Highly sensitive and selective colorimetric and off-on fluorescent probe for Cu2+ based on rhodamine derivative

Article abstract of DOI:10.1039/c0ob00553c

A new probe for Cu²⁺ based on the Cu²⁺- induced reversible ring-opening mechanism of the rhodamine spirolactam was described. It displayed a highly selective and sensitive "turn-on" fluorescent and colorimetric response toward Cu

Full text of DOI:10.1039/c0ob00553c

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Highly sensitive and selective colorimetric and off-on fluorescent probe for
Cu2+ based on rhodamine derivative†
Chunwei Yu,a,c Jun Zhang,b Rui Wanga and Lingxin Chen*a
Received 9th August 2010, Accepted 22nd September 2010
DOI: 10.1039/c0ob00553c
A new probe for Cu2+ based on the Cu2+- induced reversible
ring-opening mechanism of the rhodamine spirolactam was
described. It displayed a highly selective and sensitive “turn-
on” fluorescent and colorimetric response toward Cu2+.
Copper plays a critical positive role in several biological processes.1
However, at certain high concentrations, copper is one of the most
toxic and dangerous heavy metal elements to some organisms such
as bacteria and viruses,2 which is also confirmed to be harmful
to humans, in that it can cause neurodegenerative diseases (e.g.,
Alzheimer’s and Wilson’s diseases) probably by its involvement in
the production of reactive oxygen species.3 Consequently, a great
effort has been devoted to the development of efficient and selective
Scheme 1 Synthesis of probe 1.
methods to assess copper ions in cells and organisms.
A pH titration experiment was first evaluated as shown in Fig. S4
(ESI†), the absorbance of the probe-copper complex displayed
a plateau in the pH range from 4.0 to 8.0, and the maximum
absorbance toward the Cu2+ was obtained under pH 6.0. In the
view of sensitivity and the speed time, in our experiment, pH 6.0
was chosen as optimum experimental condition for environmental
examples. Therefore, further UV/vis and fluorescent studies
were carried out in methanol/HEPES mixed buffer solution
(methanol–water = 8/2, pH 6.0, 0.02 M HEPES).
The fluorescence spectra of 1 in the presence of different
concentrations of Cu2+ in 20% (v/v) water–methanol solution
(0.02 M HEPES, pH 6.0) were recorded (Fig. 1). Like most
of the spirocycle rhodamine derivatives,6 the free 1 displayed
a very weak fluorescence, which indicated that the spirolactam
form (Fig. 2 (bottom)) was the predominant species. When Cu2+
was added to the buffer solution of 1, a significant fluorescence
intensity with an emission maximum at 580 nm increased in a Cu2+
concentration-dependent way, which indicated the opened-ring
Fluorescent probes for Cu2+ have been extensively explored since
they allow nondestructive and prompt detection by a simple fluo-
rescence enhancement (turn-on) or quenching (turn-off) response.
Because of paramagnetic nature of Cu2+, most of the reported Cu2+
probes exhibit “on-off” signals.4 In contrast, only few examples
of “off-on” type probes are reported.5 However, in terms of
sensitivity and selectivity concerns, probes exhibiting fluorescence
enhancement upon Cu2+ complexation are favored over those
showing fluorescence quenching under Cu2+ binding. Among
the reported ‘turn-on’-type Cu2+ probes, few have nanomolar
sensitivity.5a,5b,5c Thus, it is of great interest to design and synthesis
of fluorescent probes with Cu2+ induced highly sensitive and “turn-
on” fluorescence signals.
Rhodamine dyes have been employed extensively in the con-
jugation with biomolecules owing to their excellent fluorescence
properties such as long absorption and emission wavelength,
large absorption coefficient and high fluorescence quantum yield.
Recently, various rhodamine-based turn-on fluorescent probes for
metal ions have been reported.6 The sensing mechanism of these
probes is based on the change in structure between spirocyclic
and open-cycle forms. Inspired by this platform, we envisioned a
new rhodamine-based probe 1 (Scheme 1). It showed a reversible
“turn-on” fluorescent response for Cu2+ in aqueous solution with
remarkably high sensitivity and selectivity.
Synthesis of probe 1 is shown in Scheme 1. Reaction of
rhodamine B with NH2NH2 and then glyoxal afforded 2, which
further reacted with N2S2 to give probe 1 in 80% yield.
aKey Laboratory of Coastal Environmental Processes, Yantai Institute of
Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003,
China. E-mail: lxchen@yic.ac.cn; Fax: +86 535 2109130; Tel: +86 535
2109130
Fig. 1 Emission spectra of 1 (1.0 mM) in the presence of various
concentrations of Cu2+ in 20% (v/v) water–methanol solution (0.02 M
HEPES, pH 6.0). [Cu2+]: (a) 0 to (j) 6.0 mM. Inset: Linear fluorescence
intensity (F/F0) of 1 (1.0 mM) upon addition of Cu2+ (0–0.9 mM). The
response (F) is normalized to the emission of the free probe 1 (F0).
bSchool of Tropical and Laboratory Medicine, Hainan Medical University,
Haikou 571101, China
cGraduate University of Chinese Academy of Sciences, Beijing 100049, China
† Electronic supplementary information (ESI) available: Supplementary
data. See DOI: 10.1039/c0ob00553c
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Fig.
2
(top) Reversible titration response of
1
to Cu2+ in
methanol-HEPES buffer (0.02 M, pH 6.0) (8 : 2, v/v): (a) 1 (10 mM);
(b) 1 (10 mM) with Cu2+ (50 mM); (c) 1 (10 mM) with Cu2+ (50 mM) and
then addition of EDTA (0.1 mM); (d) 1 (10 mM) with Cu2+ (50 mM) and
EDTA (0.1 mM) and then addition of 0.2 mM Cu2+. (bottom) Proposed
binding mechanism of Cu2+ with 1.
form of 1 became the main species in the examined solution, and
also a highly delocalized p-conjugated structure of 1 was formed
(Fig. 2 (bottom)). Furthermore, the F/F0 was well proportional
to the amount of Cu2+ (5.0 ¥ 10-8-9.0 ¥ 10-7 M) with a good linear
correlation (R = 0.9993). The detection limit was 3 nM (based on
S/N = 3, inset of Fig. 1). The result showed that the probe 1 was
capable of detecting both qualitatively and quantitatively of Cu2+.
The method of continuous variation (Job’s method) was used
(Fig. S1, ESI†) to determine the stoichiometry of 1–Cu2+ complex.
As expected, the result indicated that a 1 : 1 stoichiometry of Cu2+
to 1 in the complex, which was also supported by the Benesi–
Hildebrand method (Fig. S2, ESI†).7 The formation of a 1 : 1
complex was further confirmed by ESI(+)-MS analysis: an ethanol
solution containing 1 and 1 equiv. of Cu2+ (Fig. S3, ESI†) showed
a strong peak at m/z 738.06, assigned to [Cu2+ + 1 - H+]. The
association constant K was determined from the slope to be 1.7 ¥
105 M-1, corresponding to a stronger binding capability toward
Cu2+ in comparison with a tren/dansyl-appended rhodamine
based FRET probe for Cu2+ (with a K value of 7 ¥ 103 M-1),6d
or a rhodamine spirolactam derivative-based probe for Cu2+ (with
a K value of 2.08 ¥ 104 M-1).6n
To validate the selectivity of 1 in practice, some other metal ions,
such as alkali or alkaline-earth metals (Na+, Mg2+ and Ca2+) and
heavy and transition metal ions (Pb2+, Ni2+, Co2+, Zn2+, Cd2+, Ag+,
Hg2+, Cr3+ and Mn2+) were added to the solution of 1 under the
same conditions (Fig. 3). The various metal ions did not induce
any obvious color change and fluorescent enhancement, only Fe3+
caused the ignored absorption and fluorescence change. For Cu2+,
the F/F0 value was almost 100-fold, while the values for other
metal ions were less than 10-fold. From the above experimental
results, it suggested that 1 was a Cu2+-selective probe in aqueous
Fig. 3 (a) Fluorescent emission changes of 1 (1.0 mM) in the presence of
different metal ions (50 mM) in 20% (v/v) water–methanol solution (0.02
M HEPES, pH 6.0). (b) The absorption spectra of 1 (10 mM) in the absence
and presence of different metal ions (50 mM). Inset: Change in color of 1
(20 mM) with metal ions (100 mM) (from left to right): blank, Na+, Ag+,
Hg2+, Cu2+, Pb2+, Zn2+, Fe3+, Cd2+ and Ni2+. (c) Fluorescence response of
1 (1.0 mM) to 10 mM of Cu2+ or 50 mM of other metal ions (the gray bar
portion) and to the mixture of 50 mM of other metal ions with 10 mM of
Cu2+ (the black bar portion).
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condition. Finally, the competition experiments were also
carried by adding Cu2+ to the solution of 1 in the presence of
5 equiv. of other metal ions, and the results revealed that Cu2+-
induced fluorescence response was unaffected in the background
of metal ions mentioned above (Fig. 3c).
The EDTA-adding experiments were conducted to examine the
reversibility of this reaction. Addition of EDTA to the solution
containing 1 and Cu2+ diminishes the absorbance significantly,
whereas readdition of excess Cu2+ could recover the absorbance
signal (Fig. 2 (top)). The Cu2+-induced coloration and emission of
1 with Cu2+ leading to spirocycle opening of 1, as is the case for
related rhodamine-based probes.6
Similar to many reported rhodamine spirolactam-based flu-
orescent probes,6 the fluorescence enhancement response of 1
toward Cu2+ is most likely the result of the spiro ring-opening
mechanism rather than an ion-catalyzed hydrolysis reaction. The
above-mentioned EDTA experiment could serve as experimental
evidence to support this reversible spiro ring-opening mechanism.
The proposed binding mechanism of 1 with Cu2+ was shown in
the bottom of Fig. 2.
In summary, a new rhodamine derivative used as selective and
sensitive probe was developed, which could specifically recognize
Cu2+ in the aqueous buffer solution by the “naked eye”, UV/vis
and fluorescent responses. Furthermore, it also showed a “turn-
on” type of absorption and fluorescence response. The quenching
effect of water will limit the probes applicability in biological milieu
at some extent. However, by simple modified with hydrophilic
groups, we believed that this kind probe can be used for many
practical applications, including biological systems.
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This work was supported by the Department of Science and
Technology of Shandong Province of China (2008GG20005005),
the National Natural Science Foundation of China (20975089)
and the 100 Talents Program of the Chinese Academy of Sciences.
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The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 5277–5279 | 5279
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