Chemistry Reference
In-Depth Information
interactions are to be investigated and the distinct, specific, and nonspecific path-
ways of QDs into cells as well as their intracellular fate have been studied [
104
].
Extracellular targeting is typically accomplished through QD functionalization with
specific antibodies to image cell-surface receptors [
39
] or via biotin ligase-cata-
lyzed biotinylation in conjunction with streptavidin-functionalized QDs [
105
]. The
HaloTag method has just recently been combined with QDs allowing much sim-
plified protocols for cell surface labeling [
106
]. Due to their larger size, the
intracellular delivery of QDs is much more challenging compared to small organic
dyes, and accordingly, the state-of-the-art of delivery of QDs into cells and internal
labeling strategies are far behind. Although there exists no general protocol to
achieve this so far, individual solutions have been reported, that, however, need to
be empirically established in each case. Moreover, there are reports on successful
cell labeling via microinjection [
36
], electroporation [
107
], nanoinjection [
108
],
mechanochemical [
109
], or nonspecific or receptor-mediated endocytosis [
1
,
86
].
As has been recently shown, the labeling specificity and efficiency can be improved
with specifically functionalized QDs [
98
]. More sophisticated tools are needed for
labeling of specific intracellular structures outside endocytosed vesicles or imaging
of cellular reactions in the cytoplasm or the nucleus with QDs. Only a few
successful studies have been published with QDs targeted to specific cellular
locations so far [
110
]. More research is required in this respect to establish suitable
strategies. Here, ligand design also plays a crucial role for the design of stable and
small hydrophilic QDs, to minimize undesired nonspecific interactions, and to
provide the basis for further functionalization [
111
]. Positively charged peptide
transduction domains (PTDs) such as TAT (Tat peptide from the cationic domain
HIV-1 Tat), polyarginine, polylysine, and other specifically designed cell
penetrating peptides (CPPs), can be coated onto QDs to effect their delivery into
cells [
112
]. It remains to be shown whether other recently developed cell
penetrating agents like a synthetic ligand based on an
N
-alkyl derivative of
3
-cholesterylamine termed streptaphage designed for efficient uptake of strepta-
vidin conjugates by mammalian cells [
113
] or polyproline systems equipped with
cationic and hydrophobic moieties [
114
] can be adapted for QD delivery.
b
3.3
Interactions Between Chromophores and their
Microenvironment
One of the unique features of fluorophores is the general sensitivity of their
spectroscopic properties to temperature and dye local environment, i.e., matrix
polarity and proticity (hydrogen bonding ability), viscosity, pH, and ionic strength,
and also to the presence of, e.g., surfactants or serum proteins in the case of in vivo
studies as well as fluorescence quenchers such as oxygen or conjugated (bio)
molecules. Such factors need to be considered for most applications of fluorescence
ranging from analyte sensing to the characterization of cell function and behavior.
Absolute quantification from measured fluorescence signals typically requires the
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