ways to detect biological warfare agents is one of Lawrence Livermores
missions today. Detecting large quantities of a biological pathogen
is not difficult. The challenge is in detecting a few molecules
of a toxin or a few bacteria or viruses to provide the early warnings
of a biological attack.
Physicist Christine Orme
and colleagues in the Chemistry and Materials Science Directorate
are helping to understand some of the fundamental issues that underlie
biodetection as well as fulfilling other Laboratory goals. They
are performing research at minute scales in a field known as nanoscience,
which takes its name from nanometer, a billionth of a meter. The
team is examining, on an atom-by-atom and molecule-by-molecule basis,
the organization of materials on surfaces and learning how that
organization affects material properties. One of the keys
to working in nanoscience is controlling the surface and then being
able to detect what is there, says Orme.
At the nanoscale, experimental
results can be viewed only with the most powerful imaging tools.
The atomic force microscope (AFM) has been used since the mid 1980s
to produce topographic maps of nanostructures. Today, Ormes
colleagues are developing new microscopic techniques based on use
of the AFM that give even higher resolution and supply more than
just topographic data. They are also refining the spectroscopic
techniques that identify chemical bonds and supply fingerprints
The current research builds
on pioneering Livermore work in crystal growth and thin multilayers,
both of which depend on a keen understanding of material behavior
at the atomic level. Livermore has a long-standing effort in crystal
growth and characterization, born out of the need for large, ultrapure
crystals in Livermores lasers. Multilayersexceedingly
thin alternating layers of materialswere first demonstrated
more than 50 years ago. But improved fabrication technologies developed
by Livermores Troy Barbee have prompted their use as highly
reflective mirrors for telescopes as well as in a variety of optical
applications, including electron microprobes, scanning electron
microscopes, and particle beamlines in accelerators. (See S&TR,
the Sun's Secrets.)
Typical atomic force microscopy (AFM) tip and (b) nanotube tip.
With the smaller nanotube tip, it is possible to obtain much
more detailed information about a surface. AFM images of titanium
grains obtained using (c) a typical AFM tip and (d) a nanotube
In atomic force microscopy,
an extremely sharp tip senses the atomic shape of a sample while
a computer records the path of the tip and slowly builds up a three-dimensional
image. The AFM tip is positioned at the end of an extremely thin
cantilever beam and touches the sample with a force of only 1/10-millionth
of a gram, too weak to budge even one atom. As the tip is repelled
by or attracted to the sample surface, the cantilever beam deflects.
By imaging a larger or smaller area, researchers can vary the level
of magnification of an AFM image. The AFM can also be adapted to
sense a range of forces including attractive or repulsive interatomic
forces, electrostatic forces, and magnetic forces.
But even the sharp tip of
the AFM is sometimes not tiny enough for the small scale at which
the research team is working. Physical chemist Aleksandr Noy is
growing carbon nanotubes that can be used to replace the standard
AFM tip. The figure above compares a typical AFM tip and a carbon
nanotube tip. Carbon nanotubes are built of carbon hexagons that
are arrayed in a configuration resembling chicken wire. They are
1/50,000th of the width of a human hair but a hundred times stronger
than steel at one-sixth the weight. Noy can make many kinds of nanotubessingle
wall, multiwall, thick, thin, single isolated, or large arrays.
The smaller, lighter nanotube tip tracks the shape of an object
more accurately to provide more detailed information about its surface.
Noy used the nanotube-tipped
AFM to image the cucumber mosaic virus and reveal its structure
fairly clearly. AFM images contain less information than structures
revealed through x-ray diffraction techniques, but Noys image
was captured in minutes, whereas the same structure took over a
year to resolve from diffraction data. In principle, this
technology could be used to image a single virus, says Noy.
Emergency workers could compare its image with a computerized
database of known virus structures to identify it very quickly.
With the nanotube tip on
the AFM, a team led by Noy also obtained the first unambiguous visualization
of a DNA repair protein bound to DNA. By incorporating a synthetic
mutagenic molecule into DNA and tagging a repair protein with a
fluorochrome, they will be able to study the repair process in situ.
Another imaging technique
being used by physicist Thomas Huser and others is confocal microscopy.
It is based on a fluorescence microscope augmented with a pinhole
that limits the volume being probed to get rid of extraneous background
noise. Its beam can be focused to 500 nanometers. The
confocal microscope efficiently collects fluorescence emitted from
fluorescent molecules that have been excited by laser light. With
this spectroscopic technique, Huser has been able to detect single
The confocal microscope is
ideal for studying conjugated polymers, a new material that may
be used to fabricate the next generation of light-emitting diodes
(LEDs). Known as 2-methoxy, 5-(2¢-ethyl-hexyloxy)-p-phenylene-vinylene,
or MEH-PPV, the polymers are composed of a chain of benzene rings
that emit light when linked by electrodes to which voltage is applied.
The advantages of these polymers over the inorganic semiconducting
materials of todays LEDs are many: They are easier to process
on a large scale, they can be used to create ultrathin and flexible
devices, and their power consumption is lower. Last years
Nobel Prize in Chemistry was awarded for the development of conjugated
Huser has learned that the
physical configuration of the MEH-PPV molecules affects their fluorescence.
The photoluminescence of conjugated polymers depends strongly
on how they are shaped, says Huser. When they fold up into
a well-organized pattern in toluene, their shape enhances efficient
energy transfer within the molecule. As conjugated polymers begin
to be used as LEDs in electronics, some LED applications will take
advantage of the high-energy-transfer configuration while others
will benefit from the less ordered pattern for low-energy transfer.
In experiments, Huser exposed
MEH-PPV to two solvents, toluene and chloroform. In toluene, the
MEH-PPV molecules curl up tightly because, says Huser, They
dont like toluene. They try to avoid it. Spectrographic
data collected every 5 seconds show a slight flicker as the molecules
die off with exposure to oxygen and the light they emit shifts from
red to blue. In chloroform, the polymer spreads out. There is no
blue shift, the light spectrum is broader, and the light intensity
simply decays slowly with time.
Huser recently began experiments
with the confocal microscope to examine the dynamics of single molecules
of DNA. Fluorescent labeling of DNA, RNA, enzymes, and proteins
is common laboratory practice to illuminate the interactions and
functions of these important biomolecules.
At the same time, Noy has
built a whole new microscope system that combines the topographic
capabilities of the AFM and the spectroscopy of the confocal microscope.
He will be using this system to obtain even better information about
DNA repair as well as new information on how DNA is packaged.
Farms of carbon nanotubes and (b) a closeup of one
farm. Livermore is exploring the potential of such nanotube
arrays for detection applications.
of the first images of DNA repair proteins bound to DNA.
a Single Molecule
Another tool for
identifying molecular species is Raman spectroscopy, a form of light
scattering similar to fluorescence. Although Raman-scattered light
is much less intense than fluorescence, the technique is a powerful
analytical tool because the changes in wavelength of the weakly
scattered light are characteristic of the scattering material. Raman
spectroscopy can identify chemical bonds and obtain the unique fingerprint
of a molecule. Every molecule has a unique Raman spectrum, but not
every molecule fluoresces. Raman spectroscopy is one of the few
optical techniques that can identify a molecular species and determine
its chemical bonding by observing its distinct molecular vibrational
To increase the brightness
and thus the resolution of Raman-scattered light, Huser has introduced
nanometer-size gold crystals to the tip of a scanning probe microscope
in a technique known as surface-enhanced Raman spectroscopy. The
gold is negatively charged and attracts positively charged materials
such as amino acids to adhere to kinks in the crystals. Electron
density waves radiate from the corners of the gold crystals and
increase the Raman signal by a factor of a quadrillion. At the same
time, the scanning probe produces an image of the physical structure
of the sample. The combined data allow for identification of single
molecules. Unlike fluorescence, which fades with exposure to oxygen,
the increased energy from the gold particles persists.
able to characterize materials and chemical bonds at the level of
a single molecule is a whole new capability for Livermore,
says Huser. It is possible to perform Raman spectroscopy on single
DNA molecules or proteins and to look for differences between individual
cells. Using this technique, scientists also can detect and identify
the byproducts or precursors of chemical agents such as the nerve
gas sarin. This capability is important in the development of sensors
for chemical warfare agents.
Noy with the atomic forceconfocal optical microscope.
development of photoluminescence over time in the conjugated
polymer MEH-PPV, a material with multiple fluorophor segments
on a chain. (a) MEH-PPV exposed to chloroform forms an open,
irregular coil (see inset) that leads to luminescence from multiple
sites, hence the broad spectral emission. (b) MEH-PPV exposed
to toluene forms a tight coil (see inset) with strong overlap
between segments. In this conformation, only the segments with
the lowest transition energy emit light. Thus, the emission
is narrow and more structured. Once all the red fluorophors
are photodestructed, the segments with the next lowest energy
begin to emit light at slightly blue-shifted wavelengths.
Some nanoscience projects
require the careful design of surfaces to collect and organize atoms,
molecules, nanocrystals, colloids, cells, and spores. These surfaces
are known as templates or, as Noy describes them, landing
pads for toxins, proteins, and other biomolecules.
Livermore is exploring several
techniques for creating templates. Physicist Jim De Yoreo is developing
one method based on dip-pen nanolithography, which dips the tip
of the AFM into an inkwell of organic molecules to write
on an inorganic surface. As the tip moves across the surface, it
makes a pattern that has almost no topographic relief but exhibits
chemical contrast with the surrounding region. It is even possible
to create multiple ink patterns with this method. The feature size
is controlled by such factors as tip coverage, humidity, and contact
time with the substrate, or, in the case of lines, tip speed across
the substrate. Examples of patterns created using a gold-coated
mica surface for the substrate and 16-mercaptohexadecanoic acid
for the ink are shown in the figure below. This method has been
used to deposit patterns of antibodies that would attract toxins
and viruses, a first step in the development of nanostructured biosensors.
major area of research at Livermores Biology and Biotechnology
Research Program (BBRP) and elsewhere is in proteomics, the study
of proteins. Cells produce particular proteins either all the time
or as needed to prompt gene expression, that is, to turn a specific
part of the genetic code on or off. Without proteins, our DNA could
not operate properly. One of the best ways to examine the structure
of a protein is to crystallize it and then subject it to x rays
to obtain its unique diffraction pattern. During the crystallization
process, molecules come together and separate (in a process known
as nucleation) until a critical size is reached. Reaching that critical
size can take a long time, and sometimes it does not happen at all.
One goal of current proteomics work is to speed up the nucleation
process and make it more likely that proteins will crystallize.
Dip-pen lithography, using
a chemical that would prompt protein nucleation, is an option. But,
says Orme, the size scale is a challenge. Proteins are extremely
small, typically from 1 to 10 nanometers.
If we make the pens
lines smaller, they wont be visible, adds Noy. So he
and researchers in BBRP are developing a fluorescent ink for drawing
lines with the density of a single molecule. In initial tests, a
single-molecule line of the human chorionic gonadotropin (HCG) antibody
has been successfully drawn. The next step will be to attract the
Nanolaminates, the next generation
of multilayers, are also being explored as a way to accelerate the
nucleation and growth of ordered proteins. Nanolaminate structures
have been successfully synthesized with layers that are the same
small size as typical proteins. The alternating layers have different
surface charges, which prompt the proteins to adsorb in ordered
rows. In the example shown in the figure below,
a nanolaminate was dipped into a solution of the protein ATCase.
The nanolaminate was then removed, rinsed, air-dried, and imaged
with AFM using a carbon nanotube tip. The resulting extremely high
resolution of the image makes nonspherical proteins individually
distinguishable on silica stripes. An image of the same deposition
onto a homogeneous silica surface is very different, lacking any
linear order. This set of experiments was the first step in accelerating
nucleation and growing protein crystals that are suitable for x-ray
example of the benefit of surface-enhanced Raman spectroscopy.
(a) Confocal optical micrograph of 60-nanometer-diameter gold
nanocrystals loaded with just a few molecules of the laser dye
rhodamine 6G. (b) Surface-enhanced Raman spectrum of one of
the gold particles in (a) easily identifies the adsorbed rhodamine
by its characteristic Raman signature.
Schematic of dip-pen nanolithography technique. Friction force
images of (b) logos, (c) dots drawn on gold, and (d) colloid
particles adsorbed preferentially on the dots. Features are
composed of 16-mercaptohexadecanoic acid. The lines are 40 to
50 nanometers wide.
Nanoscience is finding another
application in the hands of Orme, De Yoreo, and colleagues whose
research on the growth of calcite crystals sheds new light on the
formation of bones, eggshells, and seashells.
The natural growth of organic
crystals is known as biomineralization. Biomimetics is the term
for mimicking natures building methods to make a synthetic
material. We can only learn to make better bones and teeth
if we first understand how the materials grow and interact with
biological molecules, says Orme. While there is a big
step between this fundamental research and synthesizing materials
that are truly similar to the real thing, we are part of the process
to create better materials that affect health.
calcium carbonate in the mineral form called calcite grows only
in a symmetrical, six-sided rhombohedral-shape crystal. But that
does not explain the intricate shapes found in nature, such as that
of seashells. Researchers have known for a long time that organic
molecules can influence the shape of a growing mineral crystal by
attaching themselves to it. But it took experiments at Livermore
to demonstrate the process in detail, showing how amino acids work
at the molecular level to change a growing crystal.
In the experiments, the team
added aspartate, one of the more abundant amino acids found in the
proteins of shellfish, to calcite crystals growing in solution.
Aspartate is typical of many amino acids in that it exhibits handedness,
or chirality. As the researchers monitored crystal development,
they found that the left-handed and right-handed form of the molecule
attached more strongly to opposite atomic steps. The results were
crystals that were mirror images of one another. The second figure
below illustrates how a chiral amino acid influences a growing calcite
crystal. By knowing which steps the amino acid interacted with and
using the symmetry relations of the crystal and the amino acids,
the team was able to predict the binding position of the amino acid
to the calcium carbonate step.
Comparable experiments are
just beginning on calcium phosphate, the material used by animals
to grow bones. Ultimately, experimental results may be put to myriad
uses, from potential laboratory growth of human and animal bones
to prevention of scale formation in pipes to the manufacture of
toothpasteany situation in which calcium-base crystals grow
naturally or are used.
A homogeneous silica substrate and (b) a nanolaminate of alumina
and silica were dipped into a solution of the protein ATCase.
Models show that (c) the deposition on the silica surface lacks
any linear order, but (d) proteins adsorb to the nanolaminate
in ordered rows, indicating the likelihood of growing ordered
protien crystals suitable for x-ray diffraction.
interaction of D-aspartic acid (D-Asp) with a calcite mineral
surface. (a) Model illustrating the binding of Asp to a calcite
step. (b) An atomic force microscope image of calcite steps
(0.32 nanometers high) in a solution containing D-Asp. The steps
of pure calcite are rhombohedral, but when an Asp-bearing solution
is flowed into the fluid cell, the two lower steps interact
with Asp and become curved. L-Asp binds more strongly to the
left step, and D-Asp binds more strongly to the right step.
These differences were used to deduce the binding motif. (c)
An electron microscopy image of an approximately 10-micrometer-diameter
calcite crystal nucleated on micropatterned, self-assembled
monolayers in the presence of D-Asp. The atomic step structure
in (b) is reflected in each of the three caps. (d) Crystals
nucleated in the presence of L-Asp are mirror images of those
nucleated with D-Asp.
force microscope image
(0.7 micrometers by 0.7 micrometers) of oxide grown on titanium
using a voltage applied between the tip of the atomic force
microscope and the substrate. (Image made by Livermore summer
student researchers R. Sivamani and E. Bochner.)
Science at Work
A nanostructured device is
also finding its way into tests for the Yucca Mountain project,
the nations candidate for a repository for long-term storage
of nuclear wastes. Tests of corrosion-resistant materials are being
developed that use patterns formed by writing with voltage
rather than with chemical inks. A voltage is applied between the
AFM tip and a metal or semiconductor substrate to grow oxide patterns
under the tip. In the figure above, an oxide greeting is written
into a titanium film. The dot on the i is made larger
and broader by applying a higher voltage. If the nanopatterns blur
or dissolve during testing, the change provides a very sensitive
indicator that the protective oxide film is changing.
This project is typical of
so much fundamental research performed at Livermore. Using funding
from the Laboratory Directed Research and Development (LDRD) Program,
the oxide templates were originally developed to nucleate calcium
phosphate minerals and to control protein deposition onto medical
implants. Now, the Yucca Mountain project is putting the template
to practical use. Much of the other work at Livermore to grow and
image nanostructures also started as basic research, funded either
by LDRD or by the Department of Energys Office of Basic Energy
Sciences, before finding a range of applicationsincluding
sensors that may someday be a lifesaver.
atomic force microscope (AFM), biological sensors, biomineralization,
carbon nanotubes, chemical sensors, confocal microscope, genomics,
nanolaminates, proteomics, surface-enhanced Raman spectroscopy.
information contact Christine Orme (925) 423-9509 (email@example.com).
ORME, a physicist in the Materials Science and Technology
Division of the Chemistry and Materials Science Directorate,
received a B.S. in physics from the University of California
at Berkeley. She joined the Laboratory as a postdoctoral fellow
after receiving her Ph.D. in physics from the University of
Michigan in 1995. Her background is in experimental physics
in the area of surface evolution and pattern formation during
the growth of thin films. In her thesis work, she combined
imaging with kinetic Monte Carlo simulations and continuum
modeling to deduce diffusional processes during vapor growth.
At Livermore, she uses this background to study crystal growth
from solution (rather than from vapor). She is particularly
interested in the area of biomineralization where organic
molecules substantially change the shape of inorganic crystals;
she wants to understand the formation of materials such as
shells, bones, and teeth. Recently, she has become interested
in the use of electrochemical driving forces to control electrodeposition
and corrosive processes, particularly in their application
to biomedical implants and corrosion-resistant industrial