FEATURE
ENVIRONMENTAL
BIOSENSORS A STATUS REPORT KIM R. ROGERS, CLAIRE L. GERLACH
New instruments and methods being developed show promise for continuous, in situ monitoring of toxic compounds.
T
he environmental analytical community continues to search for portable analytical techniques that can give reliable, on-site results for a variety of matrices and a host of analytes. In the 1970s, portable gas chromatographs (i) were developed that brought smaller, more rugged versions of laboratory models to newly identified hazardous waste sites. In the late 1980s, analytical chemists tapped the rich resources of clinical laboratories and developed immunochemical techniques (2) for an ever-increasing list of environmental analytes. Recently, researchers have investigated biosensors (3, 4) and ancillary technologies for environmental applications, especially those that require continuous monitoring. Biosensors are beginning to move from the proof-of-concept stage to field testing and commercialization in the United States, Europe, and Japan. Several U.S. federal agencies are evaluating the technology for studies of ecological and human exposure. Biosensors have potential for continuous and in situ applications, such as downhole or perimeter groundwater surveillance, and they are suitable for a variety of matrices including soil extracts, groundwater, blood, and urine. Some biosensors can operate in high concentrations of organic solvents (e.g., methanol and acetonitrile) and can be used for in situ monitoring of contaminated organic media or process streams that contain mixed organic wastes. They can be constructed from a wide array of immunochemicals and even genetically engineered microorganisms, and they can be configured to be reversible. The potential for environmental applications lies in the ability of biosensors to measure the interaction of pollutants with biological systems through a biomolecular recognition capability. A biosensor is made from a biological sensing element attached to a signal transducer. The sensing element can be enzymes, antibodies (as in immunosensors), DNA, or microorganisms; and the transducer may be electrochemical, optical, or acoustic (Figure 1). Electrochemical transducers measure changes in current or voltage; optical transducers measure changes in fluorescence, absorbance or reflectance; and acoustic transducers measure changes in frequency resulting from small changes in mass bound to their surface. The first biosensors were reported in the early 1960s and comprised enzymes immobilized to oxygen electrodes (5). Continued development of this kind of biosensor led to the commercializa-
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0013-936X/96/0930-486A$12.00/0 © 1996 American Chemical Society
A new biosensor to monitor explosives such as TNT and RDX has been developed by the U.S. Naval Research Laboratory. The continuous-flow immunosensor uses a clear plastic disposable immunoassay cartridge that is inserted into an analytical system (black box); measurements are converted into database format with associated signal acquisition and analysis software. (Photo courtesy Naval Research Laboratory/Research International.)
tion of various devices for such applications as the measurement of glucose in blood and the detection of glutamate, aspartame, sulfite, lactose, and ethanol in food products. Reports of enzyme-electrode biosensors continue to dominate the literature. In the environmental area, pioneering work on an antibodybased biosensor for benzo[a]pyrene was done in the 1980s at the U.S. Department of Energy's Oak Ridge National Laboratory. Advances in biochemistry, fiber optics Advances in biochemistry, molecular biology, and immunochemistry have expanded the range of biological recognition elements; and developments in fiber optics and microelectronics have expanded the capabilities of signal transducers. The durability, sensitivity, and low cost of signal transducers and the growing availability of enzymes, antibodies, and genetically engineered microorganisms that interact with environmental pollutants have contributed to the recent interest in applying biosensors to environmental monitoring. Biosensors are currently available for monitoring biochemical oxygen demand (BOD) and are in use at water treatment facilities in Europe and Japan (6). Recently, biosensors for 2,4,6-trinitrotoluene (TNT) andRDX(hexahydro-l,3,5-trinitro-l,3,5triazine) have been used at the U.S. Naval Research Laboratory. Many biosensors are on the brink of com-
mercialization, such as the Navy's continuous-flow immunosensor, which is expected to be licensed later this year. Other promising applications for environmental biosensors include groundwater monitoring, drinking-water analysis, and the rapid analysis of extracts of soils and sediments at hazardous waste sites. Biosensors are centrally located in a continuum of analytical technologies ranging from chemical sensors to bioanalytical assays (Figure 2). Although strict definitions are difficult in these often overlapping specialties, the International Union of Pure and Applied Chemistry (IUPAC) is defining biosensors as a subgroup of chemical sensors in which a biologically based mechanism is used for analyte detection (7). One characteristic of biosensors that distinguishes them from other bioanalytical methods, such as immunosassays and enzyme assays, is that the analyte tracers or catalytic products can be direcdy and instantaneously measured. For antibody-based biosensors, analyte tracers or unlabeled antibodies are directly detected in a single step, whereas for most immunoassays, an enzyme is attached to the analyte of interest and measurement of the binding of the antibody to the antigen is a multistep process. Continuous monitoring capability Another advantage that biosensors have over bioanalytical assays is that they can regenerate and reVOL.30, NO. 11, 1996/ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • 4 8 7 A
HMIhHI
Components of a biosensor A biosensor is built from a biological-sensing element attached to a physical transducer. Biosensor mechanisms that have been used for environmental applications include the use of enzymes, antibodies, and microorganisms. Theoretically, and to some extent demonstrated in practice, any of these biological recognition elements can be interfaced to electrochemical, optical, or acoustic signal transducers.
use the immobilized biological recognition element. For enzyme-based biosensors, an immobilized enzyme can be used for repeated assays rather than being discarded after each measurement; this feature allows these devices to be used for continuous or multiple assays. For antibody-based biosensors, chemical immobilization of the antibody to the signal transducer can be beneficial. In some cases, after the analyte has been measured (i.e., as a result of the antibody-analyte binding), the analyte can be stripped from the immobilized antibody and another assay done. In other cases, antibody-based biosensors have been shown to reversibly respond to chemical compounds within seconds or minutes. By contrast, immunoassays, including enzyme-linked immunosorbent assay (ELISA), are typically based on irreversible binding and are thus used only once and discarded. Because biosensors are relatively small, they can 4 8 8 A • VOL. 30, NO. 11, 1996/ ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
be used separately or as modular detectors in larger systems. For example, these devices can be used in flow injection analysis formats, as detectors for liquid chromatographic systems, or as stand-alone sensors at the end of an optical fiber or electrical cable (8,9). By optimizing the biological assay with the most appropriate transducer, it is possible to detect extremely low concentrations (JO) of a wide array of compounds of environmental concern. Biosensor mechanisms are being investigated by various government agencies, including EPA's National Exposure Research Laboratory at die Characterization Research Division in Las Vegas. These mechanisms are biocatalytic, bioaffinity, or microorganism based. Biocatalytic biosensors are based on enzymes, whereas bioaffinity biosensors are primarily based on immunochemicals (antibodies). Other biosensors use genetically engineered microorganisms (GEMs). A variety of biosensors using these sens-
ing mechanisms are being investigated for specific uses (see box on next page). Optical biosensors were identified in 1995 by the IUPAC Commission V-4 as a "new topic of interest." The IUPAC Analytical Chemistry Division noted that these biosensors "combine the exquisite selectivity of molecular recognition of bioreceptors (e.g., antibody, enzyme, nucleic acid probes) and the exceptional sensitivity of spectrochemical detection technologies" for "environmental and biomedical applications" {11). A technology still in development Environmental applications of biosensors range in their development stages from proof of concept to commercial availability. They are, however, somewhat behind biosensors for clinical and food applications. Although several biosensor-related patents are filed each year (about 40 in the United States and 40 worldwide in 1995), relatively few biosensors have been commercialized among all potential application areas. All but three of the biosensors listed in the box are in the research stages. The exceptions are the TNT and RDX immunosensors and the BOD sensors. The TNT and RDX immunosensors have been field tested by the Naval Research Laboratory {12,13) as part of a joint project with EPA Region 10 at the Umatilla Army Depot Activity site in Oregon and the Naval Submarine Base in Bangor, Wash. This project consisted of a comparison study of several methods, including commercially available immunoassay test kits, colorimetric chemical test kits, a prototype fiberoptic biosensor, and a continuous-flow immunosensor {14). Although each evaluated field memod met the basic requirements for accuracy and reliability compared with EPA standard methods, each assay type had slighuy different characteristics. For example, the colorimetric chemical assays responded equally well to nitroaromatic co-contaminants, TNT and RDX, but the immunochemical-based methods (i.e., immunoassay test kits and biosensors) responded with differing cross-reactivities depending on their antibody source. Detection limits ranged from 0.07 ug/L for the immunoassay test kits to 0.9 ug/L for the chemical test kits to 20 ug/L for the biosensor methods. The colorimetric chemical test kits were more versatile in identifying related compounds. The test kits, in general. aDDeared to be the best
One of the many types of signal transducers that can be used in biosensor technology, this acoustic transducer measures small changes in mass bound to the sensor surface. The surface transverse wave device was developed by Hewlett Packard Laboratories.
bargain for short-term monitoring projects, and the biosensor and continuous-flow immunosensor systems were most cost-effective for long-term projects such as groundwater pump-and-treat systems. These projections were derived from cost per sample versus initial investment cost. Cost per assay for the kits ranged from $50 to $75 and $8 for the biosensors; however, startup costs ranged from about $3000 for the kits to $20,000 for the biosensors. In addition, die study {14) identified several significant issues related to the application of these field methods: matrix interferences (such as nitrates and humic substances), detection capabilities for secondary and breakdown products as well as primary pollutants, and generation of contaminated waste and disposables. BOD sensors have been field tested in Japan (6, 15) and Europe {16). The short response time and high sensitivity of these microorganism-based sensors make them desirable for atmospheric and water monitoring. Japanese studies indicate that a biosensor using immobilized Trichosporon cutaneum in combination with a dissolved oxygen electrode can be used to measure BOD values in industrial waste-
JMII:i-H
Biosensors: A chemical-biological hybrid Biosensors fall in the middle of a continuum of sensors characterized by analytical assays that use chemical and biological processes. Increasingly, analytical procedures are benefitting from the sensitivity of biological constituents such as enzymes and antibodies.
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Current biosensor applications Analyte
Biochemical mechanism
Pesticides 2,4-D Carbamates Herbicides Imidazolinones Meturon, propantl Organophosphates Parathion Triazines Organic compounds Alcohols Ammonia Benzo[a]pyrene BTEX Cyanide Formaldehyde (aqueous/vapor) Organonitriles Phenol (aqueous/vapor) Polychlorinated Biphenyls RDX TNT Metals (specific) Hg, Cu Zn Biological parameters Algae Bacterial identification/ enumeration Biological oxygen demand (BOD) Biomarkers (DNA adducts) Bioremediation efficiency Indoor fungal index Phosphates Potential carcinogens Sewage contamination Toxicity3
Immunosensor, GEM sensor Enzyme sensor (inhibition) Photosystem II (Inhibition) Immunosensor Genetically engineered microorganisms (GEM) sensor Enzyme sensor (inhibition) Immunosensor Immunosensor Enzyme electrode Enzyme electrode, GEM sensor Immunosensor GEM sensor Enzyme electrode Enzyme electrode Enzyme electrode Enzyme electrode Immunosensor Immunosensor (continuous flow) Immunosensor (fiber optic, continuous flow) GEM sensor Enzyme sensor Chlorophyll fluorescence sensor Immunosensor, DNA sequence sensor GEM, bacterial, enzyme sensors Immunosensor GEM sensor Fungal growth sensor Enzyme electrode DNA intercalation sensor Marker enzyme electrodes GEM, yeast, bacterial sensors
•Toxicity of immobilized microorganisms to elements and compounds such as Hg, Ag, Cu, Zn, Pb, Cr, Co, phenols, and surfactants.
water in as little as 15 minutes. Traditional BOD measurements take five days. The BOD biosensor has been useful in process control applications for wastewater treatment in which rapid analyses are required. Finding "niche" applications Among the many possible combinations of biological recognition elements and transducers targeted to specific environmental compounds, certain combinations appear particularly promising in finding niche applications within the broad array of currently available field analytical methods. The measurement of phenolic and peroxide contaminants by using enzyme electrodes (17,18) and the detection of small molecules by evanescent wave fiber-optic technology (8,19) are two such applications. According to the Agency for Toxic Substances and Disease Registry, phenols and phenoxy acids are priority pol4 9 0 A • VOL.30, NO. 11, 1996/ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
lutants, based on the frequency of their occurrence at National Priority List sites, available toxicity data, and potential for human exposure (20). Phenolics are semivolatile aromatic hydrocarbons frequently seen in waste streams and wastewater from coal, gas, and petroleum industries. EPA's Las Vegas National Exposure Research Laboratory is supporting research efforts to determine the effectiveness of an enzyme biosensor to detect phenolic compounds in spiked and groundwater samples (9,17). This project uses an enzyme-based biosensor to monitor phenolic compounds in chromatographic effluents. Simple and potentially portable liquid chromatography systems are used to separate phenols, followed by an enzyme-electrode detector configuration. The biosensor is able to measure relative percentages of each phenolic compound present. The Naval Research Laboratory has developed a fiber-optic biosensor and a continuous-flow immunosensor that can be used to measure explosives in discrete samples or monitor process streams (19,21). The fiber-optic system is based on a competitive immunoassay performed on the fiber core of a long opticalfiber.The flow system is a displacement immunoassay with response measured by changes in the fluorescent signal in several minutes. Immunosensors such as these combine the advantages of conventional immunoassay methods with the option of obtaining real-time monitoring measurements with data integration capabilities. Laboratory confirmation is done with high-performance liquid chromatography. The Oak Ridge National Laboratory has an ongoing biosensor research and development program within its Centers for Manufacturing Technology. Among the biosensors being investigated are calorimetric microbiosensors for DNA and acetylcholinesterase (22), a DNA biosensor microchip suitable for clinical and environmental use (23), and an antibody-based biosensor (immunosensor) for monitoring benzo[a]pyrene (24) and its DNA adducts (25). Pesticide detection in Europe In addition to the research being done at U.S. regulatory agencies, private companies, and universities, considerable research on biosensors is being done in Europe (25, 26). An online pesticide analyzer based on competitive immunoassay techniques has been developed by the French firm, SERES. The instrument is being tested at Lyonnaise des Eaux, a leading French water utility. In other biosensor research in France, pesticide detection is being explored. Currently the most promising enzymebased biosensors for the detection of pesticides are those for organophosphates and carbamates and involve the inhibition of cholinesterases. Another European application of environmental biosensors is a series of herbicide sensors developed by researchers at the University of Karlruhe, Germany. These sensors use intact photosynthetic membranes as a source of photosystem II, which is inhibited by triazine and phenylurea herbicides. Researchers in Spain are refining a system that uses an enzymatic biosensor (confirmed by liquid chromatography) for monitoring organophosphate pesti-
cides (26). This biosensor system can be used to screen samples of river water for pesticides (organophosphates and their oxometabolites) and as an early warning system. Many environmental biosensors are ready to progress from the research bench to the field study stage. (A list of biotechnology market reports, including a compendium of publications about biosensors, is available on the Internet at http:// www.buscom.com/biotech.) However, as evidenced by the paucity of commercially available biosensors, overcoming the technical, regulatory, and market obstacles is not a trivial matter. These devices must compete with other, fairly well-established field analytical methods such as chemical sensors, immunoassays, and chemical test kits. In addition to the expected technical challenges—producing and packaging a durable device for field use, developing training programs, establishing regulatory acceptance procedures, adhering to quality assurance protocols—biosensors must offer new capabilities or significant improvements over existing methods to be successful in an increasingly competitive marketplace. Acknowledgment The U.S. Environmental Protection Agency, through its Office of Research and Development, funded the work involved in preparing this article. It has been subject to the agency's peer review and has been approved for publication. The U.S. government has the right to retain a nonexclusive, royalty-free license in and to any copyright covering this article. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)
(15) (16)
Budde, W. L.; Eichelberger, J. W. Anal. Chem. 1979, 51, 567A-574A. Van Emon, J. M.; Lopez-Avila, V. Anal. Chem. 1992, 64, 79A-87A. Rogers, K. R. Biosens. Bioelectron. 1995, 10, 533-41. Rogers, K. R.; Williams, L. R. Trends Anal. Chem. 1995, 14, 289-94. Clark, L. C, Jr.; Lions, C. Ann. Acad. Sci. 1962, 102, 29. Karube, I.; Nomora, Y; Arikawa, Y. Trends Anal. Chem. 1995, 14, 295-99. Bertie, J. E.; Vo-Dinh, T. Appl. Spectrosc. 1996, 50(4), 12A20A. Zhao, C. Q. et al. /. Agric. Food Chem. 1995, 43, 230815. Wang, J.; Chen, Q. Anal. Chim. Acta 1995, 312, 39-44. Bauer, C. G. et al. Anal. Chem. 1996, 68, 2453-58. Vo-Dinh, T. Oak Ridge National Laboratory, Oak Ridge, TN, personal communication, 1996. Shriver-Lake, L. C. et al. Anal. Chem. 1995, 67, 2431-35. Whelan, J. P. et al. Anal. Chem. 1992, 65, 3561-65. Craig, H. et al. In Proceedings of the Great Plains-Rocky Mountain Hazardous Substance Research Center/Waste Education and Research Consortium Joint Conference on the Environment, Albuquerque, NM, May 1996; Rocky Mountain Hazardous Substance Research Center: Manhattan, KS. Tanaka, H. et al. Water Sci. Tech., 1994, 30, 215-27. Szweda, R.; Renneberg, R. Biosens. Bioelectron., 1994, 9(1), ix-x.
Biosensors defined The field of biosensors combines elements of physical and biological sciences. Key terms and concepts in biosensor and related technologies include the following. Bioanalytic assay: An analytical method that relies on a biorecognition element (e.g., enzymes, antibodies, DNA, microorganisms, tissues). Biosensor: An analytical device composed of a biological element in intimate contact with a physical transducer, which together relate the concentration of a target analyte to a measurable signal. Bioaffinity: The process by which a protein or DNA recognizes and binds a particular target compound (one receptor, one binding event). Biocatalytic (enzyme): The process by which a protein (e.g., enzyme) dramatically increases the rate of a chemical reaction and in which the protein participates in each reaction cycle but is returned to its original state after each cycle (one biocatalyst, many reaction cycles). Cofactor: A nonprotein element or compound required for the catalytic function. Chemical probe: An analytical device that responds selectively, but not reversibly, to the concentration of a chemical species. Chemical sensor: An analytical device that selectively and reversibly responds to a concentration of a chemical species.
(17) Wang, J.; Rogers, K. R. "Detection of Phenols Using a Liquid Chromatographic System with an Enzyme-based Biosensor for Measurement of Phenolics"; EPA: Washington, DC, May 1996; EPA/600/X-96/008. (18) Wang, J. et al. In Field Screening Methods for Hazardous Wastes and Toxic Chemicals, VIP-47, Vol. 2, Proceedings of an International Symposium, Las Vegas, NV 1995; Air and Waste Management Association: Pittsburgh, PA, pp. 691-94. (19) Shriver-Lake, L. C; Ligler, E S. In Field Screening Methods for Hazardous Wastes and Toxic Chemicals, VIP-47, Vol. 2, Proceedings of an International Symposium, Las Vegas, NV, 1995; Air and Waste Management Association: Pittsburgh, PA, p. 107. (20) Agency for Toxic Substances and Disease Registry. 1993 Agency Profile and Annual Report; U.S. Department of Health and Human Services: Atlanta, GA, 1993. (21) Ligler, F. S. et al. Immunomethods 1993, 3, 122-27. (22) Thundat, T. et al. Anal. Chem. 1995, 67, 519-21. (23) Vo-Dinh, T.; Houck, K.; Stokes, D. L. Anal. Chem. 1996, 66, 3379-83. (24) Vo-Dinh, T. et al. Appl. Spectrosc. 1987, 41(5), 735-38. (25) Environmental Sensors; Bogue, R.; Pleil, J.; Tschulena, G., Eds.; Institute of Physics: Bristol, U.K., July 1995; pp. 9-11. (26) Barcelo, D.; Lacorte, S.; Marty, J. L. Trends Anal. Chem., 1995, 14, 334-40.
Kim R. Rogers is an analytical chemist and biosensor researcher at the EPA National Exposure Research Laboratory, Characterization Research Division-Las Vegas. Clare L. Gerlach is the task leader for technical transfer at Lockheed Martin Environmental Services in Las Vegas.
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