Few-Atom Silver Clusters as Fluorescent Reporters - Advanced Fluorescence Reporters in Chemistry and Biology - page 320

Fig. 9 Epifluorescence microscopy images of the amyloid fibrils of PrP 90-231 (1 M), stained at

room temperature with (a) ThT alone (10 m M) (exposure time 1.6 s), and (b) with preformed ThT-
Ag clusters (exposure time 0.02 s). ThT-Ag clusters were preformed by irradiation of aqueous
solutions of ThT (10 m M)/AgNO 3 (1 m M) at 312 nm for 3 min. Scale bars ¼ 10 m m. (c)
Photobleaching kinetics of the fibrils stained with ThT ( black line ) vs. photoactivation kinetics
of the fibrils stained with ThT-Ag clusters ( red line ). Data collected from a 5 m m 5 m m area and
normalized to the intensity measured at zero time [ 31 ]

m

Taking advantage of the well-established affinity of silver cations for proteins as
shown before in the case of nucleolin and due to the importance of nucleolin in
actual investigations [ 59 , 60 ], synthetic peptides derived from nucleolin were used
as scaffolds for the formation and protection of fluorescent silver clusters [ 57 ]. The
earliest peptide mentioned in literature as template for the formation of fluorescent
silver clusters was the histidine-rich AHHAHHAAD, but synthesis and optical
properties were only briefly described since that was not the main topic of the
publication [ 61 ]. The first detailed study of peptides as scaffold [ 57 ] describes
oligopeptides that contains 15-18 amino acids, including the most abundant amino
acids in nucleolin, like glutamic acid (E), lysine (K), and aspartic acid (D) as well as
cysteine (C), which is minor in nucleolin but it is known to bind silver [ 62 ]. The
resulting peptide allows formation of silver clusters by reducing a silver solution
(0.22 mM in peptide, 0.37 mM in AgNO 3 ) with sodium borohydride (18 mM). But
this peptide was not very effective as a stabilizer since it offers to the clusters a
chemical lifetime of only 3 days. To overcome this problem other peptides were
created. The tested peptides contain 18 amino acids, including D, C and K but also
histidine (H) and asparagine (N) or leucine (L). The peptide capable of stabilizing
silver clusters during 5 weeks had the sequence “HDCNKDKHDCNKDKHDCN”,
and the silver clusters produce fluorescence at 630 nm when excited at 400 nm.
Giving a closer look to the composition of this peptide, one can notice that it is far
from the composition of nucleolin, for instance, it contains three histidines (H) and
three cysteines (C) which are minor constituents in nucleolin ( 0.1% each) and

also three asparagines (N) ( 3% in nucleolin) [ 63 ]. Although this peptide and its

use as scaffold in the synthesis of silver clusters represent a great advance in the
field, the different composition compared to nucleolin, clearly points to the lack of
understanding of the basics of cluster synthesis and calls for more exhaustive
research to identify both, the mechanism of formation of silver clusters in peptides
and the requirements for their stabilization.
A remarkable contribution regarding the use of peptide-protected fluorescent
silver clusters in biological systems came with the insertion of fluorescent silver