A rhodamine-based fluorescent probe for detecting Hg2+ in a fully aqueous environment

Article abstract of DOI:10.1039/c3dt51279g

A water-soluble fluorescent probe for Hg²⁺ based on a rhodamine B derivative was designed and synthesized. The new probe showed reversible colorimetric and fluorescent response to Hg²⁺ in a fully aqueous solution. The probe exhibited real-time detection of Hg²⁺ with high selectivity in media containing less than 1% organic cosolvent. Furthermore, bioimaging studies indicated that the new probe was cell permeable and suitable for the real-time imaging of Hg²⁺ in living cells by confocal microscopy.

Full text of DOI:10.1039/c3dt51279g

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A rhodamine-based fluorescent probe for detecting
Hg2+ in a fully aqueous environment†
Cite this: Dalton Trans., 2013, 42, 14819
Xiaoli Chen,a Xiangming Meng,*a Shuxing Wang,a Yulei Cai,a,b Yifan Wu,a
Yan Feng,*a Manzhou Zhua and Qingxiang Guob
A water-soluble fluorescent probe for Hg2+ based on a rhodamine B derivative was designed and
synthesized. The new probe showed reversible colorimetric and fluorescent response to Hg2+ in a fully
aqueous solution. The probe exhibited real-time detection of Hg2+ with high selectivity in media contain-
ing less than 1% organic cosolvent. Furthermore, bioimaging studies indicated that the new probe was
cell permeable and suitable for the real-time imaging of Hg2+ in living cells by confocal microscopy.
Received 16th May 2013,
Accepted 29th July 2013
DOI: 10.1039/c3dt51279g
www.rsc.org/dalton
fluorescence emission. This unique structure makes rhod-
amine dyes good candidates for constructing a “turn-on”
Introduction
Hg2+ is considered one of the most dangerous and widespread fluorescent probe for Hg2+.
global pollutants, which has a high affinity for thiol groups of
To date, a lot of rhodamine-based fluorescent probes for
proteins and enzymes and causes the dysfunction of the brain, Hg2+ have been reported.7 However, most of these probes work
kidney, and central nervous system.1 On the other hand, in organic solvent or water with organic cosolvent,8 only few of
mercury ions present in soil or in effluent water can be con- them work well in aqueous buffer solutions containing less
verted into methylmercury, which will accumulate in organ- than 20% organic cosolvent.9 This will limit the application of
isms and trigger serious disorders in the human body.2 rhodamine-based probes for Hg2+ detection in biological and
Therefore, a convenient and rapid method for the deter- environmental systems. To reduce the amount of organic
mination, especially on-site or in real-time analysis, of mercury cosolvent during Hg2+ detection, the structure of rhodamine-
in biological and environmental samples is urgent.
based probes needs to be improved to give better water
Because of the simplicity and high sensitivity of fluore- solubility.
scence, fluorescent probes are regarded as the most powerful
Herein, we report a new water soluble rhodamine-based
tools for monitoring metal ions in vitro and/or in vivo biologi- probe (RNS) for Hg2+ (Scheme 1). This “turn-on” fluorescent
cally.3 A lot of fluorescent probes have been reported.4 probe is composed of two moieties: rhodamine B spirolactam
However, turn-on fluorescence probes for Hg2+ have rarely as the potential strong fluorophore and chromophore, and a
been reported, due to the fact that Hg2+ can quench the fluore- bis(2-(ethylthio)ethyl)amine (NS2) fragment as a specific and
scence of fluorophores via the spin–orbit coupling effect.5 reversible binding receptor of Hg2+ due to the thiophilic pro-
Among the compounds used as the fluorophores of probes, perties of mercury.10 For water solubility, a hydrophilic acyl-
rhodamine-based dyes have attracted much attention for their amino group was used to link the rhodamine B group and the
excellent spectroscopic properties with large molar extinction NS2 receptor. We speculated that the probe RNS would detect
coefficients and high fluorescence quantum yields.6 Further- Hg2+ in water with less or no organic cosolvent.
more, the spirocyclic forms of rhodamine derivatives are non-
fluorescent and colorless, whereas the ring-opening of these
derivatives induced by metal ions gives rise to strong
aDepartment of Chemistry, Anhui University, Hefei 230039, P. R. China.
E-mail: mengxm@ahu.edu.cn, fy70@163.com; Fax: +86-551-5107342;
Tel: +86-551-5108970
bDepartment of Chemistry, University of Science and Technology, Hefei 230026,
P. R. China
†Electronic supplementary information (ESI) available: Photophysical measure-
ments and cell imaging, NMR, crystallographic data. CCDC 938972. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3dt51279g
Scheme 1 The design of RNS for Hg2+ detection.
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(DMSO, 100 MHz, ppm), δ: 12.4, 43.6, 64.7, 97.4, 105.4, 107.7,
122.1, 123.5, 127.7, 132.4, 148.1, 151.9, 153.0, 165.3.
Experimental
All the reagents were of analytical grade and used as received.
All solvents were used after appropriate distillation or purifi- Synthesis of compound 2
cation. All reactions were magnetically stirred and monitored
A mixture of compound 1 (1.36 g, 3 mmol), chloroacetyl chlo-
by thin layer chromatography (TLC). Flash chromatography
(FC) was performed using silica gel 60 (200–300 mesh). Solu-
tions of Hg2+, Fe2+, Na+, K+, Mg2+, Mn2+, Fe3+, Cr3+, Co2+, Pb2+,
Cu2+, Cd2+, Ni2+, Ca2+, Zn2+ and Ag+ were prepared from their
perchlorate salts. 1H and H–H COSY NMR spectra were
recorded on Bruker-400 MHz spectrometers and 13C NMR
spectra were recorded on 100 MHz spectrometers. UV-vis
spectra were recorded on a Techcomp UV1000 spectrophoto-
meter. Fluorescence responses were recorded on an FL2500
spectrofluorimeter.
ride (0.5 g, 4.5 mmol) and dichloromethane (30 mL) was cooled
in an ice bath. Then Et3N (0.76 g, 7.5 mmol) was dissolved in
5 mL dichloromethane and added dropwise to the solution
with vigorous stirring. After the addition, the ice bath was kept
for about 30 min, and the color of solution changed from pink
to brown. The solvent was removed under reduced pressure to
give the crude product. The target product was recrystallized in
ethyl acetate to give 1.2 g (75%) of 2. 1H NMR (400 MHz,
DMSO, ppm), δ: 1.17 (12H, t, J = 7.03 Hz, CH3), 3.34 (8H, q, J =
7.02 Hz, CH2), 3.61 (2H, s, COCH2), 6.30 (2H, m, xanthene–H),
6.44 (4H, dd, J = 16.14 Hz, xanthene–H), 7.11 (1H, t, J = 8.26
Hz, Ar–H), 7.45 (2H, m, Ar–H), 7.94 (1H, q, J = 5.78 Hz, Ar–H).
13C NMR (DMSO, 100 MHz, ppm): δ: 12.4, 14.0, 20.7, 40.7,
43.6, 59.7, 65.1, 97.0, 104.0, 107.6, 122.6, 123.8, 128.2, 128.5,
129.1, 133.3, 148.4, 151.8, 153.0, 163.4, 164.6, 170.3.
General UV-vis and fluorescence titrations
Inorganic salt was dissolved in distilled water to afford a
10 mM aqueous solution. The 1 mM stock solution of RNS was
prepared in absolute methanol. All the measurements were
carried out according to the following procedure. To a 10 mL
volumetric flask containing 100 μL of the solution of RNS,
different amounts (10 μL–500 μL) of metal ions were added
directly with a micropipette, then diluted with buffered (pH
7.0, 20 mM HEPES (4-(2-hydroxyerhyl)piperazine-1-erhanesul-
fonic acid), 50 mM NaNO3) solution. Fluorescence measure-
ments were carried out with excitation and emission slit
widths of 5.0 and 2.5 nm and the PMT voltage and excitation
wavelength were 700 V and 520 nm, respectively.
Synthesis of compound RNS
A mixture of compound 2 (0.53 g, 1 mmol), bis(2-(ethylthio)-
ethyl)amine (0.58 g, 3 mmol), KI (0.5 mg, 0.003 mmol) and
acetonitrile (20 mL) was heated to 80 °C for 8 h and cooled to
room temperature. Then the reaction mixture was poured into
distilled water and extracted by ethyl acetate (3 × 15 mL). The
organic phase was combined, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The crude product
was purified by column chromatography by using the mixed
eluent EtOAc–PE (1 : 3) to give 0.46 g (67%) of RNS. 1H NMR
(400 MHz, DMSO, ppm), δ: 1.16 (18H, m, CH3) 2.42 (8H, q, J =
7.41 Hz, CH2SCH2), 2.62 (4H, t, J = 7.05 Hz, NCH2), 3.12 (2H, s,
COCH2), 3.33 (8H, m, NCH2CH3), 6.29 (2H, d, J = 8.69 Hz,
xanthene–H), 6.37 (2H, s, xanthene–H), 6.68 (2H, d, J =
8.81 Hz, xanthene–H), 7.13 (1H, d, J = 7.03 Hz, Ar–H), 7.47
(2H, p, J = 7.11 Hz, Ar–H), 7.94 (1H, d, J = 7.35 Hz, Ar–H), 9.04
(1H, s, NH). 13C-NMR (DMSO, 100 MHz, ppm), δ: 12.6, 14.9,
25.9, 29.4, 44.3, 54.6, 57.9, 65.9, 97.6, 104.4, 107.9, 123.4,
124.0, 128.2, 129.3, 129.4, 132.9, 148.9, 151.7, 153.6, 164.4,
169.1.
Synthesis of compound 1
As shown in Scheme 2, compound 1 was prepared according
to the literature method.11 1H NMR (CDCl3, 400 MHz, ppm): δ:
1.15 (12H, t, J = 7.02 Hz, CH3), 3.34 (8H, q, J = 7.00 Hz, CH2),
3.61 (s, 2H, NH2), 6.45–6.29 (m, 6H, xanthene–H), 7.10 (m, 1H,
Ar–H), 7.45 (m, 2H, Ar–H), 7.93 (m, 1H, Ar–H). 13C NMR
X-ray crystallography
X-ray diffraction data of RNS single crystals were collected on a
Siemens Smart 1000 CCD diffractometer. The determination
of unit cell parameters and data collection were performed
with Mo Kα radiation (λ = 0.71073 Å). Unit cell dimensions
were obtained with least-squares refinements, and all struc-
tures were solved by direct methods using SHELXS-97.13 The
other non-hydrogen atoms were located in successive differ-
ence Fourier synthesis. The final refinement was performed by
using full-matrix least-squares methods with anisotropic
thermal parameters for non-hydrogen atoms on F2. The hydro-
gen atoms were added theoretically and rode on the con-
cerned atoms. Crystallographic data reported in this
Scheme 2 Synthesis of RNS.
14820 | Dalton Trans., 2013, 42, 14819–14825
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contribution has been deposited with the Cambridge Crystallo-
Table 1 X-ray crystallography data for RNS
graphic Date Center, CCDC 938972 for RNS (ESI†).
Compound reference
RNS
Chemical formula
Formula mass
C38H50N5O3S2
688.95
Cytotoxicity assays
Crystal system
Space group
a/Å
b/Å
c/Å
α/°
β/°
γ/°
Triclinic
P1
To test the cytotoxic effect of the probe in cells over a 24 h
period, an MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazo-
lium bromide) assay was performed as previously reported.12
HeLa cells were passed and plated to ca. 70% confluence in
96-well plates 24 h before treatment. Prior to RNS treatment,
DMEM (Dulbecco’s Modified Eagle Medium) with 10% FCS
(Fetal Calf Serum) was removed and replaced with fresh
DMEM, and aliquots of RNS stock solutions (5 mM DMSO)
were added to obtain final concentrations of 10, 30, and
50 μM. The treated cells were incubated for 24 h at 37 °C
under 5% CO2. Subsequently, the cells were treated with 5 mg
mL−1 MTT (40 μL per well) and incubated for an additional
4 h (37 °C, 5% CO2). Then the cells were dissolved in DMSO
(150 μL per well), and the absorbance at 570 nm was recorded.
The cell viability (%) was calculated according to the following
equation: cell viability % = OD570 (sample)/OD570 (control) × 100,
where OD570 (sample) represents the optical densities of the
wells treated with various concentrations of RNS and OD570
(control) represents that of the wells treated with DMEM plus
10% FCS. The percentage cell survival values are relative to
untreated control cells.
ˉ
9.536(5)
11.665(5)
19.178(5)
79.850(5)
86.907(5)
67.101(5)
1934.2(14)
298(2)
Unit cell volume/Å
Temperature/K
Z
2
Number of reflections measured
Number of independent reflections
Rint
13 975
6888
0.0172
0.0634
0.2038
0.0829
0.2223
1.068
Final R1 values (I > 2σ(I))
Final wR(F2) values (I > 2σ(I))
Final R1 values (all data)
Final wR(F2) values (all data)
Goodness of fit on F2
Results and discussion
As shown in Scheme 2, the probe RNS was synthesized via four
steps from rhodamine B. The structures of RNS and the inter-
1
mediates were all confirmed by H-NMR and 13C-NMR (ESI†).
The structure of RNS was also confirmed by X-ray crystallogra-
phy analysis. The single crystals of RNS grew from anhydrous
ether–petroleum ether (60–90 °C) solution. The crystal struc-
ture of RNS is depicted in Fig. 1. The parameters associated
with the crystal data are shown in Table 1.
Fig. 2 The UV-vis absorption titration spectra of RNS (10 μM) with Hg2+
(0–80 equiv.) in methanol–water (1/99) buffer (pH 7.0, 20 mM HEPES, 50 mM
NaNO3). Inset: digital photographs of RNS and RNS in the presence of an equi-
valent amount of Hg2+ under normal light.
UV-vis and fluorescence spectra responses
The UV-vis titration of RNS with Hg2+ was carried out in
aqueous buffer solution (pH 7.0, 20 mM HEPES, 50 mM
NaNO3). As shown in Fig. 2, the solution of RNS in water was
colorless and exhibited no absorption above 500 nm in the
UV-vis spectrum, which is ascribed to the spirolactam form of
RNS. On addition of Hg2+, the solution turned from colorless
to deep red (Fig. 2, inset). A new and strong absorption cen-
tered at 568 nm appeared and was enhanced by the addition
of Hg2+, suggesting that the formation of the ring-opening of
RNS results from Hg2+ binding. Such a dramatic color change
ensures that the RNS can be used as a sensitive “naked-eye”
probe for Hg2+.
The fluorescent titrations of RNS with Hg2+ are illustrated
in Fig. 3. The free RNS solution showed almost no fluorescent
emission. The addition of Hg2+ caused the intensity of the
Fig. 1 X-ray crystal structure for RNS. Hydrogen atoms are omitted for clarity.
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Fig. 4 The 1H-NMR spectra of the RNS (a) and RNS + 1 eq. Hg2+ (b) in CD3OD–
D2O (v : v = 10 : 1).
group. The proton (g) position showed an upfield shift of
0.14 ppm, which was attributed to the increase in electron
density arising from Hg2+ ion induced opening of the spiro-
lactam ring of the RNS. H–H COSY spectra of RNS in the absence
and presence of Hg2+ showed similar results (Fig. S2 and S3,
ESI†). Taken together, Hg2+ was possibly coordinated with the
two S atoms and N atom of the receptor, and also with the two
oxygen atoms of the carbonyl group of the RNS (Fig. 4).
Ion selectivity
Metal ion selectivity studies were performed in HEPES buffer.
As shown in Fig. 5a, the addition of Hg2+ to the solution of
RNS induced an obvious fluorescence enhancement. However,
other metal ions failed to cause any change of fluorescence of
RNS even at higher concentrations. The competitive experi-
ments were carried out in presence of 1.0 equiv. of Hg2+ mixed
with 1.0 equiv. of other transition-metal ions or 50 equiv. of
alkali or alkali-earth metal ions in HEPES buffer solutions.
Fig. 5b shows that both alkali or alkali-earth metal ions (Na+,
K+, Ca2+, Mg2+) and transition-metal ions (Cr3+, Fe3+, Fe2+,
Cd2+, Mn2+, Pb2+, Zn2+, Cu2+, Ag+, Co2+, Ni2+) have little effect
on the detection of Hg2+ with RNS. The color change of RNS
water solution with different metal ions is shown in Fig. 6.
Among the metal ions tested, only Hg2+ caused dramatic
changes both in the color and in fluorescence. Therefore, RNS
can serve as a “naked-eye” probe for Hg2+ with remarkable
selectivity in water.
Fig. 3 (a) Fluorescence emission spectra of RNS (10 μM) with excitation at
520 nm, upon titration of Hg2+ (0–80 equiv.) in methanol–water (1/99) buffer
(pH 7.0, 20 mM HEPES, 50 mM NaNO3). (b) B–H plot (fluorescence at 586 nm)
of RNS with Hg2+ (F is the fluorescence intensity of RNS in the presence of Hg2+
at 586 nm, F0 is the fluorescence intensity of free RNS at 586 nm).
fluorescence at 586 nm to be enhanced dramatically (“turn-
on”). When 80 equiv. Hg2+ was added, the intensity was
increased about 1500-fold. The association constant Ka was
evaluated using a B–H plot.14 As shown in Fig. 3b, a plot of
1/(F − F0) versus 1/[Hg2+] showed a linear relationship, from
which one can estimated that RNS was bound with Hg2+ in a
1 : 1 binding stoichiometry, and the Ka was calculated to be
1.21 × 105 M−1 according to the B–H plot. The stoichiometry of
the complex of RNS with Hg2+ was also confirmed by a Job’s
plot (Fig. S1, ESI†).
pH stability and reversibility test
The effect of pH on the fluorescence emission spectra of free
RNS was tested (Fig. 7). The results showed that the solution of
RNS was nonfluorescent between pH 6 and 11. However, this
Proton NMR spectra
To further elucidate the binding mode, 1H NMR and H–H solution showed a strong and apparent fluorescence band at
COSY experiments were conducted. Fig. 4 shows the RNS’s 586 nm when the pH value was lower than 6 and increased
1H NMR change in the absence and presence of Hg2+ ions. with a decrease of the solution pH, which signified the spiro-
After addition of Hg2+, the protons (b, c) beside the S atom dis- lactam ring opening of RNS because the acyclic forms of rhod-
played apparent downfield shifts of 0.37 and 0.13 ppm, which amine B derivatives have strong fluorescence around 580 nm.
indicated the coordination of the S atom to Hg2+. Protons Thus, the pH range of 6–11 is suitable for using RNS for the
(d, e) beside the N atom also displayed downfield shifts (Δδ = detection of Hg2+ ions.
0.18, 0.24) resulting from the decrease of the electronic density
For practical applications, reversibility, the ability to regen-
of the protons, which suggested that Hg2+ was bound to the N erate the free probes from the complex, is also an important
atom of the NS2 group and the oxygen atom of the carbonyl parameter for fluorescent probes.15 To validate the reversibility
14822 | Dalton Trans., 2013, 42, 14819–14825
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Fig. 7 Fluorescence of 10 μM RNS at various pH values in methanol–water
(1/99) buffer (20 mM HEPES, 50 mM NaNO3) (λex = 520 nm, λem = 586 nm).
Fig. 5 (a) Fluorescence spectra of RNS (10 μM) upon addition of Hg2+ and
other metal ions (λex = 520 nm). (b) Fluorescence intensity at 586 nm of RNS
(10 μM) upon addition of various metal ions (black bars: RNS with other metals,
red: RNS with Hg2+ and other metals). Experimental conditions: methanol–
water (1/99) buffer (pH 7.0, 20 mM HEPES, 50 mM NaNO3), 0.50 mM of
Na+ (1), K+ (2), Ca2+ (3), Mg2+ (4); 10 μM of Cr3+ (5), Fe3+ (6), Fe2+ (7), Cd2+ (8),
Mn2+ (9), Pb2+ (10), Zn2+ (11), Cu2+ (12), Ag+ (13), Co2+ (14), Ni2+ (15), Hg2+ (16)
(λex = 520 nm).
Fig. 8 The fluorescence titration of EDTA (top to bottom: 0.0, 0.2, 0.4, 0.6, 0.8,
1.0, 1.5 and 2.0 equiv.) in the presence of equimolar RNS/Hg2+ (10 µM) in
methanol–water (1/99) buffer (pH 7.0, 20 mM HEPES, 50 mM NaNO3, λex
=
520 nm).
probe for Hg2+ and further confirmed that the fluorescent
response of RNS to Hg2+ was caused by the spiro-ring opening
rather than the ion-catalyzed hydrolysis.16
Real-time determination is another important factor for a
probe for practical application. Time course studies reveal that
the recognition response was complete immediately after Hg2+
addition without any detectable time-delay, which guarantees
RNS as an real-time detector for Hg2+ in water (Fig. S4, ESI†).
Cell imaging
The highly selective real-time response to Hg2+ makes RNS a
probable probe for Hg2+ imaging in living cells. To confirm
this possibility, fluorescence imaging experiments in living
cells were carried out. HeLa cells were incubated with a 15 μM
solution of the RNS probe for 30 min at 37 °C and then
washed with phosphate buffer solution (PBS) to remove excess
RNS. As shown in Fig. 9a, no evidence of fluorescence was
observed. After further incubation with 30 μM Hg(ClO4)2 for
another 2.5 h at 37 °C, a red fluorescence increase was
observed from the intracellular region (Fig. 9b). A bright field
image of HeLa cells treated with RNS and Hg2+ confirmed that
Fig. 6 Top: color of RNS and RNS with different metal ions. Bottom:
fluorescence (λex = 365 nm) change upon addition of different metal ions.
of complexation of RNS and Hg2+ in a fully aqueous environ-
ment, EDTA-addition experiments were performed. When
EDTA was added to the solution of the RNS–Hg2+ complex, the
fluorescence turned off gradually (Fig. 8). Further addition of
excess Hg2+ could recover the fluorescence of the solution.
These results indicated that RNS could serve as a reversible
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Notes and references
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Fig. 9 Confocal fluorescence, (a) fluorescence image of HeLa cells labeled with
15 μM RNS after 30 min of incubation at 37 °C, washed with PBS buffer.
(b) Fluorescence image of HeLa cells treated with RNS and then 30 μM
Hg(ClO4)2 aqueous solution for 2.5 h at 37 °C. (c) Bright-field image of HeLa
cells. (d) The overlay of (b) and (c).
the cells were viable throughout the imaging experiments
(Fig. 9c). Moreover, the MTT assay demonstrates that the cell
viability remains more than 95% after treatment with 10 μM
RNS after 24 h of incubation (Fig. S5, ESI†). These results
demonstrate that RNS can be used for detecting intracellular
Hg2+ with almost no cytotoxicity.
Conclusion
In summary, we have developed a fully water soluble rhod-
amine-based probe (RNS) for Hg2+ which showed real-time
and selective “turn-on” fluorescent response to Hg2+. RNS
showed reversible colorimetric and fluorescent response to
Hg2+ in an aqueous solution. RNS can work well in neutral
aqueous buffer solution containing less than 1% organic
cosolvent. Compared with rhodamine-based probes previously
reported, the amount of organic cosolvent in the detecting
media was greatly reduced. Finally, the confocal fluorescence
image confirmed that RNS is cell permeable and can be used
for monitoring intracellular Hg2+ in living cells with low
cytotoxicity.
Acknowledgements
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This work was supported by NSFC (21102001, 21102002,
21102083 and 21272223), Natural Science Foundation of
Education Department of Anhui Province (KJ2010A028,
KJ2011A018), 211 Project of Anhui University, Youth Science
Foundation of Anhui University (2009QN014A) and Doctor
Research Start-up Fund of Anhui University.
14824 | Dalton Trans., 2013, 42, 14819–14825
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