Chemistry Reference
In-Depth Information
of thumb, for molecular fluorophores, a high
e
M
value does not allow obtaining a
long emission lifetime. The fluorescence lifetimes of organic dyes, that typically
display allowed transitions between singlet states, are in the order of about 5 ns for
vis emitters and
1 ns for NIR fluorophores (Table
1
). This is too short for efficient
temporal discrimination of short-lived background fluorescence and scattered exci-
tation light. The most prominent exceptions used for bioanalytical applications are
the vis-emitting acridone dyes displaying fluorescence lifetimes in the order of
5-20 ns, that, however, require short-wavelength excitation (excitation, e.g., at
405 nm, emission at ca. 440-500 nm) [
66
] and the only recently reported UV-
absorber and vis-emitter DBO (2,3-diazabicyclo[2.2.2]oct-2-ene) with a lifetime of
ca. 300 ns in aerated water [
67
]. Due to the forbidden nature of the electronic
transitions involved, in addition to its short wavelength absorption and emission
(absorption and emission maximum at ca. 365 nm and ca. 430 nm, respectively, in
water), DBO shows very low molar absorption coefficients which reduces the
overall sensitivity. Nevertheless, advantageous for the vast majority of organic
dyes can be their typically mono-exponential decay kinetics (in a homogeneous
microenvironment), that can be exploited for the straightforward dye identification
from measurements of fluorescence lifetimes [
68
].
In comparison to conventional organic dyes shown in Fig.
2
, MLC like Ru(II)
complexes and lanthanide complexes show attractive long emission lifetimes in
conjunction with mono-exponential decay kinetics, that render them superior to
organic chromophores in this respect [
53
]. This provides the basis for the straight-
forward temporal discrimination of shorter-lived autofluorescence and scattered
excitation light from label emission with the aid of time-gated measurements,
thereby enhancing the sensitivity [
69
], and enables lifetime-based sensing. Due to
their long lifetimes in conjunction with the straightforward excitation and emission
in the visible or rarely, even in the NIR, Ru(II) complexes are common probes and
labels in lifetime-based assays and (bio)sensors and in fluorescence polarization
assays [
70
]. As the emission lifetimes of Ru(II) complexes are typically oxygen-
sensitive, these species present the most commonly used lifetime-based oxygen
sensors [
71
,
72
]. The exceptionally long luminescence lifetimes of the lanthanide
chelates (typically monoexponential decay kinetics), detailed in the previous sec-
tion, can, but must not necessarily be, oxygen-dependent [
10
,
58
]. This, in combi-
nation with “shielding ligands” like certain chelates or cryptates and narrow
emission bands makes these lanthanide fluorophores ideal candidates for all appli-
cations of time gated emission (e.g., DELFIA technology in fluoroimmunoassays)
and as energy donors in homogeneous time-resolved fluorescence assays [
10
,
73
].
Moreover, their distinct sharp emission bands can be exploited for spectral multi-
plexing applications [
74
].
Attractive for the use of QDs are their long lifetimes (typically 5 ns to hundreds
of nanoseconds), compared to organic dyes, that are typically insensitive to the
presence of oxygen. In conjunction with time-gated measurements, this provides
the basis for enhanced sensitivity [
69
]. This property can be also favorable for time-
resolved applications of FRET. The complicated size-, surface-, and wavelength-
dependent, bi- or multi-exponential QD decay behavior (Fig.
2
) can complicate
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