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Environmental To o l
C h e s t
A N E W G E N E R A T I O N O F I M M U N O A S S AY S I S S I M P L I F Y I N G E N V I R O N M E N TA L T E S T I N G .
Laura Ruth
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hen Diane Blake of Tulane University
W
was on the shores of China’s Yellow
River during the summer of 1998, she would have found it useful to
have a hand-held, all-in-one immunoassay to test for cadmium. Un-
fortunately, there was no such kit in her “tool chest” of environmental testing methods, so she used the best test available: a more awkward enzyme-linked immunosorbent assay (ELISA) that had to be performed in an open microwell plate. Many environmental scientists find themselves in Blake’s position. They need convenient, low-cost assays that can be used anywhere. Over the past few years, that need has driven the development of a new generation of rapid, inexpensive, and portable multianalyte environmental immunoassays.
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However, environmental analysis techniques are not driven by market forces alone, according to researchers. Government policies in the United States and Europe are also playing a role. For example, the U.S. Environmental Protection Agency’s (EPA’s) adoption of performance-based measurement systems—which allow different methods to be approved for a single testing application—encourages the development of various environmental methods, says Katherine Alben at the New York State Department of Health and the State University of New York–Albany. The European Union (EU) went further, specifically mandating research on environmental water testing as part of the organization’s joint research program, says Tony Turner at Cranfield University (U.K.). The details are specified in an agreement known as Framework 5, which took effect two years ago and will last another two years. Similarly, the European Commission is sponsoring the European Thematic Network on Sensors for Pollution (known as SENSPOL) to coordinate research into the development of chemical sensors, biosensors, and biomimetic systems to help reduce water pollution. Although other environmental testing techniques such as LC, GC, and MS are available, immunoassays are often preferred. “A nonexpert can run hundreds of [immunoassay] samples onsite rather than send samples to a . . . central laboratory,” says Ray Clement of the Ontario Ministry of Environment (Canada). “[And,] the use of rapid on-site immunoassays, in-site remediation, and zone mapping can save thousands to millions of dollars by avoiding unnecessary digging.” Other environmental testing methods may use less-sensitive detectors or require large samples or extraction using organic solvents, Alben adds. Fran Ligler at the U.S. Naval Research Laboratory notes, “Attempts to commercialize surface plasmon resonance [SPR] and piezoelectric . . . methods for routine environmental sample [matrixes] of ‘mud, blood, and guts’ have failed.” Such advantages have spurred the commercialization of more than 90 different ELISA kits to detect hazardous chemicals in water, food, soil, or urine by a host of companies. Detection limits for pesticides, for example, range from 14 ppb of chlordane in soil to 80 µg/L of bioresmethrin in water.
New assays continue to enter the market. EPA’s Office of Solid Waste is currently reviewing the first commercial ELISA kit for detecting multiple dioxins. This kit, from CAPE Technologies, is the only one that has been validated at low picogram-per-gram levels using real soil and ash samples. It primarily detects the 17 toxic dioxin and furan congeners (out of 210 total). One feature of the kit is its ability to identify the congeners, which makes it possible to correlate the test results with toxic equivalency values—the toxicity-weighted concentrations that are specified by most dioxin regulations. Some of the new kits for detecting multiple analytes use fresh technologies such as the rapid automatic portable fluorometer assay (RAPTOR). The RAPTOR is an optical fiber-based immunoassay that uses fluorescence detection to screen for large molecules and pathogens (Anal. Chem. 2000, 72, 739 A–748 A). The method was discovered and initially developed by Ligler and colleagues (Environ. Sci. Technol. 1998, 32, 2461–2466). While running a “quick-and-dirty” experiment to look for a plastic with specific optical properties, the researchers discovered that antibodies would adhere to a particular type of polystyrene if the plastic was soaked in an antibody solution overnight. The researchers realized that they could make a biosensor by recreating this phenomenon inside an optical fiber. The current version of the test can run up to four samples simultaneously on one of the system’s four disposable optical waveguide sensors. It was released as a commercial kit by Research International, Inc., in the fall of 2000. Any type of ELISA can be adapted to the RAPTOR format, says Ligler. Another advantage is that the fiber probes and fluorescent reagent can be reused until a positive result is obtained, making RAPTOR cost-effective for screening environmental or clinical samples for rare chemicals or pathogens, she says. In addition, the test can be automated, which allows anyone to operate it in the laboratory or the field. Compared with HPLC, SPR, and other approaches, RAPTOR has better sensitivity (1–10 ppb) because fluorescence detection has such a low background, she adds. And fluorescence detection is faster and less cumbersome than radioimmunoassays or colorimetric ELISAs. Another new technology is the flow assay sensing and test-
Finding the right test In August 1998, a working group from the Methods and Data Comparability Board began planning to build the National Environmental Methods Index (NEMI). This database catalogs the many environmental testing methods used by organizations such as the AOAC (formerly the Association of Official Analytical Chemists), the American Society for Testing and Materials, the U.S. Department of Energy, the U.S. Environmental Protection Agency (EPA), and the U.S. Geological Survey. The index should make it much easier to quickly search for or compare methods by their sensitivity, accuracy, precision, and cost, says environmental chemist Lawrence Keith. “Comparison of [the] full methods is difficult because there are thousands of methods that are typically many [10–90] pages long, are written in different formats, and are meant to be instructions for carrying out analysis, not for determining if a particular method meets an individual’s needs,” he explains. Construction of the NEMI database in phases began in 1999 and has continued through 2001. The first phase of building NEMI involved inputting EPA organic chemical testing methods widely used in contract and government labs; alpha testing of the database followed. Phase II construction and beta testing are taking place now. Among the information added in this phase is a subset of 24 commercial immunoassay methods to detect 11 organic chemicals, including the pesticide chlordane and the herbicide and wood preservative pentachlorophenol, which were compiled by Katherine Alben of the New York State Department of Health and the State University of New York–Albany.
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ing (FAST) device, which uses displacement immunochemistry technology developed by Ligler and colleagues to test for small molecules. In the FAST device, a multianalyte sample passes through an antibody-coated permeable membrane that has lowmolecular-weight, fluorescently labeled molecules bound to it. The quantity of molecules that the sample competitively displaces from the membrane is directly proportional to the concentration of analyte in the sample. Like RAPTOR, FAST has good sensitivity (e.g., 1–10 ppb for the explosives 2,4,6-trinitrotoluene and cyclonite), is fast (~1 min), and is reusable. As many as 40 positive tests can be run on a single antibody-coated membrane without adding more reagent. Since the single-analyte version of the test was commercialized—again by Research International, Inc.—it has been used primarily by EPA to monitor cleanup operations at Superfund sites. A six-analyte version is still in development. Several research groups are working on still newer immunoassays—less-expensive tests that will solve the current technical problems, including false-positive results caused by cross-reactivity of antibodies and the need to test replicate samples. The researchers also hope to design additional multi-analyte tests that can be used in the field. For example, Blake and colleagues are adapting a commercial fluorometer to be used with designer antibodies to create an inexpensive ($5000), portable (~3–4 lbs.) immunosensor to detect metal and chemical hazards such as mercury, lead, and uranium (Anal. Chem. 2001, 73, 1889–1895). In contrast, kinetic phosphorescence, the only other method for detecting the different oxidation states of uranium, costs $65,000, is not portable, and has problems with metal specificity. Blake says that her experience when testing for cadmium at the Yellow River motivated the work. Walking onto an environmental site with a briefcase-sized, automated immunoassay system is the goal of Cynthia Bruckner-Lea and a team of chemists, biologists, and engineers at the Pacific Northwest National Laboratory. The device under development uses biodetection-enabling analyte delivery system (BEADS) technology, in which analytes specifically bind to the
surface of microbeads as a sample flows over them. Because the microbeads are used only once, there is no sensor fouling, and complex sample matrixes can be analyzed, says Bruckner-Lea. Other advantages include low cost, real-time analysis, and automated handling of multiple samples. Ligler and colleagues’ new project is to develop a capillarybased displacement flow immunosensor (Anal. Chem. 1997, 69, 1961–1964). A sample injected into the antibody-coated capillary competes with a fluorescently labeled antigen for the antibody, and the amount of displaced antigen is proportional to the analyte concentration. The technique is appealing, Ligler says, because it has parts-per-trillion sensitivity and is amenable to being transferred to a rugged, inexpensive chip format. Currently, the group’s research focuses on using a single plastic chip to test for multiple explosives and making a small prototype detector. In addition, the EU has funded a multinational project to develop organic-phase immunosensors for pesticides extracted from soil. “We are focusing on commercializing an . . . immunosensor that detects two contaminants commonly found in soil and water in Europe: 2,4-D [dichlorophenoxyacetic acid] and triazines,” says Turner. The researchers are also using combinatorial and imprinting techniques to mutate antibodies to make synthetic receptors—sometimes called “plastic antibodies”. In another project, Andrea Dankwardt and colleagues at Sension GmbH (Germany) are developing new test-strip enzymatic immunoassays to detect triazines, phenylurea herbicides, polyaromatic hydrocarbons, and polychlorinated biphenyls. As the range of immunoassays grows, identifying the correct method for a particular application becomes a more pressing problem. To address it, scientists are currently building the National Environmental Methods Index database to help users search and compare environmental testing techniques (see “Finding the right test”). Nevertheless, environmental scientists are happy to have the “problem” of being able to choose from a new wealth of testing methods. Laura Ruth is a freelance writer based in Los Angeles, CA. A U G U S T 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
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