Chemistry Reference
In-Depth Information
extent by both the sensitivity of the chromophore's spectroscopic properties to the
environment and fluorophore photochemical and thermal stability [
116
]. Organic
dyes have been successfully applied for quantification in a broad variety of in vitro
fluorescence applications, but reports of analyte quantification with QD labels are
still rare. In the case of organic dyes, dye stability can be critical for all fluorescence
applications using intense light sources such as fluorescence microscopy or for
methods like in vivo fluorescence imaging, where lasers are used as excitation light
sources and measurements are performed over several days. This long term known
stability issue has been partly overcome by the synthesis of more stable dyes, see
section on thermal and photochemical stability [
94
,
134
]. Nevertheless, there is still
considerable interest in the development of brighter and more stable dyes. Of
interest are also comparative stability studies of bioanalytically relevant dyes and
labels under application-relevant conditions providing all the experimental para-
meters used including the excitation intensity or light flux reaching the sample as a
prerequisite for data reliability and comparability. In the case of generally more
photostable QDs, the recently reviewed problems still arise like photobrightening,
blinking, bluing, and also bleaching [
82
]. QD photobrightening, i.e., the increase in
emission efficiency with continuous illumination, can hamper direct quantification
and may render the use of reference standards necessary [
135
]. This QD-specific
effect is most likely related to light-induced surface passivation. The size of this
phenomenon, that often reveals a dependence on excitation wavelength and is
typically most pronounced for UV excitation [
136
], is expected to depend on the
quality of the initial QD surface passivation (i.e., the saturation of surface defects by
ligands or a passivating shell), and also on shell quality, thereby principally
reflecting the accessibility of the QD core. This can be thus exploited as a screening
test for QD quality [
80
]. In addition, the luminescence quantum yield of QDs can be
concentration-dependent [
5
], thereby yielding concentration-dependent signal fluc-
tuations, that hamper quantification. This effect depends on the bonding nature of
organic ligands to the surface atoms of nanocrystals and the related ligand- and
matrix-dependent adsorption-desorption equilibria which have been only margin-
ally investigated [
137
-
139
]. This can be critical for all applications where the
initially applied concentration of QD labels and probes changes during analysis,
especially in the case of QDs capped and stabilized with weakly bound ligands such
as many monodentate compounds. The latter processes can also result in concen-
tration-dependent fluorescence quantum yields, especially for weakly bound
ligands.
For single molecule spectroscopic applications, chromophore blinking (see
Table
1
) can be problematic. This phenomenon, that is often related exclusively
with QDs, but also occurs for organic dyes, implies that a continuously illuminated
chromophore emit detectable emission only for limited times, interrupted by dark
periods during which no emission occurs. This can be a significant disadvantage of
otherwise very attractive QDs as can be the blinking of organic dyes [
140
]. For
example, QD blinking has been reported to affect the results from bioaffinity
studies [
141
]. Another aspect that might influence the usability of QDs for quantifi-
cation lies in the fact that not all QDs in a set of QDs luminesce [
142
]. For
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