Few-Atom Silver Clusters as Fluorescent Reporters - Advanced Fluorescence Reporters in Chemistry and Biology - page 314

(Fig. 3b ). The conversion of red emitters into green emitters with time was
explained as an oxidation reaction confirmed by the addition of extra reductant,
which leads to the transformation of green emitters into red ones [ 48 ].
There are several publications describing the role of DNA bases and base
sequence on the formation of fluorescent silver clusters. The different bases have
different affinity for silver and the different base-silver cluster interaction produces
different emissions. For a given DNA strand, every particular base sequence creates
a different environment for the clusters and consequently a different emission. For
instance, Petty et al. have studied the influence of thymine (T) and cytosine (C)
bases by playing with their combinations in oligonucleotides [ 49 ]. Fluorescent
silver clusters formed in dT 12 and dT 4 C 4 T 4 have similar properties. The emission
intensity increases with pH having a midpoint at pH 9.5, which is close to the p K a of
the N3 of thymine indicating that the deprotonated thymine forms a complex with
the fluorescent silver clusters. In nitrogen atmosphere, nonfluorescent clusters are
formed, whereas the presence of oxygen allows the formation of fluorescent
species, which suggests in this case that nonfluorescent species are reduced while
fluorescent ones are oxidized. A ratio bases:Ag + of 2:1 leads to a maximum in the
emission intensity when using both templates, dT 12 and dT 4 C 4 T 4 , interpreted by the
authors as the same cluster size is stabilized by both oligonucleotides. Silver
clusters show similar excitation maxima, but different Stokes shifts, pointing out
the role of the bases and their influence on the environment of the clusters and
therefore on the optical properties of the clusters. In the case of more cytosine-rich
oligonucleotides such as dC 4 T 4 C 4 similar properties were found but an additional
emitter was formed here, a red emitter.
Dickson et al. carried out a high-throughput analysis of 12-mer DNA strands and
found that the cluster properties are highly sequence-dependent, claiming that
discrete sequences lead to well defined silver clusters sizes and hence to distinct
emission properties ranging from visible to near-IR [ 46 ]. Three long-wavelength
emitters, yellow, red and near-IR, were prepared in oligonucleotides and presented
as good candidates for their use as single molecule biolabels (Fig. 4 ). The synthesis
of each of the emitters in oligonucleotides does not follow a general procedure,
since the optimal synthetic conditions differ for every case, for instance regarding
the use of buffered or unbuffered solutions and pH closer to 5 or to 8. However, the
efforts are rewarded since silver clusters protected by oligonucleotides present
many advantages, especially those emitting in the near-IR. For instance, these
clusters can be excited with low energy, which is beneficial for the photostability
of the clusters and preserves the chemical stability of the scaffold. Moreover,
biological samples have low background fluorescence signals in the near-IR,
providing high signal-to-noise ratio. These silver clusters present large absorption
coefficients and quantum yields exceeding 30% (Table 2 ). Compared to cyanine
dyes, these emitters have higher emission intensities (1,500 and 2,500 counts/s for
cyanine dyes and clusters respectively, Fig. 5 ) and longer lifetimes (decay to 1/ e
emitters in 9 and 580 s respectively) [ 46 ]. They do not blink in relevant timescales
(0.1-1,000 ms) and the dark-state lifetime of 30

s can be reduced to less than 10 s

m m

by increasing the intensity of the excitation [ 19 ].