Technology M Solutions Biosensors for detecting pathogens gree of specificity,” says M. Allen Northrup of Cepheid (Sunnyvale, CA), which manufactures diagnostic systems that combine microfluidics and microelectronics technology with DNA analysis. The first step in nucleic acid-based analysis is to extract, purify, and concentrate target DNA, or in some cases, RNA, from the target organism. A variety of kits are commercially available for sample processing, and most procedures can take 2–4 hours. The DNA is then amplified or copied millions of times, by a process such as polymerase chain reaction (PCR), which can take as much as 2 hours. One way to speed up both steps is to go to a microscale platform. The technology can process relatively large volumes in less than 5 min and perform amplification in less than 25 min, says Northrup. At the heart of Cepheid’s microdiagnostic systems is a module that performs PCR on a 100or 25-microliter sample volume with A portable biosensor called RAPTOR Plus for detecting pathogens. rapid heating and Antibody-coated, fiber-optic probes are secured inside disposable plastic cartridges in the center hatch. Up to four target pathogens cooling. Each modcan be detected simultaneously in only 10 min. ule can detect up to four pathogens siresistance and the emergence of new multaneously. In one configuration, pathogens from increased globaliza16 independent modules are contion make it increasingly important tained in a field-portable, batteryto monitor food and water supplies operated system with real-time for microbial contamination. fluorescence detection. “For detection of pathogens, the At the front end, Cepheid is develtrend is to go toward nucleic acidoping a variety of sample-processing based systems, because that actually systems because that tends to be the measures the genome or the genes of limiting aspect in field analysis, says the organism. It gives you a high deNorthrup. One system uses disposJOEL GOLDEN, NRL
“Sensors” was THE word at this year’s Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, better known as Pittcon, held in New Orleans, LA, March 4–9, 2001. Out of 1917 abstracts in the technical program, 144 contain the key word “sensor”. Nearly all of these can be applied to environmental monitoring. Several biosensors for detecting pathogens were featured, including miniaturized nucleic acid-based systems and fiber optic-based fluoroimmunoassays. Growing antibiotic
© 2001 American Chemical Society
able cartridges and processes one sample at a time, whereas another is a flow-through, continuously operating platform, capable of processing up to eight samples in parallel. The goal is to eventually interface the flow-through device with air or water samplers for continuous monitoring of industrial processes, food and water supplies, and hospital air, says Northrup. The U.S. Naval Research Laboratory (NRL) has developed an upgraded version of RAPTOR (Environ. Sci. Technol. 2000, 34 (13), 2845–2850), called RAPTOR Plus, an automated, portable fiber-optic biosensor capable of performing four fluorescent immunoassays simultaneously in only 3–10 min. The device is now commercialized and available through Research International, headquartered in Woodinville, WA. RAPTOR is well suited for monitoring Giardia lambilia, a parasitic protozoan that can cause severe intestinal infections, in water supplies, says NRL’s George Anderson, who played a lead role in its development. Limits of detection are about 5 ⳯ 104 Giardia cysts/milliliter. Assays for other pathogens, including Staphylococcal Enterotoxin B, ricin, Bacillus anthracis, and Francisella tularensis, have also been developed for RAPTOR. “The device can be used to detect any waterborne pathogen, as long as an antibody is available for that pathogen,” says Jon Tobelmann of Research International. In addition, RAPTOR has been investigated for use in monitoring biological warfare agents in aerosols collected from remotely piloted airplanes (Anal. Chem. 2000, 72, 739A–746A). “We’ve come a long way in the last decade,” says Anderson. “In 1991 we introduced a manual, fiber-optic biosensor with single assay capability that weighed 200 pounds,” he says. RAPTOR Plus is only about 12 pounds and runs on batteries.
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RAPTOR Plus relies on antibodies able oocysts are detected. Because immobilized onto fiber-optic wavemRNA is rapidly degraded, the reguides, which are mounted into dissearchers induce production of posable plastic cartridges called mRNA using heat shock. The heat “coupons”. If an antigen specific to the shock mRNA protein (hsp70) is then antibody on the probe is present in a extracted and amplified using nuclesample, it will bind to the probe, creic acid sequence-based amplificaating an antigen–antibody complex. tion, a technique that specifically When such a complex forms, fluoresamplifies RNA molecules. Once amcently labeled antibodies, which are plified, the RNA is hybridized with housed in a separate reservoir, are inspecific DNA probes and quantified troduced. These fluorescent antibodusing electrochemiluminescence deies bind to the antigen–antibody tection. complex, completing a two-step As little as five C. parvum oocysts process. By having a two-step or per sample can be detected by the “sandwich” immunoassay, selectivity new method, but Baeumner believes is enhanced, says Anderson. In addithat they can detect even fewer. It’s tion, the probes can be reused until a difficult to prove, however, because positive result is obtained, greatly recurrently there is no way to ensure ducing how much equipment is needed in the field. Two key words to describe the new RAPTOR Plus are smaller and simpler. “We re-did the fluidics to make it more accurate and reliable,” says Tobelmann. In addition, the coupons are smaller and easier to clean than in the previous version, so crosscontamination between samples is not a problem. “It’s a simple assay when the Biosensor cassette for detecting viable Cryptosporidium parvum in antibodies are availwater samples. able,” says Anderson. Researchers at Cornell University have developed a that reference standards contain vinovel biosensor for detecting another able C. parvum oocysts. “During well-known protozoan that causes inshipment of the standards, one testinal infections, Cryptosporidium oocyst could die and we won’t be parvum. The approach is much simable to detect it,” explains pler than the U.S. EPA-approved Baeumner. And once a sample is anmethods (1622 and 1623), which call alyzed, it can’t be reanalyzed by a for immunofluorescence microscopy. different technique. “Any water treatment plant that filters The mRNA procedure can be perits water can use our biosensor,” says formed in about 3 hours, but it is not Antje Baeumner, assistant professor of quite ready for use in the field. “The biotechnology at Cornell. In contrast, problem with Crypto is that you need she says “They probably would not to analyze 10 L of water,” says have an immunofluorescence microBaeumner. That 10 L of water needs scope and someone who is trained to to be filtered, “so you do need a lab or use it.” some filtering system,” she says. The method developed by Baeumner’s group has also develBaeumner and her colleagues (Anal. oped a similar approach for detecting Chem. 2001, 73, 1176–1180) detects Escherichia coli in water samples. So C. parvum messenger RNA (mRNA) far, they claim to have had no false molecules, which are only present in positives for either organism. organisms that are alive. So, only vi—BRITT E. ERICKSON
