Characterization of a highly Cu2+-selective fluorescent probe derived from rhodamine B

Article abstract of DOI:10.1016/j.dyepig.2012.07.016

A rhodamine B derivative was synthesized and characterized as a highly selective and sensitive probe for Cu²⁺ in ethanol-water solution (2:3, v:v, pH7.4, 50 mM HEPES). A prominent fluorescence enhancement at 575 nm was observed in the presence of Cu²⁺, accompanied by the change in the absorption spectrum. Under the optimal conditions, a good linear range of 0.5-1.5 μM with a detection limit of 1.6 × 10^-7 M were obtained. Furthermore, confocal laser scanning microscopy experiments have proven that this probe is cell-permeable and can respond to changes in intracellular Cu²⁺ in living cells.

Full text of DOI:10.1016/j.dyepig.2012.07.016

Dyes and Pigments 96 (2013) 38e44
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Characterization of a highly Cu2þ-selective fluorescent probe derived from
rhodamine B
*
Chunwei Yu, Ting Wang, Ke Xu, Jing Zhao, Maohua Li, Shixing Weng, Jun Zhang
School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571101, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A rhodamine B derivative was synthesized and characterized as a highly selective and sensitive probe for
Cu2þ in ethanolewater solution (2:3, v:v, pH7.4, 50 mM HEPES). A prominent fluorescence enhancement
at 575 nm was observed in the presence of Cu2þ, accompanied by the change in the absorption spectrum.
Received 6 April 2012
Received in revised form
16 July 2012
Accepted 18 July 2012
Available online 25 July 2012
Under the optimal conditions, a good linear range of 0.5e1.5
m
M with a detection limit of 1.6 ꢀ 10ꢁ7
M
were obtained. Furthermore, confocal laser scanning microscopy experiments have proven that this
probe is cell-permeable and can respond to changes in intracellular Cu2þ in living cells.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probe
Fluorescent imaging
Rhodamine derivative
Cu2þ
Fluorescence enhancement
Living cell
1. Introduction
of highly sensitive and selective “offeon” fluorescent probes for
Cu2þ is still significant.
Fluorescence spectroscopy offers a powerful tool for sensing and
imaging trace amounts of species by virtue of its simplicity, high
sensitivity and selectivity, and instantaneous response [1e3]. In
particular, selective heavy metal ions (HTM) recognition by fluo-
rescence probes has attracted increasing interest in biological and
environmental chemistry. Among the HTM, Cu2þ plays an impor-
tant role in living systems and has an extremely ecotoxicological
impact on the human health result from its catalytic cofactor for
a variety of metalloenzymes, including superoxide dismutase,
cytochrome c oxidase and tyrosinase [4]. However, Cu2þ exhibits
toxicity under overloading conditions in that it causes neurode-
generative diseases, probably by its involvement in the production
of reactive oxygen species [4]. Owing to the biological significance
of Cu2þ, a considerable effort has been devoted to the development
of the efficient methods to detect Cu2þ, and many studies focus on
the design of fluorescent probes for the analysis of Cu2þ have been
reported [5e12]. Whereas most of the reported Cu2þ fluorescent
probes show “on-off” signal upon the binding of Cu2þ due to its
paramagnetic nature [5e8], which is not as sensitive as a fluores-
cence enhancement response [9e12]. Therefore, the development
Based on our previous research [9e14], it is necessary to choose
an efficient fluorophore in the design of fluorescent probes.
Rhodamine derivative is one of the most useful fluorophores for the
construction of artificial fluorescent probes owing to its excellent
photophysical properties [15]. In the light of the equilibrium
between the spirolactam (non-fluorescence) and the ring-opened
amide (fluorescence) of rhodamine derivatives, rhodamine-based
probes are ideal modes for in vitro detection and in vivo imaging
[16e21]. In addition, the receptor should be preliminarily consid-
ered because it is responsible for the selectivity and binding effi-
ciency of the whole probes. According to the Soft-Hard Acid-Base
theory, the probes attached the recognition moiety with N and O
atoms could show good affinity to Cu2þ. Keeping this in mind,
a new probe L was designed and synthesized as a novel cell
membrance-permeable, Cu2þ-selective probe in aqueous media
and living cells. (Scheme 1).
2. Experimental
2.1. Reagents and instruments
All chemicals were used are of analytical grade or of the highest
purity available. Rhodamine B and Hydrazine hydrate (100%) were
purchased from SigmaeAldrich. Glyoxal and other reagents were
* Corresponding author. Tel.: þ86 898 66973190; fax: þ86 898 66989173.
E-mail address: jun_zh1979@163.com (J. Zhang).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2012.07.016

C. Yu et al. / Dyes and Pigments 96 (2013) 38e44
39
obtained from Shanghai Reagent Company. All solutions were
prepared with doubleedistilled water.
Melting points were determined using a Shanghai Melting
points WRSe1B apparatus. NMR spectra were measured with
a Brucker WMe500 spectrometer, using TMS as an internal stan-
dard. The pH measurements were carried out on a PHS-3C meter.
Mass spectra were performed on a Thermo TSQ Quantum Mass
Spectrometer. Fluorescence emission spectra were conducted on
a HORIBA Fluoromax-4 spectrofluometer. UVeVis spectra were
obtained on a Beckman DUe800 spectrophotometer. Fluorescence
imaging was performed by confocal fluorescence microscopy on an
Olympus FluoView Fv1000 laser scanning microscope.
2.2. Synthetic procedure
Compounds 1 was synthesized as reported method [21].
The synthesis of compound 2: Compound 2 was synthesized as
reported procedure with some modification [22]. Under N2 atmo-
sphere, ethyl 2-aminobenzoate (1.0 mmol, 0.165 g) and hydrazine
hydrate (12.0 mmol, 0.6 mL) were mixed in 30 mL ethanol. The
mixture was refluxed for 6 h. After the reaction was finished, the
solvent was removed under reduced pressure, and then 50 mL
petroleum ether was added to the oily residue, and the precipitate
so produced was filtered and used directly. Yields: 87.2%. M.p.:
152.0e153.1 ꢂC. MS: m/z 152.09 [M þ H]þ.
Scheme 1. The synthesis route of probe L.
The synthesis of fluorescent probe L: Compound 1 (0.496 g,
1.0 mmol) and compound 2 (0.181 g, 1.2 mmol) were mixed in
30 mL ethanol and refluxed for 4 h. After cooling to room
temperature, the precipitate so obtained was washed with water
and ethanol, and then dried in vacuum. The L was obtained by
recrystallization with ethanol as pale yellow solid. Yields: 85.6%. 1H
NMR (d6-DMSO,
d
ppm): 11.64 (s, 1H), 7.99 (d, 1H, J ¼ 8.10), 7.42 (d,
1H, J ¼ 7.45), 7.82 (d, 1H, J ¼ 8.20), 7.60 (t, 1H, J ¼ 7.47), 7.54 (t, 1H,
J ¼ 7.26), 7.43 (d, 1H, J ¼ 7.85), 7.18 (t, 1H, J ¼ 7.67), 7.45 (d, 1H,
J ¼ 7.55), 6.02 (d, 1H, J ¼ 8.05), 6.53 (t, 1H, J ¼ 7.87), 6.45 (s, 2H), 6.41
(s, 2H), 6.36 (d, 2H, J ¼ 9.00), 6.33 (b, 2H), 3.32 (m, 8H, J ¼ 6.95), 1.09
(t, 12H, J ¼ 6.97). 13C NMR (d6-DMSO,
d ppm): 165.74, 164.71 (C]O),
162.76, 153.51, 152.64, 152.54, 152.34, 150.62, 149.13, 148.60, 146.02,
144.08, 134.91, 133.07, 132.84, 130.10, 129.27, 128.80, 128.58, 128.16,
127.76, 127.50, 124.11 (ArC), 123.97, 123.73 (C]N), 116.86, 115.08,
113.25, 108.67, 108.28, 105.95, 104.83, 97.97, 97.92 (ArC), 65.23,
44.12, 12.89. MS (ESI) m/z: 630.4 [M þ H]þ.
2.3. General spectroscopic methods
Fig. 1. Influences of pH on the fluorescence spetra of L (2
Cu2þ (50
M) (:) in ethanolewater solution (2:3, v:v). The pH was modulated by
adding 1 M HCl or 1 M NaOH in HEPES buffers.
mM) (-) and L (2 mM) plus
m
Metal ions, anions and fluorescent probe L were dissolved in
deionized water and DMSO to obtain 1.0 mM stock solutions,
Fig. 2. (a) UVevis spectra of L (2
mM) with different metal ions (50 mM) in ethanolewater solution (2:3, v:v, 50 mM HEPES, pH7.4). (b) UVevis spectra of L (2 mM) with different
anions (50 M) in ethanolewater solution (2:3, v:v, 50 mM HEPES, pH7.4).
m

40
C. Yu et al. / Dyes and Pigments 96 (2013) 38e44
Fig. 5. Fluorescence response of L (2
ethanolewater solution (2:3, v:v, 50 mM HEPES, pH7.4). Inset: the fluorescence at
575 nm of L (2 M).
M) as a function of Cu2þ concentrations (0.5e1.5
m
M) with various concentrations of Cu2þ in
Fig. 3. Absorbance spectra of L (2 mM) in ethanolewater solution (2:3, v:v, 50 mM
HEPES, pH7.4) in the presence of different amounts of Cu2þ
.
m
m
respectively. Before spectroscopic measurements, the solution was
freshly prepared by diluting the high concentration stock solution
to the corresponding solution. For all measurements, excitation and
emission slit widths were 4 nm and 4 nm, respectively, excitation
wavelength was 520 nm.
2.4. Cell incubation
RAW cells plated on coverslips were washed with phosphate-
buffered saline (PBS), followed by incubating with 10
(in PBS) for 30 min at 37 ꢂC, and then washed with PBS three times.
After incubating with 20
M of L for 30 min at 37 ꢂC, the cells were
mM of CuCl2
m
washed with PBS three times again.
2.5. Cytotoxicity assay
The in vitro cytotoxicity was measured by using the methyl
thiazolyl tetrazolium (MTT) assay in RAW cells. Cells were seeded
into 96-well cell culture plate at 4000/well, cultured at 37 ꢂC and 5%
CO2 for 24 h, and then different concentrations of probe L (0, 0.1, 1,
Fig. 6. Fluorescence response in ethanolewater solution (2:3, v:v, 50 mM HEPES,
pH7.4): (a) L (2 M); (b) L (2 M); (c) L (2 M)
M) with Cu2þ (50 M) with Cu2þ (50
and then addition of EDTA (100 M); (d) L (2 M) and EDTA
M) with Cu2þ (50
(100 M) and then addition of 100
M Cu2þ
m
m
m
m
m
m
m
m
m
10
24 h at 37 ꢂC under 5% CO2. Subsequently, 20
added to each well and incubated for an additional 4 h at 37 ꢂC
mM) were added to the wells. The cells were then incubated for
m
.
mL MTT (5 mg/mL) was
Fig. 4. (a) Fluorescence spectra of L (2
50
M of Cu2þ and to the mixture of 250
solution (2:3, v:v, 50 mM HEPES, pH7.4). Inset: Fluorescence response of L (2
m
M) with different metal ions (50
M individual other metal ions with 50
M) to 50
m
M) in ethanolewater solution (2:3, v:v, 50 mM HEPES, pH7.4). Inset: Fluorescence response of L (2
M of Cu2þ; (b) Fluorescence spectra of L (2
M) with different anions (50 M) in ethanolewater
M of Cu2þ and to the mixture of 250 M of Cu2þ
M individual anions with 50
mM) to
m
m
m
m
m
m
m
m
m
.

C. Yu et al. / Dyes and Pigments 96 (2013) 38e44
41
enhancement over a comparatively wide pH range of 5.0e9.0,
which is attributed to a Cu2þ-induced opening of the rhodamine
ring. Consequently, L may allow Cu2þ detection in a wide pH range.
3.2. Uvevis spectral response of L
To validate the selectivity of L in pratice, the UV/vis spectrum of
L to various metal ions and anions are illustrated in Fig. 2a and
Fig. 2b, respectively. Upon binding of Cu2þ, the absorption spectrum
shows the typical rhodamine absorption band at 556 nm, accom-
panied by a clear color change from colorless to pink. Other metal
Scheme 2. Proposed binding mode of probe L toward Cu2þ
.
ions, such as Naþ, Agþ, Ca2þ, Mg2þ, Zn2þ, Pb2þ, Hg2þ, Mn2þ, Ni2þ
,
under 5% CO2. Cells were lysed in triple liquid (10% SDS, 0.012 M
HCl, 5% isopropanol), and the amount of MTT formazan was qual-
ified by determining the absorbance at 570 nm using a microplate
reader (Tecan, Austria).
The following formula was used to calculate the inhibition of cell
growth: Cell viability (%) ¼ (mean of Abs. value of treatment group/
mean Abs. value of control) ꢃ 100%.
Cd2þ, Co2þ, Cr3þ, Fe3þ, and anions including SO42ꢁ, SCNꢁ, Acꢁ, ClOꢁ4 ,
NO3ꢁ, HPO42ꢁ, CO32ꢁ, S2ꢁ, Clꢁ and Brꢁ did not show any significant
color and spectral change under identical conditions, only Hg2þ
elicited a slight change of absorbance.
Furthermore, absorption titrations of L (2 mM) in ethanolewater
solution (2:3, v:v, 50 mM HEPES, pH7.4) was conducted (Fig. 3).
Upon the gradual addition of Cu2þ up to 5 equiv., a new absorption
band centered at 556 nm appeared with increasing intensity
evidently, clearly indicating the ring-opening process of rhodamine
B unit in L.
3. Results and discussion
3.1. pH investigation
For practical application, the spectra response of L in the
absence and presence of Cu2þ in different pH values were firstly
evaluated (Fig. 1). Under acidic conditions (pH < 5.0), ring opening
of the rhodamine occurred as a result of protonation. In the pH
5.0e9.0, no obvious characteristic emission of rhodamine could be
observed. However, the addition of Cu2þ led to the fluorescence
3.3. Fluorescence spectral response of L
Fig. 4 shows the fluorescence spectra (ex ¼ 520 nm) of L (2
mM)
measured in ethanolewater solution (2:3, v:v, 50 mM HEPES,
pH7.4) with the addition of respective metal ions and anions. As
expected, the addition of Cu2þ to the solution of L resulted in
Fig. 7. Confocal fluorescence images in RAW cells. (a) Cells incubated with 20
m
M L in PBS buffer for 30 min; (b) Brightfield image of cells shown in panel (a); (c) Cells incubated with
10
m
M Cu2þ for 30 min, washed three times, and then further incubated with 20
mM L for 30 min (ex ¼ 559 nm); (d) Brightfield image of cells shown in panel (c).

42
C. Yu et al. / Dyes and Pigments 96 (2013) 38e44
calibration plot), suggestingthat the fluorescent probe L could
sensitively detect environmentally relevant levels of Cu2þ
.
3.4. The proposed reaction mechanism
To investigate the probable complexation of L with Cu2þ, the
method of continuous variations (Job’s plot) is obtained from the L-
Cu2þ system in ethanolewater solution (2:3, v:v, 50 mM HEPES,
pH7.4), which clearly suggested the formation of 1:1 stoichiometry
between L and Cu2þ (Supporting Information, Fig. S1). Moreover,
the 1:1 stoichiometry mode is also supported by the presence of
a peak at m/z 693 corresponding to [L þ Cu2þꢁHþ]þ in the ESI-MS
spectrum of the components of the mixture of L and 1 equiv. Cu2þ
in ethanol (Supporting Information, Fig. S2). The association con-
tant for L-Cu2þ complex was further estimated to be 9.1 ꢀ 104 Mꢁ1
on the basis of the nonlinear filtting of the fluorescence titration
curve assuming a 1:1 stoichiometry by the Benesi-Hildebrand
method [17] (Supporting Information, Fig. S3).
To understand the stability of the complex formed, we have
analyzed the chemical reversibility behavior of the binding of L and
Cu2þ in the ethanolewater solution. As a consequence, upon
Fig. 8. Cell viability values (%) estimated by MTT proliferation test versus incubation
concentrations of L. RAW cells were cultured in the presence of 0e10 m
M L at 37 ꢂC.
addition of 100
(50 M) in ethanolewater solution (2:3, v:v, 50 mM HEPES, pH7.4),
mM EDTA to the mixture of L (2 m
M) and Cu2þ
m
remarkably enhanced fluorescence intensity. Upon interaction with
the color changed from pink to almost colorless, and more than 90%
fluorescence intensity of the system was quenched (Fig. 6). Then
the Cu2þ was added to the system, the signals were almost
completely recovered, and the colorless solution turned to pink.
These findings indicated that L is a reversible fluorescent probe for
Cu2þ. Thus, according to the obtained results, it is very likely due to
the metal ioneinduced ring opening of rhodamine spirolactam,
rather than other possible reactions, and the proposed binding
mode for the probe toward Cu2þ is illustrated in Scheme 2.
other metal ions and anions, a much weaker response is given
compared to Cu2þ at the same concentration (50
mM). Moreover,
the enhancement of the fluorescence intensity depending on the
addition of Cu2þ was not suppressed by subsequent addition of
other metal ions and anions (Fig. 4 inset). These results indicated
that L could selectively recognize Cu2þ in the presence of miscel-
laneous competitive metal ions and anions in ethanolewater
solution (2:3, v:v, 50 mM HEPES, pH7.4).
To further investigate the interaction of Cu2þ and L, a fluores-
cence titration experiment was carried out, as shown in Fig. 5. A
linear increase of fluorescence intensity could be observed with
increasing Cu2þ concentration over a wide range with a detection
limit of 1.6 ꢀ 10ꢁ7 M based on 3 ꢀ dblank/k (where dblank is the
standard deviation of the blank solution and k is the slope of the
3.5. Preliminary analytical application
In view of its favorable spectroscopic properties, the ability of L
to detect intracellular Cu2þ was also examined in this study. The
Table 1
Performances comparison of various off-on probes for Cu2þ derived form rhodamine.
Linear range,
m
M
LOD, nM
Testing media
Applications
Reproducibility
Ref.
0.05e0.9
0.1e1.0
0.05e0.9
0.05e4.5
NA
NA
NA
0.001e0.01
NA
NA
3
85
7
18
NA
20
NA
NA
200
NA
NA
10
Wateremethanol (8:2, v:v, pH6.0, 20 mM HEPES)
Watereethanol (4:6, v:v, pH7.0 50 mM HEPES)
Wateremethanol (2:8, v:v, pH7.0, 20 mM HEPES)
Watereethanol (9:1, v:v, pH7.0, 50 mM HEPES)
CH3CN-HEPES (4:6, v:v, pH7.4, 20 mM)
Water-CH3CN (2:8, v:v, pH7.0, buffered with HEPES)
CH3CN-HEPES (5:5, v:v, pH7.4, 20 mM)
Water-CH3CN (9:1, v:v, pH7.0, 10 mM Tris)
CH3CN-HEPES (2:8, v:v, pH7.0, 10 mM)
Water-CH3CN (1:1, v:v)
NAa
Reversible
Reversible
Reversible
Reversible
NA
Reversible
NA
NA
Reversible
NA
NA
Reversible
NA
Reversible
Reversible
NA
[9]
MCF-7 cells
HeLa cells
HeLa cells
NA
PC3 cells
NA
[10]
[11]
[12]
[16]
[18]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
This work
NA
NA
EJ cells
NA
Waster water
EJ cells
Water sample
NA
EJ cells
E coli cells
NA
NA
NA
NA
NA
NA
Methanol-HEPES (1:1, v:v, pH7.0)
1e14
0e0.005
10e300
2.0e10
NA
NA
NA
NA
5e55
NA
Water-CH3CN (1:1, v:v, pH7.1, 10 mM Tris)
Water-CH3CN (1:1, v:v, pH7.2, 10 mM Tris)
DMSO-Tris (1:9, v:v, pH7.0)
Water-CH3CN (3:2, v:v, pH7.0, 10 mM HEPES)
Water-CH3CN (1:1, v:v)
Water-CH3CN (1:1, v:v)
Water-DMSO (6:4, v:v)
CH3CN-Tris (3:1, v:v, pH7.0, 10 mM)
Water-DMF (99:1, v:v, pH5.7)
Acetate-buffer/DMF (3:3, v:v, pH3.6)
Watereethanol (4:1, v:v, pH8.5, Tris)
Ethanol-HEPES (1:1, v:v, pH7.2, 20 mM)
Water-DMSO (1:10, v:v, pH7.8, 2 mM Tris)
Watereethanol (3:2, v:v, pH7.4, 50 mM HEPES)
2
3850
280
NA
NA
NA
NA
490
6470
48
NA
NA
Reversible
NA
Reversible
Reversible
NA
0.25e25
0.1e3.0
NA
34
NA
160
HeLa cells
NA
RAW cells
NA
Reversible
0.5e1.5
a
NA: Not available.

C. Yu et al. / Dyes and Pigments 96 (2013) 38e44
43
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in the growth medium for 30 min at 37 ꢂC, which led to very weak
fluorescence as determined by laser scanning confocal microscopy
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fluorescent probe L can penetrate the cell membrane and might
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was ca. 98% in 48 h after treatment with 10
exhibiting low toxicity to cultured cells.
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The performance of the proposed probe L was compared with
some reported probes based on rhodamine B structural motif for
Cu2þ determination, as shown in Table 1. All the probes present
good selectivity for Cu2þ with signal enhancement
[9e12,16,23e40], and a few of probes possess wide quantitation
span [29,38], even down to nM LOD [9,11,29]. But some of them
need more rigorous testing media [9,36,38], and the reproductivity
[16,23,24,26,27,29,32e34,36,39,40] as well as the applicability in
living cells [9,16,23e25,27,31,34e38,40], are not investigated. Thus,
there are still numerous challenges and opportunities remaining
for development of new probes and practical applications in bio-
logical systems. Our proposed probe L based on rhodamine is easy
to prepare and presents a number of attractive analytical features
such as good selectivity, high sensitivity and wide applicability. It
can be used for rapid analysis of ultra-trace level Cu2þ in living cells
with satisfactory results.
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and anions. Moreover, it has been demonstrated that the fluores-
cent probe L can be used to detect Cu2þ in living cells. We expect
that this fluorescent probe would help to promote the studies of
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Acknowledgments
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selective turn-on fluorescent and chromogenic probe for Cu2þ and ClOꢁ
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This work was financially supported by the Natural Science
Foundation of Hainan Province of China (No. 812188), the Colleges
and Universities Scientific Research Project of the Education
Department of Hainan province of China (No. Hjkj2012-33), the
Research and Training Fundation of Hainan Medical University (No.
HY2010-004) and the Undergraduates Innovating Experimentation
Project of Hainan Medical University (No. HYCX201107).
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mosensor for the visual detection of copper and the ratiometric fluorescent
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Appendix A. Supplementary data
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Supplementary data related to this article can be found online at
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