Abstract The development of cost-effective on-site methods for environmental monitoring is instrumental to managing risks posed by environmental contamination.

Biosensors show the potential to complement both laboratory-based and field analytical methods for environmental monitoring. Although a wide range of biosensors have been reported for potential environmental applications, relatively few of these have progressed into commercial markets. Advances in areas such as toxicity-, bioavailability-, and multianalyte-screening, and incorporation as detectors in chromatographic systems could possibly widen the market and allow these techniques to be more competitive. Introduction The ability to monitor contaminants in the air, water and soil is instrumental to understanding and managing risks to human health and ecosystems. The required analytical chemistry significantly contributes to the size of this task.1 These costs are high and steadily increasing. In response to these issues, a variety of improvements have been made in laboratory-based analytical methods as well as in field analytical techniques whereby the sampling and analysis take place on-site. Incorporation of field methods into environmental monitoring processes decreases sample transportation (e.g., packaging, shipment, chain-of-custody links, etc.), and facilitates more rapid decision making.2 Field analytical and field screening methods are not, however, without limitations. For example, although these methods are relatively rapid and inexpensive, they are typically designed for specific applications that involve a narrow range of compounds. In addition, sensitivity and accuracy are often lower than what we have come to expect with classical laboratory techniques. When included as part of an integrated site study plan, however, the lower cost of field screening analyses allows a commensurate increase in the sampling frequency and can thereby reduce the overall uncertainty involved in characterization of the contaminated site.3 In many cases, a limited amount of information about the nature of pollutants is available for contaminated sites. Consequently, analytical methods used to characterize such sites must be capable of identifying expected as well as unexpected pollutants and may be required to identify compounds at low levels which contribute to a biological endpoint (e.g., endocrine disruptors, neurotoxins, etc.). Characterization of a contaminated site might best be approached using a combination of laboratory-based methods as well as field diagnostic and screening techniques. After key indicator compounds have been identified, field screening methods may be used to map their spacial distribution. After initial characterization of the site, analytical tasks associated with remediation and post-closure monitoring are required. These tasks typically require frequent and repetitive analysis at specific locations for particular compounds of interest. Because field analytical techniques, including many biosensors, are optimized to rapidly measure a single compound or class of compounds, they are particularly well suited to these tasks. A wide variety of laboratory-based biosensor techniques that could be applied to environmental measurement have been reported; and a number of these are being commercialized. These biosensor techniques must compete with other field methods such as immunoassays, chemical test kits and miniaturized laboratory techniques; many of these competitors are already commercially available and have been demonstrated at contaminated sites. In addition, biosensors must face obstacles to commercialization common to all field analytical methods. These obstacles include a limited market for analysis of individual compounds or compound classes and the inertia of historical environmental sampling and analysis practices.4 As a consequence of these obstacles, successful biosensors will likely incorporate some of the following features: sensor platforms that are versatile enough to support interchangeable recognition elements (to measure a number of analytes), miniaturization to allow automation and convenience at a competitive cost, and other capabilities not currently available such as automated, continuous and remote detection of multiple, complex organic analytes. Biosensor Technology Despite the wide variation in biosensors and biosensor-related techniques that have been introduced, the consensus definition for these devices has remained fairly constant - an analytical device composed of a biological recognition element directly

interfaced to a signal transducer which together relate the concentration of an analyte (or group of related analytes) to a measurable response.5-7 Issues that remain unresolved, however, include assay reversibility and the use of multi-step procedures that require addition of components other than the analyte of interest. A somewhat more focused way to view biosensors is as a sub-type of chemical sensors that relies on biochemical recognition.8 With respect to these views, biosensors might be placed on a continuum between chemical sensors (such as a pH electrode) and complex multi-step bioanalytical assays (such as enzyme linked immunosorbant assay, ELISA). Regardless of the specific definition used for these techniques, the critical question that biosensors must address is whether or not interfacing a particular bioassay with a signal transducer can improve the assay's characteristics or value with respect to the user's needs (in the case of this discussion, environmental monitoring applications). Biosensors with potential for environmental applications have been the subject of a number of recent reviews.6-7, 9-10 These reviews cover (with varying degrees of detail) the fundamentals of the biochemistry and the signal transduction technologies used in these devices. Our intention for this paper is to briefly review these technologies with respect to some of the practical issues related environmental monitoring. Biosensors for environmental applications have employed a wide range of biological recognition systems coupled to a similarly wide range of signal transducers. Biological recognition is accomplished using three basic mechanisms. These include biocatalytic, bioaffinity, and microbe-based systems (Figure 1). These biological recognition systems have been linked to electrochemical, optical-electronic, optical, and acoustic transducers. In theory, and as evidenced in the literature, virtually any biological recognition element can be interfaced to any of the signal transduction technologies if an appropriate operating format can be devised. Although these techniques show a wide range of characteristics (even for biosensors using similar recognition systems coupled to similar transducers) each shows certain inherent advantages and limitations with respect to environmental applications. Biochemical Recognition Biocatalysis-based biosensors. Biocatalysis-based biosensors for environmental applications depend universally on the use of enzymes. These enzyme-based biosensors primarily rely on two operational mechanisms. The first mechanism involves the catalytic transformation of a pollutant (typically from a non-detectable form to a detectable form). The second mechanism involves the detection of pollutants that inhibit or mediate the enzyme's activity. For environmental applications, the first mechanism involving catalytic transformation of the pollutant, shows certain advantages and limitations. They are simple in design and operation. Examples of this format include the use of tyrosinase for the detection of phenols11-12 and the use of organophosphate hydrolase for the detection of organophosphorus pesticides.13 These biosensors can be configured to operate continuously and reversibly. They can also be configured such that the only required reagent is the analyte of interest Inherent limitations for this type of biosensor are primarily those imposed by the nature of the enzyme itself and include the limited number of environmental pollutants which happen to be substrates for the enzyme and the relatively high detection limits (compared to those required by many environmental monitoring applications) for environmental pollutants. The detection limits for these sensors are determined by the enzyme's catalytic properties and are defined by Km and Vmax values. Biosensor formats have been devised which substantially reduce these inherent limitations. For example, in the case of the tyrosinase enzyme electrode, catalytic cycling of the enzyme intermediate between the quinone and catachol oxidation states can significantly amplify the biosensor response. This results in lower detection limits for phenols than expected from Km and Vmax values.12 Mechanisms for the tyrosinase biosensors involve the detection of phenols either through the electrochemical reduction of quinone intermediates or through oxygen consumption (O2 is a co-substrate) using a Clark electrode.14 In another example, a biosensor was designed to increase both sensitivity and the range of substrates typically measured using the tyrosinase enzyme electrode. With this biosensor, a wide range of chlorinated phenols are detected as oxidation-reduction mediators in a glucose oxidase electrode system.15 This is accomplished by chemically oxidizing the chlorophenols which

then cycle between the quinone and hydroquinone oxidation states. In this case, the quinone acts as an electron shuttle for the glucose oxidase-catalyzed oxidation of glucose. This configuration results in low nM detection limits for a number of chlorinated phenols. The other primary mechanism used in biocatalytic biosensors for environmental applications is inhibition of the enzyme by the pollutant. Inherent advantages for these formats involve the larger number of environmental pollutants, usually of a particular chemical class, that inhibit the enzyme and the low concentrations needed to affect the enzyme activity. Detection limits for biosensors based on irreversible inhibitors are usually within the range required for a variety of environmental applications.16 For example, detection limits for cholinesterase biosensors are reported to be in the g/l to ng/l range for compounds such as aldicarb, carbaryl, carbofuran, and dichlovos.17 There are several inherent limitations for biosensors based on enzyme inhibition. In addition to the analyte of interest, these assay formats require the use of substrates and in some cases, cofactors and mediators. Further, for a number of these assays, pollutants must be chemically oxidized to metabolic intermediates to show maximum sensitivities. The irreversible nature of many analyte-enzyme interactions that result in increased sensitivity also renders the biosensor inactive after a single measurement. This may not be a problem, however, for those systems that can be reactivated or that employ disposable sensing elements. Another potential limitation to this type of biosensor mechanism involves the sometimes diverse classes of pollutants that inhibit a specific enzyme. In most circumstances, this would not be expected to present problems. In some cases e.g., the co-contamination of environmental samples with organics and heavy metals, such interferences may lead to unexpected results. The interface of these devices to the contaminated matrix is critical to the successful application of biocatalytic-based biosensors, particularly for in situ applications. These matrices may range from pristine drinking water to highly contaminated industrial sludges. Although a considerable amount of work has been done with respect to use of biosensors in biological matrices (such as blood and fermentation media), little work has focused on the development of membrane barriers for the direct sampling of groundwater or pore water in sludges and saturated sediments. Bioaffinity-based biosensors. Bioaffinity-based biosensors for environmental applications primarily depend on the use of antibodies because of the availability of monoclonal and polyclonal antibodies directed toward a wide range of environmental pollutants as well as the relative affinity and selectivity of these recognition proteins for a specific compound or closely related groups of compounds.18-19 In addition to the wide range of antibodies directed toward different environmental pollutants, a range of assay formats has also been demonstrated with virtually every type of reported signal transducer. Because most small molecular weight organic pollutants in the environment have few distinguishing optical or electrochemical characteristics, the detection of stoichiometric binding of these compounds to antibodies is typically accomplished using competitive binding assay formats. Competitive immunosensor formats rely on the use of an antigentracer which competes with the analyte for a limited number of antibody binding sites. For affinity-based biosensors, this is typically accomplished in one of several ways. In one type of format, antigen-tracer competes with analyte for immobilized antibody binding sites (Figure 2). This format is often used in fluorescence-based systems. In another commonly used format, the antigen is immobilized to the signal transducer (operationally becoming the analyte-tracer) while free binding sites on the antibody, which has been previously exposed to the analyte, bind to the surface-immobilized antigen (Figure 2). Because the antibody is a relatively large molecule, its binding to the surface can be detected by signal transduction methods such as surface plasmon resonance, acoustic systems, and optical systems that measure changes in refractive index and thus, do not require an optical tag. The third commonly used format requires an indirect competitive assay and relies on the use of an enzyme-labeled antigen-tracer (Figure 2). In this format, the assay is completed in two steps. First, the enzyme-tracer competes with analyte for immobilized antibody binding sites. Then, after removal of the unbound tracer (by means of a washing step), a non-detectable substrate is catalytically converted to an electrochemically or optically detectable product. This assay format is used almost universally with electrochemical signal transduction.

Immunosensors are becoming the most frequently reported type of biosensor for environmental applications.19 Rather than expanding the envelope of fundamental understanding, however, immunosensors (and biosensors in general) for the most part represent technological advances for existing bioanalytical assays. It is important to address the issue of whether or not a biosensor shows the potential to improve the characteristics of a particular assay with respect to known or anticipated applications. Because of the wide variety of scientifically established and commercially available immunoassays (test kits), this is particularly relevant in the area of immunosensors. Although there are a variety of ways to group immunosensors based on signal transducers or format considerations, one functionally useful discriminator involves classification based on reusable/regenerable or disposable format configurations. Because immunosensors (particularly those using disposable formats) are most closely related to immunoassay test kit technology, issues which become important for this comparison are more practical in nature and involve the potential for multi-analyte capability, format versatility, assay time, assay sensitivity, system cost, assay cost, shelf life, reproducibility, ruggedness, etc. In contrast to the disposable formats, the multi-use immunosensors, which can be recharged or regenerated, offer certain advantages, particularly for use as detectors for chromatographic and flow injection analysis systems.20 For example, continuous flow and fiber optic immunosensors that have been reported for detection of explosive residues in ground water use multi-assay formats and perform comparably to chemical and immunoassay test kit methods. Initial cost estimates suggest that for a limited number of assays (e.g., < 400), the test kits are more cost-effective. For long-term monitoring projects, however, the biosensor methods which require an initial instrument investment but offer lower cost per assay would be more cost-effective.21 Nucleic acid-based affinity biosensors which may potentially be developed for environmental applications have recently been reported. Application areas for these biosensors include the detection of chemically induced DNA damage22 and the detection of microorganisms through the hybridization of species-specific sequences of DNA.23 Although results from these reports are still preliminary, they appear to offer promising avenues for further investigation. Microorganism-based biosensors. Microorganism-based biosensors tend to use one of three primary mechanisms. For the first mechanism, the pollutant is a respiratory substrate. Biosensors that detect biodegradable organic compounds measured as biological oxygen demand (BOD) are the most widely reported of the microorganism-based biosensors using this mechanism. Several of these devices are commercially available from vendors including: Nisshin Electric Co. Ltd., Tokyo; Autoteam, GmbH, Berlin; Prufgeratewrk, Medingen GmbH, Dresden; and Dr. Lange, GmbH, Berlin. The use of these devices has been incorporated into industrial standard methods in Japan.24-25 Biological oxygen demand is widely used as an indicator of the amount of biodegradable organic compounds found in industrial waste water. The standard procedure (termed BOD5) involves a 5 day incubation of the environmental or industrial water sample with an inoculum of microorganisms typically present in the waste treatment system to yield an endpoint measurement for oxygen consumption.25 The use of indicator microorganisms interfaced to signal transducers allows the measurement of the rate of organic compound metabolism rather than an endpoint; thus, data can be acquired in a significantly shorter time frame (e.g., 15 min to 1 hr), rendering this technology highly advantageous for process control applications. Although these biosensors appear to work well for in situ monitoring of industrial waste waters that result in high BOD values, they currently require improvements in several areas. The primary limitations for these methods involve the variability encountered in calibration of the biosensor response to BOD5 values. This arises from the fact that specific microbial species (used in biosensors) have characteristic substrate spectra which may or may not correspond well with the spectrum of compounds present in the sample. Additional variability results from the presence of polymers (such as protein, starch, and lipid) which must be broken down to monomers before they can be metabolized; this changes the linearity of the response over time and can make interpretation of the result problematic. Current progress on these technologies involves several areas. These areas include: the acid-induced breakdown of biological polymers prior to biosensor analysis, the selection of microorganisms with broad substrate spectra, and the introduction of

novel transduction techniques. In addition, a recent report exploring the feasibility of disposable BOD sensors suggests considerable promise for advancement in this area.26 Another mechanism used for microorganism-based biosensors involves the inhibition of respiration by the analyte of interest. Microbial respiration and its inhibition by various environmental pollutants have been measured both optically27-28 and electrochemically.29 Inherent advantages of these techniques apply primarily to the use of microorganisms as compared to isolated enzymes.24 Microorganism-based biosensors are relatively inexpensive to construct and can operate over a wide range of pH and temperature. General limitations involve the long assay times including the initial response and return to baseline. These characteristics are primarily determined by the cellular diffusion characteristics that can be modified by using genetically engineered microorganisms. The broad specificity of these biosensors to environmental toxins may be an advantage or disadvantage depending on the intended application. In this respect, these devices might be most applicable for general toxicity screening or in situations where the toxic compounds are well defined, or where there is a desire to measure total toxicity through a common mode of action. Biosensors have also been developed using genetically engineered microorganisms (GEMs) that recognize and report the presence of specific environmental pollutants. The microorganisms used in these biosensors are typically produced with a constructed plasmid in which genes that code for luciferase or -galactosidase are placed under the control of a promoter that recognizes the analyte of interest. Because the organism's biological recognition system is linked to the reporting system, the presence of the analyte results in the synthesis of inducible enzymes which then catalyze reactions resulting in the production of detectable products. With respect to environmental applications, the primary disadvantage for this type of biosensor is the limited number of GEMs which have been constructed to respond to specific environmental pollutants. Nevertheless, reported advances include the development of GEMs involving a variety of bioremediation pathways and mechanisms. GEMs that could report both the metabolic condition of the relevant microorganisms as well as the rates of pollutant breakdown could be very useful. Signal Transduction Potentiometric Transducers. The basic principle behind potentiometric measurements is the development of charge related to the analyte activity a1 in the sample through the Nernst relation: E = E0 (RT/nF)ln a1 where E0 is the standard potential for a1 = 1 mol l-1, R is the gas constant, F is the Faraday constant, T is the temperature in K, n is the total number of charges on ion, i and the signal + and - are for cations and anions, respectively. Typically, a reference electrode (inert) and one working electrode both in contact with the sample are required. The use of ion-selective membranes can make these transducers sensitive to various ions (e.g. H+, F-, I-, Cl-) in addition to gases such as CO2 and NH3, including enzyme systems that change the concentration of any of these ions or gases can result in biosensors that can measure substrates, inhibitors or modulators of the enzyme.30 The pH electrodes have been used to measure the activities of enzymes, such as penicillinase, urease, glucose oxidase and acetylcholinesterase which produce or consume protons as a results of catalysis.31 This type of configuration has several drawbacks, however. For example, the change in pH produced by the enzyme and required for detection of activity affects enzyme catalysis, potentially limiting the dynamic range of the assay. Further, the analyte response curve is dramatically influenced by the buffer capacity of the assay solution which must be adjusted in the test sample to match the reference standards. The impact of these limitations has been reduced by either measuring initial rates before the pH of the medium is substantially changed or by using a pH-stat configuration which electrochemically compensates for the enzyme-catalyzed pH change.

The main advantage of such devices is the wide concentration range for which ions can be detected, generally 10-6 - 10-1 mol/l. Their continuous measurement capability is also an interesting possibility for environmental applications. The apparatus is inexpensive, portable, and is well suited for in situ measurements. The main disadvantage is that the limit of detection in some kinds of environmental samples can be rather high (10-5 mol/l or 1 ppm) and the selectivity can be rather poor. Most potentiometric biosensors for detection of environmental pollutants have used enzymes that catalyze the consumption or production of protons. Phosphoric and carbamic pesticides can be evaluated through the use of a pH electrode that measures the activity of acetylcholinesterase.32 The activity of the enzyme is affected by the presence of pesticides, and due to enzymatic amplification, concentrations of these compounds as low as 10-9 M can be measured. In another application, heavy metals can be measured using the enzyme urease coupled to an ammonium ion sensor. Because the activity of urease is sensitive to heavy metal ions, inhibition of enzyme activity can be used to estimate the total concentration of these ions.33 Amperometric Transducers. Amperometric biosensors typically rely on an enzyme system that catalytically converts electrochemically non-active analytes into products that can be oxidized or reduced at a working electrode. This electrode is maintained at a specific potential with respect to a reference electrode. The current produced is linearly proportional to the concentration of the electroactive product, which in turn is proportional to the non-electroactive enzyme substrate. Enzymes typically used in amperometric biosensors are oxidases that catalyze the following class of reactions: Substrate + O2 ------------- Product + H2O2 As a result of the enzyme-catalyzed reaction, the substrate concentration can be determined by amperometric detection of O2 or H2O2. An example of this configuration would be an oxygen consuming enzyme coupled to a Clark electrode. The ambient oxygen concentration is then continuously monitored as it diffuses through a semi-permeable membrane and is reduced at a platinum electrode. Other common configurations include the use of oxidases specific to various substrates to produce H2O2 which is then oxidized at the electrode surface. Although these devices are the most commonly reported class of biosensors, they tend to have a small dynamic range due to saturation kinetics of the enzyme, and a large over-potential is required for oxidation of the analyte; this may lead to oxidation of interfering compounds as well (e.g., ascorbate in the detection of hydrogen peroxide). The main advantage of this class of transducer is the low cost; disposable electrodes are often used with this technique. The high degree of reproducibility that is possible for these (one time use) electrodes eliminates the cumbersome requirement for repeated calibration. The type of instrument used for these measurements is also very easy to obtain and can be inexpensive and compact; this allows for the possibility of on-site measurements. Limitations for this transducer include potential interferences to the response if several electroactive compounds can generate false current values. These effects have been eliminated, for clinical applications, through the use of selective membranes which carefully control the molecular weight or the charge of compounds which have access to the electrode. Acetylcholinesterase can also be coupled to an amperometric sensor used to detect hydrogen peroxide as described in the following reaction: Acetylcholinesterase Acetylcholine + O2 + H2O ----------------------------

 Choline + Acetate Choline Oxidase Choline + 2O2 + H2O -------------------------- Betaine + H2O2 Consequently, an amperometric biosensor for hydrogen peroxide can also be used to measure organophosphate pesticides at concentrations as low as 10-9 M.34 In addition to the use in enzyme-based biosensors, amperometric transducers have also been used to measure enzyme-labeled tracers for immunosensors. Enzymes which are commonly used include: Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (PA). Compounds of environmental interest, measured using disposable amperometric electrodes include PCBs, triazines and various toxins.35 Another type of biosensor which employs controlled potential techniques (chronoamperometry) involves detection of chemical compounds that complex with, link to, or intercalate into double stranded calf thymus DNA. These techniques show the promise for detection of genotoxic compounds which may be present in industrial effluents, waste treatment effluents, or drinking water.36 Conductance Measurements. Enzyme reactions which produce or consume ionic species will, depending on the total ionic strength of the media, change the conductance/capacitance of the solution to a greater or lesser extent. Various planar interdigitated electrode configurations have been reported as conductometric transducers for biosensors. These have been used in combination with a variety of possible enzyme systems which can confer specificity to this type of transducer. Enzymes such as urease, which catalyze the production of ionic species, have been used in these devices. Nevertheless, because the conductance is sensitive to temperature, Faradaic processes, double layer charging and concentration polarization, differential methods with internal controls must be used.37 There are a variety of possible enzyme systems which can confer specificity to this type of transducer. Urease-coated electrodes in combination with a control electrode coated with an inactive protein have been used to measure the decomposition of urea to ammonium, bicarbonate and hydroxyl ions. The primary advantage of this technique is in the use of inexpensive, reproducible and disposable sensors. The main disadvantage is that ionic species produced must significantly change the total ionic strength to obtain a reliable measurement. This requirement increases the detection limit to unacceptable levels and results in potential interferences from variability in the ionic strength of the sample. Thin film interdigitated planar conductometric electrodes have been used to measure heavy metals. Glucose oxidase, alcohol oxidase, butyril oxidase38 and urease39 have been immobilized on transducer surfaces and used as bioactive elements for detection of Ag+, Hg+2 and Pb2+. Dynamic concentration ranges of 1-100 M have been obtained for these devices. Optical measurements. Owing to the number and reliability of optical methods, a vast number of optical transduction techniques can be used for biosensor development.7-8 These may employ linear optical phenomenon, including adsorption, fluorescence, phosphorescence, polarization, rotation, interference, etc. or non-linear phenomena, such as second harmonic generation. The choice of a particular optical method depends on the nature of the application and desired sensitivities. In practice, fiber optics can be coupled with all optical techniques, thus increasing their versatility. The optical biosensor formats may involve direct detection of the analyte of interest or indirect detection through optically labeled probes. Total internal reflection fluorescence (TIRF) has been used with planar and fiber optic waveguides as signal transducers in a number of reported biosensors. In these transducers, light is propagated down a waveguide which generates an electromagnetic wave (evanescent wave) at the surface of the optically denser medium of the waveguide and the adjacent less optically dense medium. The amplitude of the standing wave decreases exponentially with distance into the lower refractive index material. The fluorescence of a fluorophore (probe) excited within the evanescent field can be collected either outside the waveguide or by coupling the emission frequencies back into the waveguide. In configurations which use TIRF the

biological sensing element is immobilized on the side rather than the end of the waveguide. This configuration is particularly useful for measuring binding events at a solid-liquid interface, because the washing steps typically used to separate bound and unbound analyte probes are not required.40 This technique has been exploited in the widely publicized fluorescence capillary filled devices and resonance mirror device. Surface plasmon resonance (SPR) has recently been used as the basis for signal transduction in biosensors. In a typical experimental set-up, incident light is reflected from the internal face of a prism in which the external face has been coated with a thin metal film. At a critical angle, the intensity of the reflected light is lost to the creation of a resonant oscillation in the electrons at the surface of the metal film. Since the critical angle is dependent on the refractive index of material present on the metal surface, this method has been used to measure the binding of antibodies to antigens immobilized at the sensor surface. This might be visualized as a multi-layer configuration consisting of quartz (prism surface) // metal // antigen // antibody and where the light reflected from the inside surface of the prism does not pass through the sample. Advantages of optical techniques involve the speed and reproducibility of the measurement. Optical transducers have been used for affinity and microbial-based biosensors, and for a few enzyme-based biosensors reported for environmental applications.41 A variety of optical immunosensors have been configured using direct and indirect formats with and without optical labels. The main drawback of optical measurements is the high cost of the apparatus. Moreover, these instruments are generally larger than is practical for on-site measurements. Among the wide range of reported optical immunosensor applications, Atrazine has been measured with SPR through an indirect format42 An antibody against Atrazine was measured using the BiacoreTM system after reaction with water samples. Low concentrations of Atrazine (0.1 ppb) were detected using a relatively simple protocol. Acoustic Transducers. The utility of the piezoelectric crystal as a mass sensor arises from the linear relationship between the change in the mass (or under some conditions, viscosity) at the crystal surface and the change in its oscillating frequency. The vibration of piezoelectric crystals produces an oscillating electric field in which the resonant frequency of the crystal depends on its chemical nature, size, shape and mass. By placing the crystal in an oscillating circuit, the frequency can be measured as a function of the mass. When the change in mass (m) is very small compared to the total mass of the crystal, the change in frequency (f) relates to m as follows: delta f = Cf2 delta m /A where f is the vibrational frequency of the crystal in the circuit, A is the area of the electrode and C is a constant determined in part by the crystal material and thickness. Piezoelectric crystals, sometimes referred to as a quartz crystal microbalances (QCM), are typically made of quartz and operate at frequencies between 1 and 10 MHz. These devices can operate in liquids with a frequency determination limit of 0.1 Hz, the detection limit of mass bound to the electrode surface is about 10-10 to 10-11 g. These transducers have been coupled with enzymes and antibodies to detect analytes, including formaldehyde, cocaine and parathion.43 Different types of waves can be generated in piezoelectric materials, using various electrode configurations on their surfaces. Surface acoustic wave (SAW) devices have been reported to detect vapors which absorb to chemically selective coatings. Although these transducers can operate at higher frequencies (i.e. 250 MHz) than the QCMs, yielding higher sensitivities, excessive signal damping prevents them from being used in liquids. Another type of piezoelectric transducer, termed a surface transverse wave (STW), operates at over 250 MHz in liquids.20 This transducer is sensitive, stable, and because it operates in liquid environments, it has promise for environmental applications. One of the main advantages for acoustic techniques is the detection, in real time, of binding reactions of chemical compounds (in gaseous form or dissolved in solution) with the solid surface of the crystal. This feature (similar to SPR) allows kinetic

evaluation of affinity interactions (typically between antibodies and antigens). In addition, the cost of the apparatus can be rather low. Limitations for this transduction method involve format and calibration requirements. Each crystal should be calibrated because its frequency depends on the crystal geometry and the immobilization technique used to coat the surface (generally gold-plated quartz) with the antigen or antibody. The main source of variability is the uniformity of the protein immobilized on the surface. Acoustic transduction has been used in a wide variety of biosensor configurations. Immobilized proteins (i.e. enzymes and antibodies) for the detection of atmospheric pollutants have proven useful in a number of applications. The first use of protein as a coating for a direct assay of a gaseous compound using a piezoelectric crystal was described by Guilbault in 1984. Formaldehyde in the concentration range 1-100 ppb was assayed in the gas phase using formaldehyde dehydrogenase.44 with no interference from other aldehydes or alcohols. In another application, parathion antibodies were immobilized on a QCM for the specific detection of this pesticide at part per billion levels in air. As long as the air contained traces of moisture the immunological reaction proceeded well in the gas phase, making possible a new generation of detectors.45 Organophosphorus nerve agents were also detected in air at part per billion levels using crystals coated with cholinesterase. In addition to biochemical advances for these devices, portable and inexpensive instruments became available in the early 1990s which were shown to perform well in field settings. More recently, both direct46 and indirect47 antibody-based piezoelectric sensors have been reported for the herbicide Atrazine and 2,4-D.48 Environmental Monitoring Reflective of the wide range of pollutants, biosensors for environmental applications also cover a broad range of compounds across a number of chemical classes. One way to organize this diverse group of techniques is by chemical class (Table 1). This means of organization gives some insights into the potential field monitoring applications for which biosensors might have the most significant impacts. For several possible reasons, pesticides account for the greatest number of reports for environmental biosensors. First, pesticides typically function by means of interacting with a specific biochemical target either as a substrate (e.g., organophosphorus insecticides/ organophosphate hydrolase) or as inhibitors (e.g., dithiocarbamate fungicides/aldehyde dehydrogenase; organophosphorus insecticides/acetylcholinesterase). In addition, a wide variety of antibodies have been developed (some of which are commercially available) toward various classes of insecticides, herbicides, and fungicides. Examples include: triazines, alachlor, aldicarb, 2,4-D and paraquat. Non-agricultural organics for which biosensors have been developed cover a broad range of chemical classes with a similar range in diversity of mechanisms for biochemical detection. Recognition elements for these biosensors include the use of enzymes, antibodies and microorganisms. With respect to environmental monitoring, one of the challenges that face these techniques involves the source of pollution which is often industrial in nature. The industrial contamination typically contains a number of industrial compounds from a variety of related and unrelated chemical classes. In many cases, field analytical methods that can measure a number of compounds without interference by breakdown products or other hazardous cocontaminants are needed. Biosensors reported for detection of environmentally significant metal ions primarily use enzymes or GEMs as recognition elements. For example, in the case of a urease enzyme-based fiber optic biosensor, a number of metal ions inhibited the biosensor response to varying degrees. The biosensor response was most sensitive to Hg2+ with a detection limit of 10 nM. In the case of the GEM-based biosensors, these devices use bacteria in which genes responsible for Hg2+ detoxification are linked to light emitting genes. The biosensor response is typically specific to Hg2+ with detection limits in the low nM range.49 As is the case for other biosensors that detect other environmental pollutants, these biosensors must also compete with well

established field methods. Field analytical techniques which detect similar metals include Field Portable X-ray Fluorescence (FPXRF) spectroscopy and anodic stripping voltammetry (ASV). The FPXRF methods are rapid and inexpensive. Detection limits for these techniques are, however, fairly high (e.g., in the low ppm range).50 ASV methods are also portable and inexpensive and have detection limits in the low ppb range.51 An area in which biosensors perhaps show the greatest diversity and potential for development involves the measurement of environmentally significant biological parameters. These biosensors are composed of biological assays which have been interfaced with various signal transducers and measure the following parameters: microorganism toxicity, enzyme inhibition, biological oxygen demand, inhibition of Photosystem II, DNA damage, and identification and enumeration of microorganisms of environmental concern. In a number of cases, the interface of a biological assay to a signal transducer has been shown to reduce the time and complexity involved with these assays. A variety of biosensors have been reported which measure compounds of environmental interest that are toxic to microorganisms. Major metabolic mechanisms monitored include: the consumption of oxygen, the evolution of protons, and the synthesis of bioluminescence enzymes genetically tied to the expression of metabolic indicators. These assays are typically sensitive to a variety of compounds representing a number of compound classes. These biosensor techniques must also compete with commercially available assays such as the Microtox System which has been extensively tested in a number of environmental settings and included as a routine tool in various monitoring programs. Similar to the chemical and immunoassay test kits, the Microtox assay is well characterized, simple to execute, and relatively reproducible if source, purity, and condition of reagents is carefully monitored. Although biosensors based on enzyme inhibition are typically demonstrated using compounds in a few specific chemical classes, recent reports have shown these enzyme biosensors to be inhibited by compounds from diverse chemical classes. Based on this phenomenon, arrays of enzymes have been used to screen for various common environmental pollutants. Pattern recognition techniques have been applied to these systems to enable identification of members of specific compound classes. Mathematical approaches to deconvoluting the relative effects of potential interferents on assay responses using multiple antibodies may also prove beneficial for use with enzyme array techniques.32a Primary limitations to the use of these systems involve the relatively few enzymes that can be included in a matrix and the complexity of assembling a number of different enzymes into a single monitoring system. The identification and enumeration of microorganisms that can pose human or environmental health problems is another area where biosensor technology may increase the speed and reduce expense associated with specific bioanalytical assays. These biosensors have focused on two mechanisms; immunochemical recognition of surface antigens and identification of DNA sequences that are unique to the organism of interest. In a recent example of an immunochemical approach, antibodies directed toward the red tide-causing plankton Alexandrium affine were immobilized to a piezoelectric sensor and used to detect this organism at concentrations as low as 102 cells/ml in sea water.52 Biosensor methods based on the identification of microorganisms through the detection of unique DNA sequences show significant promise; however, less progress toward practical methods has been achieved. Challenges for these techniques principally involve the isolation and processing of the microorganisms in the environmental sample to isolate and amplify selected DNA sequences prior to the hybridization assay. In this respect, technology borrowed from the intense efforts in micro-scale and automated gene sequencing research has application in the environmental monitoring area. For example, recently reported micro-fabricated bioelectronic chip technology was used to isolate E. coli cells from blood, electrochemically lyse the cells, and measure hybridization of specific target DNA in the lysate to capture probes immobilized to the electrode.23 In addition, multi-step genetic assays including PCR amplification and hybridization have been incorporated into "biochip" technology.53 Future Directions Biosensors and biosensor-related techniques that show potential for environmental applications must overcome a number of obstacles to become commercially viable in the highly competitive area of field analytical methods. Some of the obstacles common to all field analytical methods include: the diversity of compounds and the complexity of matrices in environmental

samples, the variability in data quality requirements among environmental programs, and the broad range of possible environmental monitoring applications. More specific to biosensor technology, these hurdles include: relatively high development costs for single analyte systems, limited shelf and operational lifetimes for pre-manufactured biorecognition components and relative assay format complexity for many potentially portable (but currently laboratory-based) biosensor systems. Nevertheless, there are a number of areas where the unique capabilities of biosensors might be exploited to meet the requirements of environmental monitoring. Advances in areas such as toxicity-, bioavailability-, and multi-pollutantscreening, could widen the potential market and allow these techniques to be more competitive. Miniaturization, reversibility and continuous operation may allow biosensor techniques to be incorporated as detectors in chromatographic systems. Because of the wide variability in environmental matrices, greater versatility in the area of sample interface would be of considerable value. For example, classical laboratory-based techniques typically employ extensive extraction and cleanup protocols. With few exceptions, these protocols yield little information concerning the potential bioavailability of the pollutant. Biosensors coupled with a series of extraction methodologies could provide valuable information in this area. Due to unique characteristics and flexibility in operational design, biosensors continue to show significant promise for use in environmental monitoring applications. Nevertheless, because of a variety of obstacles (many unique to the environmental monitoring area), the introduction (and early successes) of these devices into this commercial market will likely involve narrowly focused applications. Acknowledgments The authors gratefully acknowledge the editorial assistance of Dr. L. R. Williams. NOTICE: The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), partially funded the work involved in preparing this article. It has been subject to the EPA's peer and administrative review and has been approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use. References