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 32

nanotoxicity issue is resolved. There also exist many different instances where QDs
have been applied to biological systems. Although most of these studies are proof-
of-principle, they underline the growing potential of these reagents. QDs are very
attractive candidates for bioanalytical applications that can either exploit their
potential for spectral multiplexing, do not require strong signal amplification or
that rely on NIR fluorescence.
Apart from the advantageous properties discussed above, QDs could have a
bright future especially in the field of near infrared fluorescence imaging (NIRF),
because they show high fluorescence quantum yields in the 650-900 nm window,
may have adequate stability, good water solubility as well as large 2P action cross
sections as desired for deep tissue imaging. The only clinically approved organic
NIR fluorophore ICG (Table 1 ) suffers from a very low fluorescence quantum yield
[ 31 , 78 ], limited stability, and binding to plasma proteins. Other organic fluoro-
phores for the NIR range (with pending approval like, e.g., Cy5.5, 0.28 in

phosphate buffer solution) still possess small quantum yields compared to NIR-
emitting QDs such as CdTe (Table 1 ). In addition, QDs are attractive candidates for
the development of multifunctional composite reporters for the combination of two
or more bioanalytical imaging techniques, such as NIRF/magnetic resonance imag-
ing (MRI) [ 146 ].
Despite the promising possibilities offered by the different types of nanoparticles,
their routine use is still strongly limited by the very small number of commercially
available systems and the limited amount of data on their reproducibility
(in preparation, spectroscopic properties, and application) and comparability (e.g.,
fluorescence quantum yields, stability) as well as on their potential for quantifica-
tion. To date, no attempt has yet been published comparing differently functiona-
lized nanoparticles from various sources (industrial and academic) in a Round Robin
test, to evaluate achievable fluorescence quantum yields, and batch-to-batch varia-
tions for different materials and surface chemistries (including typical ligands
and bioconjugates). Such data would be very helpful for practitioners and would
present the first step to derive and establish quality criteria for these materials.
In addition to the practical questions linked to the application of nanoparticles,
fundamental questions such as the elucidation of quantum dot lifetime character-
istics, e.g., for lifetime multiplexing [ 147 ] and combined lifetime and spectral
multiplexing in conjunction with the development of suitable algorithms for data
analysis and for time resolved FRET have to be addressed. Other current limitations
include the comparatively large size of nanoparticles. The ligand-controlled size of
nanoparticles does not only affect their FRET efficiency but could also sterically
hamper access to cellular targets and could affect the function of labeled biomole-
cules. So far, nanoparticles for bioanalytical applications can only be prepared on a
very small scale. Commercialization of, e.g., NIR QDs requires more systematic
studies of nanoparticle nucleation and growth. This involves the control of nano-
particle surface chemistry, and the establishment of functionalization protocols. A
first useful step in this direction would be the design of a reliable and reproducible
test for the quality of surface coatings, i.e., the degree of perfection of the surface
ligand shell, as this is the most crucial parameter affecting the spectroscopic and

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