1 research outputs found
Dye Tool Box for a Fluorescence Enhancement Immunoassay
Immunochemical
analytical methods are very successful in clinical
diagnostics and are nowadays also emerging in the control of food
as well as monitoring of environmental issues. Among the different
immunoassays, luminescence based formats are characterized by their
outstanding sensitivity making this format especially attractive for
future applications. The need for multiparameter detection capabilities
calls for a tool box of dye labels in order to transduce the biochemical
reaction into an optically detectable signal. Here, in a multiparameter
approach each analyte may be detected by a different dye with a unique
emission color (covering the blue to red spectral range) or a unique
luminescence decay kinetics. In the case of a competitive immunoassay
format for each of the different dye labels an individual antibody
would be needed. In the present paper a slightly modified approach
is presented using a 7-aminocoumarin unit as the basic antigen against
which highly specific antibodies were generated. Leaving the epitope
region in the dyes unchanged but introducing a side group in positon
3 of the coumarin system allowed us to tune the optical properties
of the coumarin dyes without the necessity of new antibody generation.
Upon modification of the parent coumarin unit the full spectral range
from blue to deep red was accessed. In the manuscript the photophysical
characterization of the coumarin derivatives and their corresponding
immunocomplexes with two highly specific antibodies is presented.
The coumarin dyes and their immunocomplexes were characterized by
steady-state and time-resolved absorption as well as emission spectroscopy.
Moreover, fluorescence depolarization measurements were carried out
to complement the data stressing the different binding modes of the
two antibodies. The binding modes were evaluated using the photophysics
of 7-aminocoumarins and how it was affected in the respective immunocomplexes,
namely, the formation of the intramolecular charge transfer (ICT)
as well as the twisted intramolecular charge transfer (TICT). In contrast
to other antibody–dye pairs reported a distinct fluorescence
enhancement upon formation of the antibody–dye complex up to
a factor of 50 was found. Because of the easy emission color tuning
by tailoring the coumarin substitution for the antigen binding in
nonrelevant position 3 of the parent molecule, a dye tool box is on
hand which can be used in the construction of competitive multiparameter
fluorescence enhancement immunoassays (FenIA)