Optical enzyme-based sensors specificity

Different types of organisms such as daphnia, mussels, algae, and fish have been extensively incorporated in toxicity tests for water assessment systems [65], Most of these assays are developed as test systems with few as laboratory-based sensor systems. Membranes with their active enzyme system have also been implemented in the development of toxicity kits and sensors. An example is the MitoScan Kit (Harvard BioScience, Inc., Holliston, MA), which uses fragmented inner mitochondrial membrane vesicles isolated from beef heart (EPA, 2005 [9]). The submito-chondrial particles contain complexes of enzymes responsible for electron transport and oxidative phosphorylation. When specific toxins are in the sample, the enzyme reactions are slowed or inhibited, and these are monitored spectophotometrically at 340 mn. This is still in a bioassay test kit format but may be developed to optical sensor system. 

Another principal that has been evaluated for the construction of optically based sensors is the use of chemiluminescence. In these cases an enzyme system specific for the analyte are coupled to reactions that produce light through chemiluminescence. In principle, systems of this type could be very sensitive, first of all due to the amplification factor of enzyme reactions, and secondarily because fluorescence measurements are among the most sensitive of optical techniques. 

Widespread interest has arisen in the potential of LB films as biosensors because many believe that the incorporation of biological molecules such as enzymes will lead to novel devices. Some are exploring the deposition of biologically active molecules onto the gate electrodes or oxides of field-effect transistors, but optical sensors, probably based on fiber optics, are the most favored technique. In all cases, the aim is to couple the specificity of inter-... 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

This chapter describes a broad range of optical biosensor technologies, based on interchromophore interactions and the super-quenching phenomenon. Several specific biosensor schemes are described that are able to sense the activity of kinase, protease, lipase, and cellulase enzymes. Although the systems are disparate, the underlying sensor mechanisms are related, in that they all... 

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