Sharon Shields, a
chemist at Livermore’s BioSecurity and Nanosciences Laboratory
(BSNL), uses a time-of-flight mass spectrometer to identify the
proteins found in blood serum. These proteins are produced in response
to the presence of pathogens.
UNTIL recently, national security
was synonymous with guns, tanks, and planes. Increasingly, however,
the notion of security, especially homeland security, demands a
broader meaning that includes ways to quickly detect and identify
biological pathogens that might be unleashed by terrorists. Such
pathogens could threaten urban population centers, crops, and livestock.
Livermore’s BioSecurity and Nanosciences Laboratory (BSNL)
is proving itself a national asset in the fight against bioterrorism
by discovering new methods to detect, identify, image, and understand
pathogens such as viruses, bacteria, and their spores. The research
findings are also helping improve human health by providing a better
understanding of pathogens and molecular machines such as DNA and
proteins. In addition, BSNL researchers are contributing to the
Department of Energy’s Genomics:GTL Program, the follow-on
effort to the Human Genome Project. The goal of Genomics:GTL (formerly
called the Genomes to Life Program) is to understand the function
of proteins and how they form the machines that drive the cells.
Such information will help scientists better understand the complex
biochemical activity of microbes.
BSNL’s 62 researchers are drawn principally from Livermore’s
Chemistry and Materials Science Directorate, with significant contributions
from the Biology and Biotechnology Research Program (BBRP); Engineering;
Energy and Environment; Nonproliferation, Arms Control, and International
Security; and Physics and Advanced Technologies directorates. More
than half of the researchers are under 35 years old. “From
the start, we adopted a strategy of investing in young talent,
both from around the Laboratory and from scientists around the
nation who are just starting their careers,” says BSNL Director
Jim De Yoreo.
The center has attracted four Lawrence fellows, who are some of
the most sought after young Ph.D.s in the world. In addition, 27
students have worked with BSNL scientists over the last three years;
11 student employees are currently doing their thesis work at BSNL.
De Yoreo says the many young people create an environment
where scientists do not hesitate to try new approaches and seek
breakthroughs at the risk of failure.
Multidisciplinary research teams work at what De Yoreo terms the
intersection of biology, chemistry, and materials science. Principal
research areas are protein analysis and systems biology, bioaerosol
science, molecular recognition chemistry, physical and chemical
pathogen signatures (detection techniques), nanofabrication of
devices, and cellular- and molecular-scale measurements.
A Natural Synergy
in 1999 as the BioSecurity Support Laboratory, BSNL was reorganized
in 2003 to increase its focus on what De Yoreo calls
the “natural synergy” between nanotechnology and new
frontiers in biological research. BSNL researchers work to exploit
this synergy in three areas: sensing viruses, bacteria, and toxins;
fabricating materials from the bottom up; and understanding the
assembly and performance of protein machines and cellular systems.
takes its name from the nanometer, which is a billionth of a meter.
BSNL researchers work on the nanoscale, or single molecule
scale, to understand the organization of molecular complexes that
make up most spores, viruses, DNA, or proteins—a level that
provides unprecedented detail. (See S&TR, December
Science Gets to the Heart of Matter.)
At the nanoscale, experimental results can be viewed only with
the most powerful imaging techniques, such as atomic force microscopy
(AFM), confocal optical microscopy, and nano secondary-ion mass
spectrometry. The two microsopy techniques can even image and manipulate
single molecules, allowing researchers to study a molecule’s
structure and function. BSNL researchers’ emphasis on single
molecules differs greatly from that of traditional biological researchers,
who examine beakers full of material and infer the actions of individual
Although electron microscopes have greater resolution, specimens
must be frozen and covered with a metal film prior to imaging.
With the optical methods used at BSNL, researchers can probe live
cells to gain a much more realistic picture of their functioning
some of the research is computer simulation. For example, BSNL
scientist Andrew Quong used the Laboratory’s ALE3D code
to develop three-dimensional (3D) models that examine how epithelial
cells communicate with each other. The simulations show strong
agreement with experiments. (See S&TR, January/February
Cells in a New Way with Three-Dimensional Models.)
researchers are experts at synthesizing nanostructured materials
such as artificial membranes with nanometer-size pores, microfluidic
channels that guide the flow of single molecules for analysis,
surfaces with nanometer-scale chemical patterns, and chemical compounds
that recognize—or bind to—specific targets such as
toxin molecules. Synthesis methods include using cells as chemical
factories as well as traditional small-molecule techniques that
can produce synthetic high-affinity ligands, which bind
to pathogens and render them harmless. (See S&TR, June
2002, A Two-Pronged
Attack on Bioterrorism.)
The First Signs of Disease
One of the most powerful tools used by BSNL scientists is the mass
spectrometer, a device that measures the mass of individual molecules
to precisely identify them. The instrument is being used to understand
how cells respond when exposed to pathogens. The research is part
of a pathomics project funded by Livermore’s Laboratory Directed
Research and Development Program. The term pathomics was coined
by co-principal investigator Ken Turteltaub, a molecular biologist
in BBRP. It is the science of applying proteomics, the study of
proteins, to the discovery of certain proteins whose appearance
or increase in concentration indicates a particular pathogen is
BSNL chemist Henry Benner says the research team ultimately wants
to understand how individuals respond to pathogens, particularly
those that bioterrorists might use. Such an improved understanding
could lead to detection systems that identify an attack or disease
outbreak before the first symptoms appear. “Our goal is to
develop a technique that would analyze the proteins in a sample
of blood serum and quickly detect the presence of a pathogen long
before someone felt ill.”
project involves biologists, physicists, chemists, engineers, and
computer scientists and uses some of the most sensitive mass
spectrometers in the world. Says Benner, “This type of big,
integrated project would be difficult to duplicate anywhere else.”
researchers are focusing first on human and animal serum proteins
that are produced in response to vaccinia virus, the surrogate
for smallpox virus. They are also collaborating with other institutions
to determine how rodents can be used as model systems for human
biochemical changes. In addition, they are studying how cell cultures
can model the progression of a pathogen-caused disease.
and chemists Sharon Shields and Chris Bailey have developed a liquid
chromatography and mass spectrometry analysis system for
characterizing plasma proteins in serum. Because most intact proteins
are too large for the mass spectrometer to analyze, the team first
applies an enzyme to the serum, which cuts each protein into about
50 chunks per molecule. The liquid chromatograph then separates
and ionizes the protein chunks before they enter the mass spectrometer.
researchers use liquid chromatography to separate and ionize
chunks of protein in a sample of blood serum and then analyze
them with a mass spectrometer. The researchers hope to detect
a pattern of mass spectrometry peaks (inset) that correspond
to someone in the earliest stages of a particular bacterial
or viral attack and another pattern that corresponds to a healthy
individual. Each group of spots represents a single protein
Detecting 10,000 Proteins
notes that human blood serum can contain up to 10,000 different
proteins, many of which are unknown. “With mass spectrometry,
we analyze everything that’s in the serum. We really don’t
need to identify every protein, although we’d like to eventually
have that information. All we’re looking for is a complex
pattern—a series of mass spectrometry peaks—that corresponds
to someone who is in the earliest stages of bacterial or viral
attack and another pattern that corresponds to a healthy individual.”
Almost certainly, different pathogens will produce different patterns.
In this way, the researchers hope to accumulate a library of mass
spectrometry patterns that identify specific pathogens. In addition,
they must determine the extent to which various serum proteins
vary in both normal and diseased individuals.
goal for the research team is to discover one or a few proteins
that are a dependable signature for a pathogen’s
presence. This type of pathogen signature, based on proteins, differs
from more traditional DNA-based signatures developed by Livermore
researchers. (See S&TR, April 2004, On
the Front Lines of Biodefense.)
perfected, the BSNL mass spectrometry technique would be valuable
for detecting a bioterrorist attack, a natural outbreak of infectious
disease, and even types of cancer. Another potential application
is continual monitoring of the nation’s blood supply. Patients
receiving radiation treatment could also benefit from the technology
because different mass spectrometry patterns would indicate
damage to certain organs. A related project, led by scientists
in BBRP and Livermore’s Glenn T. Seaborg Institute, is using
biomass spectrometry of blood serum to determine whether someone
has been exposed to a dirty bomb—a crude nuclear device designed
to cause widespread dispersal of radioactive materials.
Seeing Pathogens at the Nanoscale
and characterizing proteins that reside on the surface of human
pathogens and that form their internal structures is critical
to understanding how pathogens cause disease. Such information
is also essential for developing vaccines and detectors for both
medicine and biodefense. However, despite decades of study, scientists
still have a poor understanding of many pathogens’ structural
properties. Common tools such as x-ray crystallography and electron
microscopy often cannot be used because of some pathogens’ large
size, heterogeneity, and lack of symmetry.
a result, BSNL scientists have turned to high-resolution AFM imaging
of intact and dissected pathogens and their internal structures.
AFM uses an ultrasharp tip to scan across a sample’s surface.
The resulting interactions between atoms on the surface of the
sample and those on the AFM tip are used to construct a high-resolution
image of the surface topography.
chemists Alexander Malkin, Marco Plomp, and others are using AFM
to image the proteins of intact human viruses and bacterial
spores. They are focusing on bioterrorist threat surrogates, including
the vaccinia virus, a laboratory model for smallpox virus. Vaccinia
virus is one of the largest and most complex human viruses. The
researchers are also studying several species and strains of innocuous
Bacillus spores to understand the spore structure and function
of B. anthracis, the agent of inhalation anthrax.
more than eight years, the researchers studied the molecular-scale
mechanisms of crystallization for several types of proteins, viruses,
nucleic acids, and ribosomes. Then using AFM, they imaged the high-resolution
structure of these large ensembles of macromolecules. In work on
agricultural viruses, Malkin imaged for the first time the structure
of a small virus’s capsid—the protein shell covering
the viral genome—under physiological conditions. Image resolution
approached an unprecedented 2 nanometers and clearly revealed the
individual protein capsomeres that make up the capsid. Malkin has
demonstrated that viruses from different but closely related virus
families can be differentiated by AFM on the basis of their capsid
An atomic force microscope image
of a crystalline array of turnip yellow mosaic viruses
reveals the capsid structure, which can be resolved at
Viral and Spore Structures
scientists also imaged the Herpes Simplex Virus-1, one of the most
widespread human viruses. This work demonstrated for the first
time that the internal topography of viruses could be revealed
by AFM using chemicals and enzymes to degrade particles from the
outside to the inside, revealing each layer of the virus. Images
showed the intact virus, the underlying capsid and its capsomere
components, and finally, extrusion of viral DNA.
the response of pathogens to the environment is important for understanding
pathogen lifecycles and could help scientists
develop detection systems and decontamination procedures. The researchers
have visualized both hydrated and dehydrated samples of vaccinia
virus. AFM images show that the surface of the hydrated form bristles
with 30-nanometer protein protrusions that had never been previously
described for pox viruses. A membrane surrounds the viral core,
which consists of 16-nanometer-diameter filaments containing double-stranded
DNA. AFM visualization of intact viruses and their internal structures
allows researchers to model the complex internal architecture of
a large human virus.
and Plomp recently began visualizing bacterial spores. They resolved
the surface structure of a B. atrophaeus spore coat and
found that, in hydrated form, the top surface layer consists of
regular arrays of rodlike crystalline structures that fold when
dehydrated. In a study
of two other Bacillus species, they found striking differences
in spore structure. One
species (B. thuringiensis) had an outer spore coat
formed by a hexagonal honeycomb crystalline structure, whereas
the other (B. cereus)
had an outer rodlike structure and an underlying honeycomb structure.
researchers are also pioneering a new approach called AFM-based
immunolabeling. In this work, they use monoclonal antibodies synthesized
to bind to targeted viral and spore proteins. The research is conducted
in collaboration with scientists from the Oakland Children’s
Hospital Research Institute and the National Institute of Allergy
and Infectious Diseases.
mapping of surface proteins using monoclonal antibodies is a powerful
tool for examining the surface topology of pathogens.
Each bound monoclonal antibody defines one specific site on the
antigen’s surface. In this way, AFM can determine the location
of proteins on a pathogen’s surface—information that
will help scientists develop vaccines, detection systems, bioforensic
methods, and decontamination procedures.
images resolve the shape and surface features of a Bacillus
atrophaeus spore. (a) A hydrated spore is magnified in
(b), showing a surface consisting of arrays of rodlike structures
that fold when dehydrated (c).
New Kinds of Sensors
of BSNL’s most important research goals is developing
fast, sensitive, and accurate instruments to detect and identify
a wide range of pathogens. In the area of airborne pathogen detection,
Livermore researchers have worked with colleagues at the University
of California (UC) at Davis to develop the bioaerosol mass spectrometer
(BAMS). BAMS combines advanced laser desorption and ionization
techniques with mass spectrometry, and its sensitivity is two to
three times greater than that of other laser ionization techniques.
In addition, BAMS's response time is fast—it can identify
a single airborne particle in about 100 milliseconds. (See S&TR, September
Every Second Counts: Pathogen Identification in Less Than a Minute.)
researchers are working to shrink pathogen sensors to the size
of a semiconductor chip for bioterrorism and health-care applications.
BSNL physical chemist Alex Noy and graduate student Alex Artyukhin
are developing a new kind of biosensor that is based on a lipid-coated
nanotube, the first ever manufactured. In effect, the sensor is
a tiny but mechanically resilient “molecular wire” designed
to detect pore-forming bacterial toxins. These toxins, which are
large proteins, are secreted by the bacteria and insert themselves
into outer membranes of host cells. The 2-nanometer-wide holes
created by the proteins rupture the cell and kill it.
construct the biosensor, the researchers start with a carbon nanotube—a
rolled-up, single layer of graphite. If the tube is rolled in a
certain orientation, it becomes a semiconductor,
a material that allows electrons to flow under certain conditions.
Because of their electrical properties, semiconductors make excellent
the help of microfabrication expert Olgica Bakajin, the researchers
coat the nanotubes with a 5-nanometer-thick, dual-layer membrane
made of phospholipids. The result is an insulated wire that mimics
a cell membrane. “Our idea was to construct something like a shielded cable that
would be a good electrical detector,” says Artyukhin. With
electrodes attached to both ends of the nanotube and a voltage
applied, the minuscule sensor can detect pathogen toxins that typically
puncture a hole in a cell membrane or an artificial one. Any pathogen
toxins in the immediate environment would insert themselves into
the membrane. The punctures they create would allow ions to rush
in, touch the bare nanotube, and immediately change the voltage.
researchers recently added a polymer layer between the carbon nanotube
and the lipid bilayer, to confer electrical stability
and increase the tube’s diameter. Many proteins require more
room to function when they protrude through a membrane. The polymer
layer can be reapplied to make several layers, each 1 nanometer
thick. The researchers are experimenting with a five-layer nanotube.
perfected, the biosensor would be cheap to manufacture because
it could be fabricated by the thousands, much like semiconductor
chips in clean rooms. “Our biosensor is extremely simple,” says
Artyukhin. “It doesn’t need lasers or other sophisticated
equipment to function.” The device would be ubiquitous and
function as a “biological smoke detector.”
type of biosensor is based on a lipid-coated nanotube: (a)
longitudinal and (b) transverse sections. The sensor is
designed to detect bacterial toxins such as the protein a-hemolysin,
which pokes 2-nanometer-wide holes in cell membranes. The biosensor
starts with a rolled-up carbon nanotube that is coated with
a layer of polymer molecules and then a bilayer of phospholipids
that mimic a cell membrane. With electrodes attached to both
ends of the nanotube and a voltage applied, the minuscule sensor
can detect pathogen toxins that puncture a hole in the membrane.
A Close Look at Packaged DNA
is central to a research effort headed by Noy that examines how
DNA is packaged inside the cell nucleus so that it is a small
fraction of its uncoiled size. He cites famed baseball player Yogi
Berra, who once said, “You can observe a lot just by watching.”
mediate DNA packaging in all organisms,” says Noy.
Packaging protects DNA from physical damage and from free radicals,
which are extremely reactive molecules. Different species package
DNA differently. For example, mammalian sperm DNA is wrapped into
dense toroids, like a pile of rope. But the mechanisms of how different
proteins package DNA into distinct shapes are poorly understood.
is focusing on the role of AbF2, a protein in yeast mitochondria—the
cellular organelles that produce energy. AbF2’s role is to “scrunch” DNA
into a much more compact size. In collaboration with graduate student
Ray Friddle and researchers at UC Davis, Noy has acquired AFM images
of the DNA-AbF2 molecule. The images show
how the AbF2 binds to DNA and reveals the DNA–protein complex
making repeated bends of 102 degrees.
then wondered whether the bends were important to compaction,” Noy
says. To find the answer, he constructed a computer model of the
molecule, including the 102-degree bends. The model fit perfectly
with data obtained from the images and showed that the bending
indeed causes compaction. “When we put certain bends into
DNA, it naturally folds into a compact shape,” he explains.
important lesson from this research is that studying and imaging
a single molecule yields significant rewards. “When
we use the single molecule technique," says Noy, "we get both binding
information and the binding mechanism—the 102-degree bends.
Traditional methods give binding information and then force us
to deduce the
new piece of data about a protein’s structure and function
helps efforts to detect, identify, and treat disease, says Noy. “We
need to know what bad microbes do. The first rule is ‘Know
Progressive images from atomic force microscopy show the compaction
of DNA in yeast caused by a protein called AbF2.
Probing Inside a Cell
physicist Thomas Huser and his colleagues Chris Hollars, Chad Talley,
Anthony Esposito, and Steve Lane have developed optical
probes that enable nondestructive characterization and identification
of cells and their functions at the nanometer scale. These probes
use Raman scattering—the inelastic scattering of light by
molecular bonds. Raman scattering is one of the few optical techniques
that can identify a molecule by observing its distinct vibrational
fingerprints as the molecule scatters laser light. It also provides
a more accurate representation than common fluorescent labeling
of biomolecules. “With
fluorescent techniques, we have to introduce fluorescent molecules
to the biomolecules of interest,” says Huser. “Raman
is the intrinsic signal from the native material.”
Scientists are using gold particles
measuring 50 nanometers in diameter as cell pH sensors.
The particles are coated with molecules of mercaptobenzoic
acid, which changes its Raman spectrum in response to changes
in the chemical environment.
uses the confocal microscope, which is based on a fluorescence
microscope augmented with a pinhole that limits the volume being
probed and thus decreases background noise. The confocal microscope
efficiently collects the scattered light emitted from molecules
that have been excited by laser light. With this technique, Huser
can perform Raman spectroscopy on single cells and look for differences
one drawback is that Raman spectra are quite weak. To increase
the brightness and resolution of Raman-scattered light, Huser attached
nanometer-size gold crystals to molecules or cells. The method,
known as surface-enhanced Raman spectroscopy, increases the signal
by a factor of a quadrillion (1 x 1015) and
vastly improves the sensitivity of the measurements.
nanoparticles about 50 nanometers in diameter serve as tiny detectors
that “report” on the environment they’re
in through Raman scattering. The particles are covered with molecules
of mercaptobenzoic acid. Depending on pH, this molecule changes
its Raman spectrum. “In essence, we’ve created an intracellular
pH nanosensor that reacts to changes in its chemical environment,” says
Huser. If a cell undergoes changes as a result of external stimuli,
its pH will usually change in response.
possible application of this technique is studying the pH of cancer
cells. Although tumors tend to be more acidic than normal
tissue, the pH inside individual cancer cells is still mostly unknown. “We
want to compare the pH of cancer cells to the exterior cell environment
and to normal cells,” Huser says. “We’d also
like to see if cancer cell pH changes in response to different
chemotherapy agents.” Another possibility is to place the
nanoparticles just outside the cells to signal the presence of
certain proteins belonging to pathogens.
and his colleagues are applying their expertise in a Genomics:GTL
project, using nanoprobes to study how microbes clean up the environment
by digesting toxic molecules. “Microbiologists would like
to obtain much more detailed information about how some microbes
assimilate toxic materials,” he says.
and Hollars are also part of a new effort, headed by Bailey, to
a class of mysterious proteins called prions. When misfolded,
prions can attack healthy cells. Prions cause mad cow disease,
technically known as bovine spongiform encephalopathy, and humans
can contract a similar form, known as variant Creutzfeldt–Jacob
disease. In sheep, prions cause a degenerative disease called scarpie.
collaboration with the U.S. Department of Agriculture, the team
is developing techniques to look for prions in sheep blood serum.
One approach is to add fluorescent molecules that would bind to
any prions. The serum is then run through microfluidic channels
that are 100 micrometers wide, 500 micrometers long, and 0.5
micrometer deep. An optical microscope, sensitive to fluorescence,
would detect any prions.
chemist Chad Talley (left) and physicist Thomas Huser use a
fluorescent microscope with a wide field of view to image single
Meeting the Vision
any standard, says De Yoreo, BSNL is meeting its goals to become
a formidable resource for advancing national biosecurity, improving
human health, and understanding the molecular machinery of life.
Increasing funding from sponsors, a growing number of publications
in major peer-reviewed journals, and deepening scientific understanding
of pathogens and biomolecules all speak to its success. The next
few years should bring even greater understanding of life processes
at the nanoscale.
Key Words: atomic force microscopy (AFM), biodefense, BioSecurity
and Nanosciences Laboratory (BSNL), bioterrorism, DNA, Genomics:GTL
Program, nanoparticles, nanoscale, nanotube, pathogens, pathomics,
prions, proteomics, signatures, smallpox.
For further information contact Jim De Yoreo
(925) 423-4240 (firstname.lastname@example.org).
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