BIOSENSORS FOR ENVIRONMENTAL MONITORING - II

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 co-contaminants 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

^{Source: U.S EPA}