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    Bionanoscience at LLNL

    Science in Support of National Objectives

    PLS conducts bionanocience research projects that apply nanoscience and nanotechnology to

    cutting-edge problems in biophysics, life, and materials science. We focus on developingnovel detection methods and platforms for a variety of national security interests ranging from

    nuclear nonproliferation to biosecurity applications. Our research is focused on developing:

    1. Probe microscopy techniques for biosciences and biosecurity applications2. Functional self-assembly in one-dimensional bionanosystems3. Carbon nanotube-based membranes for molecular-scale filtration and separation

    applications

    4. Ultrafast microfluidic mixing devices for protein-folding studies5.

    Ultrasensitive optical spectroscopy and microscopy

    1. Probe Microscopy Techniques for Biosciences and Biosecurity Applications

    PLS is developing probe microscopy techniques to assemble a nanotechnology toolbox for

    biosciences and biosecurity applications. We are using probe microscopy techniques to betterunderstand structure-function relationships and the life cycle of microbial and cellular

    systems. We are also studying the mechanism of biominerization and biologically-inspiredfabrication of nanostructures and nanodevices. Our work also entails probing and measuring

    chemical and biological interactions on a single molecule level with chemical force

    microscopy.

    Structure-Function Relationships and the Life Cycle of Microbial and Cellular Systems

    Elucidating the molecular structure and architecture of human pathogen surfaces is essentialto understanding mechanisms of pathogenesis, immune response, physicochemical

    interactions, and environmental resistance so that we can develop countermeasures againstbioterrorist agents. We are investigating the architecture, proteomic structure, and function of

    pathogens through a combination of high-resolution in vitro atomic force microscopy (AFM)and AFM-based immuno-labeling with threat-specific antibodies. This work provides a

    foundation for identifying structures of pathogens that could lead to the development of

    vaccines, detection and attribution technologies and improved decontamination systems.

    We have demonstrated, using various species of bacterial spores, strikingly different species-

    and formulation- dependent crystalline structures of the spore coat appear to be a consequence

    of crystallization mechanisms that regulate the assembly of the spore coat. We also mapped

    the proteomic structures of cell surfaces and revealed molecular-scale structural dynamics of

    single germination spores and a cell outgrowth during the germination process. These results

    could enable the development of targeted pathogen-specific therapeutic countermeasures,

    diagnostics, bioforensics, and vaccines for pathogen biodefense.

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    Left: High-resolution atomic force microscopy image of the rodlet layer covering the outer

    coat ofBacillus atrophaeus spore. The scale bar is 50 nm. Right: The development of a

    dormantBacillus atrophaeus spore into a live vegetative cell (grey) was captured with in

    vitro AFM.

    References

    Plomp, M., T. J. Leighton, K. E. Wheeler, H. D. Hill, and A. J. Malkin, In Vitro High-

    Resolution Structural Dynamics of Single Germinating Bacterial Spores,Proc. Natl. Acad.

    Sci. 104: 9644-9649 (2007).

    Plomp, M., T. J. Leighton, K. E. Wheeler, and A. J. Malkin, The High-Resolution

    Architecture and Structural Dynamics ofBacillus spores,Biophys. J. 88: 603-608 (2005).

    [top]

    Mechanism of Biominerization

    Understanding of the physical mechanisms by which biological systems use small molecules

    and macromolecules to control crystallization can provide insights into methods of

    synthesizing crystalline structures for applications across a wide range of technologies.

    Moreover, developing this understanding also presents a potential opportunity for creatingnew strategies towards synthesis of novel therapeutic agents for controlling pathogenic

    crystallization. For the past decade, we have been combining in situ AFM and molecular

    modeling to reveal the underlying principles, energetic factors, and stereochemical

    relationships that enable the biological control of inorganic molecular assembly of various

    model systems including calcium oxalate monohydrate (COM), a main constituent of human

    kidney stones. We obtained the first molecular-scale views of COM modification by two

    urinary constituentscitrate (figure below) and osteopontinand found that, while both

    molecules inhibit the growth kinetics and modify growth shape, they do so by attacking

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    different faces on the COM crystals. The results have significant implications for kidney stone

    disease therapy.

    Molecular-scale views of calcium oxalate monohydrate (COM) modification by citrate (image

    size = 6 micrometers). Left: Atomic force microscopy (AFM) image showing COM grows ondislocation hillocks. Center: Molecular modeling reveals that citrate interacts strongly with

    specific steps on existing crystal face by stereochemical match. Right: AFM image displaying

    altered morphology due to strong interaction between citrate and COM steps. The growth

    hillock has been changed from triangular to disc-like shape.

    References

    Qiu, S. R., A. Wierzbicki, C. A. Orme, A. M. Cody, J. R. Hoyer, G. H. Nancollas, S. Zepeda,

    and J. J. De Yoreo, Molecular Modulation of Calcium Oxalate Crystallization by

    Osteopontin and Citrate,Proc. Natl. Acad. Sci. 101, 1811-1815 (2004). (Cover Article)

    Qiu, S. R., A. Wierzbicki, E. A. Salter, S. Zepeda, C. A. Orme, J. R. Hoyer, G. H. Nancollas,

    A. M. Cody, and J. J. De Yoreo, Modulation of Calcium Oxalate Monohydrate

    Crystallization by Citrate through Selective Binding to Atomic Steps,J. Am. Chem. Soc. 127,

    9036-9044 (2005).

    [top]

    Biologically-Inspired Fabrication of Nanostructures and Nanodevices

    The use of macromolecular scaffolds for hierarchical organization of molecules and materials

    is a common strategy in living systems. For example, in proteins complexes, micrometer-scalestructures are generated from nanometer-scale building blocks possessing high-density

    functionality. We are mimicking this strategy by creating nanoscale chemical templates to

    direct the organization of engineered macromolecules and complexes, such as DNA, RNA,

    proteins, and viruses. These building blocks then serve as scaffolds for the assembly of

    materials and hierarchical organization of macromolecules such as metallic andsemiconductor nanocrystals or artificial-light harvesting complexes. These efforts not only

    provide well-controlled systems for developing a fundamental understanding of the physical

    principles governing the macromolecular assembly processesthey also offer exploratory

    routes to define a new technology for device fabrication of ultradense multicomponent

    architectures, such as signature-based, chemical and biological sensors that are effective

    against a wide range of known targets.

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    Atomic force

    microscopy

    images of

    biologically

    driven

    fabrication on

    nanostructures

    on chemicaltemplates.

    A.Functionalize

    d alkyl thiolmolecules

    i.e.,maleimide

    terminated

    (left) and

    nitrotriacetic

    acid (NTA)terminated

    (right) alkyl

    thiolline

    and dot

    patterns (line

    width = ca. 25

    nanometers).

    They are

    fabricated viananografting

    on atomically

    flat Ausubstrates.B. 2D

    assembly ofCowpea

    Mosaic Virus

    (CPMV) on

    atomically flat

    mica surfaces.

    C. 1D CPMV

    assembly fully

    covered on

    Ni-NTA linepatterns

    fabricated via

    a route similar

    to A. The

    figure showsthe single line

    of CPMVparticles.

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    D. Assembled

    RNA aptamer

    catalyzed by

    hexagonal Pd

    nanoplates

    assemble on

    2D chemical

    templateswhere RNA

    catalysts arecovalently

    immobilized.TEM inset

    image showssingle

    hexagonal Pd

    nanoplate.

    References

    Huang, Y., C. Y. Chiang, S. K. Lee, Y. Gao, E. L. Hu, J. J. De Yoreo, and A. M. Belcher,Programmable Assembly of Nanoarchitectures Using Genetically Engineered Viruses,

    Nano Lett.5, 1429-1434 (2005).

    Cheung, C. L., S.-W. Chung, A. Chatterji, T. Lin, J. E. Johnson, S. Hok, J. Perkins, and J. J.

    De Yoreo, Directed Self-Assembly of Virus Particles at Chemical Templates, J.A.C.S.128,

    10801-1807 (2006).

    [top]

    Chemical and Biological Interactions on a Single Molecule Level

    We are exploiting the nanoscale precision and manipulation capabilities of atomic force

    microscopes to measure, characterize, and map nanoscale interactions with chemical forcemicroscopy (CFM). CFM is a scanning probe microscopy technique that uses a tip of a

    scanning probe microscope modified with a specific chemical functionality to detect andprobe specific interactions with surface chemical groups. We are using CFM on a variety of

    systems ranging from probing interactions of chemical functional groups with single carbon

    nanotubes to measuring interactions between biological molecules, as well as between

    biological molecules and cell surfaces. Recent highlights include using CFM to quantify the

    strength of single and multiple bonds for interactions of multivalent cancer drugs with their

    targets, and measurement of interactions of a single functional group with a carbon nanotubesurface.

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    Left: Chemical force microscopy measurement of the affinity of a multivalent antibodyconstruct to the surface-immobilized targets (MUC1 peptides). Polymer tethers link

    individual antibody fragments to the AFM tip surface.Right, top: A representative force vs distance trace showing different parts of the

    measurement: Cantilever touches the sample surface in region I, pulls away from thesurface at region II, ruptures the antibodyprotein bond at III, and returns to the

    undeflected state at IV.Right, bottom: Dynamic force spectra measured for the rupture of one-, two-, and three-

    peptide-antibody bonds. These measurements provided the first-ever experimental prooffor the prediction of Markovian model of multivalent bond strength (solid lines).

    References

    Sulchek, T. A., R. W. Friddle, and A. Noy, Strength of Multiple Biological Bonds, Biophys.

    J. 90, 4686-4691 (2006).

    Sulchek, T. A., R. W. Friddle, K. Langry, E. Lau, H. Albrecht, T. V. Ratto, S.J. DeNardo, M.

    Colvin, and A. Noy, Dynamic Force Spectroscopy of Parallel Individual M ucin1AntibodyBonds,Proc. Natl. Acad. Sci. USA,102, 16638-16643 (2005).

    [top]

    2. Functional Self-Assembly in One Dimensional Bionanosystems

    One-dimensional nanoscale materials have unique properties that we can use to create

    functional devices and nanostructures. These nanostructures could combine material andelectronic properties of nanotubes and nanowires with the sophisticated functionality of

    biological machines. We are concentrating on using carbon nanotubes and silicon nanowiresas one-dimensional self-assembly scaffolds to create biomimetic supramolecular structures for

    potential use as advanced embedded nanoscale sensors and as a broad platform for detectionand translation of biological signals.

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    We have recently created a new bionano architecture, i.e., a one-dimensional lipid bilayer that

    consists of a functional continuous lipid membrane wrapped around an inorganic nanowire.

    Our current efforts are focused in the following areas: (1) we are continuing to study the

    fundamental processes that govern self-assembly in one-dimensional systems, specifically the

    role of substrate curvature in determining the fundamental properties of the self-assembled

    lipid and polymer layers; (2) we are working on integrating biological channels in nanotube

    and nanowire devices with the goal of creating a new generation of biomimetic interfaces for

    advanced detection technologies.

    Left: A scanning confocalmicroscopy image of a 1D

    bilayer assembled on asingle carbon nanotube.

    Right: A schematicrepresentation of a 1D

    bilayer structure.

    References

    Artyukhin, A. B., M. Stadermann, R. W. Friddle, P. Stroeve, O. Bakajin, and A. Noy,

    Controlled Electrostatic Gating of Carbon Nanotube FET Devices,Nano Lett.6, 2080-2085

    (2006).

    Huang, S.-C., A. B. Artyukhin, Y. Wang, J.-W. Ju, P. Stroeve, and A. Noy, Persistence

    Length Control of the Polyelectrolyte Layer-by-Layer Self-Assembly on Carbon Nanotubes.

    J. Am. Chem. Soc. 127, 14176-14177 (2005).

    [top]

    3. Development of Carbon Nanotube-Based Membrane for Filtration and

    Separation Applications

    Carbon nanotubes are an excellent platform

    for the fundamental studies of transportthrough channels commensurate with

    molecular size. Water transport throughcarbon nanotubes is also believed to be

    similar to transport in biological channelssuch as aquaporins.

    We have developed a process to

    microfabricate a membrane with sub-2-

    nanometer, aligned carbon nanotubes asideal atomically-smooth pores. The measured gas flow through carbon nanotubes in this

    membrane exceeds predictions of the Knudsen diffusion model by more than an order ofmagnitude. The measured water flow exceeded values calculated from continuum

    hydrodynamics models by more than three orders of magnitude and is comparable to flowrates extrapolated from molecular dynamics simulations and measured for aquaporins.

    Artist's

    vision of

    methane

    molecules

    traveling

    through a

    carbon

    nanotube.

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    We are currently investigating the fundamentals of mass transport through carbon nanotubes

    and exploring applications that exploit these unique nanofluidic phenomena. The extremely

    high permeabilities of these membranes, combined with their small pore size, may enable

    energy efficient filtration and eventually decrease the cost of water desalination and of

    separations of industrial gases and biomolecules.

    References

    Holt, J. K., H. G. Park, Y. Wang, M. Stadermann, A. B. Artyukhin, C. P. Grigoropoulos, A.Noy, and O. Bakajin, Fast Mass Transport through Sub-2nm Carbon Nanotubes,Science

    312, 1034-1037 (2006). (Cover Article)

    [top]

    4. Development of Ultrafast Microfluidic Mixing Devices for Protein Folding

    Studies

    We are developing microfluidic mixers for use in studying protein folding. These mixers

    allow us to measure protein-folding kinetics at fast timescales using a range of spectroscopic

    techniques: fluorescence resonance energy transfer (FRET), tryptophan fluorescence, and

    circular dichroism. By piecing together the complementary information that these techniques

    provide, we are trying to understand the conformational changes that occur during the first

    milliseconds of folding.

    Using mixers compatible with synchrotron radiation circular dichroism spectroscopy, westudied transiently populated collapsed unfolded proteins. The results indicate a -structure

    content of the collapsed unfolded state of about 20% compared to the folded protein. Thissuggests that collapse can induce secondary structure in an unfolded state without interfering

    with long-range distance distributions characteristic of a random coil, a situation previouslyfound only for highly expanded

    unfolded proteins.

    Using mixers made out of fused

    silica, we demonstrated that thesubmillisecond protein-folding

    process referred to as collapseactually consists of at least two

    separate processes. We observedthe ultraviolet fluorescence

    spectrum from naturally occurringtryptophans in three well-studied

    proteinscytochrome c,

    apomyoglobin, and lysozymeas afunction of time in a microfluidic

    mixer with a dead time of ~20 microseconds. We attributed the first process to hydrophobic

    collapse and the second process to the formation of the first native tertiary contacts.

    Recently designed mixers with a mixing time of 1 1 s with sample consumption on the

    order of femtomoles are currently being used for FRET and tryptophan fluorescence studies.

    References

    Schematic

    of the

    ultrafast

    mixer

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    Lapidus, L. J., S. Yao, K. S. McGarrity, D. E. Hertzog, E. Tubman, and O. Bakajin, Protein

    Hydrophobic Collapse and Early Folding Steps Observed in a Microfluidic Mixer,

    Biophysical Journal99, 218-224 (2007).

    Hoffmann, A., A. Kane, D. Nettels, D. E. Hertzog, P. Baumgrtel, J. Lengefeld , G. Reichardt,

    D. A. Horsley, R. Seckler, O. Bakajin, and B. Schuler, Mapping Protein Collapse with

    Single Molecule Fluorescence and Kinetic Synchrotron Radiation Circular Dichroism

    Spectroscopy,Proc. Nat. Acad. Sci.104,105-110 (2007).

    [top]

    5. Development of Ultrasensitive Optical Spectroscopy and Microscopy

    We are developing ultrasensitive optical spectroscopy and microscopyincluding single

    molecule fluorescence spectroscopy, surface-enhanced Raman spectroscopy, and micro-Raman spectroscopy of molecules, biological cells, and crystalsto enable the development

    of detailed molecular descriptions of cellular processes. There are three theme areas in ourresearch. First, we are studying the structure, function, interactions, and dynamics of the

    multiprotein machines involved in DNA replication and repair. Using solution-based, single-

    molecule spectroscopy, we have studied the motion of the polIII -subunit DNA sliding

    clamp (-clamp) on DNA and demonstrated that the clamp not only acts as a tether, but also

    a placeholder.

    Second, our goal is to obtain a quantitative description of entire biological networks of

    interacting molecules and to describe emergent properties of the systems. We are developing

    the capability to obtain quantitative information on the interactions and dynamics of proteins

    and study the pathogenicity of selected pathogens in real time and at the single cell level.

    Third, we are developing methods for measuring intracellular concentrations of a wide variety

    of analytes using surface-enhanced Raman scattering from functionalized metallic

    nanoparticles. Surface-enhanced Raman spectroscopy (SERS) allows sensitive detection ofchanges in the state of chemical groups attached to single nanoparticles. We have tested a

    nanoscale pH meter in a cell-free medium, measuring the pH of the solution immediately

    surrounding the nanoparticles.

    References

    Miller, A. E., A. J. Fischer, T. Laurence, C. W. Hollars, R. J. Saykally, J. C. Lagarias, and T.

    Huser, Single-Molecule Dynamics of Phytochrome-Bound Fluorophores Probed byFluorescence Correlation Spectroscopy,Proc. Natl. Acad. Sci. USA103, 11136-11141

    (2006).

    Contact: Alex Malkin [bio], 925-423-7817, [email protected]