L. 2007. Supported Lipid Bilayer/Carbon Nanotube Hybrids. Nature Nanotechnology 2, 185.
Walczak, W., Pipalia, N., Soni, M., Faruqi, A.F., Ralph, H., Maxfield, F.R. and Webb, B.L. 2006. Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays. Combinatorial Chemistry & High Throughput Screening 6:711-718.
Pipalia, N., Hao, M., Mukherjee, S., and Maxfield, F.R. 2007. Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect. Traffic 8:130-141.
Gong, L.-W., de Toledo, A. & Lindau, M. 2007. Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore. Nature Cell Biology 9: 915-922.

Progress 01/01/06 to 12/31/06

Outputs
Extension of the Core-shell ratiometric sensor concept to smaller 17nm diameter pH sensors. Injection of fluorescent nanoparticles into hippocampal neurons by single cell electroporation. Evidence for endo-exocytosis of fluorescent silica nanoparticles. Development and analysis of Ca+2 core-shell ratiometric sensors. Single pixel minimal potentiostat designed, fabricated, tested and characterized reveals fA noise. New design of hybrid chip to measure cellular exocytosis. Microelectrode arrays modified with a charged polymer brush enhance dopamine specificity. L-Glutamate oxidase modified electrodes show high sensitivity towards L-glutamate. Optimized conditions for synthesize addressable branched DNA. Y-Shaped DNP(3)-DNA ligands prepared for studies of cell activation. DNP(3)-DNA ligands found to bind with same affinity and cause clustering of IgE-receptors on cells, but stimulated signaling found to depend on length. DNA hydrogels developed and characterized. Demonstration of electrochemical sensing with carbon nanotube transistors. Development of methods for the reliable growth and derivatization of nanotubes. Creation and imaging of nanotube transistor-supported lipid bilayer hybrids. Measurement of the permeability of membrane-bound proteins at nanotube barrier. Demonstration of high co-transfection efficiency in reverse transfection. Demonstration of Tyr kinase activities of multiple src isoforms. Simultaneous detection of fusion pores and fluorescence using transparent microelectrodes. Tetracysteine-based FRET reporter of SNARE complex conformation constructed. Electrochemical detection of exocytosis after IgE-mediated stimulation of peritoneal mast cells.

Impacts
The Nanoscale Cell Biology Program of the NBTC is aimed at approaching a mechanistic understanding of cellular function with nanoscale precision. To achieve this aim new nano- and microscale tools are developed and applied. Microfabricated devices are also developed to probe cellular function in highly parallel manner for proteomics and drug testing. The approaches include nanoparticles, nanofabricated devices and molecular design. All projects involve interdisciplinary teams with PIs, post-docs and students from many different disciplines supplemented by outside collaborations on the national and international level.

Publications

Yao, J., D.R. Larson, H.D. Vishwasrao, W.R. Zipfel and W.W. Webb. 2005. Blinking and Non-Radiant Dark Fraction of Water-Soluble Quantum Dots in Aqueous Solution. PNAS 102(40), 14284-14289.
Burns, A., P. Sengupta, T. Zedayko, B. Baird and U. Wiesner. 2006. Core-Shell Fluorescent Silica Nanoparticles for Chemical Sensing: Moving towards Single Particle Laboratories. Small, 2(6),723-726.
Sun, G., C.J. Fecko, R.B. Nicewonger, W.W. Webb and T.B. Begley. 2006. DNA-Protein Cross-Linking: Model Systems for Pyrimidine-Aromatic Amino Acid Cross-Linking. Organic Letters 8(4),681-683.
Muldoon, L.L., P. Tratnyek, P.M. Jacobs, N.D. Doolittle, G.A. Christoforidis, J. Frank, M. Lindau, P.R. Lockman, S. Manninger, Y. Qiang, A.M. Spence, S.I. Stupp, M. Zhang and E.A. Neuwelt. 2006. Imaging and nanomedicine for diagnosis and therapy in the CNS: Report of the eleventh annual blood-brain barrier disruption consortium meeting. American Journal of Neuroradiology 27:715-721.
Webb, W.W. 2005. Impossible problems. Biotechniques 38(4), 515-515.
Dombeck, D.A., L. Sacconi, M. Blanchard-Desce and W.W. Webb. 2005. Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy. Journal of Neurophysiology 94, 3628-3636.
Sacconi, L., D.A. Dombeck and W.W. Webb. 2006. Overcoming photodamage in second-harmonic generation microscopy: Real-time optical recording of neuronal action potentials. PNAS 103(9), 3124-3129.
Rhoades, E., T.F. Ramlall, W.W. Webb and D. Eliezer. 2006. Quantitation of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy. Biophysical Journal 90(12),4692-4700.
Larson, D.R., J.A. Gosse, D. Holowka, B. Baird and W.W. Webb. 2005. Temporally Resolved Interactions Between Antigen-Stimulated IgeE Receptors and Lyn Kinase on Living Cells Revealed by Fluorescence Cross-correlation Spectroscopy. J. Cell Biology 171(3),527-536.
Korlach, J., C. Reichle, T. Muller, T. Schnelle and W.W. Webb. 2005. Trapping, deformation and rotation of giant unilamellar vesicles in octode dielectrophoretic field cages. Biophys J. 89,554-562.
Larson, D.R., M.C. Johnson, W.W. Webb and V.M. Vogt. 2005. Visualization of Retrovirus Budding with Correlated Light and Electron Microscopy. PNAS 102(43),15453-15458.
Kim, Y-R; Paik, H-J; Ober, C.K.; Coates, G.W.; Mark, S.S.; Ryan, T.E.; Batt, C.A. 2006. Real-time analysis of enzymatic surface-initiated polymerization using surface plasmon resonance (SPR). Macromolecular Bioscience 6(2),145-152.
Senaratne, W., Sengupta, P., Jakubek, V., Holowka, D., Ober, C.K. and Baird, B. 2006. Self-assembled monolayer functionalized surface arrays for investigating immune cell signaling. JACS 128(17),5594-5595.
Senaratne, W., Andruzzi, L. and Ober, C.K. 2005. Self-Assembled Monolayers and Polymer Brushes in Biotechnology: Current Applications and Future Perspectives. Biomacromolecules 6,2427-2448.
Zhou, X., Park, J-Y, Huang, S., Liu, J. and McEuen, P.L. 2005. Band Structure, Phonon Scattering and the Ultimate Performance of Single-Walled Carbon Nanotube Transistors. Physical Review Letters 95, 146805.
Larrimore, L., Nad, S., Zhou, X., Abruna, H. and McEuen, P.L. 2006. Probing Electrostatic Potentials in Solution with Carbon Nanotube Transistors. Nano Letters 6, 1329.
Liu, H., C. Reccius and H. Craighead. 2005. Single Electrospun Regioregular Poly (3-hexylthiophene) Nanofiber Field-Effect Transistor. Applied Physics Letters 87, 253106-1:253106-3.
Hafez, I., K. Kisler, K. Berberian, G. Dernick, V. Valero, M.G. Yong, H.G. Craighead and M. Lindau. 2006. Electrochemical imaging of fusion pore openings using electrochemical detector (ECD) arrays reveals slow diffusion of catecholamines near the cell surface. Proc. Natl. Acad. Sci. 102:13879-13884.
Pipalia, N.H., Huang, A., Ralph, H., Rujoi, M. and Maxfield, F.R. 2006. Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells. J. Lipid Res. 47:284-301.

Progress 01/01/05 to 12/31/05

Outputs
Publications in top ranked journals document many achievements in studying cellular and subcellular function on the nanoscale as well as the nanobiotechnological devices that were developed. Intracellular tracking of GluR4 using quantum dots revealed rapid movement with frequent stops and reversal of direction. However, time spectra of Quantum Dots in solution revealed totally dark fractions of QDot batches that ranged from 5 to 70 percent. Fluorescent silica core/shell CU Dots were developed into a sensor platform, by integrating environmentally sensitive fluorophores at the particle surface with a different fluorescent core (patent pending). Ratiometric concentration data such as pH can thus be obtained from single particles in confocal or TIRF microscopy. Acicular nanostructure antennae are developed to enhance locally electromagnetic fields of laser illumination in a 20 nanometer diameter focal volume. Designs were developed using computer modeling and initial experiments performed. In exocytosis properties of single fusion pores with 1-2 nm diameter are investigated by patch amperometry and it was demonstrated that electrochemical imaging of single fusion pore openings can be performed using microfabricated electrochemical detector arrays. A CMOS chip was developed capable of measuring currents with 1 pA sensitivity and 1 ms time resolution using pixel sizes of 15um. This design is suitable to measure single excocytotic events amperometrically and assess modulation of quantal size by various drugs. Polymer brush or self-assembled monolayers are used to improve electrode performance and to immobilize enzymes to detect other biological compounds. To investigate nanoscale cross-linking geometries of mast cell IgE receptors, Y-DNAs with dinitrophenyl (DNP) hapten groups at the ends are designed. Subtle differences were observed for different strand lengths when binding and stimulatory activity was investigated, indicating that the geometry of cross-linking may be elucidated with these constructs. Supported lipid bilayers formed over carbon nanotube field effect transistors (FETs) shift the voltage sensitivity of the FET. Reverse transfection arrays adapted to a high throughput format are developed. Programs were designed to print DNA constructs in 96-well microplates. Tyrosine kinase was measured by anti-phosphotyrosine immunostaining in cells transfected with 7 different v-src proteins in each well of a 96-well plate and the level of activity correlated with the known kinase activity of the various v-src proteins. The approaches and devices to study protein-protein interactions and fusion pore openings, single molecule and vesicle tracking using nanoparticles and nanosensors, geometric requirements for IgE receptor cross-linking, use of nanotube detectors, and high throughput screening using reverse transfection will be further developed and applied to study cell function on the nanoscale.

Impacts
In the Nanoscale Cell Biology Program of the NBTC several interdisciplinary teams employ nano- and microscale tools with the ultimate goal to achieve a mechanistic understanding of cellular function with nanoscale precision. A complementary goal of other teams in the program is to develop nano- and microfabricated devices that probe cellular function in highly parallel devices for proteomics and drug testing. The approaches include nanoparticles, nanofabricated devices and molecular design. They involve PIs, post-docs and students from many different disciplines and outside collaborations on the national and international level.

Publications

Ow, H., DR Larson, M Srivastava, BA Baird, WW Webb, U. Wiesner. 2005. Bright and Stable Core-Shell Fluorescent Silica Nanoparticles. Nano Lett., 5 (1), 113-117.
Baumgart, T., S. Das, W. W. Webb and J. T. Jenkins. 2005. Membrane elasticity in giant vesicles with fluid phase coexistence. Biophys J. 89(2), 1067-1080.
Dernick, G., L.-W.Gong, L. Tabares, G. Alvarez de Toledo & M. Lindau. 2005. Patch amperometry: high-resolution measurements of single vesicle fusion and release. Nature Methods 2:699-708.
Gong, L.W., G. Di Paolo, E. Diaz, G. Cestra, E. Diaz, M. Lindau, P. De Camilli and D. Toomre. 2005. Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion. Proc. Natl. Acad. Sci. 102:5204-5209.
Gong, L.-W., G. Di Paolo, E. Diaz, E. Diaz, M. Lindau, P. De Camilli and D. Toomre. 2005. PIP Kinase Type Ig Regulates Dynamics of Large Dense-Core Vesicle Fusion. Proceedings of the National Academy of Sciences of the United States of America, 102, no. 14: 5204.
Li, Y., Cu, Y.T.H. and Luo, D. 2005. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nature Biotechnology 23, 885-889 (01 Jul 2005).
Moran-Mirabal, J.M., Edel, J.B., Meyer, G.D., Throckmorton, D., Singh, A.K. and Craighead, H.G. 2005. Micrometer-Sized Supported Lipid Bilayer Arrays for Bacterial Toxin Binding Studies through Total Internal Reflection Fluorescence Microscopy. Biophysical Journal, 89, 296-305.
Levene, M.J., D.A. Dombeck, R.P. Molloy, K.A. Kasischke, R.M. Williams, W.R. Zipfel and W.W. Webb. 2004. In vivo multiphoton microscopy of deep brain tissue. Journal of Neurophysiology 91, 1908-1912.
Levene, M.J., D.A. Dombeck, R.M. Williams, J. Skoch, G.A. Hickey, K.A. Kasischke, R.P. Molloy, M. Ingelsson, E.A. Stern, J. Klucken, B.J. Backskai, W.R. Zipfel, B.T. Hyman and W.W. Webb. 2004. In vivo multiphoton microscopy of deep tissue with gradient index lenses. Photonics West 2004, San Jose, CA, Proceedings of SPIE.

Progress 01/01/04 to 12/31/04

Outputs
A large number of publications documents the wealth of information that was obtained on cellular and subcellular function as well as the nanobiotechnological devices that were developed. This includes publications in Nature and Science as well as patent disclosures. In addition, exciting new approaches have been developed that have not been published yet. Two-photon FRET measurements of labeled retrovirus Gag protein in living cells revealed interactions that are required for membrane targeting and budding. Total internal reflection FRET microscopy combined with electrochemical imaging of vesicle fusion will be employed in studies of exocytosis using labeled SNARE proteins. Intracellular injection of quantum dots by single cell electroporation (SCE) for intracellular tracking of glutamate receptors has been achieved and will be presented at the Neuroscience Meeting 2004. The submission has been among the 3% selected for a lay summary in the books for the public press. Development of CU Dots was continued, which have significant negative charge and may be superior for injection by SCE. Histamine release is stimulated by multivalent antigen cross-linking of mast cell surface IgE receptors. To investigate specific nanoscale cross-linking geometries, Y-DNA with 1-3 conjugated dinitrophenyl (DNP) hapten groups were designed. IgE binding affinity increases dramatically from DNP1 to DNP3-Y-DNA and multivalent versions are effective stimuli. With these tools geometric determinants of cross-linking will be characterized. Stimulation is thought to involve membrane rafts. In membranes with raft-like phases coupling of curvature, line tension and fluid phase coexistence was demonstrated and analyzed in a cover story publication in Nature. Many drugs, from L-Dopa used to treat Parkinsons disease to amphetamines like ecstasy, modulate the amount of catecholamines released from single vesicles. For high throughput drug testing an electrochemical pixel CMOS test chip with shared regulated cascode amplifier circuit was fabricated at MOSIS and test measurements performed. Such devices may be equipped with carbon nanotube transistors integrated with a micro/nanofluidic system. Preliminary results show electrical signals as individual molecules pass a nanotube detector. Novel surface chemistries were developed for reverse transfection arrays and the technology adapted to a high throughput format. In microspots printed with a Green Fluorescent Protein expression vector high transfection efficiency and the capacity to perform high throughput screening were demonstrated. From GFP we will proceed to screening of multiple target proteins.

Impacts
The main objectives of the program Nanoscale Cell Biology are development and application of micro-/nanofabricated tools to understand cellular function and to probe cellular function in highly parallel devices for proteomics and drug testing. Currently, a total of seven projects are part of this program. The topics investigated range from single molecule detection to regeneration of neurons using implantable electrode arrays. They involve PIs, post-docs and students from many different disciplines and outside collaborations on the national and international level.

Publications

Foquet, M., J. Korlach, W. R. Zipfel, W. W. Webb and H. G. Craighead. 2004. Focal Volume Confinement by Submicrometer-Sized Fluidic Channels. Analytical Chemistry 76(6), 1618-1626.
Larson, D. R., Y. M. Ma, V. Vogt and W. W. Webb. 2003. Direct Observation of Retrovirus Assembly with FRET, Fluorescence Correlation Spectroscopy, and Single Particle Tracking. Microscopy & Microanalysis 2003, San Antonio, TX, Microsc Microanal 9 (Suppl 2), 1146-1147.
Kissler, K., I. Hafez, A. Dias and M. Lindau. 2003. Simultaneous Electrochemical and Fluorescence Imaging of Single Exocytotic Events in Chromaffin Cells. Biophys. J., 84:207a.
Larson, D., Y. M. Ma, V. M. Vogt and W. W. Webb. 2003. Observation of Retrovirus Gag-Gag Interactions in vivo with Multiphoton FRET and Fluorescence Correlation Spectroscopy. Journal of Cell Biology 162(7), 1233-1244.
Hartmann, J., S. Scepek, I. Hafez and M. Lindau. 2003. Differential regulation of exocytotic fusion and granule- granule fusion in eosinophils by Ca2+ and GTP analogs. J. Biol. Chem., 278, 44929-44934.
Lindau, M. 2003. Synaptotagmin function illuminated. J.Gen.Physiol. 122: 251-254.
Hafez, I., A. Stolpe and M. Lindau. 2003. Compound exocytosis and cumulative fusion in eosinophils. J. Biol. Chem. 278: 44921-44928.
Mosharov, E.V., L.W. Gong, B. Khanna, D. Sulzer and M. Lindau. 2004. Intracellular Patch Electrochemistry: Regulation of Cytosolic Catecholamines in Chromaffin Cells Mosharov. J. Neurosci., 23:5835-5845.
Li, Y., Y.D. Tseng, S.Y. Kown, L. dEspaux, J.S. Bunch, P.L McEuen and D. Luo. 2004. Controlled assembly of dendrimer-like DNA. Nature Materials, 3, 38-42.
Mark, S.S., Sandhyarani, N., Zhu, C., Campagnolo, C., Batt C. 2004. ADendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface. Langmuir. 20:6808-17 2004
Webb, B.L., Diaz, B., Martin, G.S. and Lai, F. 2003. A Reporter System for Reverse Transfection Cell Arrays; J. Biomol. Screening 8:620-623.

Progress 01/01/03 to 12/31/03

Outputs
The project has led to a number of publications that demonstrate feasibility for all the approaches that are involved. For single molecule tracking high resolution has been demonstrated and the use of non-bleachable nanoparticles in living animals has been demonstrated. Electrochemical detectors were patterned on the surface of a microscope cover glass for simultaneous fluorescence imaging and electrochemical imaging of quantal release from chromaffin cells. Two publications partly supported by NBTC appeared, which provide insight into the regulation of vesicular transmitter content by drugs such as the L-Dopa (used in Parkinsons disease treatment) and reserpine (which inhibits transmitter uptake as do amphetamines). A first and second generation combined nanotube/microfluidics systems were designed to test the feasibility of single molecule detection. For this purpose submicron channels were etched directly underneath the nanotube such that DNA could flow past the CNT detector one at a time. Preliminary efforts have been made to bring the molecule very close to the CNT using dielectrophoretic trapping and also to functionalize the CNT with specific binding sites. In addition, a new process for depositing oriented nanofibers was developed using a scanned electrospinning approach. A software package was created with a graphic interface for sequence optimization and structure prediction. Several sequence design rules were empirically deduced. Several low energy structures were determined as potential candidates for future designs. The approaches for synthesizing Y-DNA were further explored. Previous work showed that the crayfish motor axons can recover functionality in a remarkably short time after experiencing the functionally and anatomically disruptive lesion. Prototypes of array electrodes bonded to polyimide, and preliminary electrical and mechanical measurements have proved to be quite promising. In short, polyimide appears to be a suitable substrate for nanofabricating complex but flexible electrode arrays.

Impacts
The projects in this program are directed towards understanding cellular function on nanoscale dimensions and to develop micro- and nanofabricated instrumentation to investigate cellular function. They also aim at the development of micro- and nanofabricated devices that perform highly parallel measurements of cellular nanoscale events for the purpose of drug testing and drug development.

Publications

Tabares, L., M. Lindau, and G. Alvarez De Toledo. 2003. Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry. Biochem Soc Trans. 31:837-41.
Lindau, M. and G. Alvarez de Toledo. 2003. The Fusion Pore. Biochim. Biophys. Acta, Mol. Cell Res. 1641: 167-173.
Larson, D.R., W.R. Zipfel, R.M. Williams, S.W. Clark, M.P. Bruchez, F.W. Wise and W.W. Webb. 2003. Water Soluble Quantum Dots for Miltiphoton Flourescence Imaging in Vivo. Science 300:1434-1436.
Dernick, G. Alvarez de Toledo, G. and Lindau, M. 2003. Exocytosis of Single Chromaffin Granules in Cell-free Inside-out Membrane Patches. Nature Cell Biol. 5:358-362.
Kissler, K., I. Hafez, A. Dias and M. Lindau. 2003. Simultaneous Electrochemical and Fluorescence Imaging of Single Exocytotic Events in Chromaffin Cells. Biophys. J. In press.
Lindau, M. et al. 2003. Compound exocytosis and cumulative fusion in eosinophils. J. Biol. Chem. In press.
Li, Y., Y.D. Tseng, S.Y. Kown, L. dEspaux, J.S. Bunch, P.L McEuen and D. Luo. 2003. Controlled assembly of dendrimer-like DNA. Nature Materials (accepted, pending revision).
Hartmann, J. S. Scepek, I. Hafez and M. Lindau. 2003. Differential regulation of exocytotic fusion and granule- granule fusion in eosinophils by Ca2+ and GTP analogs. J. Biol. Chem. In press.

Progress 01/01/02 to 12/31/02

Outputs
Investigations in the six individual projects include conformational changes of single proteins, utilization of biomolecular conformational changes for applications such as nanobiosensors, development of electrochemical biosensors, cell biological functions on the nanoscale, and processing of drugs in an organism as simulated with a nanofabricated chip. The program is unified by a very significant overlap in the technical as well as the biological aspects of the different projects and has strong connections to the other research programs of the NBTC. Five projects continued and one new project was initiated during the past year.

Impacts
The projects in this program focus on the development of methods and devices to analyze minute amounts of biological molecules.

Publications

Russell, R., I.S. Millett, M.W. Tate, L. W. Kwok, S.M. Gruner, S.G.J. Mochrie, S. Doniach, D. Herschlag and L. Pollack. 2002. Rapid Compaction During RNA Folding, Proc. Natl. Acad. Sci, USA, 99, p.4266-4271.
Dias, A.F., G. Dernick, V.Valero, M. G. Yong, C. D. James, H. G. Craighead and M. Lindau. 2002. An Electrochemical Detector Array to Study Cell Biology on the Nanoscale, Nanotechnology 13: 285-289.
Thompson, R.E., D.R. Larson and W.W. Webb. 2002. Precise Nanometer Localization Analysis for Individual Fluorescent Probes, Biophys J. 82(5), 2775-2783.
Viravaidya, K. and M. L. Shuler. 2002. The Effect of Various Substrates on Cell Attachment and Differentiation of 3T3-F442A Preadipocytes, Biotechnology Bioengineering 78:454-458.
Viravaidya, K. and M. L. Shuler. 2002. The Prediction of Naphthalene Bioaccumulation Using An Adipocyte Cell Line Model, Biotechnology Progress 18:174-181.
Sin, A., G.T. Baxter, M. L. Shuler 2001. Animal on a Chip: A Microscale Cell Culture Analog Device for Evaluating Toxicological and Pharmacological Profiles, Proceedings of SPIE - Microfluidics and BioMEMS, Vol. 4560, pp. 98-101, October 21-24, San Francisco, 2001.
Heikal, A.A., S. T. Hess, E. D. Sheets, and W. W. Webb. 2002. Mutation-Photophysics Relationships in Intrinsically Fluorescent Proteins, Femtochemistry and Femtobiology:Ultrafast dynamics in Molecular Science, A. Douhal and J. Santamaria (Editors), World Scientific, Singapore, pp 777-781.

Progress 01/01/01 to 12/31/01

Outputs
The projects in this program are developing different approaches for analysis of minute amounts of biological molecules to understand function at various levels of complexity. The phenomena presently investigated in the five individual projects include protein conformational changes, the use of such changes for applications such as nanobiosensors, the development of electrochemical biosensors, the investigation of cell biological functions on the nanoscale, and processing of drugs in the organism modeled on a nanofabricated chip. The program is unified by a very significant overlap in the technical as well as the biological aspects of the different projects and has strong connections to the other programs of the NBTC: Bioselective Surfaces - the development of electrochemical biosensors depends on appropriate surface chemistry and the growth of cells on the nanodevices will rely on strong interactions with that program. Molecular Templates - the local stimulation of cells from the immune system will be combined with the detection of secreted material (e.g. serotonin). The localization of enzymes on biosensors is closely related. Molecular Filtration - the fabrication of microchannel devices will provide means to develop several aspects involving the local application of soluble stimuli in Projects 2-4 and will be crucial for Project 2 which envisions multi-barreled micropipettes leading to the sealed membranes.

Impacts
(N/A)

Publications

Pollack, L., M. W. Tate, A.C. Finnefrock, C. Kalidas, S. Trotter, N.C. Darnton, L. Lurio, R.H. Austin, C. A. Batt, S.M. Gruner and S.G.J. Mochrie. 2001. Time Resolved Collapse of a Folding Protein Observed with Small Angle X-Ray Scattering, Phys. Rev. Lett. 86: 4962-4965.
James, C.D., R. Davis, M. Meyer, A. Turner, S. Turner, G. Withers, L. Kam, G. Banker, H. G. Craighead, M. Isaacson, J. N. Turner and W. Shain. 2000. Aligned Microcontact Printing of Micrometer-Scale Poly-L-Lysine Structures for Controlled Growth of Cultured Neurons on Planar Microelectrode Arrays, IEEE Transactions on Biomedical Engineering, 47, 17-21.

Progress 01/01/00 to 12/31/00

Outputs
Microfabricated Rapid Fluid Mixer for Protein Folding Experiments - Micro-flow cells that are compatible with the brilliant x-ray beams available at third generation x-ray sources like the Advanced Photon Source have been developed. Using these devices we were able to time resolve the initial stages of the collapse of proteins in the process of folding triggered by a sudden dilution of denaturing detergent using small angle x-ray scattering. Experiments on the protein B-lactoglobulin show that transition from the unfolded to the folded state occurs via collapse from unfolded to compact structures on the millisecond time scale. Using 'pink' beam, x-ray exposure times of tens of seconds provide good signal to noise. Since the protein flows through the microfabricated mixing devices at rates of only 10 Ul per minute, these short exposure times keep sample consumption and thus the amount of protein required at a very low level. Sensors for Detecting Neurotransmitters - A method has been developed to generate ca. 50 nm diameter liposomes as measured by dynamic light scattering. Surface plasmon resonance is being used to examine the ability of such liposomes to spontaneously form bilayers on hydrophobic surfaces. These surfaces are prepared by treating a gold surface with long chain alkylthiols, including linear C12, C16, and C18 primary thiols. Nanofabricated Detector Arrays to Probe Cellular Functions on the Nanoscale - In experiments on chromaffin cells releasing epinephrine and norepinephrine, it was demonstrated that exocytosis of single vesicles with dimensions of 100-300 nm could be clearly measured as discrete capacitance steps in recordings from cell attached patches and these experiments allowed the direct determination on the concentration of catecholamine in single vesicles. Very recently these experiments were extended to excised inside-out patches and it was shown that exocytosis and transmitter release cn thus also be studied in-vitro. Using Nanotechnology to Predict Efficacy and Toxicity of Drugs - A new microscale prototype has been fabricated and its microfluidics have been tested and verified. A microscale pump for this device has been constructed. This device incorporates 5 compartments: (1) a gas exchange module for exchange of O2 and CO2 that consist of an ultrathin silicon elastomer membrane, (2) a lung chamber, (3) an 'other tissue' chamber, (4) a liver chamber and (5) a fat chamber. The various types of tissue are modeled by incorporating cultured cells (2-5). Using appropriate culture conditions preadipocytes attached to a polylysine coated chamber made of silicon were successfully differentiated into mature fat cells. This compartment provides a site for bioaccumulation of some environmental pollutants, such as PCBs and other hydrophobic drugs or chemicals.

Impacts
(N/A)

Publications

No publications reported this period