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 6

medical diagnosis and imaging. Increasingly used chromophore-doped particle
labels (4) and materials based on conjugated polymers [ 22 ] are beyond the scope
of this review. The optical properties of such chromophore-doped particles are
controlled by the parent chromophores or dopants, and the surface modification and
labeling strategies presented here for the QDs labels can also be typically applied to
these systems.

2 Properties of Molecular and Nanoparticular Labels
and Reporters

2.1 Spectroscopic Properties

The relevant spectroscopic features of a chromophore include the spectral position,
width (FWHM: full width at half height of the maximum), and shape of its
absorption and emission bands, the Stokes shift, the molar absorption coefficient
(

F F ).
The Stokes shift equals the (energetic) difference (in frequency units) between the
spectral position of the maximum of the lowest energy absorption band (or the first
excitonic absorption peak in the case of QDs) and the highest energy maximum of
the luminescence band. This quantity determines the ease of separation of excitation
from emission and the efficiency of emission signal collection. It can also affect the
degree of spectral crosstalk in two- or multi-chromophore applications such as
FRET or spectral multiplexing and the amount of homo-FRET (excitation energy
transfer between chemically identical chromophores) occurring, e.g., in chromo-
phore-labeled (bio)macromolecules that can result in fluorescence quenching at
higher labeling densities [ 23 , 24 ]. The product of

e M ), and the photoluminescence efficiency or fluorescence quantum yield (

e M at the excitation wavelength

( F F , that is termed brightness ( B ), presents a frequently used measure for

the intensity of the fluorescence signal obtainable upon excitation at a specific
wavelength or wavelength interval and is thus often used for the comparison of
different chromophores. A value of B below 5,000 M 1 cm 1 renders a label
practically useless for most applications [ 25 ]. Further exploitable chromophore
properties include the luminescence or fluorescence lifetime (

l ex ) and

t F ), that determines,
e.g., the suitability of a label for time-gated emission [ 4 ], time-resolved fluores-
cence immunoassays [ 26 - 28 ], and lifetime multiplexing [ 5 ], and the emission
anisotropy or fluorescence polarization. The latter quantity, that presents a measure
for the polarization of the emitted light, reflects the rotational freedom or mobility
of a chromophore in the excited state and provides information on the orientation
distributions of fluorescent moieties or on the size of molecules (hydrodynamic
radius) via the measurement of the rotational correlation time [ 4 ]. This can be
exploited, e.g., for the study of enzyme activity, protein-peptide and protein-DNA
interactions, and ligand-receptor binding studies in homogeneous solution.