BIOCATALYSIS BASED BIOSENSORS 4 ENVIRONMENTAL MONITORING

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 phenols^11-12 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 K_m and V_max 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 K_m and V_max 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 µg/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 antigen-tracer 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., < class="epaFooterText">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.

Source: U.S EPA