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
The detection limits for these sensors are determined by the enzyme's catalytic properties and are defined by Km and Vmax values. ELISA). Biosensors with potential for environmental applications have been the subject of a number of recent reviews. They are simple in design and operation.8 With respect to these views. 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. and acoustic transducers. a wide range of chlorinated phenols are detected as oxidation-reduction mediators in a glucose oxidase electrode system. These enzyme-based biosensors primarily rely on two operational mechanisms. This results in lower detection limits for phenols than expected from Km and Vmax values. For example. shows certain advantages and limitations. These include biocatalytic. A somewhat more focused way to view biosensors is as a sub-type of chemical sensors that relies on biochemical recognition.6-7. The second mechanism involves the detection of pollutants that inhibit or mediate the enzyme's activity. environmental monitoring applications). In theory. Regardless of the specific definition used for these techniques. 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. Biological recognition is accomplished using three basic mechanisms. virtually any biological recognition element can be interfaced to any of the signal transduction technologies if an appropriate operating format can be devised.14 In another example.interfaced to a signal transducer which together relate the concentration of an analyte (or group of related analytes) to a measurable response. Biochemical Recognition Biocatalysis-based biosensors. however. and as evidenced in the literature. in the case of the tyrosinase enzyme electrode. the first mechanism involving catalytic transformation of the pollutant.5-7 Issues that remain unresolved. Biosensor formats have been devised which substantially reduce these inherent limitations. The first mechanism involves the catalytic transformation of a pollutant (typically from a non-detectable form to a detectable form). and microbe-based systems (Figure 1). 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. include assay reversibility and the use of multi-step procedures that require addition of components other than the analyte of interest. a biosensor was designed to increase both sensitivity and the range of substrates typically measured using the tyrosinase enzyme electrode. catalytic cycling of the enzyme intermediate between the quinone and catachol oxidation states can significantly amplify the biosensor response.15 This is accomplished by chemically oxidizing the chlorophenols which
.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. With this biosensor. bioaffinity. Biosensors for environmental applications have employed a wide range of biological recognition systems coupled to a similarly wide range of signal transducers. Our intention for this paper is to briefly review these technologies with respect to some of the practical issues related environmental monitoring. optical-electronic. 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.13 These biosensors can be configured to operate continuously and reversibly. These biological recognition systems have been linked to electrochemical. Biocatalysis-based biosensors for environmental applications depend universally on the use of enzymes. For environmental applications. 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. optical. 9-10 These reviews cover (with varying degrees of detail) the fundamentals of the biochemistry and the signal transduction technologies used in these devices.
this would not be expected to present problems. In some cases e. In this format.17 There are several inherent limitations for biosensors based on enzyme inhibition. The other primary mechanism used in biocatalytic biosensors for environmental applications is inhibition of the enzyme by the pollutant. In addition to the analyte of interest. this is typically accomplished in one of several ways. such interferences may lead to unexpected results. however.
. the quinone acts as an electron shuttle for the glucose oxidase-catalyzed oxidation of glucose. Competitive immunosensor formats rely on the use of an antigentracer which competes with the analyte for a limited number of antibody binding sites. In most circumstances. a non-detectable substrate is catalytically converted to an electrochemically or optically detectable product. First. the detection of stoichiometric binding of these compounds to antibodies is typically accomplished using competitive binding assay formats. do not require an optical tag. pollutants must be chemically oxidized to metabolic intermediates to show maximum sensitivities. bind to the surface-immobilized antigen (Figure 2). In this case. little work has focused on the development of membrane barriers for the direct sampling of groundwater or pore water in sludges and saturated sediments. a range of assay formats has also been demonstrated with virtually every type of reported signal transducer. and dichlovos. particularly for in situ applications. these assay formats require the use of substrates and in some cases. The third commonly used format requires an indirect competitive assay and relies on the use of an enzyme-labeled antigen-tracer (Figure 2). This may not be a problem. The irreversible nature of many analyte-enzyme interactions that result in increased sensitivity also renders the biosensor inactive after a single measurement. that inhibit the enzyme and the low concentrations needed to affect the enzyme activity. carbofuran. Inherent advantages for these formats involve the larger number of environmental pollutants. Although a considerable amount of work has been done with respect to use of biosensors in biological matrices (such as blood and fermentation media). These matrices may range from pristine drinking water to highly contaminated industrial sludges.16 For example. the enzyme-tracer competes with analyte for immobilized antibody binding sites. Because most small molecular weight organic pollutants in the environment have few distinguishing optical or electrochemical characteristics. acoustic systems. Another potential limitation to this type of biosensor mechanism involves the sometimes diverse classes of pollutants that inhibit a specific enzyme. This configuration results in low nM detection limits for a number of chlorinated phenols. 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. detection limits for cholinesterase biosensors are reported to be in the µg/l to ng/l range for compounds such as aldicarb. This format is often used in fluorescence-based systems. This assay format is used almost universally with electrochemical signal transduction. In another commonly used format. its binding to the surface can be detected by signal transduction methods such as surface plasmon resonance. which has been previously exposed to the analyte. for those systems that can be reactivated or that employ disposable sensing elements. Further. Detection limits for biosensors based on irreversible inhibitors are usually within the range required for a variety of environmental applications. the co-contamination of environmental samples with organics and heavy metals. For affinity-based biosensors. Bioaffinity-based biosensors. and optical systems that measure changes in refractive index and thus.18-19 In addition to the wide range of antibodies directed toward different environmental pollutants. In one type of format. the assay is completed in two steps. usually of a particular chemical class. for a number of these assays. Because the antibody is a relatively large molecule.then cycle between the quinone and hydroquinone oxidation states.g. antigen-tracer competes with analyte for immobilized antibody binding sites (Figure 2).. The interface of these devices to the contaminated matrix is critical to the successful application of biocatalytic-based biosensors. carbaryl. the antigen is immobilized to the signal transducer (operationally becoming the analyte-tracer) while free binding sites on the antibody. after removal of the unbound tracer (by means of a washing step). cofactors and mediators. Then.
Ltd. offer certain advantages.20 For example. format versatility. Microorganism-based biosensors. The primary limitations for these methods involve the variability encountered in calibration of the biosensor response to BOD5 values. and lipid) which must be broken down to monomers before they can be metabolized. they appear to offer promising avenues for further investigation. Current progress on these technologies involves several areas. Microorganism-based biosensors tend to use one of three primary mechanisms. etc.. Berlin. Although there are a variety of ways to group immunosensors based on signal transducers or format considerations. assay sensitivity. assay cost. ruggedness. the multi-use immunosensors. Several of these devices are commercially available from vendors including: Nisshin Electric Co. starch.g. the test kits are more cost-effective. immunosensors (and biosensors in general) for the most part represent technological advances for existing bioanalytical assays. assay time. The use of these devices has been incorporated into industrial standard methods in Japan. shelf life.24-25 Biological oxygen demand is widely used as an indicator of the amount of biodegradable organic compounds found in industrial waste water. Initial cost estimates suggest that for a limited number of assays (e. These areas include: the acid-induced breakdown of biological polymers prior to biosensor analysis. the selection of microorganisms with broad substrate spectra. 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.21 Nucleic acid-based affinity biosensors which may potentially be developed for environmental applications have recently been reported. one functionally useful discriminator involves classification based on reusable/regenerable or disposable format configurations. In contrast to the disposable formats. Because immunosensors (particularly those using disposable formats) are most closely related to immunoassay test kit technology. system cost. however..g. and Dr. 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. 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. Lange. thus. 15 min to 1 hr).25 The use of indicator microorganisms interfaced to signal transducers allows the measurement of the rate of organic compound metabolism rather than an endpoint.23 Although results from these reports are still preliminary. however. Because of the wide variety of scientifically established and commercially available immunoassays (test kits). For the first mechanism. Medingen GmbH. For long-term monitoring projects. this is particularly relevant in the area of immunosensors. GmbH. the biosensor methods which require an initial instrument investment but offer lower cost per assay would be more cost-effective. Prufgeratewrk. 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.Immunosensors are becoming the most frequently reported type of biosensor for environmental applications. issues which become important for this comparison are more practical in nature and involve the potential for multi-analyte capability.19 Rather than expanding the envelope of fundamental understanding. Berlin. < 400). which can be recharged or regenerated. GmbH. Additional variability results from the presence of polymers (such as protein. Autoteam. the pollutant is a respiratory substrate. 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. reproducibility. Dresden. this changes the linearity of the response over time and can make interpretation of the result problematic. data can be acquired in a significantly shorter time frame (e. Tokyo.. 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. particularly for use as detectors for chromatographic and flow injection analysis systems. they currently require improvements in several areas. 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. and the introduction of
Further.30 The pH electrodes have been used to measure the activities of enzymes. In addition.26 Another mechanism used for microorganism-based biosensors involves the inhibition of respiration by the analyte of interest. respectively. GEMs that could report both the metabolic condition of the relevant microorganisms as well as the rates of pollutant breakdown could be very useful. glucose oxidase and acetylcholinesterase which produce or consume protons as a results of catalysis. Nevertheless. urease. the change in pH produced by the enzyme and required for detection of activity affects enzyme catalysis. R is the gas constant. F-. These characteristics are primarily determined by the cellular diffusion characteristics that can be modified by using genetically engineered microorganisms. the presence of the analyte results in the synthesis of inducible enzymes which then catalyze reactions resulting in the production of detectable products. including enzyme systems that change the concentration of any of these ions or gases can result in biosensors that can measure substrates. such as penicillinase. a recent report exploring the feasibility of disposable BOD sensors suggests considerable promise for advancement in this area. Biosensors have also been developed using genetically engineered microorganisms (GEMs) that recognize and report the presence of specific environmental pollutants. potentially limiting the dynamic range of the assay. 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. For example. reported advances include the development of GEMs involving a variety of bioremediation pathways and mechanisms. or where there is a desire to measure total toxicity through a common mode of action. I-. 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. F is the Faraday constant.29 Inherent advantages of these techniques apply primarily to the use of microorganisms as compared to isolated enzymes. With respect to environmental applications. 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. a reference electrode (inert) and one working electrode both in contact with the sample are required. In this respect.
. n is the total number of charges on ion.are for cations and anions.novel transduction techniques. Microbial respiration and its inhibition by various environmental pollutants have been measured both optically27-28 and electrochemically.31 This type of configuration has several drawbacks. Because the organism's biological recognition system is linked to the reporting system. Signal Transduction Potentiometric Transducers.g. The use of ion-selective membranes can make these transducers sensitive to various ions (e. Cl-) in addition to gases such as CO2 and NH3. the primary disadvantage for this type of biosensor is the limited number of GEMs which have been constructed to respond to specific environmental pollutants. Typically. General limitations involve the long assay times including the initial response and return to baseline. i and the signal + and . 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. inhibitors or modulators of the enzyme. these devices might be most applicable for general toxicity screening or in situations where the toxic compounds are well defined. however. H+.24 Microorganism-based biosensors are relatively inexpensive to construct and can operate over a wide range of pH and temperature. T is the temperature in K. The broad specificity of these biosensors to environmental toxins may be an advantage or disadvantage depending on the intended application.
This electrode is maintained at a specific potential with respect to a reference electrode.10-1 mol/l. These effects have been eliminated. The high degree of reproducibility that is possible for these (one time use) electrodes eliminates the cumbersome requirement for repeated calibration.. concentrations of these compounds as low as 10-9 M can be measured. The apparatus is inexpensive. this allows for the possibility of on-site measurements. generally 10-6 . The current produced is linearly proportional to the concentration of the electroactive product. disposable electrodes are often used with this technique. and a large over-potential is required for oxidation of the analyte. the substrate concentration can be determined by amperometric detection of O2 or H2O2. Phosphoric and carbamic pesticides can be evaluated through the use of a pH electrode that measures the activity of acetylcholinesterase. 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. for clinical applications. Most potentiometric biosensors for detection of environmental pollutants have used enzymes that catalyze the consumption or production of protons. inhibition of enzyme activity can be used to estimate the total concentration of these ions. and is well suited for in situ measurements. The type of instrument used for these measurements is also very easy to obtain and can be inexpensive and compact. In another application. which in turn is proportional to the non-electroactive enzyme substrate. ascorbate in the detection of hydrogen peroxide). Because the activity of urease is sensitive to heavy metal ions.The main advantage of such devices is the wide concentration range for which ions can be detected. heavy metals can be measured using the enzyme urease coupled to an ammonium ion sensor. 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. 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.g. and due to enzymatic amplification. The main advantage of this class of transducer is the low cost. Limitations for this transducer include potential interferences to the response if several electroactive compounds can generate false current values. Although these devices are the most commonly reported class of biosensors. 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. Their continuous measurement capability is also an interesting possibility for environmental applications. they tend to have a small dynamic range due to saturation kinetics of the enzyme.33 Amperometric Transducers. portable. Other common configurations include the use of oxidases specific to various substrates to produce H2O2 which is then oxidized at the electrode surface. this may lead to oxidation of interfering compounds as well (e. through the use of selective membranes which carefully control the molecular weight or the charge of compounds which have access to the electrode.32 The activity of the enzyme is affected by the presence of pesticides. Acetylcholinesterase can also be coupled to an amperometric sensor used to detect hydrogen peroxide as described in the following reaction: Acetylcholinesterase Acetylcholine + O2 + H2O ----------------------------
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. These techniques show the promise for detection of genotoxic compounds which may be present in industrial effluents. Enzymes such as urease.34 In addition to the use in enzyme-based biosensors. which catalyze the production of ionic species. fiber optics can be coupled with all optical techniques. or non-linear phenomena. Various planar interdigitated electrode configurations have been reported as conductometric transducers for biosensors. polarization. because the conductance is sensitive to temperature. reproducible and disposable sensors. fluorescence. This requirement increases the detection limit to unacceptable levels and results in potential interferences from variability in the ionic strength of the sample. or intercalate into double stranded calf thymus DNA. Glucose oxidase. bicarbonate and hydroxyl ions. Compounds of environmental interest. The optical biosensor formats may involve direct detection of the analyte of interest or indirect detection through optically labeled probes. such as second harmonic generation.37 There are a variety of possible enzyme systems which can confer specificity to this type of transducer. In practice.7-8 These may employ linear optical phenomenon. In these transducers. These have been used in combination with a variety of possible enzyme systems which can confer specificity to this type of transducer.35 Another type of biosensor which employs controlled potential techniques (chronoamperometry) involves detection of chemical compounds that complex with. Hg+2 and Pb2+. waste treatment effluents. Dynamic concentration ranges of 1-100 µM have been obtained for these devices.36 Conductance Measurements. Total internal reflection fluorescence (TIRF) has been used with planar and fiber optic waveguides as signal transducers in a number of reported biosensors. Owing to the number and reliability of optical methods. differential methods with internal controls must be used. Optical measurements. depending on the total ionic strength of the media. etc. alcohol oxidase. 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. triazines and various toxins. The primary advantage of this technique is in the use of inexpensive. The choice of a particular optical method depends on the nature of the application and desired sensitivities. thus increasing their versatility. change the conductance/capacitance of the solution to a greater or lesser extent. Thin film interdigitated planar conductometric electrodes have been used to measure heavy metals. link to. The main disadvantage is that ionic species produced must significantly change the total ionic strength to obtain a reliable measurement. double layer charging and concentration polarization. Enzymes which are commonly used include: Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (PA). an amperometric biosensor for hydrogen peroxide can also be used to measure organophosphate pesticides at concentrations as low as 10-9 M. including adsorption. amperometric transducers have also been used to measure enzyme-labeled tracers for immunosensors. a vast number of optical transduction techniques can be used for biosensor development. Faradaic processes. rotation. phosphorescence. butyril oxidase38 and urease39 have been immobilized on transducer surfaces and used as bioactive elements for detection of Ag+. interference.• Choline + Acetate Choline Oxidase Choline + 2O2 + H2O --------------------------• Betaine + H2O2 Consequently. 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. The amplitude of the standing wave decreases exponentially with distance into the lower refractive index material. In configurations which use TIRF the
. or drinking water. have been used in these devices. Nevertheless. Enzyme reactions which produce or consume ionic species will. measured using disposable amperometric electrodes include PCBs.
A is the area of the electrode and C is a constant determined in part by the crystal material and thickness. Surface plasmon resonance (SPR) has recently been used as the basis for signal transduction in biosensors. Surface acoustic wave (SAW) devices have been reported to detect vapors which absorb to chemically selective coatings. 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.biological sensing element is immobilized on the side rather than the end of the waveguide. it has promise for environmental applications. using various electrode configurations on their surfaces. are typically made of quartz and operate at frequencies between 1 and 10 MHz. sometimes referred to as a quartz crystal microbalances (QCM). The vibration of piezoelectric crystals produces an oscillating electric field in which the resonant frequency of the crystal depends on its chemical nature. including formaldehyde. At a critical angle. the frequency can be measured as a function of the mass. these instruments are generally larger than is practical for on-site measurements.41 A variety of optical immunosensors have been configured using direct and indirect formats with and without optical labels. stable. When the change in mass (m) is very small compared to the total mass of the crystal. 250 MHz) than the QCMs. cocaine and parathion. Moreover. termed a surface transverse wave (STW). By placing the crystal in an oscillating circuit. This configuration is particularly useful for measuring binding events at a solid-liquid interface. 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. this method has been used to measure the binding of antibodies to antigens immobilized at the sensor surface. 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. operates at over 250 MHz in liquids. Among the wide range of reported optical immunosensor applications. because the washing steps typically used to separate bound and unbound analyte probes are not required. 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. 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. yielding higher sensitivities. Low concentrations of Atrazine (0. Another type of piezoelectric transducer. and for a few enzyme-based biosensors reported for environmental applications. One of the main advantages for acoustic techniques is the detection. of binding reactions of chemical compounds (in gaseous form or dissolved in solution) with the solid surface of the crystal. Acoustic Transducers. and because it operates in liquid environments. excessive signal damping prevents them from being used in liquids. Optical transducers have been used for affinity and microbial-based biosensors. size. Since the critical angle is dependent on the refractive index of material present on the metal surface. Although these transducers can operate at higher frequencies (i. incident light is reflected from the internal face of a prism in which the external face has been coated with a thin metal film. in real time.e. viscosity) at the crystal surface and the change in its oscillating frequency.43 Different types of waves can be generated in piezoelectric materials.20 This transducer is sensitive.1 ppb) were detected using a relatively simple protocol.1 Hz. Advantages of optical techniques involve the speed and reproducibility of the measurement. These devices can operate in liquids with a frequency determination limit of 0. This feature (similar to SPR) allows kinetic
.40 This technique has been exploited in the widely publicized fluorescence capillary filled devices and resonance mirror device. the detection limit of mass bound to the electrode surface is about 10-10 to 10-11 g. The main drawback of optical measurements is the high cost of the apparatus. In a typical experimental set-up. shape and mass. These transducers have been coupled with enzymes and antibodies to detect analytes. Piezoelectric crystals.
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 1984.4-D.44 with no interference from other aldehydes or alcohols. The biosensor response was most sensitive to Hg2+ with a detection limit of 10 nM. pesticides typically function by means of interacting with a specific biochemical target either as a substrate (e. enzymes and antibodies) for the detection of atmospheric pollutants have proven useful in a number of applications. both direct46 and indirect47 antibody-based piezoelectric sensors have been reported for the herbicide Atrazine and 2. these devices use bacteria in which genes responsible for Hg2+ detoxification are linked to light emitting genes.45 Organophosphorus nerve agents were also detected in air at part per billion levels using crystals coated with cholinesterase.e. As long as the air contained traces of moisture the immunological reaction proceeded well in the gas phase. biosensors for environmental applications also cover a broad range of compounds across a number of chemical classes. Limitations for this transduction method involve format and calibration requirements. For example.g. alachlor. and fungicides. The main source of variability is the uniformity of the protein immobilized on the surface.g. 2. Acoustic transduction has been used in a wide variety of biosensor configurations. making possible a new generation of detectors. in the case of a urease enzyme-based fiber optic biosensor. dithiocarbamate fungicides/aldehyde dehydrogenase. field analytical methods that can measure a number of compounds without interference by breakdown products or other hazardous cocontaminants are needed.48 Environmental Monitoring Reflective of the wide range of pollutants. antibodies and microorganisms. In addition. This means of organization gives some insights into the potential field monitoring applications for which biosensors might have the most significant impacts. Formaldehyde in the concentration range 1-100 ppb was assayed in the gas phase using formaldehyde dehydrogenase. these biosensors must also compete with well
. 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.evaluation of affinity interactions (typically between antibodies and antigens). In another application.. organophosphorus insecticides/acetylcholinesterase). the cost of the apparatus can be rather low. The biosensor response is typically specific to Hg2+ with detection limits in the low nM range. Immobilized proteins (i. parathion antibodies were immobilized on a QCM for the specific detection of this pesticide at part per billion levels in air. With respect to environmental monitoring. Biosensors reported for detection of environmentally significant metal ions primarily use enzymes or GEMs as recognition elements. In many cases. In addition to biochemical advances for these devices. pesticides account for the greatest number of reports for environmental biosensors. In addition. a number of metal ions inhibited the biosensor response to varying degrees. 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. For several possible reasons. More recently.4-D and paraquat. Recognition elements for these biosensors include the use of enzymes. In the case of the GEM-based biosensors. aldicarb. one of the challenges that face these techniques involves the source of pollution which is often industrial in nature. organophosphorus insecticides/ organophosphate hydrolase) or as inhibitors (e.49 As is the case for other biosensors that detect other environmental pollutants. First. portable and inexpensive instruments became available in the early 1990s which were shown to perform well in field settings. The industrial contamination typically contains a number of industrial compounds from a variety of related and unrelated chemical classes. Examples include: triazines.. a wide variety of antibodies have been developed (some of which are commercially available) toward various classes of insecticides. herbicides. One way to organize this diverse group of techniques is by chemical class (Table 1).
immunochemical recognition of surface antigens and identification of DNA sequences that are unique to the organism of interest. These biosensors are composed of biological assays which have been interfaced with various signal transducers and measure the following parameters: microorganism toxicity. 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. In a recent example of an immunochemical approach. purity. Based on this phenomenon.52 Biosensor methods based on the identification of microorganisms through the detection of unique DNA sequences show significant promise. the evolution of protons. electrochemically lyse the cells. In this respect. biological oxygen demand. 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.g. recently reported micro-fabricated bioelectronic chip technology was used to isolate E. recent reports have shown these enzyme biosensors to be inhibited by compounds from diverse chemical classes. multi-step genetic assays including PCR amplification and hybridization have been incorporated into "biochip" technology. enzyme inhibition. however. in the low ppm range). and measure hybridization of specific target DNA in the lysate to capture probes immobilized to the electrode.23 In addition. arrays of enzymes have been used to screen for various common environmental pollutants. inhibition of Photosystem II. Major metabolic mechanisms monitored include: the consumption of oxygen. and relatively reproducible if source.50 ASV methods are also portable and inexpensive and have detection limits in the low ppb range. however.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. The FPXRF methods are rapid and inexpensive. Although biosensors based on enzyme inhibition are typically demonstrated using compounds in a few specific chemical classes. DNA damage. In a number of cases.. A variety of biosensors have been reported which measure compounds of environmental interest that are toxic to microorganisms.51 An area in which biosensors perhaps show the greatest diversity and potential for development involves the measurement of environmentally significant biological parameters. and the synthesis of bioluminescence enzymes genetically tied to the expression of metabolic indicators. For example. fairly high (e. Detection limits for these techniques are. and identification and enumeration of microorganisms of environmental concern. These assays are typically sensitive to a variety of compounds representing a number of compound classes. Some of the obstacles common to all field analytical methods include: the diversity of compounds and the complexity of matrices in environmental
. 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. technology borrowed from the intense efforts in micro-scale and automated gene sequencing research has application in the environmental monitoring area. Similar to the chemical and immunoassay test kits. simple to execute. and condition of reagents is carefully monitored. These biosensors have focused on two mechanisms. less progress toward practical methods has been achieved. the interface of a biological assay to a signal transducer has been shown to reduce the time and complexity involved with these assays.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. coli cells from blood.established field methods. Field analytical techniques which detect similar metals include Field Portable X-ray Fluorescence (FPXRF) spectroscopy and anodic stripping voltammetry (ASV). 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. the Microtox assay is well characterized. Pattern recognition techniques have been applied to these systems to enable identification of members of specific compound classes. 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.
. because of a variety of obstacles (many unique to the environmental monitoring area).samples. Nevertheless.S. More specific to biosensor technology. Williams. and multi-pollutantscreening. It has been subject to the EPA's peer and administrative review and has been approved for publication. Advances in areas such as toxicity-. biosensors continue to show significant promise for use in environmental monitoring applications. Acknowledgments The authors gratefully acknowledge the editorial assistance of Dr. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use. reversibility and continuous operation may allow biosensor techniques to be incorporated as detectors in chromatographic systems. and the broad range of possible environmental monitoring applications. Due to unique characteristics and flexibility in operational design. For example. Miniaturization. there are a number of areas where the unique capabilities of biosensors might be exploited to meet the requirements of environmental monitoring. Environmental Protection Agency (EPA). classical laboratory-based techniques typically employ extensive extraction and cleanup protocols. these protocols yield little information concerning the potential bioavailability of the pollutant. greater versatility in the area of sample interface would be of considerable value. could widen the potential market and allow these techniques to be more competitive. NOTICE: The U. the introduction (and early successes) of these devices into this commercial market will likely involve narrowly focused applications. Nevertheless. these hurdles include: relatively high development costs for single analyte systems. bioavailability-. Biosensors coupled with a series of extraction methodologies could provide valuable information in this area. the variability in data quality requirements among environmental programs. through its Office of Research and Development (ORD). partially funded the work involved in preparing this article. R. L. 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. Because of the wide variability in environmental matrices. With few exceptions.