New fluorescent and colorimetric chemosensors based on the rhodamine detection of Hg2+ and Al3+ and application of imaging in living cells

Article abstract of DOI:10.1016/j.dyepig.2013.02.002

Employing the "OffeOn" switching of the spirocyclic moiety in Rhodamine B derivatives, three sensors were designed and characterized as new fluorescent probes for detecting Hg²⁺ and Al³⁺ in the environment, respectively. The first probe exhibited chromogenic and fluorogenic selectivity to detection of Hg²⁺ in methanol-H₂O (4:6, v/v, HEPES, pH 7.0). The first and second probes displayed fluorescence intensity enhancement following Hg²⁺ coordination with limits of detection for Hg²⁺ at the ppb level. The limit of detection based on 3 blank/k was calculated to be 2.5 × 10^-8 M and 4.2 × 10^-8 M, respectively. The third probe contained a benzyl group which resulted in better selectivity for Al³⁺. Job's plot clearly suggested the formation of 1:1 complexation behavior between the three probes and Hg ²⁺ or Al³⁺. The three probes can be used as a fluorescent probe for monitoring Hg²⁺ or Al³⁺ in living cells with satisfying results, which demonstrates the value of the probes in practical applications in environmental and biological systems.

Full text of DOI:10.1016/j.dyepig.2013.02.002

Dyes and Pigments 98 (2013) 42e50
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
New fluorescent and colorimetric chemosensors based on the
rhodamine detection of Hg2þ and Al3þ and application of imaging in
living cells
,
a
a
a
a
a,
Fanyong Yan a , Meng Wang , Donglei Cao , Ning Yang , Yang Fu , Li Chen
*
*
,
Ligong Chen b
a State Key Laboratory of Hollow Fiber Membrane Materials and Processes, Key Lab of Fiber Modification & Functional Fiber of Tianjin, Tianjin Polytechnic
University, Tianjin 300387, China
b School of Chemical Engineering, Tianjin University, Tianjin 300072, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Employing the “OffeOn” switching of the spirocyclic moiety in Rhodamine B derivatives, three sensors
were designed and characterized as new fluorescent probes for detecting Hg2þ and Al3þ in the envi-
ronment, respectively. The first probe exhibited chromogenic and fluorogenic selectivity to detection of
Hg2þ in methanol-H2O (4:6, v/v, HEPES, pH 7.0). The first and second probes displayed fluorescence
intensity enhancement following Hg2þ coordination with limits of detection for Hg2þ at the ppb level.
The limit of detection based on 3 blank/k was calculated to be 2.5 ꢀ 10ꢁ8 M and 4.2 ꢀ 10ꢁ8 M,
respectively. The third probe contained a benzyl group which resulted in better selectivity for Al3þ. Job’s
plot clearly suggested the formation of 1:1 complexation behavior between the three probes and Hg2þ or
Al3þ. The three probes can be used as a fluorescent probe for monitoring Hg2þ or Al3þ in living cells with
satisfying results, which demonstrates the value of the probes in practical applications in environmental
and biological systems.
Received 7 December 2012
Received in revised form
31 January 2013
Accepted 1 February 2013
Available online 16 February 2013
Keywords:
Fluorescent sensor
Mercury
Aluminum
Rhodamine
Phthalimido acids
Cellular imaging
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Many detection methods for the quantitative analysis of Hg2þ in
water samples include atomic absorption spectroscopy, cold vapor
The design of chemosensors for the detection of heavy and
transition metal ions are particularly attractive, because these ions
play important roles in living systems and have an extremely toxic
impact on the environment [1e4]. Among transition-metal ions,
mercury is considered as one of the most dangerous cations for the
environment. Mercury can accumulate in the human body, its high
affinity for thiol groups in proteins and enzymes lead to the
dysfunction of cells and consequently causing health problems,
such as prenatal brain damage, serious cognitive and minamata
disease [5e7]. Al3þ widely exists within the environment due to
acidic rain and human activities. The increase of Al3þ concentration
in the environment is not only detrimental to growing plants but
also damages many biological functions. Excessive exposure of the
human body to Al3þ leads to a wide range of diseases, such as
Alzheimer’s disease, Parkinson’s disease, etc. [8e10].
atomic fluorescence spectrometry, and gas chromatography, which
usually require complicated, multistep sample preparation and
sophisticated instrumentation [11e14]. Fluorescence analysis offers
significant advantages over other methods for metal ion detection
and measurement due to its simplicity, high sensitivity, low cost
and instantaneous response [15].
In recent years, progress in the area of chemosensors has
contributed significantly to the development of a variety of Al3þ
sensors based on triazole, Schiff’s base and coumarin Refs. [16e18].
Unfortunately, some of the Al3þ sensors have disadvantages such as
poor water-solubility or UV light excitation [8,19,20]. So few Al3þ
sensors have been reported up to now. Mercury-selective fluores-
cent sensors have been reported in the past where in most cases the
presence of mercury can cause fluorescence quenching of the fluo-
rophores via the spin orbit coupling effect or complicated synthesis
methods [21e23]. Therefore, overcoming these disadvantages in the
development of excellent sensors for the sensitive and selective
determination of Hg2þ and Al3þ is a challenging task.
In the form of proteins, amino acids comprise the second largest
componentotherthanwaterofhumanmuscles,cellsandothertissues.
* Corresponding authors. Tel.: þ86 22 83955766; fax: þ86 22 83955016.
E-mail addresses: yfytju@yahoo.com (F. Yan), chenlis11@yahoo.com.cn (L. Chen).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.02.002

F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
43
As amino acids have both a primary amine group and a primary
carboxyl group, these chemicals can undergo most of the reactions
associated with these functional groups [24]. These include amide
bond formation and decarboxylation for the carboxylic acid group. The
combination of these functional groups allowed amino acids to be
effective ligands for metal-amino acid chelates. Because of their bio-
logical significance, amino acids are also used to chelate metal cations
in order to improve the ability of a probe to detect metals [25e30].
Due to the structure and behavior of rhodamine and related
derivatives [31e33], rhodamine can make an ideal model to use in
constructing chelation-enhanced fluorescence “OffeOn” switch
sensors for metal ions. Without cations, these rhodamine-based
chemosensors exist in a spirocyclic form which is colorless and
non-fluorescent. The addition of a specific metal ion leads to spi-
rocycle opening via a reversible coordination or an irreversible
chemical reaction [34e39], resulting in intense fluorescence
emission and development of pink color. However, the rhodamine-
based derivatives may lead to self-quenching and fluorescence
detection errors because of excitation back-scattering effects [40].
Thus, it is important to have sensors with improved properties. In
our previous work, we have reported an excellent rhodamine-based
fluorescent sensor for Hg2þ in aqueous solution [41]. Compared with
the fluorescent probe RheC which had been reported by our group,
RW1 exhibited much better fluorescence selectivity toward Hg2þ
and bio-compatibility, and much lower detection limit
(2.5 ꢀ 10ꢁ8 M). Herein, we synthesized the rhodamine chemo-
sensors based spirolactam derivatives RW1-3, which incorporate
two kinds of phthlandione derivatives and two kinds of amino acids.
They would be suitable for introducing O atom. However, O atom
will provide even more binding sites that might be a choice to be
parts of a selective receptor for the selective recognition of Hg2þ
[42e44]. At the same time, we envisaged different phthlandione
derivatives and amino acids can affect the fluorescence intensity of
probes and the metal ion-selective recognition.
was utilized to measure the pH values of aqueous solutions. IR data
were taken in KBr disks on TENSOR37 Fourier-Transform Infrared
Spectrometer. 1H NMR and 13C NMR measurements were per-
formed with a Varian Inova-400 MHz spectrometer with TMS as an
internal standard and CDCl3 as solvent. ESI-MS measurements were
carried out on Waters QT of-Micro instrument.
2.2. Reagents
All reagents used were purchased from commercial suppliers
and used without further purification. The solutions of metal ions
were performed from their nitrate and chloride salts. Double-
distilled water was used throughout the experiments. HEPES
buffer solutions (10 mM, pH 7.0) were prepared in water.
2.3. Synthesis procedure
Synthesis of RW1 (Scheme 1): Rhodamine ethylenediamine was
synthesized as reported method [45]. Phthalic anhydride (0.59 g,
4 mmol) and alanine (0.35 g, 4 mmol) were dissolved in dry toluene
(20 mL), and the mixture were heated under reflux for 4 h; after
removal of the toluene, the residue was dissolved in thionyl chlo-
ride (10 mL) and refluxed for 3 h. Rhodamine ethylenediamine
(1.73 g, 4 mmol) was dissolved in dichloromethane (20 mL) in a
100 mL flask. An excess of triethylamine was added and dichloro-
methane (15 mL) solutions of 2-methylphthalimido acetyl chloride
was added to this solution. The reaction mixture was stirred 2 h at
0e5 ꢂC, after drying under reduced pressure, and pale yellow solid
was observed. The target material was further purified by column
chromatography with CH3OH/CH2Cl2 (1:5 v/v) to give 1.5 g of 1 in
52% yield. 1H NMR (CDCl3, 400 MHz)
d
¼ 7.95 (s, 1H), 7.90 (m, 2H),
7.79 (m, 2H), 7.29-7.40 (m, 3H), 7.05 (d, J ¼ 7.2 Hz, 1H), 6.46 (d,
J ¼ 8.8 Hz, 1H), 6.32-6.38 (m, 4H), 6.27 (d, J ¼ 8.4 Hz, 1H), 4.91(q,
J ¼ 7.2 Hz, 1H), 3.34 (m, 10H), 2.96 (m, 2H), 1.74 (t, J ¼ 7.2 Hz, 3H),
1.17 (t, J ¼ 6.8 Hz, 12H). 13C NMR (CDCl3, 100 MHz)
d
¼ 168.93,
2. Experimental
167.69, 154.12, 153.24, 153.07, 149.00, 148.93, 134.21, 132.70, 129.88,
128.49, 128.23, 128.06, 128.03, 123.39, 122.39, 108.31, 97.12, 64.72,
2.1. Instruments
48.09, 43.68, 40.20, 38.35,13.76,11.59. IR (KBr, n
/cmꢁ1): 3425, 3003,
2856, 1673, 1612, 1460, 1224, 1118, 976. ESI-MS (M þ Hþ): m/
z ¼ 686.33. Anal. Calcd for C41H43N5O5: H, 6.32; C, 71.80; N, 10.21.
Found: H, 6.25; C, 70.77; N, 10.16.
Fluorescence spectra were measured on a Hitachi F-4500
spectrofluorimeter with quartz cuvette (path length ¼ 1 cm). The
absorption spectra measurements were observed by a Purkinje
General TU-1901 UV/Vis spectrometer. A pH-10C digital pH meter
RW2 and RW3 were prepared with the similar synthetic method
to RW1. Compound RW2: yield: 43%. 1H NMR (CDCl3, 400 MHz)
Scheme 1. Synthesis of RW1-3.

44
F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
d
¼ 8.05 (s,1H), 7.29e7.45 (m, 3H), 7.06 (d, J ¼ 7.6 Hz,1H), 6.26e6.45
(m, 6H), 4.91 (q, J ¼ 7.2 Hz, 1H), 3.74 (q, J ¼ 6.8 Hz, 1H), 3.36 (q,
J ¼ 7.2 Hz, 8H), 3.25 (m, 2H), 2.97 (d, J ¼ 7.6 Hz, 1H), 1.73 (d,
J ¼ 7.2 Hz, 3H), 1.17 (t, J ¼ 6.8 Hz, 12H). 13C NMR (CDCl3, 100 MHz)
d
¼ 167.76, 153.98, 153.23, 152.99, 149.07, 148.90, 137.71, 132.83,
129.80, 129.17, 128.84, 128.01, 123.39, 122.51, 108.35, 104.52, 97.53,
65.17, 54.80, 44.14, 40.96, 38.48, 33.81, 12.14. IR (KBr, n
/cmꢁ1): 3426,
3004, 2858, 1673, 1615, 1457, 1220, 1112, 979. ESI-MS (M þ Hþ): m/
z ¼ 824.17. Anal. Calcd for C41H39Cl4N5O5: H, 4.77; C, 59.79; N, 8.50.
Found: H, 4.54; C, 58.89; N, 8.19.
Compound RW3: yield: 47%. 1H NMR (CDCl3, 400 MHz)
d
¼ 7.95
(s, 1H), 7.89 (m, 4H), 7.79 (m, 4H), 7.28e7.42 (m, 4H),7.05 (d,
J ¼ 7.6 Hz, 1H), 6.27-6.46 (m, 6H), 4.92 (q, J ¼ 7.6 Hz, 1H), 3.34 (m,
10H), 2.96 (t, J ¼ 7.2 Hz, 2H), 1.74 (t, J ¼ 6.8 Hz, 2H), 1.17 (q,
J ¼ 6.8 Hz, 12H). 13C NMR (CDCl3, 100 MHz)
d
¼ 170.44, 167.41,
163.11, 153.84, 153.27, 153.04, 148.99, 139.84, 137.17, 132.90, 129.57,
128.72, 128.63, 128.47, 128.18, 128.03, 127.80, 126.74, 123.89,
122.18, 108.35, 104.23, 97.65, 65.88, 55.47, 44.35, 39.07, 33.73,
12.58. IR (KBr,
n
/cmꢁ1): 3422, 3001, 2852, 1672, 1616, 1459,
1228, 1112, 974. ESI-MS (M þ Hþ): m/z ¼ 762.36. Anal. Calcd
for C47H47N5O5: H, 6.22; C, 74.09; N, 9.19. Found: H, 6.14; C, 72.93;
N, 9.02.
Fig. 1. (a) UVevis absorption of RW1, RW2 and RW3 (10
m
M) in 4:6 CH3OH/HEPES
M Hg2þ or Al3þ. (b) RW1 (10
M) as a selective
M) in in 4:6 CH3OH/HEPES buffer (v/v, 10 mM,
buffer (v/v, 10 mM, pH 7.0) with 10
m
m
naked-eye chemosensor for Hg2þ (10
pH 7.0).
m
2.4. Cell incubating imaging
Images of the human hepatocyte cell line (HL-7702 cells) were
cultured in (Invitrogen) supplemented with 10% Fetal Bovine
Serum (FBS, Invitrogen) in an atmosphere of 5% CO2 at 37 ꢂC. One
day before imaging, cells were placed in 6-well flat-bottomed
plates. Immediately, before the experiments, the HL-7702 cells
were exposed to the probe RW1 (2.5 ꢀ 10ꢁ5 mol Lꢁ1) for 30 min at
room temperature to allow the probe to permeate into the cells and
supplementing cells with 2.5 ꢀ 10ꢁ5 mol Lꢁ1 Hg(ClO4)2 in PBS at
37 ꢂC under 5% CO2 for another 30 min. The cells were washed with
PBS three times and then imaged. The fluorescence imaging of
intracellular was observed under an Olympus IX71 inverted fluo-
rescence microscopy with 40 ꢀ objective lens (excited with green
pH 7.0) solution. Upon the gradual addition of Al3þ up to 1 equiv., a
new absorption band centered at 556 nm appeared with increasing
intensity, accompanied by a clear color change from colorless to
pink.
In addition, the selectivity profiles of RW1, RW2 and RW3 for
metal cations were investigated by fluorimetric experiments. The
fluorescence spectra were obtained by excitation of the rhoda-
mine fluorophore at 525 nm. Both a red color and the fluores-
cence characteristics of rhodamine B appear when upon the
addition of Hg2þ to a colorless solution of RW1 and RW2 (Fig. 2a).
In the presence of other metal ions, such as alkali and alkaline
earth metal ions (Kþ, Naþ, Mg2þ, Ca2þ) and other transition metal
light). The HL-7702 cells only incubated with 10
at 37 ꢂC under 5% CO2 was as a control.
mM RW1 for 30 min
ions (Mn2þ, Ni2þ, Co2þ, Cu2þ, Zn2þ, Cd2þ, Agþ, Pb2þ, Al3þ, Fe3þ
,
Auþ, Au3þ), there were no evident fluorescence intensity
enhancement (Fig. 2b). Apparently, due to Hg2þ-induced signifi-
cant fluorescence enhancement, Hg2þ could be distinguished
from other metal ions. RW3 showed only a very weak fluores-
cence in the absence of metal ions. The addition of Al3þ resulted
in remarkably enhanced fluorescence intensity. Under the same
3. Results and discussion
We take advantage was made of phthalic anhydride as an
amino-protecting group, appropriate to introduce oxygen atoms,
which are involved in the binding of metal ions. Then, due to the
phthalimido acid chlorides possessing high reactivity, nucleo-
philic substitution can be prone to occur with rhodamine
ethylenediamine.
condition, additions of other metal ions including Kþ, Naþ, Mg2þ
Ca2þ, Mn2þ, Ni2þ, Co2þ, Cu2þ, Zn2þ, Cd2þ, Agþ, Pb2þ, Al3þ, Fe3þ
,
,
Auþ and Au3þ did not cause any discernible changes. Since the Ce
C
p bonds of the benzene ring disappear due to the presence of
3.1. Spectral characteristics
Al3þ. Each of the resulting singly occupied carbon p-orbitals in-
teracts with two of the three empty 3p-orbitals of aluminum (the
The binding behavior of RW1, RW2 and RW3 toward different
ones parallel to the aromatic ring plane). Each s CeC bond also
cations, such as Agþ, Ca2þ, Cd2þ, Co2þ, Al3þ, Cu2þ, Hg2þ, Kþ, Pb2þ
,
interacts with the Al3þ empty 3s-orbital, and the remaining 3p-
orbital [46]. The bonding orbitals formed by the 5d orbitals of
Hg2þ are completely filled, resulting in a closed-shell electronic
configuration. So the benzene rings of RW3 have better selectivity
of electron-deficient Al3þ (3s0 3p0) than completely fill 5d orbitals
Hg2þ (5d10 6s0).
Mg2þ, Mn2þ, Naþ, Ni2þ, Fe3þ, Auþ, Au3þ and Zn2þ ions were
investigated by UVevis and fluorescence spectroscopy. Among the
various metal ions examined (10 equiv.), RW1 and RW2 showed
highly selective “OffeOn” absorption enhancement only with Hg2þ
at methanol-H2O (4:6, v/v, HEPES, pH 7.0) solution (Fig. 1). As
shown, almost all the metal cations including the free RW1 and
RW2 exhibit very little absorbance at 556 nm. However, Hg2þ
resulted in a prominent change, which can be ascribed to the spi-
rolactam bond cleavage followed by the formation of a delocalized
xanthene moiety of the rhodamine group. Interestingly, Al3þ also
yielded a significant absorption (Fig. 1a) enhancement with RW3
which contains the benzyl group at methanol-H2O (4:6, v/v, HEPES,
Achieving high selectivity toward the sensors over the other
competitive species coexisting in the sample is a very important
feature to evaluate the performance of a fluorescence chemosensor.
In certain environmental samples, such as river and seawater, the
concentrations of some prevalent toxic metal ions are significantly
higher, therefore competition experiments with high concentra-
tions of the above-mentioned metal ions (10 equiv.) were carried

F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
45
Fig. 3. Fluorescent intensity of RW1 (bule bars), RW2 (yellow bars) and RW3 (red bars)
(10 M) to various cations in 4:6 CH3OH/HEPES buffer (v/v, 10 mM, pH 7.0). The front
m
3 bars represent the emission intensities of RW1, RW2 and RW3 in the presence of
other cations (10 equiv.), respectively. The back 3 bars represent the emission in-
tensities that occur upon the subsequent addition of 10 m
M of Hg2þ or Al3þ to the
above solution, respectively. (lex ¼ 525 nm, lem ¼ 581 nm). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
RW3] of 20
ions from 0 to 1 (Fig. 4, inset). From the Job’s plot, the absorbance
m
M and changing the mole fraction of Hg2þ/Al3þ
of RW1-2/RW3 approached
a maximum when the molar
fraction of Hg2þ/Al3þ was 0.5, indicating a 1:1 stoichiometry
was most possible for the binding mode of the RW1-2/RW3 and
Hg2þ/Al3þ
.
The complexation of Hg2þ by RW1 and RW2 were also investi-
gated by means of fluorescence titration in methanol-H2O (4:6, v/v,
HEPES, pH 7.0) solution. Addition of increasing concentrations of
Hg2þ ions to the solutions of RW1 (Fig. 5a) and RW2 (Fig. 5b)
resulted in the gradual increase of the fluorescence intensity with
emission peak at 581 nm, reaching saturation at addition of 1.5
equiv. Hg2þ. Nonlinear least-squares fitting of the titration profiles
based on the 1:1 binding model strongly support the 1:1 stoichi-
Fig. 2. (a) Fluorescent spectra of RW1, RW2 and RW3 (10
buffer (v/v, 10 mM, pH 7.0) with 10
M Hg2þ or Al3þ. (b) Fluorescence responses of
RW1, RW2 and RW3 (10 M) to various cations (25 mM) at 581 nm in 4:6 CH3OH/
mM) in 4:6 CH3OH/HEPES
m
ometry of RW1 (Fig. 5a, inset) and RW2 (Fig. 5b, inset) with Hg2þ
,
m
and the binding constant was calculated to be 2.1 ꢀ 107 and
HEPES buffer (v/v, 10 mM, pH 7.0). Inset: Pictures of RW1, RW2 and RW3 as a selective
4.4 ꢀ 105, Respectively [47]. The equation used was:
naked-eye chemosensors (bottom) and the visual fluorescence emissions by using a
UV lamp (365 nm) (top) for Hg2þ or Al3þ
.
ꢀ
Ylim ꢁ Y0
CM
CL KSCL
1
Y ¼ Y0 þ
1 þ
þ
out. The competition experiments revealed that the Hg2þ/Al3þ
-
2
ꢁꢂ
ꢃ
ꢄ
ꢅ
induced fluorescence response was unaffected in by 10 equiv. of
environmentally relevant alkali or alkaline-earth metals, such as
Naþ, Kþ, Mg2þ and Ca2þ. In addition, the other transition metal ions
(Mn2þ, Ni2þ, Co2þ, Cu2þ, Zn2þ, Cd2þ, Agþ, Pb2þ, Al3þ, Fe3þ, Auþ,
Au3þ) did not interfere with the Hg2þ/Al3þ-induced fluorescence
(Fig. 3). Obviously, all of these results confirmed that our proposed
chemosensor RW1-2/RW3 has remarkably high selectivity toward
Hg2þ/Al3þ ions over other competitive cations in the water
medium.
2
1=2
CM
1
CM
CL
ꢁ
1 þ
þ
ꢁ 4
;
CL KSCL
where Y is the recorded fluorescent intensity, Y0 is the start value
without addition of ions, Ylim is the limiting value, CM is the ions
concentration, and CL is the sensor concentration.
The above results indicate that the sensor RW1 exhibited a su-
perior binding capability toward the Hg2þ ions relative to the
sensor RW2, which can be attributed to additional chloride atoms
in the case of RW2. This observation along with lower binding
constant of RW2 suggests that the chloride atom plays an impor-
tant role in the extent of the binding with Hg2þ. The chlorine atom
led to the heavy atom effect and the electrophilic effect which can
lead to a decrease in fluorescence intensity [48].
To gain more insight into the properties and mechanism of RW3
toward Al3þ, fluorescence titration with Al3þ was recorded. As seen
from Fig. 5c, upon sequential addition of Al3þ, an emission band
peaked at 581 nm significantly increased in fluorescence intensity,
possibly because of the rhodamine ring-opening process, and the
fluorescence intensity increased up to 1.3 equiv. and then no
changes were observed up to 2.0 equiv. Based on 1:1 stoichiometric
relationship, a binding constant of 3.9 ꢀ 105 Mꢁ1 was obtained from
Figs. 4 and 5 explain the fluorescent and absorption titrations of
RW1 and RW2 with Hg2þ in methanol-H2O (4:6, v/v, 10 mM, HEPES,
pH 7.0) solution. On addition of Hg2þ ions (1e20
mM), the solution
of RW1 and RW2 turned from colorless to pink, and the absorbance
was significantly enhanced with a new peak appearing at around
556 nm (Fig. 4), clearly suggesting the formation of the ring-opened
form of RW1 and RW2 as a result of Hg2þ binding. Compared to
RW1, the enhancement in the UV absorption (lmax ¼ 556 nm) of
RW2 from 180 fold under to 60 fold.
To understand the recognition abilities of RW1-2/RW3 toward
Hg2þ/Al3þ ions, the Job’s plot analysis for the absorbance was
conducted to determine the binding stoichiometry of the RW1-2/
RW3 and Hg2þ/Al3þ ions complex, by according to the continuous
variations with a total concentration of [Hg2þ/Al3þ] þ [RW1-2/

46
F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
Fig. 5. Fluorescence spectra of RW1 (a), RW2 (b) and RW3 (c) (10 mM) in 4:6 CH3OH/
HEPES buffer (v/v, 10 mM, pH 7.0) upon addition of different amounts of Hg2þ and Al3þ
ions (0e2 equiv.). lex ¼ 525 nm. Inset: Top: Fluorescence at 581 nm as a function of
Hg2þ and Al3þ concentration, indicating a 1:1 metal-ligand ratio; bottom: Fluorescence
intensity at 581 nm for RW1, RW2, and RW3 as a function of the concentration of Hg2þ
ion.
Fig. 4. Absorption spectra of RW1 (a), RW2 (b) and RW3 (c) (10 mM) in 4:6 CH3OH/
HEPES buffer (v/v, 10 mM, pH 7.0) upon addition of different amounts of Hg2þ and Al3þ
ions (0e2 equiv.). Inset: Job’s plots at 556 nm (RW1, RW2 and RW3).
a nonlinear least-squares fit according to a 1:1 binding stoichiom-
etry (Fig. 5c, inset).
Hg2þ/Al3þ ions with different concentrations ranged from 0 to
10
recorded to generate a calibration curve. When Hg2þ concentra-
tion added was from 0.5 to 10 M, an almost perfect linearity
was found between fluorescence intensity of RW1-2/RW3 and
mM, the fluorescence intensity of RW1-2/RW3 at 581 nm was
The high sensitivity of RW1-2/RW3 for Hg2þ/Al3þ ions
might be used to generate a calibration curve for quantitative
measurement of Hg2þ/Al3þ ions in aqueous solution. By adding
m

F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
47
Hg2þ/Al3þ concentration (Supporting data, Figs. S1eS3). The limit
of detection (LOD) was attained of 2.5 ꢀ 10ꢁ8 M, 4.2 ꢀ 10ꢁ8 M and
2.9 ꢀ 10ꢁ8 M, respectively, based on 3 ꢀ dblank/k (where dblank is
the standard deviation of the blank solution and k is the slope of
the calibration plot). The results indicated that the RW1-2/RW3
chemosensor could sensitively detect environmentally relevant
levels of Hg2þ/Al3þ
.
3.2. pH investigation
To apply the probe in complex environments, the stabilities of
the two probes were tested in the absence and presence of Hg2þ
at different pH values. Fig. 6 shows that for free RW1 in the pH
range of 3.0e4.0, the emission increased as the pH decreased the
ring-opening of the rhodamine framework took place due to the
strong protonation. When pH > 4.0, It can be seen that the
fluorescence was of very low intensity and indicating no signif-
icant ring-opening. However, after the addition of Hg2þ, the
emission increased substantially between pH 4.0 and 8.0. Thus,
RW1 can detect Hg2þ ions with a wide pH range (4.0e8.0)
because in this region RW1 with Hg2þ induces a remarkable
fluorescence “OffeOn”, whereas RW1 without Hg2þ does not
lead to such change. Under identical condition, RW2 and RW3
show similar behavior. In the pH range from 5.0 to 8.0, RW1-2/
RW3 can respond to Hg2þ/Al3þ without any interference by
protons.
3.3. Theoretical mechanism
Thus, in accordance with the 1:1 stoichiometry, the sensors are
the most likely to be chelated with metal ions via its O atoms
(Fig. 7, inset) [49,50]. On the other hand, reversibility of target ion
binding is an important quality in a probe. Consequently, we also
investigated the reversibility of the RW1 and RW2-Hg2þ binding
by a simple titration methodology with Na2S. Na2S was intro-
duced because of its sulfide ion has a strong binding ability toward
thiophilic Hg2þ. Addition of Na2S to the RW1 and RW2-Hg2þ
complex led to an expected decrease of fluorescence intensity
(Fig. 7). The solution returned to its original colorless state, which
reveals that the reversibility due to the chelation-induced ring
opening of rhodamine spirolactam, rather than other possible
reactions.
Fig. 7. Reversible investigation of RW1 and RW2 for Hg2þ with addition of S2ꢁ
.
IR titration results clearly supported the proposed ring-
opening mechanism. As demonstrated in Fig. S4 (Supporting
data), the carbonyl absorption of RW1 at 1670 cmꢁ1 shifted to
a lower frequency at 1633 cmꢁ1 upon the addition of Hg2þ
,
indicating that the carbonyl oxygen coordinated with Hg2þ
resulting in the spirocycle ring opening. A similar behavior was
observed for RW2 and RW3, as shown in Fig. S4 (Supporting
data).
The 13C NMR spectra of RW1-3 recorded in the absence or
presence Hg2þ/Al3þ ions clearly show the involvement of carbonyl
oxygen of RW1-3 in complex formation. The reduction in the 13C-
resonance at near 65 ppm confirms the opening of spirolactam ring
upon complex formation with Hg2þ/Al3þ ions [51] (Supporting
data, Figs. S5eS10).
3.4. Preliminary analytical application
In view of the above-mentioned advantages of the sensor
RW1, bioimaging applications of RW1 for monitoring of Hg2þ
ions in living cells were then carried out. After HL-7702 cells
were incubated with RW1 (10 mM) in culture medium for 30 min
at 37 ꢂC, and no fluorescence of RW1 inside the living HL-7702
cells was observed (Fig. 8b). After three times washing with
Fig. 6. Fluorescence intensity (581 nm) of RW1 RW2 and RW3 (10
CH3OH/HEPES buffer (v/v, 10 mM, pH 7.0) of different pH in the absence and presence
of Hg2þ or Al3þ (10 mol Lꢁ1).
m
mol Lꢁ1) in 4:6
PBS buffer, The cells were then supplemented with 10
mM
m
Hg(NO3)2 in the growth medium for another 30 min at 37 ꢂC, a

48
F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
Fig. 8. Fluorescence images of Hg2þ in HL-7702 cells with 10
m
M solution of RW1 in ethanol-PBS (1:99, v/v) buffer for 30 min at 37 ꢂC, bright-field transmission image (a and c) and
fluorescence image (b and d) of HL-7702 cells incubated with 0 mM, 10 m
M of Hg2þ for 30 min, respectively.
bright fluorescence was observed from within the cell (Fig. 8d).
A bright-field transmission image of cells treated with RW1 and
Hg2þ confirmed that the cells were viable throughout the im-
aging experiments (Fig. 8a, c). It is proven that RW1 is
cell-permeable and primarily little toxic to the cell culture. These
results demonstrated that RW1 may be used for detecting Hg2þ
in biological samples. The fluorescence imaging experiments of
RW2 and RW3 are shown in the supporting information
(Figs. S11eS12) [50,52e66].
shows a number of attractive characteristics such as good selec-
tivity, high sensitivity and wide applicability. RW1 can be used for
rapid analysis of trace level Hg2þ in living cells with satisfactory
results.
To evaluate cytotoxicity of RW1 was taken as an example to
perform a assay on HL-7702 cells with dye concentrations from 0 to
10
mM. A cytotoxicity screen (Fig. 9) was conducted for four dye
concentrations: 0
m
M, 0.1 M, 1 M, 10 M. The cellular viability
m
m
m
estimated was ca. 97% in 24 h after treatment with 10
exhibiting low toxicity to HL-7702 cells.
mM of RW1,
3.5. Method performance comparison
Table 1 summarizes the performance of typical Hg2þ fluorescent
chemosensors and highlights their applications. Most of the probes
present coordination mode was 1:1 with Hg2þ [67,68,70,72e77].
Although all the probes have good selectivity for Hg2þ, a few of
probes possess a wide quantitative range [68,70,72,74,75,78], even
down to nM LOD [72,74,75]. However some of the probes require
more rigorous testing media [68,69,71], and the reproductivity
[68e70,72,73,75e77] as well as the applicability in living cells
[67,68,72e74,76e78], are investigated. RW1 based on rhodamine
Fig. 9. Cell viability values (%) estimated by MTT proliferation test versus incubation
concentrations of RW1. HL-7702 cells were cultured in the presence of 0e10 mM RW1
at 37 ꢂC.

F. Yan et al. / Dyes and Pigments 98 (2013) 42e50
49
Table 1
Performances comparison of various “OffeOn” probes for Hg2þ
.
Reagents
Linear
range,
LOD,nM
Coordination
mode
(probe:ion)
Testing media
Applications
Reproducibility
NA
Ref.
[67]
m
M
Rhodamine derivative
NAa
NA
1:1
Water/CH3CN(1:1,
Rat Schwann
cells
Hela cells
NA
v/v pH ¼ 7 NaAceHAc)
Rhodamine derivative
Rhodamine derivative
Rhodamine derivative
0e25
NA
0.08e10
80
500
40
1:1
1:2
1:1
100% CH3CN
Reversible
Reversible
Reversible
[68]
[69]
[70]
100% THF
Water/ethanol
NA
(1:1, v/v pH ¼ 7.24 TriseHCl)
BINOL derivative
Rhodamine derivative
NA
0e0.35
NA
8.25
1:2
1:1
100% CH3CN
NA
NA
Reversible
[71]
[72]
MethanoleHEPES buffer
(30:70, v/v, 20 mM HEPES, pH 7.0)
CH3CNeH2O (4:1, v/v;
HL-7702 cells
Rhodamine derivative
NA
NA
1:1
HeLa cells
Reversible
[73]
10
m
M tris HCl buffer; pH ¼ 7.0)
Rhodamine derivative
1,8-anthrancene
1e28
0.2e2
1.41
0.79
1:1
1:1
1:99 ethanol/H2O (v/v, pH 7.0)
K 562 cells
NA
NA
Reversible
[74]
[75]
Water containing 2% (v/v) of DMSO
disulfonamide
Rhodamine derivative
NA
NA
NA
NA
1:1
1:1
20 mM, MeCNewater solution,
95:5,v/v, pH ¼ 7.2)
HeLa cells
A549 cells
Reversible
Reversible
[76]
[77]
Rhodamine derivative
1 ꢀ 10ꢁ2 mol Lꢁ1 HEPES,
1 ꢀ 10ꢁ1 M NaClO4, pH ¼ 7.4,
50% ethanol, v/v)
Rhodamine derivative
Rhodamine derivative
0.1e1
0.1e3
100
25
2:1
1:1
WatereDMF solution
Rat Schwann
cells
Reversible
Reversible
[78]
(1/1, v/v, pH ¼ 7.0 NaAceHAc)
CH3OH/HEPES buffer
This work
of RW1
(4:6, v/v, 10 mM, pH 7.0)
a
Na: Not available.
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solid waste (MSW) incineration. Environ Sci Technol 2010;44:7994e5.
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4. Conclusion
In conclusion, rhodamine derivatives RW1-2/RW3 were syn-
thesized by the one-step condensation between Rhodamine ethyl-
enediamine with phthalimido acid chlorides and behave as
selective fluorescent and colorimetric sensors for Hg2þ/Al3þ in
aqueous solution. Complexation of the Hg2þ/Al3þ ions open the
spirolactam ring of rhodamine moieties to give specific color change
as well as fluorescence enhancement at 581 nm. Most importantly,
due to the presence of chlorine atoms, RW2 has a lower binding
constant than RW1 for the Hg2þ. In addition, Moreover, the benzyl
group can make RW3 better identify Al3þ. The living cell imaging
experiments further demonstrate the value of RW1-3 in the prac-
tical applications in biological systems.
[8] Liu YW, Chen CH, Wu AT. A turn-on and reversible fluorescence sensor for
Al3þ ion. Analyst 2012;137:5201e3.
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separation for recovering aluminum from waste toner cartridges. Environ Sci
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Acknowledgments
The authors thank the financial supports from the National
Natural Science Foundation of China (50973084), Science and
Technical Development Foundation of Colleges and Universities,
Tianjin, China (20071214).
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trations in Canadian shield streams along vegetation and pond-age gradients.
Environ Sci Technol 2009;43:5605e11.
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fluorescent chemodosimeter to monitor mecury/methylmercury species
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Appendix A. Supplementary data
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sensing of pyrophosphate in 100% aqueous solution by a system comprised of
rhodamine B compound and Al3þ complex. Analyst 2010;135:2079e84.
[17] Maity DR, Daraju TG. Conformationally constrained (Coumarin-Triazolyl-
Bipyridyl) click fluoroionophore as a selective Al3þ sensor. Inorg Chem 2010;
49:7229e31.
[18] Kang J, Zhang YM, Huang ZX, Han L. Directly oxidized chemiluminescence of
2-Substituted-4,5-di(2-Furyl)-1H-imidazole by acidic potassium permanga-
nate and its analytical application for determination of albumin. J Fluoresc
2011;21:1607e15.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.dyepig.2013.02.002.
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