A new rhodamine-based "off-on" fluorescent chemosensor for Hg (II) ion and its application in imaging Hg (II) in living cells

Article abstract of DOI:10.1007/s10895-012-1065-x

A novel rhodamine derivative (Rh-C), synthesized by the reaction of rhodamine ethylenediamine and cinnamoyl chloride, was evaluated as a chemoselective Hg²⁺ ion sensor. Addition of Hg²⁺ to an ethanol aqueous solution of the Rh-C resu

Full text of DOI:10.1007/s10895-012-1065-x

J Fluoresc (2012) 22:1249–1256
DOI 10.1007/s10895-012-1065-x
ORIGINAL PAPER
A New Rhodamine-Based “Off-On” Fluorescent Chemosensor
for Hg (II) Ion and its Application in Imaging Hg (II)
in Living Cells
Fanyong Yan & Donglei Cao & Meng Wang & Ning Yang &
Qiuhua Yu & Linfeng Dai & Li Chen
Received: 11 January 2012 /Accepted: 28 May 2012 /Published online: 14 June 2012
#
Springer Science+Business Media, LLC 2012
Abstract A novel rhodamine derivative (Rh-C), synthe-
sized by the reaction of rhodamine ethylenediamine and
cinnamoyl chloride, was evaluated as a chemoselective
Hg2+ ion sensor. Addition of Hg2+ to an ethanol aqueous
solution of the Rh-C resulted in a color change from color-
less to obvious pink color together with distinctive changes
in UV–vis absorption spectrum and fluorescence spectrum.
However, other common alkali-, alkaline earth-, transition-
and rare earth metal ions induced no or minimal spectral
changes. The interaction of Hg2+ and sensor Rh-C was
proven to adopt a 1:1 binding stoichiometry and the recog-
nition process is reversible. The chemosensor displayed a
linear response to Hg2+ in the range of 0.4–5 μM with a
detection limit of 7.4×10−8 M. The sensor Rh-C was also
successfully applied to the imaging of Hg2+ in HL-7702
cells.
nervous system, kidney, and endocrine system diseases re-
sult from a series of biological effects [7–9]. Thus, much
attention has been focused on developing Hg2+ fluorescent
chemosensors with excellent sensitivity and selectivity,
quick response time and easy signal detection [10–16].
The rhodamine-based chemosensors can react specifically
with specific metal ions to induce a concomitant change of
their photochemical properties (excitation/emission wave-
length, fluorescence intensity, and so forth). Alteration in
molecular structure between non-fluorescent of spirocyclic
and fluorescent ring-open conformations of rhodamine frame-
work is employed as the detection mechanism [17–22].
Since Hg2+ is a heavy metal ion with 5d10 6s0 electronic
configuration, the oxygen atom or the nitrogen atom of the
imino moiety might be a proper binding site when incorpo-
rated into rhodamine fluorophore [23–27]. The increase of
the O, N atom has the potential to enhance the selective and
sensitivity of the resulting chemosensors toward Hg2+.
Herein,we synthesized a rhodamine-based fluorogenic che-
mosensor (Rh-C), for selective response to Hg2+ in aqueous
media and in living cells. Further, upon chelation of Hg2+,
Rh-C will change to a fluorescent ring-opened form, which
could be detected by the naked eye.
.
.
Keywords Hg (II) ion Rhodamine derivative Fluorescence
.
.
.
enhancement Chemosensor Fluorescent image living cells
Introduction
Mercury is a highly toxic and widespread global pollutant
[1–4]. The mercuric ion, Hg [II], combines with both inor-
ganic and organic ligands, which can readily penetrate
through biological membranes even at very low concentra-
tions [5, 6]. A wide variety of symptoms, including central
Experiments
Materials and Instruments
:
:
:
:
:
:
All the reagents were purchased from commercial suppliers
and used without further purification. All the common
chemicals were of analytical grade. Solvents were purified
by standard procedures. All reactions were monitored by
TLC (thin-layer chromatography) with detection by UV.
The solutions of Na+, K+, Mg2+, Mn2+, Ca2+, Ba2+, Fe3+,
F. Yan D. Cao M. Wang N. Yang Q. Yu L. Dai
L. Chen (*)
State Key Laboratory of Hollow Fiber Membrane Materials
and Processes, Key Lab of Fiber Modification & Functional
Fiber of Tianjin, Tianjin Polytechnic University,
Tianjin 300160, China
e-mail: chenlis11@yahoo.com.cn

1250
J Fluoresc (2012) 22:1249–1256
Scheme 1 Synthetic routes of
the probe Rh-C
Zn2+, Pb2+, Cu2+, Cd2+, Ag+ and Hg2+ were prepared from
their nitrate salts. Doubly distilled deionized water was used
throughout the experiment.
ethylenediamine (Rh-E) was facilely synthesized in high yield
according to the procedure as published in the literature [28].
Rhodamine ethylenediamine (Rh-E) (2.52 g, 5.19 mmol)
and triethylamine (4 mL) were dissolved in 25 mL dry
dichloromethane. After cooling to 0 °C in ice bath, to the
solution was added dropwise a solution of acinnamoyl chlo-
ride (0.86 g, 5.19 mmol) in 20 mL of dichloromethane over
10 min. The resulting mixture was stirred for 2 h at room
temperature. The recation mixture was then evaporated and
the crude product was purified by column chromatography
(silica gel, petroleum ether: ethyl acetate 1: 1, v/v). The
The absorption spectra were acquired on a Purkinje Gen-
eral TU-1901 UV–vis spectrometer. Fluorescence spectra
measurements were performed on a Hitachi F-4500 Fluores-
cence spectrophotometer at room temperature. NMR spectra
were measured on a Bruker 300 MHz spectrometer with
chemical shifts reported in ppm (in CDCl3; TMS as internal
standard). Electrospray ionization (ESI) mass spectra were
conducted by ABI-057-TY4675 instrument. The pH measure-
ments were carried out on a PHS-3W pH meter. The masses of
the samples and solvents were determined by a Mettler Toledo
AB204-N analytical balance with an accuracy of 0.0001 g.
Fluorescence images experiments were carried out with an
Olympus IX71 inverted fluorescence microscope.
1
yield was 72 %. HNMR(CDCl3, 300 MHz): δ07.98 (dd,
1H), 7.57 (s, 1H), 7.51 (m, 2H), 7.47 (dd, 2H), 7.35 (m, 3H),
7.08 (dd, 2H), 6.47 (d, J08.9Hz, 2H), 6.30–6.39(m, 5H),
3.18–3.34 (m, 12H), 1.19 (t, J07.1Hz, 12H). 13C NMR
(CDCl3, 75 MHz): δ0170.15, 165.82, 153.85, 153.28,
140.10, 135.18, 132.84, 130.40, 129.32, 128.66, 128.45,
128.21, 127.84, 123.93, 122.83, 121.38, 108.39, 97.89,
65.82, 44.43, 41.24, 40.17, 12.56. IR (KBr, ν/cm−1): 3427,
3006, 2854, 1672, 1615, 1459, 1220, 1118, 979. ESI-MS
(M+H+): m/z0614.33 (C39H42N4O3).
Synthesis of Probe Rh-C
The newly synthesized rhodamine B derivative (Rh-C) was
prepared in high yield (Scheme 1). Compound rhodamine
General UV–vis and Fluorescence Spectra Measurements
2 mM of each inorganic salt (NaNO3, KNO3, Mg(NO3)2•6H2O,
Mn(NO3)2•4H2O, Ca(NO3)2, Ba(NO3)2, Zn(NO3)2•6H2O, Fe
(NO3)2•6H2O, Pb(NO3)2, Cu(NO3)2•3H2O, Cd(NO3)2•2H2O,
AgNO3 and HgNO3•0.5H2O) was dissolved in distilled water
to afford 2×10−3 mol•L−1 aqueous solution. A 2.0×
10−3 mol•L−1 stock solution of Rh-C was prepared in absolute
ethanol. All the measurements were made according to the
following procedure. To 10 mL glass tubes containing differ-
ent amounts of metal ions, proper amounts of the solution of
Rh-C was added directly with micropipette, then diluted with
buffered (HEPES 20 mM, pH07.0) water-ethanol (7/3, v/v) to
10 mL, then the absorption and fluorescence sensing of metal
ions were run. In selectivity experiments, the test samples
were prepared by placing the appropriate amounts of metal
ion stock solution into 10 mL solution of Rh-C (10 μM). All
samples were prepared at room temperature, shaken for 10 s
and waited for 10 min before test. Fluorescence measurements
Fig. 1 Fluorescence intensity of Rh-C (10 μM) in the absence and
presence of 1 equiv. Hg2+ in water -ethanol solution (30:70, v/v) at
different pH. The pH modified by adding 75 % HClO4 or NaOH
(10 %). Excitation: 535 nm, emission: 586 nm

J Fluoresc (2012) 22:1249–1256
1251
Fig. 2 UV–vis absorption
spectra of Rh-C (10.0 μM)
obtained during the titration by
Hg2+ (0-2 equiv.) in buffered
(HEPES 20 mM, pH07.0)
water-ethanol (7/3, v/v) at room
temperature. Inset: a The color
change of Rh-C (10 μM)+2
equiv. Hg2+ in solution, b Job’s
plot of changes of absorbance at
562 nm, the total concentration
of [Hg2+]+[Rh-C] was 20 μM
were carried out with excitation and mission slit width of 10
and 5 nm and excitation wavelength was 535 nm.
fluorescence imaging of intracelluar Hg2+ was observed
under inverted fluorescence microscope (IX71, Olympus,
Japan) with a 40×objective lens (excited with green light).
The HL-7702 cells only incubated with 10 μM Rh-C for
0.5 h at 37 °C under 5 % CO2 was as a blank control.
Experimental Details for Cell Imaging Experiments
The HL-7702 cells (human hepatocyte cell line) were cul-
tured in DEME (Invitrogen) supplemented with 10 % FBS
(Invitrogen). One day before imaging, the cells were seeded
in 6-well flat-bottomed plates. The next day, the HL-7702
cells were incubated with 10 μM sensor Rh-C for 0.5 h at
37 °C in humidified environment of 5 % CO2 and then
washed with phosphate-buffered saline (PBS) three times
before incubating with 10 μM Hg(NO3)2 for another 0.5 h,
cells were rinsed with PBS three times again, then the
Determination of Binding Constants
The binding constant was calculated from the absorption
intensity titration curves according to Benesi-Hildebrand
equation as follow.
1
1
1
¼
þ
A ꢀ A0 KaðAmax ꢀ A0Þ½cꢁ Amax ꢀ A0
A and A0 is the absorbance of Rh-C solution in the
presence and absence of Hg2+; respectively; Amax is the
saturated absorbance of Rh-C in the presence of excess
amount of Hg2+; [c] is the concentration of Hg2+ ions added
(mol L−1). The association constant values Ka is calculate by
the ratio intercept/slope.
Results and discussion
Synthesis and Structural Characterization of Rh-C
Rh-C was facilely synthesized from rhodamine B by
acylation reaction, as summarized in Scheme 1. Its
structure was confirmed by HNMR, 13CNMR and MS
data.
A solution of Rh-C in ethanol is colorless and weakly
fluorescent, indicating that the spirolactam form of Rh-C
1
Fig. 3 UV–vis spectra of Rh-C (10.0 μM) upon addition of 2.0 equiv.
of Hg2+ and 10 equiv. other metal ions (Ag+, Ba2+, Ca2+, Cd2+, Cu2+
,
Fe3+, K+, Mg2+, Mn2+, Na+, Pb2+ and Zn2+) in buffered (HEPES
20 mM, pH07.0) water-ethanol (7/3, v/v)

1252
J Fluoresc (2012) 22:1249–1256
Fig. 4 Benesi-Hildebrand plot
(absorbance at 562 nm) of Rh-C
using 1:1 stoichiometry for
association between Rh-C
and Hg2+
exists predominantly. The characteristic peak of the 9-
carbon of Rh-C near 66 ppm in the 13CNMR spectrum also
supports this consideration [29]. The chemosensing behavior
of Rh-C was investigated by UV–vis and fluorescence
measurements.
opening of rhodamine took place because of the strong
protonation. No obvious fluorescence emission of Rh-C
was observed between pH 5 and 12, suggesting that the
compound is insensitive to pH and that the spirolactam form
is still preferred in this condition. However,the addition of
Hg2+ led to the fluorescence enhancement over a compara-
tively wide pH range (5.0–9.0), which is attributed to open-
ing of the rhodamine ring. The results suggest that Rh-C was
insensitive to pH near 7.0 and could work in physiological
pH conditions with a very low background fluorescence
[30]. Therefore, further UV–vis and fluorescent studies were
carried out in buffered (HEPES 20 mM, pH07.0) water-
ethanol (7/3, v/v) at room temperature.
Effect of pH Value
For practical application, the appropriate pH conditions for
successful operation of the sensor were evaluated. The
effects of pH on the fluorescence response of Rh-C obtained
without and with Hg2+ in water-ethanol (7/3, v/v) are shown
in Fig. 1. For free Rh-C, at acid conditions (pH<5), the ring
Fig. 5 Fluorescence emission
spectra of Rh-C (10.0 μM) in
buffered (HEPES 20 mM, pH0
7.0) water-ethanol (7/3, v/v)
upon the addition of Hg2+
(0–2 equiv.) with an excitation
of 535 nm. Inset: a fluorescence
enhancement at 586 nm as a
function of Hg2+ concentration,
b Job’s plot of changes of
fluorescent intensity at
586 nm, the total concentration
of [Hg2+]+[Rh-C] was 10 μM

J Fluoresc (2012) 22:1249–1256
1253
Fig. 6 Reversibility of Hg2+ coordination to Rh-C (10 μM) by Na2S in
buffered (HEPES 20 mM, pH07.0) water-ethanol (7/3, v/v). The top
lines represent the fluorescence enhancement that occurs after addition
of 1 equiv. of Hg2+. The bottom lines represent the fluorescence
intensity of free Rh-C and the fluorescence intensity decrease that
occurs after addition 1 equiv. of Na2S to a solution containing the
[Rh-C-Hg2+] species. Four cycles of on/off by Hg2+/Na2S addition are
depicted in this plot.
Fig. 7 Fluorescent spectra of Rh-C (10.0 μM) upon addition of 2.0
equiv. of Hg2+ and 10 equiv. other metal ions (Ag+, Ba2+, Ca2+, Cd2+
,
Cu2+, Fe3+, K+, Mg2+, Mn2+, Na+, Pb2+ and Zn2+) in buffered (HEPES
20 mM, pH07.0) water-ethanol (7/3, v/v)
K+, Mg2+, Mn2+, Na+, Pb2+ and Zn2+ did not show any
significant spectral change except Fe3+ and Cu2+ caused
a little bit of enhancement under identical conditions
(Fig. 3). These results suggested that Rh-C could serve
as a “naked-eye” chemosensor selective for Hg2+ in
neutral buffered media.
The method of continuous variations (Job’s plot)
obtained from the Rh-C+Hg2+ system in buffered (HEPES
20 mM, pH07.0) water-ethanol (7/3, v/v), When molar
fraction of Hg2+ was 0.5, the absorbance at 562 nm got to
maximum (Fig. 2.Inset (b)), indicating that forming a 1:1
complex between Rh-C and Hg2+, which was confirmed by
the Benesi-Hildebrand method [32].
UV–vis Spectral Reponses of Rh-C
The absorption spectra of Rh-C with varying Hg2+ concen-
trations in buffered (HEPES 20 mM, pH07.0) water-ethanol
(7/3, v/v) were recorded, as shown in Fig. 2. Like most of
the spirocycle RhB derivatives, When no Hg2+ was added to
the solution, free Rh-C was colorless and exhibited almost
no absorption peak in the visible wavelength range
(>400 nm) due to the closed spirolactam ring. In the
presence of 2 equiv. of Hg2+, the absorbance was en-
hanced obviously and a new peak at 562 nm was
observed, accompanied by a clear color change from
colorless to pink. This enhancement in absorbance can
be ascribed to the clear formation of the ring-opened
amide form of Rh-C upon Hg2+ ions binding [19, 31].
The color of Rh-C also change from colorless to pink
(Fig. 2.Inset (a)), indicating that Rh-C can serve as a
“naked-eye” sensor for Hg2+ in aqueous solution. Other
metal ions, such as Ag+, Ba2+, Ca2+, Cd2+, Cu2+, Fe3+,
Fig. 8 Competition of Rh-C (10.0 μM) for Hg2+ (2equiv.) upon
addition of other metal ions (10equiv.) in buffered (HEPES 20 mM,
pH07.0) water-ethanol (7/3, v/v). △F0F–F0, (F0 and F were the
fluorescence intensity of Rh-C in the absence and presence of Hg2+).
Excitation: 535 nm, emission: 586 nm
Scheme 2 Probable complexation mechanism of Rh-C with Hg2+

1254
J Fluoresc (2012) 22:1249–1256
Fig. 9 Fluorescence images of
Hg2+ in HL-7702 cells with
5 μM solution of Rh-C in
Ethanol-PBS (1 : 99, v/v) buffer
for 30 min at 37 °C, Bright-field
transmission image (a, c) and
fluorescence image (b, d) of
HL-7702 cells incubated with
0 μM, 5 μM of Hg2+ for
30 min, respectively
Linear fitting of the titration profiles using Benesi-
Hildebrand plot based on a 1:1 binding mode results in a
good linearity (Fig. 4), which strongly support the 1:1
binding stoichiometry of Rh-C and Hg2+, and the binding
constant was calculate to be 3.81×104 M−1[33].
demonstrated that the probe can quantitatively determine
Hg2+ at the environmentally relevant level.
The Proposed Reaction Mechanism
In addition, the Na2S-adding experiments were conducted to
examine the reversibility of this reaction as shown in Fig. 6.
Firstly, the addition of Hg2+ to the free Rh-C solution makes
the fluorescence increase significantly. Secondly, the addi-
tion of Na2S could immediately restore the initial fluores-
cent intensity and the color of free Rh-C due to the Kd value
of 10-50 M2 for Hg2+ at a standard condition in the form of
[HgS2]2−. The stability of Rh-C has been test by add Hg2+
and Na2S alternately. After four cycles, the fluorescence
intensity of Hg-Rh-C quenched a little compared to the
original fluorescent intensity. Thus, the probe Rh-C may
be used repeatedly by adding Na2S. It was also confirmed
that the response of Rh-C to Hg2+ was reversible rather than
a cation-catalyzed reaction.
Thus, based on the 1:1 binding mode and the re-
versible behaviour between Rh-C and Hg2+, a possible
coordination mode for Rh-C with Hg2+ was proposed
(Scheme 2). Rh-C chelates with Hg2+ via two O atoms
of two carbonyl groups, forming the complexation that
requires the opening of the spiroring, which caused the
recovery of fluorescence of rhodamine. It is very likely
due to the chelation–induced ring opening of rhoda-
mine spirolactam, rather than other possible reactions
[25, 34].
Fluorescence Spectral Responses of Rh-C
Fluorescence titrations of Rh-C with Hg2+ in buffered
(HEPES 20 mM, pH07.0) water-ethanol (7/3, v/v) were
then performed. As shown in Fig. 5, free Rh-C showed a
very weak band in the range 500–700 nm due to the spi-
rocyclic structure. Upon addition gradual increase of Hg2+
to the solution of Rh-C (10 μM), a significant enhancement
in fluorescence intensity at 586 nm was observed following
excitation at 535 nm and gradual increased with Hg2+ con-
centrations. Meanwhile the solution showed an orange
fluorescence.
Fitting of the Job’s plot evaluated from the fluores-
cent spectra of Rh-C and Hg2+ at 586 nm gave rise to a
1: 1 stoichiometry for the Rh-C-Hg2+ complex(Fig 5
Inset (b)), which consistent with the results of absortion.
The linear response of the fluorescence intensity toward
Hg2+ was obtained in Hg2+ concentration range of 0.4–
5 μM (Fig 5 Inset (a)). And the limit of detect ion
(LOD) was obtained of 7.4×10−8 M, which was calcu-
lated based on 3 δ/k (δ is the standard deviation of the
measured intensity of the blank solution and k is the
slope of the plot in the inset of Fig. 5). The results

J Fluoresc (2012) 22:1249–1256
1255
Acknowledgements 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).
Selectivity and Completion
The selectivity of Rh-C for Hg2+ was further observed in the
fluorescent spectra. As expected, Rh-C exhibited excellent
fluorescence selectivity towards Hg2+ over all other alkali
and alkaline earth metal ions, transition and heavy metal
ions except for a little bit of fluorescence enhancement for
Fe3+ (Fig. 7). And, for reliable application, we can add F− to
mask Fe3+ caused the fluorescence. This finding indicated
that Rh-C could selectively recognize Hg2+ in ethanol
aqueous condition.
References
1. Clarkson TW, Magos L, Myers GJ (2003) The toxicology of
mercury-current exposures and clinical manifestations. New Engl
J Med 3(49):1731–1737
2. Harris HH, Pickering IJ, George GN (2003) The chemical form of
mercury in fish. Science 301(5637):1203
Then competitive experiments were conducted in the
presence of 2.0 equiv. of Hg2+ mixed with 10.0 equiv.
of various cations in the solution of Rh-C (10 μM),
respectively. No significant variation in fluorescence
intensity was found by comparison with that without
other metal ions besides Hg2+ expect for Ag+ and Zn2
3. Nolan EM, Lippard SJ (2008) Tools and tactics for the optical
detection of mercuric ion. Chem Rev 108(9):3443–80
4. Wang Q, Kim D, Dionysiou DD, Sorial GA, Timberlake D (2004)
Sources and remediation for mercury contamination in aquatic
systems–a literature review. Environ Pollut 131(2):323–336
5. Fitzgerald WF, Lamgorg CH, Hammerschmidt CR (2007) Marine
biogeochemical cycling of mercury. Chem Rev 107(2):641–662
6. Nendza M, Herbst T, Kussatz C, Gies A (1997) Potential for
secondary poisoning and biomagnification in marine organisms.
Chemosphere 35(9):1875–1885
+
caused a little decrease (Fig. 8). All these indicate that
the selectivity of Rh-C for Hg2+ over other competitive
cations is high.
7. Grandjean P, Weihe P, White RF, Debes F (1998) Cognitive
performance of children prenatally exposed to “safe” levels of
methylmercury. Environ Res 77(2):165–172
Bioimaging Applications of Rh-C in HL-7702 Cells
8. Nolan EM, Lippard SJ (2005) MS4, a seminaphthofluorescein-
based chemosensor for the ratiometric detection of Hg (II). J Mater
Chem 15:2778–2783
9. Hoyle I, Handy RD (2005) Dose-dependent inorganic mercury
absorption by isolated perfused intestine of rainbow trout, Onco-
rhynchus mykiss, involves both amiloride-sensitive and energy-
dependent path ways. Aquat Toxicol 72(1–2):147–159
10. Quang D, Kim JS (2010) Fluoro- and Chromogenic chemodosim-
eters for heavy metal ion detection in solution and biospecimens.
Chem Rev 110(10):6280–6301
Bioimaging applications of Rh-C for monitoring of Hg2+
ions in living cells were then carried out. HL-7702 cells
were incubated with Rh-C (5 μM) in culture medium for
30 min at 37 °C, and very weak fluorescence of Rh-C inside
the living HL-7702 cells was observed (Fig. 9b). After three
times washing with PBS buffer, The cells were then supple-
mented with 5 μM Hg(NO3)2 in the growth medium for
another 30 min at 37 °C,a bright fluorescence was observed
from the intracellular (Fig. 8d). A bright-field transmission
image of cells treated with Rh-C and Hg2+ confirmed that
the cells were viable throughout the imaging experiments
(Fig. 9a, c). It is proved that Rh-C is cell-permeable and
primarily little toxic to the cell culture. These results
demonstrated that Rh-C may be used for detecting Hg2+ in
biological samples.
11. Lee MH, Wu JS, Lee JW, Jung JH, Kim JS (2007) Highly sensitive
and selective chemosensor for Hg2+ based on the rhodamine fluo-
rophore. Org Lett 9(13):2501–2504
12. Zheng H, Qian ZH, Xu L, Yuan FF, Lan LD, Xu JG (2006)
Switching the recognition preference of rhodamine B spirolactam
by replacing one atom: design of rhodamine B thiohydrazide for
recognition of Hg (II) in aqueous solution. Org Lett 8(5):859–861
13. Feng LH, Chen ZB (2007) Screening mercury (II) with selective
fluorescent chemosensor. Sensor Actuat B- Chem 122(2):600–604
14. Wu DY, Huang W, Lin ZH, Duan CY, He C, Wu S, Wang DH
(2008) Highly sensitive multi-responsive chemosensor for selec-
tive detection of Hg2+ in natural water and different monitoring
environments. Inorg Chem 47(16):7190–7201
Conclusion
15. Zhan XQ, Qian ZH, Zheng H, Su BY, Lan Z, Xu JG (2008)
Rhodamine thiospiro-lactone highly selective and sensitive revers-
ible sensing of Hg (II). Chem Commun 16:1859–1861
16. Huang HJ, Xu Y, Qian X (2009) A rhodamine-based Hg2+ sensor
with high selectivity and sensitivity in aqueous solution: a NS2-
containing receptor. J Org Chem 74(5):2167–2170
17. Xiang Y, Tong AJ, Jin PY, Ju Y (2006) New fluorescent rhodamine
hydrazone chemosensor for Cu (II) with high selectivity and sen-
sitivity. Org Lett 8(13):2863–2866
18. Huang W, Song C, He C, Lv G, Hu X, Zhu X, Duan C (2009)
Recognition preference of rhodamine-thio spirolactams for mercu-
ry (II) in aqueous solution. Inorg Chem 48(12):5061–5072
19. Kwon JY, Jang YJ, Lee YJ, Kim KM, Yoon J et al (2005) A highly
selective fluorescent chemosensor for Pb2+. J Am Chem Soc 127
(28):10107–10111
In summary, we have developed a novel easily available
turn-on fluorescent sensor Rh-C based on a rhodamine
conjugate. It selectively responds to Hg2+ by chromo-
and fluorogenic changes and also facilitates “naked-eye”
detection of Hg2+. The background metal ions showed
small or no interference with the detection of Hg2+ ion.
The chemosensor displayed a linear response to Hg2+ in
the range of 0.4–5 μM with a detection limit of 7.4×
10−8 M. Moreover, fluorescence microscopy experiments
establish that Rh-C can be used for detecting Hg2+
within living cells.

1256
J Fluoresc (2012) 22:1249–1256
20. Huang W, Zhu X, Wu DY, He C, Hu XY, Duan CY (2009)
Structural modification of rhodamine-based sensors toward highly
selective mercury detection in mixed organic/aqueous media.
Dalton Trans 47:10457–10465
21. Chartres JD, Busby M, Riley MJ, Davis JJ, Bernhardt PV (2011) A
turn-on fluorescent iron complex and its cellular uptake. Inorg
Chem 50(18):9178–9183
22. Huang W, Wu DY, Duan CY (2010) Conformation-switched che-
mosensor for selective detection of Hg2+ in aqueous media. Inorg
Chem Commun 13(2):294–297
27. Bhalla V, Tejpal R, Kumar M (2010) Rhodamine appended ter-
phenyl: a reversible “off-on” fluorescent chemosensor for mercury
ions. Sensor Actua B-Chem 151(1):180–185
28. Zhang X, Shiraishi Y, Hirai T (2007) Cu (II) selective green
fluorescence of a rhodamine-diacetic acid conjugate. Org Lett 9
(24):5039–5042
29. Anthoni U, Christophersen C, Nielsen P, Puschl A, Schaumburg K
(1995) Structure of red and orange fluorescein. Struct Chem
3:161–165
30. Nolan EM, Lippard SJ (2003) A “turn-on” fluorescent sensor for
the selective detection of mercuric ion in aqueous media. J Am
Chem Soc 125(47):14270–14271
23. Suresh M, Shrivastav A, Mishra S, Suresh E, Das A (2008) A
rhodamine-based chemosensor that works in the biological system.
Org Lett 10(14):3013–3016
31. Wang HG, Li YP, Xu SF, Li YC, Zhou C, Fei XL et al (2011)
Rhodamine-based highly sensitive colorimetric off-on fluorescent
chemosensor for Hg2+ in aqueous solution and for live cell imag-
ing. Org Biomol Chem 9:2850–2855
24. Zhou Y, Zhu CY, Gao XS, You XY, Yao C (2010) Hg2+-selective
ratiometric and “off on” chemosensor based on the azadiene pyr-
ene derivative. Org Lett 12(11):2566–2569
25. Hu ZQ, Lin CS, Wang XM, Ding L, Cui CL, Liu SF, Lu HY
(2010) Highly sensitive and selective turn-on fluorescent chemo-
sensor for Pb2+ and Hg2+ based on a rhodamine-phenylurea con-
jugate. Chem Comm 46:3765–3767
26. Yang H, Zhou Z, Huang K, Yu M, Li FY, Yi T, Huang C
(2007) Multisignaling optical- electrochemical sensor for Hg2+
based on a rhodamine derivative with a ferrocene unit. Org
Lett 9(23):4729–4732
32. Benesi HA, Hildebrand JH (1949) A spectrophotometric investi-
gation of the interaction of iodine with aromatic hydro carbons. J
Am Chem Soc 71(8):2703–2707
33. Conner KA (1987) Binding constants-the measurement of molec-
ular complex stability. Wiley, New York
34. Soh JH, Swamy MK, Sook KK, Suki K, Sang HL, Yoon J (2007)
Rhodamine urea derivatives as fluorescent chemosensors for Hg2+
.
Tetrahedron Lett 48:5966–5969