Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison - Advanced Fluorescence Reporters in Chemistry and Biology - page 18

nanoparticle stabilization in aqueous solution can be accomplished by coating the
particles with sterically demanding surface ligands such as polyethyleneglycol
(PEG) [ 76 ].
Electrostatically stabilized nanoparticles are usually much smaller than steri-
cally stabilized ones. Since this is favorable for most applications in the life
sciences, electrostatic stabilization strategies are recommended if small nanoparti-
cles in low ionic strength buffers are to be used. However, these particles tend to
aggregate in solutions of high ionic strength such as biological matrices. Sterically
stabilized nanoparticles are mostly too large to enter cells, but are less likely to
aggregate. A compromise can be reached by using smaller, but nevertheless still
bulky, charged polyelectrolytes such as polyethyleneimine (PEI) [ 40 ], or an addi-
tional amphiphilic inorganic shell like silica [ 41 , 42 ] which can be further functio-
nalized using standard silica chemistry.
It is difficult to predict the effect of surface functionalization on the optical
properties of nanoparticles in general. Surface ligands have only minor influence on
the spectroscopic properties of nanoparticles, the properties of which are primarily
dominated by the crystal field of the host lattice (e.g., rare-earth doped nanocrys-
tals) or by plasmon resonance (e.g., gold nanoparticles). In the case of QDs, the
fluorescence quantum yield and decay behavior respond to surface functionaliza-
tion and bioconjugation, whereas the spectral position and shape of the absorption
and emission are barely affected.
2.3 Thermal and Photochemical Stability
Aside from spectroscopic considerations, one of the most important features of a
fluorescent label or reporter is its stability under application-relevant conditions.
This includes typically used solvents such as buffers, cell medium, or other
supports, the presence of oxygen and typical reagents such as dithiothreitol
(DTT), common temperatures as well as typical excitation wavelengths, and exci-
tation light fluxes over routinely used detection times. The latter parameter is also
linked to the detection method employed with certain fluorophores being suitable
only for specific applications. In any case, chromophore stability is of crucial
relevance for the achievable sensitivity and limit of detection, especially in single
molecule experiments, and for contrast in fluorescence imaging. Blinking, that is
the interruption of the photoluminescence of continuously illuminated QDs or
organic dyes by dark periods, is relevant for single molecule applications and is
briefly discussed in section 3.7 .
Organic dyes like fluorescein and TRITC and the majority of NIR fluorophores
suffer from poor photostability [ 77 ]. In addition, many NIR dyes, such as clinically
approved indocyanine green (ICG) reveal poor thermal stability in aqueous solution
[ 78 ]. Moreover, the presence of ozone can result in dye decomposition as observed
for Cy5 [ 79 ]. In the last years, many organic dyes like the Alexa dyes have been