years, experts in terrorism have been warning that a terrorist attack
with biological agents is not a question of if but when.
As recent events have proved, when is now.
For almost a decade, researchers
at Lawrence Livermore, working on the when-is-now premise, have
been developing systems that can rapidly detect and identify biological
agents, including pathogens such as anthrax and plague. (For more
background on Livermores research against bioterrorism, see
the Threat of Biological Weapons, and May
Bioterrorism.) Among such systems are the Handheld Advanced
Nucleic Acid Analyzer (HANAA) and the Autonomous Pathogen Detection
Although HANAA and APDS are
of different sizes and made for different situations, they have
a common purpose: to get results, fast. Lawrence Livermore biological
scientist Richard Langlois explains, There are any number
of laboratory tests available right now to analyze pathogens. They
all require getting a sample and then transporting it to a laboratory
for processing. Our systems use new instrumentation and methods
that provide faster and more timely results, on the spot. Faster
results mean the responders can act quickly and begin treatment
the size of a brick, the HANAA biodetection system can be held in
one hand and weighs less than a kilogram. The system was designed
for emergency response groups, such as firefighters and police,
who are often first on the scene at sites where bioterrorism may
have occurred. Each handheld system can test four samples at onceeither
the same test on four different samples or four different tests
on the same sample. HANAA can provide results in less than 30 minutes,
compared with the hours to days that regular laboratory tests typically
The process of detecting
and identifying whats in a sample works like this. The operator
prepares the samples by putting them in a liquid buffer and adding
chemicals. A tiny disposable plastic tube holding about 0.02 milliliter
of the prepared liquid is then inserted into the system. Many copies
of a samples DNA are needed to analyze it and identify its
makeup. HANAA uses a technique called the polymerase chain reaction
(PCR), which amplifies agent-specific DNA fragments to a detectable
level. In PCR, an aqueous sample is heated close to the boiling
point and then cooled many times (40 times in HANAA). Every time
the DNA is heated, the two intertwined strands of DNA unwind and
come apart. As the sample cools down, the DNA makes a copy of itself.
Thus, at the end of each cycle, the amount of DNA is doubled.
To detect the DNA in a sample, a synthesized DNA probe tagged with
a fluorescent dye is introduced into the sample before it is inserted
into the heater chamber. Each probe is designed to attach to a specific
organism, such as anthrax or plague. Thus, the operator must have
an idea of what substances might be involved. The system doesnt
test for all unknowns, says Langlois. A responder has
to decide what kinds of pathogens to test for ahead of time and
set up the system accordingly. If that organism is present
in the sample, the probe attaches to its DNA, which is then amplified
during the PCR process, releasing the fluorescent tag. HANAA measures
the samples fluorescence and the presence (or absence) of
the targeted organism.
One of the big breakthroughs
for the handheld system involved the design of a small silicon heater
chamber for the heating and cooling cycle, a concept developed at
Livermore by Allen Northrup, a former Laboratory scientist. The
commercial thermocyclers used for standard laboratory tests are
pretty big, ranging from the size of a microwave oven to a large
desk, notes Langlois. A typical large thermocycler takes
about 3 minutes to cycle through one heating and cooling cycle,
so a complete analysis requires 2 to 3 hours. In the HANAA
system, the thermal cycling process occurs in tiny silicon heater
chambers, micromachined by Livermores Center for Microtechnology.
Each chamber has integrated heaters, cooling surfaces, and windows
through which detection takes place. Because of the low thermal
mass and integrated nature of the chambers, they require little
power and can be heated and cooled more quickly than conventional
units. The mini-chambers typically cycle from about 55°C to
95°C and back to 55°C in about 30 seconds.
Using this technique, the
HANAA system could, in principle, detect as few as 10 individual
bacteria in one-hundredth of a milliliter in less than 30 minutes.
The system has the potential of saving many lives by saving timeanthrax,
for example, is highly treatable if detected early.
The Laboratory has a cooperative
research and development agreement for HANAA with Environmental
Technologies Group (ETG), a chemical and biological detector company
and subsidiary of Smiths Industries, based in Baltimore, Maryland.
ETG expects to have a commercial version of HANAA available early
this year. Ron Koopman, special projects manager for the Chemical
and Biological National Security Program at Livermore, notes that
HANAA is essentially ready to go at this critical juncture because
of the forward-thinking efforts begun in the previous decade. A
number of people recognized the vulnerability of the country to
bioterrorism a long time ago, he says. In 1996, although
bioterrorism seemed far away and was something we hoped would never
happen, the Laboratory and members of the defense community decided
to invest in the research, just in case. Thanks to that investment,
we now have something to put in the hands of people to protect us
all, something that can help during the current crisis and in the
Autonomous Pathogen Detection System is capable of continuous,
automated, 24-hour monitoring for pathogens, with results reported
every 30 minutes.
Bio Smoke Detector
Whereas HANAA can be hand-carried
to sites at which an attack is suspected to have happened, the APDS
is stationed in one place for continuous monitoring and is designed
to work much like a smoke detector, but for pathogens. When fully
developed, the APDS could be placed in a large area such as an airport,
a stadium, or a conference hall. The system will sample the air
around the clock and sound an alarm if pathogens are detected.
The important point
here is that the system would be fully automated, stresses
Langlois. The system will collect and prepare the samples,
do the analysis, and interpret the results, all without human assistance.
Livermore is testing the
second APDS prototype, which is about the size and shape of a lectern
or mailbox. The APDS-II consists of an aerosol collector, a sample
preparation subsystem, and two subsystems for detecting and analyzing
the samples: one based on PCR and the other based on flow cytometry,
which uses antibodies to identify pathogens. The final system
will double-test each sample to decrease the likelihood of false
positives and increase the reliability of identification,
aerosol collector, which was designed by Vern Bergman and Don Masquelier
at Livermore, gathers an air sample every 30 minutesthe length
of time it takes to complete a sample analysis. A built-in fan pulls
in the air, which passes through a glass tube containing water.
The water traps any particles in the air, and the resulting fluid
is pumped to the next stage for sample preparation and testing.
The flow-through PCR subsystem
for the APDS includes a Livermore-designed thermocyclermuch
like the thermocycler in HANAAalong with a sequential injection
analysis system. This analysis system performs all the necessary
PCR sample preparation functions, such as mixing the sample with
PCR reagents, delivers the resulting liquid sample to the thermocycler,
and decontaminates the thermocycler chamber and fluid delivery tubes
to prepare for the next run.
For the flow-cytometry subsystem,
small capture beads that are 5 micrometers in diameter
are coated with antibodies specific to the target pathogens. The
beads are color coded according to which antibodies they hold. Once
the pathogens attach to their respective antibodies, more antibodiesthose
labeled with a fluorescent dyeare added to the mix. A labeled
antibody will stick to its respective pathogen, creating a sort
of bead sandwichantibody, pathogen, and labeled antibody.
The beads flow one by one through a flow cytometer, which illuminates
each bead in turn with a laser beam. Any bead with labeled antibodies
will fluoresce. The system can then identify which agents are present,
depending on the color of the capture bead. Right now, we
use seven bead types to detect four agents simultaneously with controls,
says Langlois. The next step is to increase the number of detectable
pathogens to 20 or 30. Ultimately, the researchers expect to be
able to test for a hundred pathogens simultaneously in a single
Langlois and the APDS team
hope that, within the next year or two, the system will be ready
to put in place wherever needed. Ultimately, notes Langlois, numerous
detector systems could be linked together in a network connected
to an emergency response center to protect a complex of buildings
or a city.
from the flow-cytometry subsystem of the Autonomous Pathogen
Detection System (APDS) using seven color-coded capture
beads coated with antibodies specific to the target pathogens.
The pathogens attach to their respective antibodies and then
to more antibodies added to the sample mixture that are labeled
with fluorescent dyes. When the beads pass one by one through
the flow cytometers laser beam, any bead with labeled
antibodies will fluoresce, and the APDS identifies the pathogens
present, depending on the color of the capture bead.
Faster the Better
handheld, immediate testing to autonomous and continuous testing,
HANAA and APDS are two of many systems Livermore is developing to
help the nation fight bioterrorism. With HANAA, emergency responders
can get answers on the scene in less than half an hour. With APDS,
no human direction will be necessary, and the system will perform
on its own, completely self-contained, monitoring 24 hours a day,
7 days a week. What ties these approaches together is the
ability to analyze a sample quicklywithin 30 minutes or lessand
do it on site, concludes Langlois. Getting the answer
quickly is important. In the case of a biological attack, the sooner
we know what bioagent were dealing with, the sooner treatment
can start for those affected. Systems such as these have the potential
for saving many lives.
Key Words: anthrax,
Autonomous Pathogen Detection System (APDS), biodetectors, biological
warfare agents, bioterrorism, DNA analysis, flow cytometry, Handheld
Advanced Nucleic Acid Analyzer (HANAA), pathogens, polymerase chain
information contact Richard Langlois (925) 422-5616 (firstname.lastname@example.org).