Few-Atom Silver Clusters as Fluorescent Reporters - Advanced Fluorescence Reporters in Chemistry and Biology - page 317

Additional studies confirmed the sequence dependency on the generation of
fluorescent silver clusters when using scaffolds of 19 bases, with a shape of a
hairpin or containing two single-stranded (C or G rich) sequences bound by two
base pairs (Table 2 ). In this case, the authors claim that based on mass spectra, silver
binds with comparable affinities to chemically similar sites on C and G bases. The
reason for the differences in the emission peaks might be the unique local environ-
ment offered by different base stacking between the two strands or by distinct of the
strand, since the incorporation of silver in the loop of the hairpin is more difficult
than in the open geometry. The discrepancy regarding the silver-oligonucleotide
affinity described by various groups could be also explained considering the
different relative concentrations used in the experiments. Authors claiming higher
affinity of silver for cytosine have used 0.5 silver per nucleotide, whereas authors
claiming similar affinities for cytosine and guanine have used 0.29 silver per
nucleotide and the preferences of silver might be different at different ratios [ 43 ,
44 , 51 , 52 ].
The fluorescence properties and stability of silver cluster in hairpins were
reported to be related to the number of cytosines in the loop (varying from 3 to 12
cytosines) [ 53 ]. However, as a general rule, red emitters were brighter than green
emitters. Further specific studies on the 9C loop show that the red emitter corresponds
to Ag 13 :DNA and the green emitter correlates with Ag 11 :DNA. A great advantage of
silver clusters encapsulated in hairpins is that they are bright enough to be imaged by
using an epifluorescence microscope.
The high fluorescence quantum yield of DNA-encapsulated silver clusters

( 30%) [ 46 , 50 ] makes them good candidates as fluorophores in cell imaging.

Aiming the application for cell imaging, the DNA-encapsulated silver clusters were
prepared as described before but using a longer strand, a 24-mer oligocytosine
(dC 24 ), linked to a protein, avidin. The presence of avidin does not affect the chemical
or photophysical stability of the clusters (emission maximum at 634 nm, excitation at
580 nm, fluorescence lifetime of about 2.86 ns and remarkable brightness).
First results in this research showed that biotinylated fixed cells became fluores-
cent upon staining with avidin-dC 24 -Ag clusters (Fig. 7a , b). However, in the case
of living cells, loading with avidin-dC 24 -Ag clusters produces bright spots, indicat-
ing endocytosis (Fig. 7c ). The results were quite different when using an antibody,
heparin sulfate (HS) instead of avidin. HS-dC 24 -Ag clusters can penetrate the cells
when incubated at 37 C showing fluorescent nuclei (Fig. 7d-f )[ 50 ].

F F >

4.2 Proteins and Peptides

A commonly used staining method for the cell nucleolus is based on silver
nanoparticles [ 54 ]. The proteins of the nucleolus, such as nucleolin, are known to
have high affinity to silver ions due to their amino-terminal domain. Subsequent
reduction leads to the formation of the silver nanoparticles stain. In spite of all the
efforts, a general and definitive conclusion regarding the attraction between silver