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April 8, 2002
Volume 80, Number 14
CENEAR 80 14 pp. 26-28
ISSN 0009-2347
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Symposium looks at a variety of sensors for detecting biological and chemical weapons


SMART DUST Small silicon particles such as the ones shown here have been demonstrated for standoff detection of volatile organic compounds.
When news of mail tainted with Bacillus anthracis spores broke last October, no one knew how widespread the contamination would be. Although the extent turned out to be fairly limited, 22 people--five of whom died--were infected with either inhalation or cutaneous anthrax. The events drove home the fact that chemical and biological terrorism are not merely abstract notions. They are real and need to be dealt with.

One challenge for people dealing with the threat of terrorism is detecting the agents--sensitively, selectively, and rapidly. That topic was addressed at a daylong symposium held at Pittcon last month, where scientists discussed a variety of sensors being developed for biological and chemical weapons. David R. Walt, a chemistry professor at Tufts University, organized the symposium last spring--long before the events of Sept. 11, 2001, and the subsequent anthrax scare. "This symposium is not a case of jumping on the bandwagon," he told the audience.

Walt told C&EN that even if he had arranged the symposium in the wake of everything that happened last fall, the speaker lineup would probably have been the same. For each session, he selected a lead-off speaker to establish a context. "It was pretty tough to wean this program to as few people as there are," Walt told C&EN. "I looked for people I knew were going to give good talks and were going to cover some neat technology."

David R. Franz--vice president of the Chemical & Biological Defense Division at Southern Research Institute in Frederick, Md., and former commander of the U.S. Army Medical Research Institute of Infectious Diseases at Fort Detrick, Md.--set the stage for the other talks in the session that focused on sensors for biological weapons.

Franz said that in 1996, when he was still in the Army, the Department of Defense was "the only game in town" looking at chemical and biological weapons. At that time, Defense was looking for ways to protect U.S. troops but didn't really see a need to make a major investment in civilian defense. That's not true anymore. Billions of dollars for civilian defense are being requested for the 2003 budget. The money will be spent on a threat from which five people died, Franz pointed out. "That says something about the unknown nature of the threat," he said.

Franz sees biological and chemical weapons as very different beasts. He described chemical weapons as a "hazmat problem" and biological weapons as a "public health problem."

An important consideration, Franz said, is whether the goal is to be able to detect agents in order to warn before an attack or to be able to detect in order to treat after an attack. Detecting to warn is difficult because of the sensitivity required.

Franz recommended that any investment be intended to serve dual purposes. "Think public health," he said, "because we may never have another biological event." Discoveries and technologies funded as a defense against terrorism can help in the general public health arena.

Bernadette Johnson of the Massachusetts Institute of Technology's Lincoln Laboratory described the need for integrated systems, that is, systems with more than one type of sensor. She said the detection requirements differ depending on the goal--deterrence, protection, treatment, or decontamination.

8014paired_array 8014paired_array
BEFORE AND AFTER After being exposed to active substances, the chromatophores in the array (left) change their size (right).
is going to provide sufficient information to protect, it must provide a low rate of false negatives and false positives, Johnson said. In addition, it must be able to accurately determine the agent and the source. A number of difficulties complicate the detection of biological agents, she pointed out. First, the particles are small aerosols. They can have nonspecific signatures, especially if they are encapsulated. Also, competition from the background can swamp the signal from any biological weapon.

Most detection systems involve staged methods, Johnson said. First a trigger sensor causes a sample to be collected for an identification assay. By that point, the opportunity to detect to warn is pretty much gone. In addition, Johnson said, "no one will trust a single assay," so a confirmatory, preferably orthogonal, or independent, assay must be included.

The trigger sensor should be broad spectrum, Johnson said, because discrimination is not essential at that point. However, she said, the trigger sensor should not trigger an alarm for nonbiological particles. It should be flexible so that it can operate in different environments and against different backgrounds. One example of a technology used in a trigger sensor is particle fluorescence. Two-channel fluorescence (ultraviolet and visible) can usually discriminate between dirt particles and bioagents, Johnson said.

Other speakers described specific technologies for bioagent detection. Raymond P. Mariella Jr., director of the Center for Microtechnology at Lawrence Livermore National Laboratory, talked about immunoassays and DNA assays for biological weapons detection. The Livermore team has been building a series of autonomous pathogen detection systems since 1995, based on immunoassays that run on a flow cytometer. The current version runs 10 simultaneous assays based on the Luminex flow cytometer, including assays for B. anthracis, Yersinia pestis (the bacterium that causes plague), and Francisella tularensis (tularemia), as well as simulants, and could be expanded to include even more assays.

To achieve a sensitivity of less than one particle per liter of air, the Livermore system uses a large aerosol collector that collects several hundred liters of air per minute. Because typical biological background levels are widely variable, the Livermore team views trigger sensors as a stand-alone resource that could rapidly warn against a massive release rather than as part of an integrated system, Mariella told C&EN.

Using silicon micromachining, the Livermore group made a battery-powered, handheld thermal cycler for real-time DNA analysis using the polymerase chain reaction. The performance of the small instrument is comparable to a conventional instrument, Mariella said. But the detection sensitivity using the micromachined thermal cycler has reached the lower limits of reliability because of the Poisson sampling statistics involved. So the Livermore team has developed a microfluidic purifier and concentrator. Ultimately, however, the size of the autonomous system will be limited by the aerosol collector, he said.

Philip N. McFadden, associate professor of biochemistry and biophysics at Oregon State University, described a cell-based biosensor that he calls the SOS (for stochastic optical signals) cytosensor. This sensor uses living cells known as chromatophores, generally taken from fish, to determine whether an active substance is present. These cells change their appearance in response to toxins. This alteration can involve changes in both the shape and color of the cells. "Some cells seem to be more sensitive to one type of toxin than another," McFadden told C&EN. "One particular type of cell can respond in many different ways depending on what the toxin is."

Currently, McFadden knows that these cells respond to toxins, but he doesn't really know why. "It's clear that a comprehensive understanding is down the road, but a practical use of the cells is here," he said. "We've been making the assumption that we can use the cells by detecting their changes as a practical method for detecting active substances. Then, in a few instances, we are investigating the mechanisms of precisely how the toxins affect the cells." McFadden told C&EN that his group is testing sensors made from both a single type of chromatophore and arrays of various chromatophores.

AN IMPORTANT POINT, McFadden told C&EN, is that his sensor is not intended to identify or even quantify compounds. "It can give information on quantity. It can give hints on composition, but its main purpose is as an alarm," he said. "My primary goal is to develop a broadly sensitive activity detector."

McFadden's group has developed three generations of sensors--dubbed Mercury, Gemini, and Apollo--using these chromatophores. Mercury is a lab-based instrument that is high throughput with multiple wells. It resembles an immunoassay plate reader, he said. The Gemini and Apollo systems, in contrast, are integrated, portable instruments. The cells are kept in a self-contained chamber of about a cubic inch. So far, they have not attempted to miniaturize the electronics.

McFadden finds that the cells respond more strongly to the blends of toxins that pathogenic organisms produce than to purified toxins. "Our view is that a living pathogenic organism produces a blend of toxins that in combination exert a powerful effect on the cell," he said. McFadden believes that these sensors can help unravel some of the complexities. "We're presently dissecting the various components of these pathogenic broths, and the cytosensor is a nearly ideal instrument for scoring whether one fraction or another has an active component in it," he said.

"MS can detect anything, but it can also detect everything. We need to separate the anything from the everything."
detect activity rather than structural information, which distinguishes them from other sensors, McFadden told C&EN. "The activity of a substance is how it affects a living system. The conventional way of determining activity is to expose an animal to a substance and look for symptoms of toxicity," McFadden said. "Our system is a surrogate for animal testing that is portable and operable by the type of personnel who are familiar with operating instruments."

So far, the sensors have been used primarily in the lab. McFadden has done some testing away from the lab but still in indoor arenas. "We know the instrument is transportable and will operate. There's no doubt that the hardware can be transported. The challenge is to transport and store living cells. That's where we've been investing our efforts" to make the instrument suitable for field use.

A more chemical approach to biological detection was described by Wayne Bryden of the Applied Physics Laboratory at Johns Hopkins University. He talked about using miniaturized time-of-flight mass spectrometry (MS; see page 34) for bioagent detection.

Bryden characterized a strength of MS as also a weakness. "MS can detect anything, but it can also detect everything. We need to separate the anything from the everything."

MS has several advantages as a sensor, Bryden said. MS is rapid--total time to detection is only five minutes, including preparation--and sensitive. MS is used to look at proteins and other high-mass biomarkers. Bryden showed several examples of what MS can do. For example, different Bacillus species were distinguished based on the spectra of spores. Bryden and coworkers are also working to incorporate tandem MS, which will provide more detailed structural information.

False alarm rates are still a problem to be dealt with, Bryden noted. A detector that takes a reading every five minutes and has a 1% false alarm rate will trigger three false alarms per day--an unacceptable level, Bryden said.

Turning to chemical weapons detection, Walt said in his introduction that an ideal detector would be able to measure everything, cost nothing, be easily manufactured, and be ubiquitous--an ambitious goal indeed. Cross-reactive sensors are necessary, Walt said, as well as what he called "smarter sensors" that would not so much identify a substance as answer the question "Is it bad"?

Michael J. Sailor, a chemistry and biochemistry professor at the University of California, San Diego, described his group's efforts in making sensors using porous silicon. The sensors are based on color changes that occur under different chemical conditions. One sensor incorporates a catalyst that hydrolyzes P–F bonds, a characteristic of the class of nerve agents that includes sarin and soman. When the P–F bond is hydrolyzed, hydrofluoric acid is produced, which etches the silicon surface and causes a change in the optical properties (C&EN, June 12, 2000, page 12).

Sailor also described sensors made from nanostructured "smart dust." "We take these films, and instead of putting them on chips, we break them up into really small dust particles. We structure the films so that they will return very specific wavelengths of light at very specific angles. These films turn colors based on what's in them." Using the particles, a helium-neon laser, and a telescope, Sailor and his colleagues were able to detect ethanol, acetone, and toluene vapor from a distance of 20 meters--the first step in using the smart dust for detection at a distance, or "standoff" detection.

VISIBLE CHANGE The right side of this porous silicon wafer has been exposed to HF, which etches the silicon and changes its optical properties.
More traditional biosensors, which use enzymes to detect, are also being developed for chemical agent detection. Ashok Mulchandani, a professor of chemical and environmental engineering at the University of California, Riverside, described the development of biosensors that incorporate the enzyme organophosphorus hydrolase, which catalyzes the hydrolysis of bonds between phosphorus and fluorine, oxygen, cyanide, or sulfur. The enzyme-catalyzed reactions are specific and significantly faster than chemical hydrolysis. The hydrolysis products can be detected using a number of transduction methods, including electrochemistry and optical spectroscopy.

The enzymatic biosensors can also be used to detect and identify organophosphate pesticides. These compounds, which are readily available commercially, are used as simulants for chemical warfare agents in experiments. However, because these compounds are so readily available and are also neurotoxic, Mulchandani noted that perhaps we should be more concerned about them being used as terrorist weapons than less accessible agents such as sarin or soman.

TO IMPROVE the specificity of the biosensors, Mulchandani and his coworkers are using directed evolution to mutate the enzymes. The mutants respond differently to various compounds. Arrays of these mutants can be combined with pattern recognition to distinguish between compounds. These mutations don't need to be near the enzyme's active site, Mulchandani said.

By combining organophosphorus hydrolase with microchip separations, one can also use the biosensors to identify chemical warfare agents and organophosphorus pesticides. Mulchandani and his coworkers are working on lab-on-a-chip technology that will detect both explosives and chemical nerve agents. They have received a grant from the Department of Justice to develop the technology.

Andrew McGill, head of the functional materials and devices section at the Naval Research Laboratory, also talked about pattern recognition. The sensors he described are based on sorption vapor detection. In these sensors, an analyte-selective coating is placed on a resonant transducer such as a surface acoustic wave device. Shifts in the device resonant frequency that occur when target compounds are sorbed to the polymers coating the surface allow the detection of vapors and gases. Arrays of devices coated with different sorptive polymers are needed to generate "nose" prints of gases. These patterns, when combined with pattern recognition techniques, allow identification of target analytes.

McGill and coworkers improved the performance of the devices by switching from polymers with oxygen in the backbone (which can adsorb water) to polycarbosilanes, with increasing densities of active sites for nerve agents. They enhanced the sensor performance by an order of magnitude simply by "tweaking" the backbone and the arrangement of the functional groups, McGill said.

A prototype of the chemical agent detector, called pCAD, has already been taken into the field, McGill said. The array responds on the millisecond timescale, reaches an equilibrated response in less than half a second, and returns to baseline quickly as well. The real-time detection limits of the device have been demonstrated to be at miosis concentration levels. Miosis is the first clinical effect that nerve agents cause, which results in a constriction of pupils at extremely low concentrations and could affect pilots. McGill and coworkers hope to shrink the pCAD from its current cigarette box size to a smaller matchbox-size detector, called the Beaglette, which could be used as a disposable device.

Array sensors are "dumb" without training, McGill pointed out. The interferences expected for a given application are an important part of that training. The range of potential interferences increases greatly when the application is homeland defense rather than military. "The answer," McGill said, "is not a single technology, but orthogonal technologies."

Walt reiterated that point in summing up the symposium. Developing detectors for terrorist weapons will require a multidisciplinary effort, he commented. "There are vexing problems still facing us, such as speed and sensitivity."

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