A rhodamine/BODIPY-based fluorescent probe for the differential detection of Hg(ii) and Au(iii)

Article abstract of DOI:10.1039/c3cc48436j

We described the design and synthesis of a molecular sensor based on a rhodamine/BODIPY platform that displayed differential fluorescence responses towards Hg²⁺ and Au³⁺ and demonstrated its utility in intracellular ion imaging.

Full text of DOI:10.1039/c3cc48436j

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DOI: 10.1039/C3CC48436J
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A Rhodamine/BODIPYꢀbased fluorescent probe for the differential
detection of Hg(II) and Au(III)††
‡
‡
a
Erman Karakuꢁ, Muhammed Üçüncü and Mustafa Emrullahoğlu,*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
We described the design and synthesis of a molecular sensor
extremely difficult to construct a molecular sensor that
based on a rhodamine/BODIPY platform that displayed 50 differentiates between the Hg and Au species. To our
2+
3+
2
+
3+
differential fluorescence responses towards Hg and Au
and demonstrated its utility for intracellular ion imaging.
knowledge, in literature there is only one example of a
3
+
2+
fluorescent probe that can differentiate between Au and Hg .
That fluorescent probe, reported by Dong et al., operates through
a single emission mode and the differentiation is highly
1
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In recent years, the construction of fluorescent molecular sensors
2d
for the detection of metal ion species has received a great deal of 55 dependent on the sensing conditions.
1
attention. To date a large number of molecular sensors have been
designed and developed, the majority of which are singleꢀion
responsive and present no great challenge to researchers.
Compared to singleꢀion responsive molecular sensors, however,
Obviously, there is a high demand for the development of
molecular sensors that can differentiate multiple analytes of a
2
+
3+
similar chemical nature (e.g. Hg and Au ). In addition, smallꢀ
molecule fluorescent sensors allowing the intracellular
the construction of multiꢀion responsive molecular sensors with 60 monitoring of multipleꢀions via differential responses are of high
2
multiple emission modes are extremely challenging. Molecular
sensors displaying differential responses towards multiple ions
are indispensable for designing molecular logic gates and
necessity for realꢀtime cell imaging studies.
Herein, we present the design, synthesis, spectral properties,
and cell imaging studies of RhSꢀBOD, a new “turnꢀon” multiꢀ
3
,4
2+
3+
molecular keypad lock devices.
The challenge of multiple analyte recognition presents several 65 differentiated on the basis of distinct fluorescence responses.
fluorescent probe that allows the Hg and Au species to be
detection strategies. Incorporating multiple binding motifs onto a
single sensing molecule, or alternatively, combining different
transducing units (chromophore/fluorophore), allows for rapid
RhSꢀBOD constitutes a boronꢀdipyrromethene dye (BODIPY)
and a spirocyclic rhodamine dye covalently attached to each
other. RhSꢀBOD was prepared in a reasonable yield (25%,
overall) by the synthetic route outlined in Scheme S1 (see ESI†)
2
access to molecular sensors with multiple emission modes.
1
13
We envisaged that incorporating both a chemosensor and a 70 and the structure of RhSꢀBOD was confirmed by HꢀNMR, Cꢀ
chemodosimeter onto a single molecule could provide a suitable
sensing platform for the differential detection of metal species.
On the basis of this hypothesis, we constructed a molecular
sensor possessing two different fluorophore units chemically
integrated with each other. Both fluorophore units were elegantly
designed to be nonꢀemissive (i.e., “off”) in their initial states and
are expected to turn on respectively in response to the metal
species of interest. To the best of our knowledge, molecular
sensors based on this novel approach haven’t been covered in
literature.
NMR, and mass spectroscopy.
Et
2
N
O
NEt
2
Et
2
N
O
NEt
2
F
F
F
N
F
N
N
B
N
N
B
N
N
N
7
8
5
0
S
O
ex=525 nm
λ
no emission
no emission
RhS-BOD
RhO-BOD (control probe)
Scheme 1 Structure of RhSꢀBOD and RhOꢀBOD
2
+
3+
Ionic species of mercury (Hg ) and gold (Au ) share several
similarities in terms of coordination properties. As both metal
species show high affinities to thiols, they have the potential to
interact with sulfur bearing biomolecules such as enzymes,
proteins, and DNA. As a result, these metal species can disturb a
Importantly, the novel molecular sensor, RhSꢀBOD, was
designed in such a way that both of the fluorophore units are nonꢀ
emissive before the addition of any metal species. As can be seen
from the structure of the probe shown in Scheme 1, the C=N
functionality on the BODIPY core diminishes the BODIPY
emission potentially because of a nonꢀradiative deactivation
process of the excited state through rapid isomerization of the
C=N group. Similarly, the rhodamine fluorophore is nonꢀ
emissive because the rhodamine dye exists in the ring closed
isomeric form.
8
5
5,6
series of cellular processes that lead to toxicity in humans. In
recent years, a variety of wellꢀdesigned fluorescent probes highly
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3+
specific for Hg and Au ions have been developed, most of
which are built on the exploitation of the thiophilic and
90
7,8
alkynophilic behavior of these metal species.
Despite
impressive advances, many of those probes suffer from cross
affinity. Because of their similar coordination properties, it is thus
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DOI: 10.1039/C3CC48436J
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+
The sensing behavior of RhSꢀBOD towards the addition of
different metal species was studied using UVꢀVis and
fluorescence spectroscopy. As shown in Fig.S1 (see ESI†), the
The probe was highly selective towards Hg and showed no
3
+
spectral response to any other metals ions, except for Au ions.
Upon the addition of Au , the nonꢀemissive probe solution
3+
UV/Vis spectrum of free RhSꢀBOD (CH CN/HEPES 1:1, pH 55 immediately turned to a strong green emissive solution that could
3
5
0
5
0
7.0) exhibits a single absorption band at 527 nm, which belongs
to BODIPY core. As the rhodamine core is in the ring closed
isomeric form, we expect no absorption bands for the rhodamine
derivate. However, the addition of Hg (1 equiv.) to RhSꢀBOD
led to the appearance of a new strong absorption band at 554 nm, 60 as controlled using TLC. The formation of a green emissive
be easily monitored by the naked eye under the UV lamp. A
green emission was a clear evidence for the existence of an
emissive BODIPY derivative. This suggestion was also supported
2
+
3+
from the outcome of the reaction of RhSꢀBOD mediated by Au ,
1
1
2
which was assigned to a ring opened rhodamine derivative.
compound, BODIPYꢀAL, could be easily monitored from the
The fluorescence spectra of RhSꢀBOD displayed a similar
spots on the TLC plate (Fig. S21, ESI†).
The fluorescence sensing behavior of RhSꢀBOD towards Au
2
+
3+
behavior towards the addition of Hg (Fig.1). Initially, when
excited at 525 nm there were no emission bands in the
was comprehensively surveyed upon excitation at 470 nm and
fluorescence spectrum of RhSꢀBOD. However, upon the addition 65 525 nm. As shown in Fig.S11 (ESI†), the fluorescence spectrum
2
+
3+
of Hg , a new emission band with a maximum at 585 nm
appeared and the intensity of this band gradually increased with
of (RhSꢀBOD/Au , (1:1)) displays an emission band at 506 nm
when excited at 470 nm, a characteristic emission band of a
BODIPY fluorophore. On the other hand, the same probe solution
2
+
increasing concentration of the Hg (Fig.1). The increase in
emission intensity showed a linear relationship towards the
3+
(RhSꢀBOD/Au ) when exited at 525 nm displays a different
2+
addition of Hg in the range of 0ꢀ0.3 ꢁM. The minimum amount 70 emission band at 585 nm, which is supposed to belong to the ring
2
+
of Hg was evaluated to be 8.0 nM under these conditions
Fig.S10, ESI†). The response of the probe towards the addition
opened isomer of the rhodamine core (Fig.S13b, ESI†). The
fluorescence emission intensity at both wavelengths increased
(
2+
3+
Hg was immediate and the emission intensity became saturated
linearly with an increasing concentration of Au over a wide
2
+
when 1 equiv. of Hg were added, creating an enhancement
factor of over 50ꢀfold.
concentration range (Fig.2 and Fig.S14). The response of RhSꢀ
3+
75 BOD towards Au was fast (<1 min.) and the emission intensity
3+
1
1
1
4
2
0
8
6
4
2
0
became saturated when 2 equiv. of Au was added. In addition,
the detection limit measured at both wavelengths was at nM level
(
65 nM (λ =585 nm), and 10 nM (λ =506 nm)).
em em
2+
Hg
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0
500
50
00
350
00
50
200
50
100
(a)
(b)
7
0
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4
60
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20
10
0
3+
3
Au
3+
Au
2
540
560
580
600
620
640
660
680
700
Wavelength (nm)
2
5
0
1
85
2+
Fig. 1 a) Fluorescence spectra of RhSꢀBOD (5.0 ꢁM) + Hg (0.05 to
5.0 ꢁM) in 1:1 CH CN/HEPES buffer (pH=7.0), (λex: 525 nm).
50
2
3
0
480
500
520
540
560
580
600
620
640
540
560
580
600
620
640
660
680
700
Wavelength (nm)
Wavelength (nm)
2
+
3
As expected, in the presence of Hg , RhSꢀBOD displayed the
optical features of the rhodamine chromophore. During the
3+
Fig. 2 Fluorescence titration spectra of RhSꢀBOD (5.0 ꢁM) + Au (0ꢀ
1
3
0.0 equivalent) in 1:1 CH CN/HEPES buffer (pH=7.0) a) λex=470 nm, b)
2
+
process of adding Hg , no other accompanying emission bands
were noticed in the emission spectrum that might belong to the
BODIPY dye, indicating that the BODIPY was still in a sleep
(“off”) mode.
90
λex=525 nm.
3
+
The fluorescence response of RhSꢀBOD toward Au (1
equiv.) in the presence of the other metal ions (10 equiv.) was
explored in order to assess the possible interference by other
metal ions. As shown in Fig.S16 (see ESI†), the tested metal ions
35
40
45
50
2
+
To check the reversibility of the Hg sensing process, the
2+
highly emissive probe solution preꢀtreated with Hg (RhSꢀ
3+
2+
95
displayed no interference with the detection of Au ions.
BOD/Hg ) was subsequently treated with a cyanide ion source
KCN or NH CN) (Fig.S7, ESI†). The probe solution
2+
As discussed before, the addition of Hg to RhSꢀBOD
triggers a spiroꢀring opening reaction and results in the formation
of a highly emissive rhodamine derivative. Throughout the
(
4
immediately lost its color and its emission, thus showing the
sensing process is based on a reversible metalꢀligand coordination
2+
2
+
addition of Hg to RhSꢀBOD, the BODIPY core continues to be
00 nonꢀemissive because the C=N moiety was still preserved.
process. The binding stoichiometry of the Hg / RhSꢀBOD
association was determined by Job’s plot from both the UVꢀVis
absorption and fluorescence data (Fig.S9, ESI†). Both of the plots
1
3+
However, the addition of Au to the probe solution preꢀtreated
2+
2+
2
+
with Hg (RhSꢀBOD/Hg ) resulted in an immediate change in
revealed a 1:4 ratio of the Hg ions associating with each
molecule of the probe.
the emission color from orange to green. Evidently, in the
2+
3+
presence of Hg and Au , RhSꢀBOD hydrolyzes to give a green
05 emissive BODIPY derivative, BODIPYꢀAL, which dominates
the emission color of the probe solution (Scheme 2).
We further investigated the selectivity profile of RhSꢀBOD in
response to other metal ions. For all other metal cations, such as
1
2
+
+
2+
2+
2+
+
2+
+
+
2+
2+
2+
Cu , Ag , Zn , Pb , Ni , Na , Mg , Li , K , Pd , Fe , Co ,
2+
2+
2+
3+
3+
RhOꢀBOD, the oxygen bearing derivative of RhSꢀBOD, was
used as the control probe to clarify the nature of the sensing
Cd , Ca , Ba , Fe and Cr , no detectable change in the
emission intensity for RhSꢀBOD were observed (Fig.S5, ESI†).
2
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DOI: 10.1039/C3CC48436J
3
+
process. Under the same sensing conditions, RhOꢀBOD
displayed no response towards any metal species, indicating the 45 ions, and a single emission mode for the detection of Hg ions.
BOD exhibits a dual emission mode for the detection of Au
2
+
indispensable role of the sulfur functionality in the detection of
both metal species.
We thank Đzmir Institute of Technology (ĐZTECH) and
TUBĐTAK for financial support.
5
Notes and references
a
Department of Chemistry, Faculty of Science, Đzmir Institute of
5
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0
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0
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0
5
0
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0
Technology (IZTECH), Urla 35430, Đzmir, Turkey. Eꢀmail:
mustafaemrullahoglu@iyte.edu.tr,
†
Electronic Supplementary Information (ESI) available: Synthesis and
characterization of all compounds, and all data for UVꢀVis and
fluorescence titrations. See DOI: 10.1039/b000000x/
‡ These authors contributed equally to this work
1
0
†
† Dedicated to the memory of Prof. Dr. Ayhan S. Demir (1950ꢀ2012)
1
.
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Kim and J. Yoon, Chem. Rev., 2012, 112, 1910; (d) G. Ulrich, R.
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1
5
(e) N. Boens, V. Leen and W. Dehaen, Chem. Soc. Rev., 2012, 41,
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2+
Scheme 2 Response of RhSꢀBOD towards the addition of Au and Hg
2.
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We next assessed the ability of RhSꢀBOD to operate within
living organisms. To our delight, RhSꢀBOD showed the same
sensing behavior in living cells. Human A549 lung
adenocarcinoma cell lines were incubated with the probe (5 ꢂM)
for 40 min. and then followed by the addition of metal species.
With the aid of fluorescence microscopy, the differential turnꢀon
response towards Au and Hg was clearly monitored in the
cells (Fig.3). The images taken before and after the addition of
the metal species displayed a distinct fluorescence change
consistent with the results observed in the solution.
2
0
(
(
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3
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Fig. 3 a) Fluorescence image of A549 cells treated with only RhSꢀBOD
95
3+
(
(
(
(
5ꢁM); b) image of cells treated with probe (5ꢁM) and Au (5ꢁM)
3+
λ
λ
ex=470 nm); c) image of cells treated with probe (5ꢁM) and Au (5ꢁM)
ex=525 nm); d and h) Image of cells treated with DAPI for 15 min
control); d) merged image of frame b and d; f) merged image of frame c
2+
and d; g) image of cells treated with probe (5ꢁM) and Hg (5ꢁM) 100
(λex=525 nm); i) merged image of frame g and h.
To close, we have presented the synthesis, spectral properties,
and biological application of RhSꢀBOD, a new type of
2+
3+
105
fluorescent probe for the differential detection of Hg and Au .
2+
3+
4
0
This novel probe features excellent selectivity for Hg and Au .
2
+
3+
Detection of Hg and Au is realized through two distinct
2
+
fluorescence changes resulting from Hg /ligand coordination or
from the hydrolysis of the C=N moiety catalyzed by Au . RhSꢀ
3+
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3