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Chapter 14 : Bioreporters, Biosensors, and Microprobes
This chapter stresses on techniques that are significantly different from other molecular techniques in that they are suitable for complex environments inhabited by a diverse collection of bacteria. While there are several bioreporter genes that might be used, bioreporters that make use of light for bioreporting have significant advantages. The use of either bioluminescent or fluorescent bioreporters is now an established technology, and the uses have expanded greatly over the years. Bioreporters for bioluminescent and fluorescent gene products are described in this chapter. The advantages of bioluminescent bioreporters lie primarily in the relative ease of light measurement. A discussion of the weaknesses of bacterial bioreporters and the means by which these techniques may be improved is provided in the chapter. There are now several fluorescent proteins that can be used as bioreporters in bacterial cells, but the first successful one was green fluorescent protein (GFP). The development of microprobes for the examination of microbial environments has proceeded rapidly thanks to innovative construction techniques. Microprobes have been described for ammonium, nitrate, oxygen, denitrification (by nitrous oxide production), and sulfate reduction. In surface plasmon resonance (SPR), the surface of the waveguide is coated with a thin layer of gold. Multigene analysis will have a substantial impact on the understanding of genetic control. In the area of biosensors, the trend toward miniaturization and commercialization will continue. It is expected that fieldable biosensors will have a great impact on biowarfare monitoring and long-term ecological studies.
Genes and chemical intermediates involved in the bioluminescence reaction of Vibrio fischeri.
The chromophore of Aequorea GFP. Amino acids 65, 66, and 67 of GFP form a cyclical structure by an autocatalytic reaction. This chromophore is the source of the bright fluorescence seen with this protein. The dotted lines delineate the separate amino acids in the chromophore.
Cross-section of a typical microprobe. Reprinted from reference 55 with permission from the publisher.
Biosensors. (Left) A generalized scheme for a biosensor. Interaction of the target analyte with the biological component results in a signal, which is transmitted to the transducer. The transducer senses the signal and converts it to an electrical signal. (Right) A DNA biosensor. The hybridization event brings the labeled DNA in contact with the transducer, a CCD camera. The CCD camera detects beta emission from 32P decay and converts it to an electrical signal.
The principle of surface plasmon resonance. The sensing surface is on the opposite side of the metal film from the illuminated surface. Here an antibody-antigen-type biosensor is shown, with the sensing surface incorporated into a flow cell. The light source can be either polarized or laser light or an electron stream. The photodetector must be able to record subtle changes in light intensity.
Commercially available fluorescent proteins