408
A. Roda . P. Pasini . M. Guardigli
as spots on a glass slide, obtaining a biochip with an array containing up to 1 600 spots.
The glass slide was inserted in a flow cell in which the immunoreactions took place
and, after addition of the chemiluminescent substrate, the CL signal was acquired by
means of a CCD imaging device. The biochip was used to develop model systems for
the simultaneous detection and quantitation of triazine, trinitrotoluene and 2,4-D in
water samples based on immunoassays either in direct (antibody-immobilized) or
indirect (hapten-immobilized) formats. Both formats proved to be able to detect and
quantitate the analytes, offering the possibility of many regeneration cycles of the
biochip. This device could thus represent a potential analytical tool for performing
multiple analyses on one sample in parallel, also including the identification of the
analytes.
The development and optimization of a chemiluminescent ELISA for the simultaneous determination of 2,4-D in multiple samples, using innovative solid supports
represented by gold-coated surfaces and glass capillaries with CCD imaging of the
chemiluminescent signal has been reported (Dzgoev et al.1997). The authors observed
that such solid supports offered advantages with respect to other supports used in the
development of multiarray devices such as the "flat-wells" obtained by thick-film technology' but the analytical sensitivity was still lower than that of the single sample determination assay.
The perception of immunoassay has changed in the past decade from considering
it to be an environmental analytical alternative to accepting it as a reliable quantitative method. Luminescent immunoassays represent a valuable alternative to the conventional radiometric or colourimetry ones. They are more sensitive than colourimetry
immunoassays, and can be as sensitive as radioimmunoassays, without all the problems related to storage, handling and disposal of radioactive materials. This feature,
combined with the rapidity of response and the possibility of performing simultaneous
analysis of several samples in a 96- or 384-well microtitre plate format, makes these
assays suitable for the development of HTS analytical methods and for extensive automation. New refinements of immunoassay technology, such as biochip-based methods and multianalyte immunoassays, while still in research, will certainly take their
place among other environmental monitoring techniques of the future.
17.3
Luminescent Recombinant Cell-Based Biosensors in
Environmental Analysis
A different bioanalytical approach has been made possible by the use of recombinant
DNA techniques in analytical chemistry, which led to the development of luminescent
whole-cell biosensors. Luminescence-based reporter genes have been used to develop
efficient bioassays for the specific detection of several inorganic and organic compounds, which are applied especially in the environmental analysis field (Kohler et al.
2000). These biosensors are based on the ability of genetically engineered microorganisms (bacteria and yeast) or mammalian cells to emit visible light in response to
specific substances. Such transformed cells are constructed by recombinant DNA techniques, using a plasmid vector containing the luminescent reporter gene under transcriptional control of a specific gene sequence, whose expression is regulated very
precisely. The presence of the analyte induces the expression of the specific gene se-
A. Roda . P. Pasini . M. Guardigli
as spots on a glass slide, obtaining a biochip with an array containing up to 1 600 spots.
The glass slide was inserted in a flow cell in which the immunoreactions took place
and, after addition of the chemiluminescent substrate, the CL signal was acquired by
means of a CCD imaging device. The biochip was used to develop model systems for
the simultaneous detection and quantitation of triazine, trinitrotoluene and 2,4-D in
water samples based on immunoassays either in direct (antibody-immobilized) or
indirect (hapten-immobilized) formats. Both formats proved to be able to detect and
quantitate the analytes, offering the possibility of many regeneration cycles of the
biochip. This device could thus represent a potential analytical tool for performing
multiple analyses on one sample in parallel, also including the identification of the
analytes.
The development and optimization of a chemiluminescent ELISA for the simultaneous determination of 2,4-D in multiple samples, using innovative solid supports
represented by gold-coated surfaces and glass capillaries with CCD imaging of the
chemiluminescent signal has been reported (Dzgoev et al.1997). The authors observed
that such solid supports offered advantages with respect to other supports used in the
development of multiarray devices such as the "flat-wells" obtained by thick-film technology' but the analytical sensitivity was still lower than that of the single sample determination assay.
The perception of immunoassay has changed in the past decade from considering
it to be an environmental analytical alternative to accepting it as a reliable quantitative method. Luminescent immunoassays represent a valuable alternative to the conventional radiometric or colourimetry ones. They are more sensitive than colourimetry
immunoassays, and can be as sensitive as radioimmunoassays, without all the problems related to storage, handling and disposal of radioactive materials. This feature,
combined with the rapidity of response and the possibility of performing simultaneous
analysis of several samples in a 96- or 384-well microtitre plate format, makes these
assays suitable for the development of HTS analytical methods and for extensive automation. New refinements of immunoassay technology, such as biochip-based methods and multianalyte immunoassays, while still in research, will certainly take their
place among other environmental monitoring techniques of the future.
17.3
Luminescent Recombinant Cell-Based Biosensors in
Environmental Analysis
A different bioanalytical approach has been made possible by the use of recombinant
DNA techniques in analytical chemistry, which led to the development of luminescent
whole-cell biosensors. Luminescence-based reporter genes have been used to develop
efficient bioassays for the specific detection of several inorganic and organic compounds, which are applied especially in the environmental analysis field (Kohler et al.
2000). These biosensors are based on the ability of genetically engineered microorganisms (bacteria and yeast) or mammalian cells to emit visible light in response to
specific substances. Such transformed cells are constructed by recombinant DNA techniques, using a plasmid vector containing the luminescent reporter gene under transcriptional control of a specific gene sequence, whose expression is regulated very
precisely. The presence of the analyte induces the expression of the specific gene se-