A simple yet effective fluorescent probe for detecting and imaging mercury ions in cells

Article abstract of DOI:10.1039/c4ra16267f

We have synthesized rhodol hydrazide (RDH) as a simple fluorescent probe for detecting Hg²⁺. The probe can be applied in nontoxic solvents (EtOH and H₂O). The probe has high selectivity and sensitivity to Hg²⁺ at pH 6-8. In addition, the probe has a superior capacity to resist interference from other ions. Both fluorescence intensity and absorbance have a linear relationship with the concentration of Hg²⁺, which ensured the precise detection of Hg²⁺. Furthermore, we have studied the intracellular Hg²⁺ imaging behavior of the probe on mammalian cells, which indicated that the probe can be applied to monitor Hg²⁺ within biological samples, especially in mammalian cells.

Full text of DOI:10.1039/c4ra16267f

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A simple yet effective fluorescent probe for
detecting and imaging mercury ions in cells†
Cite this: RSC Adv., 2015, 5, 20634
a
a
a
b
a
Yan-Ru Zhang, Qing-Rong Wang, Peng Su, Fei Zhao, Jun Huang
and Bao-Xiang Zhao*a
2+
We have synthesized rhodol hydrazide (RDH) as a simple fluorescent probe for detecting Hg . The probe
2+
2
can be applied in nontoxic solvents (EtOH and H O). The probe has high selectivity and sensitivity to Hg at
pH 6–8. In addition, the probe has a superior capacity to resist interference from other ions. Both
2+
Received 12th December 2014
Accepted 6th February 2015
fluorescence intensity and absorbance have a linear relationship with the concentration of Hg , which
2+
2+
ensured the precise detection of Hg . Furthermore, we have studied the intracellular Hg imaging
DOI: 10.1039/c4ra16267f
behavior of the probe on mammalian cells, which indicated that the probe can be applied to monitor
2+
www.rsc.org/advances
Hg within biological samples, especially in mammalian cells.
interference from other ions and displays low toxicity, remark-
able membrane permeability and high sensitivity to Hg in
1
. Introduction
2
+
Mercury is a highly toxic material which is distributed widely in living cells.
the environment. It can damage brain, kidney, central nervous
1
–5
system, mitosis and endocrine system. Seriously, mercury can
cause irreversible harm to people because it can easily enter
organic cells and cannot be excreted from body via metabo-
2
. Results and discussion
6–10
lism.
organisms via the food chain, though it is present in low
Moreover, mercury can accumulate gradually in 2.1. Synthesis and characterization
RDH was synthesized in three steps from 3-(diethylamino)
phenol and o-phthalic anhydride (Scheme 1). The structure of
RDH was conrmed by H NMR, C NMR and HRMS. No
absorbance and emission peaks of RDH were observed in the
absence of Hg because RDH was in a spiro ring-close state.
When Hg was added to the solution of RDH, the typical
absorbance (525 nm) and emission (553 nm) of rhodol acid
apparently appeared. This result could be attributed to the
hydrolyzation of rhodol hydrazide producing rhodol acid,
which was conrmed by HRMS (Fig. S1, ESI†). Rhodol acid was
in a spiro ring-open state, forming a conjugated system, and
thus the typical absorbance and emission were observed. This
detection mechanism is consistent with the rhodamine hydra-
6,7
concentrations in the surrounding. Therefore, developing
2
+
new techniques for the detection of Hg is of great signicance.
Fluorescence probes have attracted considerable interest
because they have high selectivity and sensitivity, and low
1
13
2+
11–14
detection limits.
2+
As uorophores, xanthenes are highly favorable owing to
their excellent photochemical properties, including excellent

uorescence intensities, high molar extinction coefficients and
15,16
tolerance to photobleaching.
Thus, numerous uorescent
chemosensors based on xanthenes have been designed for the
detection of metal ions. Most of these uorescent probes are
based upon the principle of uorescence enhancement (turn-
17–23
on) when they encounter specic metal ions.
In this work, we designed and synthesized rhodol hydrazide
2+ 29
zide method for the detection of Hg .
2
+
(
RDH) for the detection of Hg . RDH shows higher selectivity to
2
+
2+
Hg than Cu and other ions, although many rhodamine
hydrazide derivatives have been reported for the detection of
2+
24–28
Cu .
In addition, RDH has a superior capacity to resist
a
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, P.R. China. E-mail: bxzhao@sdu.edu.cn; Fax:
+
86 531 8856 4464; Tel: +86 531 8836 6425
b
Institute of Developmental Biology, School of Life Science, Shandong University, Jinan
2
50100, P.R. China
†
Electronic supplementary information (ESI) available: Characterization and
2+
some spectra of probe. See DOI: 10.1039/c4ra16267f
Scheme 1 Synthesis of RDH and detection mechanism for Hg .
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Fig. 2 Fluorescence intensity of the probe (10 mM) in ethanol/HEPES
buffer (1 : 9, v/v, pH ¼ 7.4) with representative cations and anions (100
Fig. 1 Effect of pH on the fluorescence intensity of RDH. Probe:
1
0 mM, ethanol/HEPES buffer (1 : 9, v/v, pH ¼ 7.4), lex: 525 nm.
2+
2+
3+
3+
2+
2+
2+
2+
3+
+
mM), including Hg , Zn , Fe , Cr , Co , Ca , Cd , Ba , Al , Ag ,
+
2+
+
2+
2+
2+
ꢀ
2ꢀ
2ꢀ ꢀ ꢀ
2 4
, H PO , AcO ,
K , Mg , Na , Ni , Pb , Cu , Cl , HPO
4
, SO
4
ꢀ
4ꢀ
3
HCO , and HSO (lem: 553 nm).
2.2. Effect of pH and response time
To evaluate the effect of pH on the uorescence of RDH, we
investigated the uorescence spectra of RDH at various pH
values from 3 to 8. As shown in Fig. 1, no uorescence of the free
probe is observed in the pH range of 3–8. With the addition of
2
+
Hg , the uorescence intensity increased dramatically in bio-
logical conditions (pH 6–8). This result indicates that RDH is
2
+
suitable for the imaging of Hg in biological systems. A time
course of the uorescence of RDH (10 mM) was investigated
2
+
upon the addition of Hg (10 equiv.) (Fig. S2, ESI†). The uo-
rescence intensity reached equilibrium in about 2 h.
2
.3. Selectivity and interference
Selectivity is a crucial parameter to evaluate the properties of
uorescent probes. Therefore, we measured the selectivity of

2
+
2+
3+
3+
0
Fig. 3 The relative fluorescence intensity profiles (F/F ) of the probe
RDH towards various metal ions (namely, Hg , Zn , Fe , Cr ,
Co , Ca , Cd , Ba , Al , Ag , K , Mg , Na , Ni , Pb , and
Cu ) and ordinary anions (namely, Cl , Br , HPO
AcO , HCO
tion spectroscopy. The uorescence data indicate that the RDH
(
1
10 mM) in ethanol/HEPES buffer (1 : 9, v/v, pH ¼ 7.4) in the presence of
2
+
2+
2+
2+
3+
+
+
2+
+
2+
2+
2+
+
3+
2+
00 mM Hg and interfering ions ((1) blank; (2) Ag ; (3) Al ; (4) Ba ; (5)
2
+
ꢀ
ꢀ
2ꢀ
ꢀ
, H
2
PO
4
,
2+
2+
2+
3+
2+
3+
2+
2+
4
Ca ; (6) Cd ; (7) Co ; (8) Cr ; (9) Cu ; (10) Fe ; (11) Ni ; (12) Pb
;
;
ꢀ
ꢀ ꢀ
+
2+
+
2+
ꢀ
ꢀ
ꢀ
3 4
, and HSO ) by uorescence and UV-vis absorp- (13) K ; (14) Mg ; (15) Na ; (16) Zn ; (17) AcO ; (18) Cl ; (19) H PO
2
4
ꢀ
2ꢀ
2ꢀ
2ꢀ
(20) HCO
3
; (21) HPO
4
; (22) HSO
4
; (23) SO ) (lex: 525 nm).
4
2
+
probe has a higher selectivity to Hg over other ions (Fig. 2). In
particular, the RDH probe shows insignicant response towards
2
+
Cu , although many rhodamine hydrazide analogues have 2.4. Fluorescence and UV-absorption titration
2
+
24–28
been reported as Cu uorescence probes.
The UV-vis
The uorescence (Fig. 4) and UV-absorption (Fig. S4, ESI†)
titrations were performed in a mixed solution of HEPES and
EtOH (9 : 1, v/v, pH ¼ 7.4). The free probe showed no uores-
cent emission upon excitation at 525 nm. The characteristic
spectra (Fig. S3, ESI†) showed that a new absorption at 525
2
+
nm was observed in the presence of Hg . In contrast, other ions
2
+
did not cause this obvious spectral change. Therefore, Hg can
be distinguished from other ions by UV-vis absorption spec-
troscopy. Moreover, the uorescence change of the probe

uorescent emission at 553 nm of rhodol was observed in the
2+
presence of Hg , and the uorescence intensity increased
dramatically upon the addition of Hg , from 0 to 130 mM. The
2
+
towards Hg was negligible in the presence of coexisting ions.
The interference from other common ions was expressed with
the ratio of IRDH+Hg2++ion/IRDH+Hg2+ that varied from 0.79 to 1.11
in a nite range (Fig. 3). The abovementioned results suggest
that the probe not only has an excellent selectivity but also can
resist interference from other ions.
2+
2+

uorescence intensity peaked at 110 mM (Hg ) and enhanced
by about 550 fold compared to that of the free probe.
Moreover, the RDH probe showed an excellent linearity
2+
between uorescence intensity and the concentration of Hg
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Fig. 4 Fluorescence spectra of reaction solution of the probe (10 mM)
in ethanol/HEPES buffer (1 : 9, v/v, pH ¼ 7.4) with different concen-
2+
trations of Hg (lem: 553 nm). (Inset-left) Fluorescence intensity at Fig. 5 Fluorescence images of the application of the probe to detect
2
+
2+
5
53 nm upon the addition of Hg (from 0 to 13 eq); (inset-right) Hg in living HeLa cells. (a1–4) Bright-field view; (b1–4) fluorescent;
2+
linearity of the fluorescence intensity and the concentration of Hg
(c1–4) overlay image of (a1–4) and (b1–4). (1) HeLa cells incubated
with 2 mM of the probe for 30 min at 37 ꢂC. (2–4) HeLa cells were
pretreated with Hg at the indicated concentrations (2 : 2.5 mM; 3 : 5
from 0 to 10 eq.
2+
ꢂ
mM; 4 : 10 mM) for 30 min at 37 C before incubating with the 2 mM
2
+
probe under the same conditions.
from 15 to 100 mM. The detection limit for Hg was calculated
ꢀ8
to be 3.14 ꢁ 10 M according to the formula 3s/k (s is the
2
+
standard deviation of ten blank solutions without Hg and k is
the slope of uorescence titration calibration equation). The
2
+
results indicated that RDH exhibited high sensitivity to Hg
and can quantitatively detect Hg in a wide range of concen-
2
+
trations. As shown in Fig. S4 (ESI†), the absorbance increased
2
+
with the addition of Hg , and there was linearity between the
2
+
absorbance of RDH and the concentration of Hg from 15 to
00 mM. The two linear relationships of uorescence and UV-
1
2
+
absorbance ensure the precise detection of Hg .
2
+
2
.5. Imaging of intracellular Hg and evaluation of
cytotoxicity
To verify the potential biological applications of RDH, we
2
+
studied intracellular Hg imaging with the probe in mamma-
lian cells (Fig. 5). Incubating HeLa cells with 2 mM of RDH probe
ꢂ
for 30 min at 37 C showed very weak uorescence that could be
2
+
negligible. When HeLa cells were pretreated with 2.5 mM of Hg
Fig. 6 The viability of HeLa cells after incubation with different
with the same treatment of the probe, uorescence was visible. concentrations of probe (2 and 5 mM) or with DMSO 0.1% (v/v) (control)
It provided visual evidence that the probe can permeate cells for 6 h measured by SRB assay and illustrated in the labelled column.
2
+
Results are presented as mean ꢃ SE (n ¼ 3).
and inform the intracellular existence of Hg . Moreover, the
uorescence intensity revealed a remarkable enhancement with

2
+
increasing concentration of Hg (5, 10 mM). It suggested that
2
+
the probe RDH can be applied to monitor Hg within biological
samples, especially in mammalian cells.
3. Materials and methods
We used an SRB assay to detect the viability of HeLa cells
incubated with different concentrations of RDH probe for 6 h
Fig. 6) and found no distinct decline compared to controls.
Furthermore, even when RDH probe concentration was as high
as 5 mM, which is more than 2 times higher than the normal
concentration (2 mM) used in cell imaging, the viability of HeLa
cells remained at a normal value. Therefore, the RDH is non-
toxic to cells and could be used for clinical applications in
living organisms.
3.1. Instruments and materials
HRMS spectra were recorded on a Q-TOF6510 spectrograph
(
1
13
(
Agilent). H NMR (300 MHz) and C NMR (75 MHz) spectra
were recorded on a Bruker Avance 300 spectrometer using
DMSO as the solvent and tetramethylsilane (TMS) as an internal
standard. Fluorescent measurements were recorded on an F-
7000 (Hitachi) luminescence spectrophotometer, and UV-vis
spectra were recorded on a U-4100 Spectrometer (Hitachi).
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The pH was measured by the use of a PHS-3C digital pH-meter 3.3. Preparation of solution for detection
(

YouKe, Shanghai). Images of HeLa cells were captured with a
uorescent microscope (Nikon ECLIPSE TE 2000-U, Japan).
Resorcinol, 3-(diethylamino)phenol, o-phthalic anhydride,
ꢀ2
2+
2+
2+
3+
Stock solutions (10 M) of the ions (including Hg , Zn , Fe ,
3+
2+
2+
2+
3+
+
+
2+
+
2+
2+
Cr , Co , Ca , Cd , Ba , Al , Ag , K , Mg , Na , Ni , Pb ,
ꢀ
ꢀ
2ꢀ
ꢀ ꢀ
ꢀ
ꢀ
Cl , Br , HPO
4
, H PO , AcO , HCO , and HSO ) were
prepared for detection by dissolving them in deionized water.
The probe was dissolved in ethanol as stock solution (10 M).
The solvent was composed of ethanol and HEPES buffer (v/v,
2 4 3
4
methanesulfonic acid, hydrazine hydrate, and all the reagents
were purchased from commercial suppliers and used without
further purication. The solutions of metal ions were prepared
from KNO
Al(NO $9H
ꢀ3
3
,
NaNO
3
,
Ca(NO
Cr(NO
3
)
2
$4H
2
O, Mg(NO
O, Ni(NO
3
3
)
)
2
$6H
$6H
2
2
O,
O,
1
: 9, pH ¼ 7.4). The excitation slit was 3 nm and the excita-
3
)
3
2
O, Ba(NO
3
)
2
,
3
)
3
$9H
2
2
tion wavelength was 525 nm. The emission slit was 13 nm and
the emission wavelength was 553 nm.
Cd(NO ) $4H O, Fe(NO ) $9H O, AgNO3, Co(NO ) $6H O,
Pb(NO ) , Cu(NO ) $3H O, Zn(NO ) $6H O and HgCl with
3
2
2
3 3
2
3 2
2
3
2
3 2
2
3 2
2
2
deionized water. Deionized water was used throughout the
absorption and uorescence determinations. All the samples
were prepared at room temperature, shaken for 10 s and
allowed to stand for 5 h before UV-vis and uorescence deter-
mination. HEPES buffer solutions (pH 7.4) were prepared with
3
.4. Cell culture and imaging
HeLa cells were cultured in Dulbecco's modied Eagle's
medium (DMEM, Gibco) containing 10% calf bovine serum
ꢂ
(HyClone) in dishes maintained at 37 C in humidied air with
5% CO
2
. For uorescence imaging, cells at the desired cell
20 mM HEPES and adjusted with aqueous sodium hydroxide.
concentration were seeded into 24-well plates, and then incu-
2
+
bated with fresh DMEM with different concentrations of Hg
0, 2.5, 5, 10 mM) for 30 min. The cells were washed twice with
(
3
.2. Synthesis of probe RDH
PBS buffer before staining experiments. Then, the cells were
incubated with DMEM containing 2 mM of the RDH probe dis-
solved in DMSO (DMSO : DMEM < 2 : 1000, v/v) for 30 min.
Aer washing twice with PBS, the cells were imaged under a
uorescence microscope (Nikon ECLIPSE TE 2000-U, Japan).
2
-(4-Diethylamino-2-hydroxy-benzoyl)-benzoic acid (com-
pound 1). Compound 1 was synthesized according to a previous
report.
30
0
0
0
3
-(Diethylamino)-6 -hydroxy-3H-spiro[isobenzofuran-1,9 -
xanthen]-3-one (compound 2). Compound 1 (1.2 g, 3.83 mmol)
and benzene-1,3-diol (0.43 g, 3.39 mmol) were dissolved in 30
3.5. Cytotoxicity
mL methanesulfonic acid. The mixture solution was stirred at The RDH probe was dissolved in DMSO as a stock solution. HeLa
ꢂ
9
0 C for 9 h. The reaction mixture was poured into 200 mL of cells were cultured in DMEM (Gibco) containing 10% calf bovine
ꢂ
ice-water and adjusted to neutral with saturated sodium serum in dishes maintained at 37 C in humidied air with 5%
carbonate solution. The abovementioned solution was extracted CO . Cells were harvested with 0.25% trypsin (Sangon Biotech) in
2
with dichloromethane (100 mL ꢁ 3). The organic layers were PBS and seeded in 96-well plates. When cells covered 60–70% of
combined, washed with distilled water (50 mL ꢁ 3), dried over the plate surface, the culture was replaced with fresh DMEM
anhydrous magnesium sulfate, and then ltered. The containing 0.1% DMSO (as control) or the RDH probe at different
dichloromethane solvent was removed under reduced pressure. concentrations (2 and 5 mM). Cell viability was evaluated by a
The residue was puried by silica gel column chromatography sulforhodamine B (SRB) assay. Aer incubation for 6 h, cells were
ꢂ
(
PE : EA ¼ 2 : 1) to obtain 460 mg of red solid (yield: 28.4%).
xed with 10% cold trichloroacetic acid (TCA) at 4 C for 1 h. The
0
0
0
2
-Amino-3 -(diethylamino)-6 -hydroxyspiro[isoindoline-1,9 - stationary liquid was then removed and washed ve times with
xanthen]-3-one (RDH). Compound 2 (400 mg, 1.07 mmol) was deionized water. Then, the air-dried plates were stained with SRB
dissolved in EtOH (30 mL) and NH NH $H O (100 mL). The solution for more than 10 min in a shaker (QILINBEIER Orbital
2
2
2
reaction solution was heated and kept under reux for 4 h. The Shaker TS-100, China) at room temperature. The superuous SRB
ethanol was removed under reduced pressure and the aqueous was washed away with 1% acetum and plates were air-dried and
layer was extracted with dichloromethane (100 mL ꢁ 3). The subsequently 100 mL of 10 mM unbuffered Tris base (pH 10.5) was
organic solution was washed with distilled water (50 mL ꢁ 3), added to every well with HeLa cells to dissolve the bound dye and
dried over anhydrous magnesium sulfate and then ltered. The mixed for 5 min on a microtiter plate shaker. Optical density (OD)
solvent was removed under reduced pressure and the crude was measured by the use of a microplate reader (TECAN Nano-
product was puried by column chromatography on silica gel Quant Innite M200PRO, Austria) at a wavelength of 540 nm aer
(
dichloromethane : methanol ¼ 5 : 1) to obtain 350 mg of white plates were shaken at room temperature for 10 min. The OD
1
solid (yield: 87.5%). H NMR (300 MHz, DMSO) d 9.75 (s, 1H), values of probe-incubated wells were compared and normalized to
.82–7.70 (m, 1H), 7.54–7.39 (m, 2H), 7.02–6.93 (m, 1H), 6.59 (d, those of controls treated with 0.1% DMSO to calculate the relative
7
J ¼ 2.1 Hz, 1H), 6.45–6.29 (m, 5H), 4.31 (s, 2H), 3.36–3.29 (m, cell viability. We used 3 parallel wells in every measurement and
1
3
4
1
1
1
3
4
H), 1.08 (t, J ¼ 6.9 Hz, 6H). C NMR (75 MHz, DMSO) d 165.32, the results are expressed as the average of triplicate assays.
58.07, 152.74, 152.69, 151.66, 148.19, 132.44, 129.47, 128.21,
27.92, 127.62, 123.42, 122.21, 111.67, 110.12, 108.05, 105.21,
02.29, 97.44, 64.66, 43.62, 40.35, 40.07, 39.79, 39.52, 39.24,
8.96, 38.68, 12.41. HRMS: m/z [M + H] calcd for C24
4. Conclusions
+
H
23
N
3
O
3
:
A simple yet effective uorescent probe (RDH) was developed
for the detection of Hg . The probe exhibited an excellent
2
+
02.1817, found: 402.1815.
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2
+
selectivity to Hg in friendly solvents (EtOH and H
2
O). Both 13 S. Y. Kim and J. I. Hong, Org. Lett., 2007, 16, 3109.

uorescence intensity and absorbance have linear relationships 14 (a) Y. Xiang, A. J. Tong, P. Y. Jin and Y. Ju, Org. Lett., 2006, 13,
2
+
with the concentration of Hg . Moreover, the probe can be
applied to monitor Hg in mammalian cells with remarkable
membrane permeability and non-toxicity to the cells.
2863; (b) H. N. Kim, S. W. Nam, K. M. K. Swamy, Y. Jin,
X. Q. Chen, Y. Kim, S. J. Kim, S. Park and J. Yoon, Analyst,
2011, 136, 1339.
2
+
15 G. Sivaraman, B. Vidya and D. Chellappa, RSC Adv., 2014, 4,
3
0828.
Acknowledgements
1
6 X. Q. Chen, T. Pradhan, F. Wang, J. S. Kim and J. Yoon,
This study was supported by the National Basic Research
Program of China (2010CB933504).
Chem. Rev., 2012, 112, 1910.
17 G. G. Hou, C. H. Wang, J. F. Sun, M. Z. Yang, D. Lin and
H. J. Li, Biochem. Biophys. Res. Commun., 2013, 439, 459.
1
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2
2
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