BIOAFFINITY BASED BIOSENSORS 4 ENVIRONMENTAL MONITORING

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.