Few-Atom Silver Clusters as Fluorescent Reporters - Advanced Fluorescence Reporters in Chemistry and Biology - page 319

Fig. 8 Emission from formaldehyde-fixed NIH3T3 cells loaded with 100 mM silver nitrate for
20 h. (a) Fluorescence image; the inset is the intensity profile along the line drawn across the cell.
(b) Merge of (c) and (d). (c) Emission from RNASelect fluorescence ( green channel ); (d)
Emission from silver clusters ( red channel ); Scale bar 30 m m[ 57 ]

silver ions to produce well dispersed and stable solutions of protein-conjugated
fluorescent silver clusters [ 58 ]. The protein-protected silver clusters show emission
at 680 nm when excited at 500 nm. The molar ratio of CHT:Ag + :BH 4 was
1:10:100, where the large excess of NaBH 4 provokes two adverse effects in the
system. A first consequence of the excess of NaBH 4 , is that the reductant cleaves
disulfide bonds inducing denaturation of the protein. Partial reconstitution of the
protein was achieved with efficient oxidation, by dialyzing the sample against water
of pH 8.0-8.5 for 24 h in aerated conditions. The functional integrity of the protein
was confirmed by studying the enzymatic activity on a substrate Ala-Ala-Phe 7-
amido-4-methyl coumarin. The enzymatic activity of CHT-Ag was retarded by
2.8 times compared to the activity of native CHT. The origin of the retard might be
the reduction and oxidation processes since reconstituted CHT presents similar
delays. This indicates that silver clusters are not located in the enzymatic active site
of CHT. A second event caused by the excess of sodium borohydride is the increase
in the pH and a subsequent aggregation of the protein. However, the aggregation
might be reversible by lowering the pH with an exhaustive dialysis against pure
water.
Prior to these findings, in 2005, fluorescent silver clusters in combination with a
fluorophore, thioflavin T (ThT), were already used to image proteins [ 31 ]. Silver
clusters were prepared by photoreduction at

330 nm of solutions containing ThT

and Ag + . The emission band centered at

450 nm grew by fivefold when ThT:Ag +
was 100:1 and by 50-fold when the ratio was 1:1 (Fig. 9a , b), suggesting the enor-
mous effect of silver. The authors proposed that the emission observed is originated
by both, intrinsic formation of fluorescent silver clusters (when the samples were
irradiated at 500 W/cm 2 ) and by metal-enhanced fluorescence of ThT (with irradi-
ation at 1 W/cm 2 ). Amyloid fibrils stained with ThT-Ag clusters present a time-
dependent increase of fluorescence with no photobleaching after 24 h of illumina-
tion at 475 nm (500 W/cm 2 ) in contrast to ThT-stained fibrils, which have a rapid
decay of fluorescence (Fig. 9c ). With this method, the authors managed also to
image a single fibril and claim that the luminescence produced by ThT-Ag clusters
is at least 100-fold higher than the luminescence reported in photoreduced silver
clusters formed in water solution with dendrimers [ 16 ]orinAg 2 O films [ 9 ].