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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).
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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).
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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).
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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).
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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).
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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)
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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).
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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]