CHAPTER 17 • Luminescence for the Analysis of Organic Compounds in Natural Waters
407
of bound antibody is detected by applying a suitable potential to the electrode, in order to obtain the luminollabel electro chemiluminescence. An innovative aspect of this
device with respect to other 2,4-D immunosensors reported in the literature is the novel
immobilization procedure used to obtain the 2,4-D antigen-coated glassy carbon electrode, which allowed multiple (up to 50 times) regenerations of the immunosensor.
This immunosensor was able to detect as low as 0.21lg rl of free 2,4-D, which is close
to the accepted level in drinking water in the European Union (O.lllg rl).
A chemiluminescent competitive multianalyte immunoassay based on a combination of mono- and polyclonal antibodies was developed for the detection of three crossreacting s-triazine pesticides (atrazine, terbuthylazine and ametryn) in water samples
(Samsonova et al. 1999). Detection of single s-triazines using immunological methods is a difficult task, because even monoclonal antibodies against such small analytes
are often unable to efficiently discriminate between structurally similar molecules. The
simultaneous use of three polyclonal and two monoclonal antibodies with different
specificities towards s-triazines allowed for the development of a multianalyte assay
in which antibody cross-reactivity is exploited to identify and quantify the three
analytes. The antibodies were immobilized in separate wells of an eight -well microtitre
strip, and horseradish peroxidase, which was detected with the enhanced chemiluminescence reaction, was used as a label for antigens. Measurement of the chemiluminescence intensities was performed by using a small, battery-powered portable
luminometer, which is of practical importance from the on-site monitoring point of
view. The data obtained with the chemiluminescent immunoassays were processed
using a neural network, and the best parameters for the correct identification of an
individual s-triazine in a mixture and the estimation of its amount were found. When
this assay was applied to environmental samples containing various s-triazine mixtures, it was able to correctly classify the analytes in most cases, suggesting that it can
be proposed as an alternative field test for multianalyte environmental monitoring.
In all the luminescent immunoassays described above, luminescence measurements
were performed using conventional photomultiplier tube-based luminometers or timeresolved fiuorometers. Imaging devices, which are able not only to measure the intensity but also to evaluate the spatial distribution of the luminescence emission from a
sample, represent a significant improvement in luminescence measurement. These
devices allow for the simultaneous measurement of the luminescent signals from a
whole 96- or 384-well microtitre plate, thus being more rapid than conventional
luminometers that measure light output well by well or strip by strip. They also represent the detectors used for the development of multiarray affinity devices, in which
several different biospecific reagents are immobilized in an array arrangement on a
suitable surface. This sensor design allows for the parallel processing of many immunoassays, allowing simultaneous determination of different analytes in the same
sample, using reagents volumes much smaller than those needed to perform separate
immunoassays for each analyte of interest. Quantitation of the analyte content of the
sample is performed by using image analysis techniques: the localization of the CL
signal on the target surface and its intensity are related to the identity and the concentration of the analyte, respectively.
The development of a parallel affinity sensor array based on chemiluminescent
labels for the detection of environmental contaminants in water has been reported
(Weller et al. 1999). The required reagents (antibodies or haptens) were immobilized
407
of bound antibody is detected by applying a suitable potential to the electrode, in order to obtain the luminollabel electro chemiluminescence. An innovative aspect of this
device with respect to other 2,4-D immunosensors reported in the literature is the novel
immobilization procedure used to obtain the 2,4-D antigen-coated glassy carbon electrode, which allowed multiple (up to 50 times) regenerations of the immunosensor.
This immunosensor was able to detect as low as 0.21lg rl of free 2,4-D, which is close
to the accepted level in drinking water in the European Union (O.lllg rl).
A chemiluminescent competitive multianalyte immunoassay based on a combination of mono- and polyclonal antibodies was developed for the detection of three crossreacting s-triazine pesticides (atrazine, terbuthylazine and ametryn) in water samples
(Samsonova et al. 1999). Detection of single s-triazines using immunological methods is a difficult task, because even monoclonal antibodies against such small analytes
are often unable to efficiently discriminate between structurally similar molecules. The
simultaneous use of three polyclonal and two monoclonal antibodies with different
specificities towards s-triazines allowed for the development of a multianalyte assay
in which antibody cross-reactivity is exploited to identify and quantify the three
analytes. The antibodies were immobilized in separate wells of an eight -well microtitre
strip, and horseradish peroxidase, which was detected with the enhanced chemiluminescence reaction, was used as a label for antigens. Measurement of the chemiluminescence intensities was performed by using a small, battery-powered portable
luminometer, which is of practical importance from the on-site monitoring point of
view. The data obtained with the chemiluminescent immunoassays were processed
using a neural network, and the best parameters for the correct identification of an
individual s-triazine in a mixture and the estimation of its amount were found. When
this assay was applied to environmental samples containing various s-triazine mixtures, it was able to correctly classify the analytes in most cases, suggesting that it can
be proposed as an alternative field test for multianalyte environmental monitoring.
In all the luminescent immunoassays described above, luminescence measurements
were performed using conventional photomultiplier tube-based luminometers or timeresolved fiuorometers. Imaging devices, which are able not only to measure the intensity but also to evaluate the spatial distribution of the luminescence emission from a
sample, represent a significant improvement in luminescence measurement. These
devices allow for the simultaneous measurement of the luminescent signals from a
whole 96- or 384-well microtitre plate, thus being more rapid than conventional
luminometers that measure light output well by well or strip by strip. They also represent the detectors used for the development of multiarray affinity devices, in which
several different biospecific reagents are immobilized in an array arrangement on a
suitable surface. This sensor design allows for the parallel processing of many immunoassays, allowing simultaneous determination of different analytes in the same
sample, using reagents volumes much smaller than those needed to perform separate
immunoassays for each analyte of interest. Quantitation of the analyte content of the
sample is performed by using image analysis techniques: the localization of the CL
signal on the target surface and its intensity are related to the identity and the concentration of the analyte, respectively.
The development of a parallel affinity sensor array based on chemiluminescent
labels for the detection of environmental contaminants in water has been reported
(Weller et al. 1999). The required reagents (antibodies or haptens) were immobilized