Chemistry Reference
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respect to small fluorescent labels and reporters, here, lanthanide chelates are to be
favored, yet depending on the respective application, they may encounter problems
with respect to accomplishable sensitivity. In the case of organic dyes, an increas-
ingly common multiplexing approach implies the use of donor-acceptor dye combi-
nations (so-called tandem dyes or energy-transfer cassettes) that exploit FRET to
increase the spectral separation of absorption and emission and thus to tune the Stokes
shift [
6
]. A typical example of a four color label system consists of a 5-carboxy-
fluorescein (FAM) donor attached to four different fluorescein- and rhodamine-type
acceptors (e.g., JOE, TAMRA, ROX) via a spacer such as an oligonucleotide. FRET
dye-labeled primers and FRET-based multiplexing strategies are the backbone of
modern DNA analysis enabling e.g. automated high speed and high throughput DNA
sequencing and the development of robust multiplex diagnostic methods for the
detection of polymerase chain reaction (PCR) products. With suitably designed
systems, even intracellular dual FRET measurements using a single excitation
wavelength were described [
123
]. Although broadly used, the limitations of organic
dyes for FRET applications discussed in the previous section nevertheless also
hamper the efficiency of these FRET-based multiplexing systems. This can be
overcome by multiwavelength excitation using different lasers, which is becoming
affordable due to progress in laser technology. This approach has been already
successfully used in flow cytometry with the independent detection of 12 different
analytes being reported using organic labels and state-of-the art cytometers [
126
].
The unique flexibility in excitation and the very narrow and symmetric emission
bands simplifying color discrimination render QDs ideal candidates for spectral
multiplexing at a single excitation wavelength. Accordingly, there are many reports
of the use of QDs as labels inmultiplexed assays or immunohistochemistry or imaging
applications requiring multiplexing [
6
,
39
]. Although rarely discussed, despite their
very attractive spectroscopic features, the simultaneous detection and quantification of
several different analytes with QD labels can also require spectral decomposition
procedures of measured signals, as has been recently demonstrated for a multiplexed
fluoroimmunoassay for four different toxins [
127
]. The importance of spectral unmix-
ing for QD multiplexing was recently evaluated and demonstrated [
128
].
3.5.2 Lifetime Multiplexing
Multiplexing can also be performed by making use of the fluorophore-specific decay
behavior, measured at a single excitation and single emission wavelength, to dis-
criminate between different fluorophores. This approach requires sufficiently differ-
ent lifetimes of the chromophores. With a single exception, lifetime multiplexing, as
well as a combined spectral and lifetime discrimination have only been realized with
organic chromophores [
129
]. This is most likely, related to the fact that the need for
monoexponential decay kinetics was often assumed for this application. Meanwhile,
successful lifetime multiplexing has been also reported both for a mixture of a QD
and an organic dye and for a mixture of two different QDs [
5
] despite the multi-
exponential decay kinetics of the QDs. This may pave the road for future
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