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 commercialization 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 antibody-based biosensor for benzo[a]pyrene was done in the 1980s at the U.S. Department of Energy's Oak Ridge National Laboratory.
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) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) have been used at the U.S. Naval Research Laboratory. Many biosensors are on the brink of commercialization, 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 directly 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.
Because biosensors are relatively small, they can 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 (10) 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 the 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 sensing 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).
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 fiber-optic biosensor, and a continuous-flow immunosensor (14).
Although each evaluated field method met the basic requirements for accuracy and reliability compared with EPA standard methods, each assay type had slightly 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 0.07µg/L for the immunoassay test kits to 0.9µg/L for the chemical test kits to 20µg/L for the biosensor methods. The colorimetric chemical test kits were more versatile in identifying related compounds.
The test kits, in general, appeared to be the best 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, the 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 wastewater 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.
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 optical fiber. 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).
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 pesticides (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.
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