10.1002/anie.201801653
Angewandte Chemie International Edition
COMMUNICATION
Inspired by the aggregation-induced emission (AIE)
phenomenon,[9] the desired fluorogenic Ag+ detection
accompanied by an aggregation process, can be rationally
achieved. Unlike conventional dyes, a typical AIE dye has a
flexible structure and emits faintly when molecularly dissolved. In
the aggregated state, the dye emits strongly. The fluorescence in
responses to molecular states has been explored as a general
way to design fluorogenic probes for metal ions, small molecules
and enzymes.[10] In this work, we designed a tetrazole-tagged AIE
luminogen, TPE-4TA, to sense Ag+ in a fluorogenic manner. Many
1/2H-tetrazole compounds have been known to ‘precipitate’ out
Ag+ from a solution efficiently.[11] The resulting insoluble tetrazole-
silver complexes are infinite metal-coordination polymers, with
silver atoms binding in mono-, bi- or tri-dentate formats to the N
atoms of the tetrazole ring.[11] For TPE-4TA, the anion tetrazolate
acts as Ag+-specific targeting group to trigger aggregation, while
the core tetraphenylethene (TPE) endows aggregation-induced
fluorescence (Fig. 1b).
Figure 2. Characterization of the fluorogenic aggregation process. (a)
Fluorescence of TPE-4TA (5 µM) by stepwise addition of Ag+ in DI-water; (b)
Plot of intensity at 504 nm in (a) as a function of [Ag+]/[TPE-4TA]; (c) DLS and
(d) SEM characterization of the fluorescent solution; (e) Fluorescence of TPE-
4TA (5 µM) mixed with metal ions (20 µM, including Na+, K+, Ca2+, Mg2+, Mn2+
,
TPA-4TA (X=H) was synthesized in a 21% total yield over three
steps (Fig. S1). TPA-4TA (X=H) showed modest solubility (~10-4
M) in DI-water and was highly aqueous soluble (>0.05 M) when
formulated in the salt form (X=Na+).
Zn2+, Cu2+, Fe2+ Fe3+, Ni2+, Au+, Pb2+, Cd2+, Pd2+ and Co2+) in phosphate
aqueous solution (pH 7.4); (f) Fluorescence of TPE-4TA (10 µM) at 504 nm
against the pH values of the phosphate aqueous solution in the absence or
presence of Ag+ or Hg2+ (10 equiv.) respectively. Excitation: 368 nm.
When molecularly dissolved in aqueous solution, TPE-4TA is
non-fluorescent, attributing to free rotational motions of phenyl
rings which activated non-radioactive pathways.[9] As expected,
the addition of Ag+ lighted up a fluorescence peaked at 490530
nm by 368 nm excitation. The turn-on response was instant. A
stoichiometric study indicated that the fluorescence increased by
the step-wise addition of Ag+ (Fig. 2a). By plotting the intensity at
504 nm against the [Ag+]/[TPE-4TA] ratio, a linear relationship
with R2 = 0.996 was established in the range of 4015000 nM for
[Ag+] (Fig. 2b). With a further increase of [Ag+], the fluorescence
reached a steady plateau. To be noticed the linearity and the 1:1
mole ratio of Ag+ to the tetrazolate group on approaching the
plateau indicated that the sensing process correlated well with a
stoichiometric metal-organic coordination-driven assembly.[12]
The limit of detection for Ag+ was estimated to be 2.3 nM, i.e., 0.25
µg/L (S/N = 3, n = 12), which is about 40 times better than the
colorimetric method using dithizone (10 µg/L), and is amongst that
of the best fluorescence-based methods.[13]
The fluorogenic Ag+ detection was next evaluated in the presense
of interfering factors including different metal ions, pH, and
common silver-binding reagents. Most metal ions cannot turn on
the fluorescence of TPE-4TA (Fig. 2e and Fig. S7). Hg2+ lighted
up a weak fluorescent signal, which may result from a similar
Hg2+-tetrazolate coordination.[11b] The Ag+/Hg2+ coordination-
induced fluorogenic detection were then evaluated at different pH
values (Fig. 2f). At pH≤4, the tetrazole moiety of a pKa 45 was
mainly in the protonated form.[14] The protonated TPE-4TA likely
aggregated in aqueous solution and thus elicited blue emission
peaked at 470 nm. At pH≥5, the probe was fully dissolved and
gave a dark background. The Ag+-induced fluorescence thus
became significant. At pH>6, the maximum turn-on response kept
at a stable level, indicating that it offered a robust Ag+
quantification method from neutral to highly basic solutions. In the
case of Hg2+ detection, the fluorogenic response was only
observed within pH 58.
The fluorogenic sensing was accompanied by formation of nano-
sized aggregates. The dynamic light scattering (DLS) analysis
suggested that aggregates were formed with a good batch-to-
batch reproducibility in solutions and the size distributions varied
with the mole ratio of TPE-4TA to Ag+ (Table. S1). As an example,
the mixture ([TPE-4TA], 5 µM; [AgNO3], 25 µM) gave a size of
efficient diameter ~50 nm (Fig. 2c). After evaporation of the
solvent, nano-sized particles (d = 530 nm) were also observed
under electron microscopy (Fig. 2d and Fig. S4). Element
mapping confirmed the clustered nanoaggregates consisting of
Ag, N and C atoms (Fig. S6). The fluorescent aggregates ([TPE-
4TA], 5 µM; [Ag+], 150 µM) had a negative zeta potential of -15
to -40 mV, likely due to the anionic tetrazolate groups of TPE-4TA
being exposed at the aggregate-solution interface. The colloid
gave no noticeable precipitation on-shelf for over three months.
The aggregates are hardly re-dissolved in common solvents,
including DMSO, even at reflux conditions, likely attributing to the
strong tetrazolate-silver interactions. These results supported a
good colloidal stability of these fluorescent aggregates.
The interference tests included reagents used in silver stains
and amino acids which are reported to host Ag+ in the in-gel
staining of proteins (Fig. S8). All of these interfering reagents did
not trigger the fluorescence turn-on of TPE-4TA. When Ag+ were
bound to the reagents beforehand, TPE-4TA molecules were also
able to snatch off (or co-aggregated with) Ag+ from all the pre-
formed Ag+-bounded complexes included in the test and lighted
up, except Ag+-bounded cysteine complexes, likely due to the
strong thiol-Ag+ bonding.[7] The results suggested the feasibility to
combine the Ag+ sensing with biological silver staining methods.
Following the flow chart in Fig. 3a, we achieved an efficient
fluorescent silver staining of proteins after SDS polyacrylamide
gel electrophoresis (SDS-PAGE). For comparison, gels were also
stained by conventional silver nitrate stain[8] or SYPRO Ruby
fluorescent stain.[15] In the tests, a commercialized protein ladder
consisting of 14 proteins served as a library of samples. In the
fluorescent silver-AIE stain, all the protein bands were clearly
visible under a UV lamp, correlating well with the bands stained
This article is protected by copyright. All rights reserved.
Xie, Sheng
