Sensing Evil

Worst-case scenarios don't come much uglier than the plume of an aerosolized biowarfare agent infiltrating a city. What happens then? Do alarms ring, evacuations and vaccinations begin? Or will anyone even know what the cloud contains? The answer could depend on efforts to improve molecular recognition systems that identify biowarfare agents in the air, water, or food. Problems of accuracy and efficiency that have dogged such technologies for decades are approaching resolution, but even then, the real test will remain: to expose a given threat with such speed that "detect-to-treat" becomes "detect-to-warn." The devices are known as biosensors, a term with many uses in the past. Nowadays, it refers to sensors that are capable of yielding extremely sensitive and specific measurements of contamination by incorporating a biological constituent, such as an antibody, enzyme, nucleic acid, even single or multiple cells. The constituent binds to a given analyte--in this case, a biowarfare agent--and a fluorescent dye is often used for detection. Simultaneous processing of multiple, different analytes has already been achieved, and within a year or two, biosensors might be miniaturized to hand-held size. Ultimately, air quality sensors might even be pinned on a lapel. But the technology is still far from perfected, and the economic argument for civilian applications of it might not be strong enough to warrant big spending increases in the private sector. Nor does next year's flurry of counterbioterrorism funding by the US government demonstrate faith in quick solutions to the shortcomings of biosensors. Under President George W. Bush 's proposed 2003 budget, the Department of Defense's (DoD) Chemical and Biological Defense Program will receive $933 million (US), an almost 85% increase. But its biosensor funding will rise by just 7%, to $34.3 million. The other DoD group that funds military biosensor research, the Defense Advanced Research Projects Agency (DARPA), will get a $433 million boost to $2.68 billion. Yet its biosensor dollars will decrease by $5 million, to $25 million. This failure to greatly escalate biosensor spending reflects the unlikelihood that sensors will soon be perfected to continuously monitor air quality and to identify, with few false alarms, any of the growing array of biowarfare agents that could occupy an approaching particulate cloud. Unfortunately, without such capabilities, even a visible cloud's threat will remain unconfirmed until the damage has begun. BOTTOM LINE BLUES While the military need for biosensing is independent of marketplace constraints, research and development in the private sector is hampered by low demand. Biochemist and medical diagnostician Alan Louie says the key to creating profitable biosensor products--which also could enhance biodefense--is a perceived need for timely information. Louie is head of applied biotechnology for Tiax (formerly the management and technology consultancy Arthur D. Little). He explains that biosensors could speed up detection of, say, cholesterol levels in a patient or pesticide levels in a crop, giving results in a matter of hours rather than days for conventional assays. But such information isn't time-critical, which provides little incentive for undertaking the high costs of development and regulatory compliance. By far the dominant application of biosensors to date is personal blood glucose monitoring for diabetes, which appeared in the late-1980s. "That has been the one success story," Louie remarks. "The question after that has been how to take the technology to the next level." Researchers have struggled since the 1960s with numerous obstacles that have prevented widespread use of biosensors. Among them are: insufficient sensitivity and selectivity in many cases; the need to assemble arrays of biosensors and integrate them with associated technologies such as electronics, fluidics, and separation; and the inherent instability of biological molecules. 1 For biodefense, an array of biosensors that use laser-induced fluorescence to detect biological aerosol in a cloud is state of the art in "fence-line monitoring." Such a system typically triggers an air-to-liquid sample collection. The aerosol is exposed to strips that turn color if positive, similar to a home pregnancy test. All this can be done within a half-hour, but DNA analysis usually is required for high-confidence results, which takes another half-hour. 2 Much DARPA research is aimed at speeding up the process. For example, one project would replace a fluid-based system with miniaturized mass spectrometry. Also, a biosensor based on multitarget probes is being developed, using a new range of antibodies and designer small molecules to bind specific agents, and cellular and tissue-based sensors. 3 Neal T. Sleszynski, vice president of research and development for Precision Research, a biotech in Kenosha, Wis., worries that much of the military research is inappropriate. "A lot of it is lab technology that people are trying to adapt to field use," says Sleszynski, whose company has been funded by the DoD and other federal agencies to develop biosensors. First, the nonportable lab equipment poses a strategic problem, he explains. Second, stand-off technology must be developed to indicate an approaching threat. "If you're a soldier deployed in the field, you have to have some warning that you've got to start sampling." One of the most advanced biodefense efforts to date is a commercially available fiber optic biosensor called RAPTOR that can detect and quantify explosives, toxins, bacteria, and viruses in water, soil, meat, and blood or other clinical fluids. Developed largely through the Naval Research Laboratory in Washington, DC, the proprietary rights are now held by Research International of Woodinville, Wash. Company president Chuck Jung says the device is heading into mass production through a collaboration with British Aerospace and Engineering. It weighs just over 12 pounds (5.6 kilograms) and is approximately the size of a car battery, but Jung estimates that a hand-held version will be available within about a year. RAPTOR can simultaneously process four analytes, typically producing results within seven to 12 minutes, while the hand-held model will be capable of 12 to 16 simultaneous assays. The device includes a cooling canister to keep the biological materials stable, even in desert conditions. This permits recycling of unbound antibodies, an important cost savings if reagent-based biosensors are ever to become widespread. "We can run up to 30 assays in the same coupon [optical probe container] and if nothing is found, we can reuse all the antibodies," Jung declares. Anthony P.F. Turner, a microbiologist who is editor-in-chief of Biosensors and Bioelectronics and head of the UK's Cranfield University, led a research group that developed a device similar to RAPTOR. However, their invention monitors antibody binding by using enzyme labels with electrochemical detection. This technology is being incorporated into the UK Ministry of Defense's development of an integrated biological detection system for battlefield use against biowarfare agents. It includes an air sampler coupled to the immunosensors for detecting airborne agents. A container that fits onto a four-ton military vehicle houses a suite complete with meteorological, communications, observation and crew rest facilities. Turner points out that stability of the molecules remains a key issue in battlefield conditions. "It may not be possible to maintain a supply chain for labile components, or it may be necessary to store devices for long periods along a supply route and to withstand climatic extremes," he notes. "We are working on synthetic receptors with greater stability. We have designed, for example, molecularly imprinted polymers for a range of toxins and some microorganisms. In addition to much greater stability, these receptors can be as selective as antibodies but have much larger sensitivity ranges." Another way to improve sensitivity and specificity is to use a nucleic acid-based methodology, but the extra time needed for amplification is compounded by a difficulty in determining whether DNA samples are alive or dead, says Tiax's Louie. He adds that any DNA-based method must be somehow sensitive to manufactured genetic hybrids that combine, say, the infectiousness of one constituent with the toxicity of another. Photo: © 2001 Research International Inc. THE RAPTOR: A fiber optic biosensor that can detect and quantify explosives, toxins, bacteria and viruses. THE REAGENTLESS OPTION Biosensors that don't need a reagent are another possibility. Ann E. Grow, vice president and chief technology officer of San Diego biotech startup Biopraxis, is developing a patented platform technology based on Raman spectroscopy. Called specific transduction, the method relies on optical fingerprinting. A chip that can carry thousands of immobilized biomolecules is incubated in a sample, and Raman spectroscopy is used to analyze the unique molecular structure of the crossreactions. It works for chemicals, biologicals, and explosives. The company now intends to develop a biochip for eight to 10 targets. "From my perspective, there are all sorts of problems in working with nonspecific transduction," Grow comments. She warns that with fluorescence-based technologies, "You get a lot of false positives and false negatives, and that has really hurt commercial prospects." Louie agrees that the public is generally intolerant of any equipment that gives false readings. He notes that development of a reagent-based "biosmoke alarm" to monitor air quality would require the ability to discriminate between biowarfare agents and nonpathogenic agents such as biopesticides, or opportunistic pathogens in humans. But he also expresses reservations about methods that employ spectroscopy. "There are inherent difficulties in physical methods of identifying biological materials," he explains. For example, the analyte must be grown in a medium until it is well defined, which takes time. It might be that only an increase in the public perception of a threat would create enough demand to spur private-sector funding aimed at transforming detect-to-treat into detect-to-warn. The question now is, will solutions arise from federal funding before the public has cause to feel afraid? Steve Bunk (sbunk@the-scientist.com) is a contributing editor. References 1. A.P.F. Turner, "Biosensors: Past, present, and future," Cranfield University, 1996. (www.cranfield.ac.uk/biotech) 2. B. Johnson, "Integrated systems for the detection, discrimination, and identification of biological agents," abstract, Pittcon 2002, March 17-22, 2002. 3. Defense Advanced Research Projects Agency, "Fiscal Year 2003 Budget Estimates," February 2002. (www.darpa.mil/body/pdf/FY03BudEst.pdf)

Sensing Evil

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Steve Bunk

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July 2002

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