A BODIPY-based colorimetric and fluorometric chemosensor for Hg(ii) ions and its application to living cell imaging

Article abstract of DOI:10.1039/c2ob25589h

A new monostyryl boron dipyrromethene derivative (MS1) appended with two triazole units indicates the presence of Hg²⁺ among other metal ions with high selectivity by color change and red emission. Upon Hg²⁺ binding, the absorption band of MS1 is blue-shifted by 29 nm due to the inhibition of the intramolecular charge transfer from the nitrogen to the BODIPY, resulting in a color change from blue to purple. Significant fluorescence enhancement is observed with MS1 in the presence of Hg ²⁺; the metal ions Ag⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, K ⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb ²⁺, and Zn²⁺ cause only minor changes in the fluorescence of the system. The apparent association constant (K_a) of Hg ²⁺ binding in MS1 is found to be 1.864 × 10⁵ M ^-1. In addition, fluorescence microscopy experiments show that MS1 can be used as a fluorescent probe for detecting Hg²⁺ in living cells.

Full text of DOI:10.1039/c2ob25589h

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 5410
www.rsc.org/obc
PAPER
A BODIPY-based colorimetric and fluorometric chemosensor for Hg(II) ions
and its application to living cell imaging†
Mani Vedamalai and Shu-Pao Wu*
Received 20th March 2012, Accepted 30th May 2012
DOI: 10.1039/c2ob25589h
A new monostyryl boron dipyrromethene derivative (MS1) appended with two triazole units indicates the
presence of Hg2+ among other metal ions with high selectivity by color change and red emission. Upon
Hg2+ binding, the absorption band of MS1 is blue-shifted by 29 nm due to the inhibition of the
intramolecular charge transfer from the nitrogen to the BODIPY, resulting in a color change from blue to
purple. Significant fluorescence enhancement is observed with MS1 in the presence of Hg2+; the metal
ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+ cause only minor
changes in the fluorescence of the system. The apparent association constant (Ka) of Hg2+ binding in
MS1 is found to be 1.864 × 105 M−1. In addition, fluorescence microscopy experiments show that MS1
can be used as a fluorescent probe for detecting Hg2+ in living cells.
Numerous molecular probes using different receptors and fluo-
Introduction
rescent units have been developed for Hg2+ detection. Because
Hg2+ is known as a fluorescence quencher due to spin–orbit
The development of chemosensors for detecting biologically and
environmentally important metal ions, such as Cu2+, Zn2+, Hg2+,
and Pb2+, has attracted much attention. Mercury is one of the
most toxic heavy metal elements and exists in three forms:
elemental, inorganic, and organic mercury. Mercury ions have
high affinity for thiol groups in proteins, leading to the malfunc-
tion of cells and consequently causing many health problems in
the brain, kidney, and central nervous system. Its accumulation
in the body results in a wide variety of diseases, such as prenatal
brain damage; serious cognitive and motion disorders; and
Minamata disease.1 In order to detect mercury ions in biological
and environmental samples, the design of highly selective and
sensitive mercury sensors has been an important issue.
coupling,7 most fluorescent chemosensors detect Hg2+ through a
fluorescence quenching. Due to sensitivity concerns, fluorescent
chemosensors detecting metal ions using fluorescence enhance-
ment are more easily monitored than those using fluorescence
quenching. This paper reports on a newly designed monostyryl
boron dipyrromethene (BODIPY) based fluorescent enhance-
ment Hg2+ chemosensor, based on intramolecular charge transfer
(ICT). When Hg2+ binds to the chemosensor, it blocks the ICT
mechanism, giving rise to a color change and fluorescence
enhancement of BODIPY.
In this study, a monostyryl BODIPY-based fluorescent chemo-
sensor (MS1) containing two triazole units was designed for
metal ion detection (Scheme 1). MS1 was blue and exhibits
weak fluorescence. Binding metal ions to the chemosensor
blocks the ICT mechanism and results in a color change and fluo-
rescence enhancement of BODIPY. The metal ions Ag+, Ca2+,
Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+,
Pb2+, and Zn2+ were tested for metal ion binding selectivity with
MS1, but Hg2+ was the only ion that caused a red emission upon
binding with MS1. The fluorescence microscopy experiments
also demonstrated that MS1 can be used as a fluorescent probe
for detecting Hg2+ in living cells.
In general, several traditional methods2 for the detection of
mercury ions in various samples have been developed, including
atomic absorption–emission spectroscopy,3 inductively coupled
plasma mass spectroscopy (ICPMS),4 and inductively coupled
plasma–atomic emission spectrometry (ICP-AES).5 Although
these methods are quantitative, most of these methods require
expensive instruments and are not good for on-site analysis.
Recently, more attention has been focused on the development
of fluorescent chemosensors for the detection of Hg2+ ions.6
Result and discussion
Department of Applied Chemistry, National Chiao Tung University,
Hsinchu, Taiwan, Republic of China. E-mail: spwu@mail.nctu.edu.tw;
Fax: +886-3-5723764; Tel: +886-3-5712121-ext56506
Synthesis of MS1
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra of compounds 2, 3, 4, 5, and MS1; ESI-MS of MS1–
Hg2+. See DOI: 10.1039/c2ob25589h
The synthesis of the fluorescent probe, MS1, is outlined in
Scheme 1. Mono formylated dipyrromethane (1) was
5410 | Org. Biomol. Chem., 2012, 10, 5410–5416
This journal is © The Royal Society of Chemistry 2012

View Online
Scheme 1 Synthesis of MS1.
synthesized according to the procedure found in the literature.9
Compound 2 was obtained by a Wittig reaction of (4-nitro-
benzyl)triphenyl phosphonium bromide and mono formylated
dipyrromethane to form a double bond between pyrrole and
nitrobenzene. In the next step, compound 2 was transformed into
a BODIPY skeleton by a stepwise reaction; first, dipyrromethane
was oxidized to form dipyrromethene by DDQ, followed by
dipyrromethene conversion into a BODIPY in the presence of
boron trifluoride. Further reduction of compound 3 using iron
powder gave compound 4. The reaction of compound 4 with
propargyl bromide in the presence of potassium carbonate
yielded compound 5. MS1 was obtained by treatment of com-
pound 5 with picolyl azide under click chemistry conditions.
The absorption spectrum of MS1 displays an absorption
peak centered at 606 nm with a molar extinction coefficient of
6.2 × 104 M−1 cm−1. The absorption maximum of MS1 has
about a 100 nm red shift in comparison to that of the standard
BODIPY dye.8 This red shift was assigned to a substitution of
an amino styryl group at the “3” position of the BODIPY group.
Cation sensing selectivity
The sensing ability of MS1 was tested by mixing it with metal
ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mg2+,
Mn2+, Ni2+, Pb2+, and Zn2+. Qualitatively, Hg2+ was the only ion
that caused a visible color change (from blue to purple) and red
fluorescence from MS1 (Fig. 1). Other metal ions led to no sig-
nificant change in the fluorescence of MS1. Quantitative absorp-
tion and fluorescence spectra of MS1 were taken in the presence
of several transition metal ions. Hg2+ was the only metal ion that
caused a significant red emission (Fig. 2). During Hg2+ titration
with MS1, the absorption band at 606 nm was shifted to 577 nm
(Fig. 2). This caused a visible color change from blue to purple.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5410–5416 | 5411

View Online
Fig. 1 Colorimetric change (top) and fluorescence change (bottom) of
MS1 (4 μM) with 60 μM of individual cations.
Fig. 3 Fluorescence response of MS1 (4 μM) to the addition of Hg2+
(60 μM) or 150 μM of other metal ions (black bars) and to the mixture
of other metal ions (150 μM) with 60 μM of Hg2+ (gray bars) in aceto-
nitrile–water (v/v = 9/1, 2.5 mM Hepes, pH 7.0) solutions. The
excitation wavelength is 550 nm.
Fig. 4 Job plot of Hg2+–MS1 complexes in acetonitrile–water
(v/v = 9 : 1, 2.5 mM Hepes, pH 7.0) solutions. The monitored wavelength
was 650 nm. The total concentration of the sensor and Hg2+ ion was 8 μM.
Fluorescence enhancement caused by the mixture of Hg2+ with
most metal ions was similar to that caused by Hg2+ alone.
A smaller fluorescence enhancement was observed when Hg2+
was mixed with Co2+ or Fe3+. This indicates that only Co2+ and
Fe3+ compete with Hg2+ for binding with MS1. Most of the
other metal ions do not interfere with the binding of MS1 with
Hg2+.
In order to understand the binding stoichiometry of MS1–
Hg2+ complexes, Job plot experiments were carried out. In
Fig. 4, the emission intensity at 650 nm was plotted as a function
of the mole fraction of MS1 under a constant total concentration.
Maximum emission intensity was reached when the mole frac-
tion was 0.5. These results indicate a 1 : 1 ratio for MS1–Hg2+
complexes, in which one Hg2+ ion was bound with one MS1.
Further, the formation of 1 : 1 MS1–Hg2+ complex was
confirmed using ESI-MS in which the peak at m/z 929.9 indi-
cates a 1 : 1 stoichiometry for MS1–Hg2+ complexes (see
Fig. S11 in ESI†). The apparent association constant was calcu-
lated from Fig. 5 by using nonlinear regression analysis and was
found to be 1.864 × 105 M−1. The detection limit of MS1 as a
Fig. 2 Absorption (top) and emission (bottom) changes of chemo-
sensor MS1 (4 μM) in the presence of various equivalents of Hg2+ in
acetonitrile–water (v/v = 9 : 1, 2.5 mM Hepes, pH 7.0) solutions.
During Hg2+ titration with MS1, a new emission band centered
at 650 nm formed (Fig. 2). After adding 15 equivalents of Hg2+,
the quantum yield of the emission band was Φ = 0.327, which is
65 fold higher than that of MS1, with Φ = 0.005. These obser-
vations indicate that Hg2+ is the only metal ion that readily binds
with MS1, causing significant fluorescence enhancement and
permitting highly selective detection of Hg2+.
To study the influence of other metal ions on Hg2+ binding
with MS1, we performed competitive experiments in the pres-
ence of Hg2+ (60 μM) with other metal ions (150 μM) (Fig. 3).
5412 | Org. Biomol. Chem., 2012, 10, 5410–5416
This journal is © The Royal Society of Chemistry 2012

View Online
upon the addition of Hg2+. The down-field shifts upon Hg2+
coordination are due to a decrease in electron density induced by
Hg2+. This indicated that Hg2+ binding occurs mainly through
the nitrogen at the triazole ring. The proton signals (Hj and Hk)
showed up-field shifts upon the addition of Hg2+. This indicated
that Hg2+ binds to the amine attached to the phenyl ring and
Hg2+ binding affects the ring current at the phenyl ring. The
proton signals (Hn, Ho, Hp & Hq) at the pyridine were slightly
influenced by Hg2+ binding. This showed weak interactions
between Hg2+ and the pyridines. These observations revealed
that Hg2+ binding with MS1 was mainly through one amine at
the phenyl ring and two nitrogens at two triazole units. Hg2+
also had weak interactions with two nitrogens at pyridine
moieties.
Fig. 5 Benesi-Hildebrand plot of the Hg2+–MS1 complexes in aceto-
nitrile–water (v/v = 9 : 1, 2.5 mM Hepes, pH 7.0) solutions. The moni-
tored emission wavelength was 650 nm.
Living cell imaging
MS1 was also applied to living cell imaging. For the detection
of Hg2+ in living cells, HeLa cells were cultured in DMEM sup-
plemented with 10% FBS at 37 °C and 5% CO2. Cells were
plated on 14 mm glass coverslips and allowed to adhere for
24 hours. HeLa cells were treated with 2 μM Hg(BF4)2 for
30 min and washed with PBS for three times. Then cells were
incubated with MS1 (2 μM) for 30 min and washed with PBS to
remove the remaining sensor. The images of the HeLa cells were
obtained using a fluorescence microscope. Fig. 8 shows the
images of HeLa cells with MS1 after the treatment of Hg2+. The
overlay of fluorescence and bright-field images reveal that
the fluorescence signals are localized in the intracellular area,
indicating a subcellular distribution of Hg2+ and good cell-
membrane permeability of MS1.
Conclusions
Fig. 6 Fluorescence intensity (650 nm) of MS1 (4 μM) (■), and after
addition of Hg2+ (60 μM) ( ) in an acetonitrile–water (v/v = 9 : 1,
2.5 mM buffer) solution as a function of different pH values. The exci-
tation wavelength was 550 nm.
In summary, the new fluorescence chemosensor MS1 exhibits a
high affinity and selectivity for Hg2+ ions over competing metal
ions. Fluorescence was significantly enhanced by chemosensor
MS1 in the presence of Hg2+, and the addition of Ag+, Ca2+,
Cd2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Ni2+, Pb2+, or
Zn2+ barely affected the fluorescence. This BODIPY-based Hg2+
chemosensor also provides an effective method of Hg2+ sensing
in living cell imaging.
fluorescent sensor for the analysis of Hg2+ was determined from
the variation of fluorescence intensity as a function of the con-
centration of Hg2+ (see Fig. S12 in the ESI†). It was found that
MS1 has a detection limit of 0.226 μM, which allows micro-
molar concentrations of Hg2+ to be detected.
A pH titration of MS1 was performed to investigate a suitable
pH range for Hg2+ sensing. As depicted in Fig. 6, the emission
intensities of metal-free MS1 were very low. After mixing
MS1 with Hg2+, the emission intensity at 650 nm remained
a maximum in the pH range of 3.0–7.0. Above pH 7.5, the
emission intensity decreased. This indicates poor stability of the
MS1–Hg2+ complexes at high pH values.
Experimental section
General
All reagents were obtained from commercial sources and used as
received without further purification. UV-vis spectra were
recorded on an Agilent 8453 UV-vis spectrometer. Fluorescence
spectra were recorded in a Hitachi F-4500 spectrometer.1H and
13C NMR spectra were recorded on a Bruker DRX-300 NMR
Spectrometer, Varian AS500 Unity Innova Spectrometer and
Varian VNMRS 600 NMR Spectrometer.
To gain a clearer understanding of the structure of MS1–Hg2+
1
complexes, H NMR spectroscopy (Fig. 7) was employed. Hg2+
is a heavy metal ion and can affect the proton signals that are
close to Hg2+ binding.9 In the 1H NMR spectra of MS1, the
proton (Hl, triazole) signal at 7.75 ppm showed down-field shifts
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5410–5416 | 5413

View Online
Fig. 7 1H NMR spectra of MS1 (5 mM) in the presence of different concentrations of Hg2+ in CD3CN.
Synthesis
Synthesis of 1-formyl-5-phenyldippyromethane (1). Com-
solution was stirred at room temperature for 30 min. Compound
1 (500.2 mg, 2 mmol) dissolved in dry THF (10 mL) was added
dropwise to the mixture. The reaction mixture was heated at
66 °C for 12 h. Then solvents were removed under reduced
pressure, and the crude product was purified by on column
chromatography (hexane–ethyl acetate, 5 : 1) to give a compound
2 as a red solid. Yield: 70%, 517 mg. Melting point 163–164 °C.
1H NMR (CD3OD): δ = 8.14 (d, J = 9 Hz, 2H), 7.57 (d, J =
9 Hz, 2H), 7.17–7.31 (m, 6H), 6.80 (d, J = 16.5 Hz, 1H), 6.67
(dd, J = 1.5 Hz, 2.7 Hz, 1H), 6.33 (d, J = 3.6 Hz, 1H), 6.00
pound 1 was obtained in modest yield by treating 5-phenyl-
dipyrromethane with benzoyl chloride and DMF under dry N2.10
Synthesis of 1-[2-(4-Nitro-phenyl)-vinyl]-5-phenyl-4,6-dipyrro-
methane (2). Potassium tert-butoxide (281 mg, 2.5 mmol)
was added to a solution of (4-nitrobenzyl)triphenyl phos-
phonium bromide (1.002 g, 2.1 mmol) in dry THF (30 mL). The
5414 | Org. Biomol. Chem., 2012, 10, 5410–5416
This journal is © The Royal Society of Chemistry 2012

View Online
8.4 Hz, 2H), 7.40 (d, J = 16.2 Hz, 1H), 7.00 (d, J = 4.8 Hz, 1H),
6.97 (d, J = 4.8 Hz, 1H), 6.72 (d, J = 3.6 Hz, 1H), 6.70 (d, J =
8.4 Hz, 2H), 6.47 (dd, J = 1.8 Hz, 3.3 Hz, 1H), 4.10 (s, br, 2H).
13C NMR (CD2Cl2): δ = 159.8, 149.5, 141.3, 140.9, 138.3,
137.5, 134.6, 134.1, 133.0, 130.8, 130.3, 130.2, 128.6, 126.5,
126.3, 118.3, 116.5, 115.1, 114.6. MS (EI): m/z (%) = 385
(100.0), 384 (30.1), 364 (20.6), 288 (4.1), 192 (4.8).
HRMS (EI): calcd for C23H18BF2N3 385.1562; found:
385.1559.
Synthesis of compound 5. Propargyl bromide (0.174 mL, 80%
solution in toluene, 1.6 mmol) and potassium carbonate
(276.4 mg, 2 mmol) were added to a solution of compound 4
(269.6 mg, 0.7 mmol) in acetone (5 mL). The reaction mixture
was refluxed for two days. The solvent was evaporated under
reduced pressure, and the crude product was purified by column
chromatography (ethyl acetate–hexane, 1 : 5) to give compound
5 as a violet solid. Yield 87%, 281.5 mg. Melting point
Fig. 8 Hg2+-treated HeLa cell images. (Top left) Bright field image;
(Top right) fluorescence image; and (Bottom) merged image.
1
156–157 °C. H NMR (CD3CN): δ = 7.76 (s, 1H), 7.49–7.67
(m, 9H), 7.16 (d, J = 4.8 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.01
(d, J = 9.0 Hz, 2H), 6.76 (d, J = 3.9 Hz, 1H), 6.53 (dd, J = 2.1
Hz, 3.9 Hz, 1H), 4.25 (d, J = 2.4 Hz, 4H), 2.56 (t, J = 2.4 Hz,
2H). 13C NMR (CD3CN): δ = 160.4, 149.7, 142.0, 141.8, 139.0,
137.9, 134.9, 134.5, 133.9, 131.4, 131.1, 130.3, 129.3, 127.2,
119.5, 118.3, 117.3, 115.4, 115.3, 79.9, 74.0, 40.8. MS (ESI):
m/z = 462.1 [M + H]+; HRMS (ESI): calcd C29H22BF2N3
[M + H]+ 462.1953; found 462.1944.
(t, J = 3.0 Hz, 1H), 5.75 (d, J = 3.3 Hz, 1H), 5.73 (dd, J =
1.8 Hz, 2.1 Hz, 1H), 5.45 (s, 1H). 13C NMR (CD3OD): δ =
146.9, 146.7, 144.2, 139.0, 133.9, 131.4, 129.6, 129.2, 127.5,
126.7, 125.3, 125.0, 120.2, 118.2, 112.8, 110.3, 108.1, 107.8,
45.5. MS(FAB): m/z
= 369. HRMS (FAB): calcd for
C23H19N3O4: 369.1477; found 369.1481.
Synthesis of MS1. Picolyl azide (160.9 mg, 1.2 mmol),
CuSO4·5H2O, (15.0 mg, 10 mol%), and sodium ascorbate
(30.0 mg, 20 mol%) were added to a solution of compound 5
(277.3 mg, 0.6 mmol) in THF–H2O (7 : 3, v/v; 15 mL) under
nitrogen. The solution was stirred at room temperature for 12 h.
A saturated ammonium chloride solution (20 mL) was added to
the reaction mixture, and the organic phase was extracted with
dichloromethane (100 mL, 3×). The combined organic extracts
were dried with anhydrous MgSO4. The solvent was evaporated
under reduced pressure, and the crude product was purified by
column chromatography (dichloromethane–methanol, 20 : 1) to
give compound MS1 as a dark violet solid. Yield 71%,
311.1 mg. Melting point 94–95 °C. 1H NMR (CD3CN): δ = 8.50
(d, J = 5.0 Hz, 2H), 7.76 (s, 2H), 7.69–7.72 (m, 3H), 7.40–7.59
(m, 9H), 7.26 (dd, J = 5.0 Hz, 7.0 Hz, 2H), 7.14 (d, J = 8.0 Hz,
2H), 7.06 (d, J = 5.0 Hz, 1H), 6.93–6.96 (m, 3H), 6.68 (d, J =
4.0 Hz, 1H), 6.48 (dd, J = 2.5 Hz, 3.8 Hz, 1H), 5.47 (s, 4H),
4.75 (s, 4H). 13C NMR (CD3CN): δ = 161.6, 156.6, 151.4,
151.2, 146.2, 143.2, 141.9, 139.0, 138.8, 138.7, 135.7, 135.1,
134.6, 132.1, 131.7, 131.3, 130.0, 127.2, 126.2, 125.2, 124.8,
123.7, 120.3, 118.9, 117.7, 114.9, 56.6, 47.6. MS (ESI): m/z =
730.2 [M + H]+. HRMS (ESI): calcd for C41H34BF2N11
[M + H]+ 730.3138; found 730.3146.
Synthesis of compound 3. 2,3-Dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ; 318 mg, 1.4 mmol) dissolved in CH2Cl2
(50 mL) was added to a solution of compound 2 (443 mg,
1.2 mmol) in CH2Cl2 (100 mL) under nitrogen, and the mixture
was stirred for 1 h. It was then treated with Et3N (3.0 mL) and
BF3·OEt2 (4.0 mL) for 3 h. The solvent was evaporated under
reduced pressure, and the crude product was purified by column
chromatography (ethyl acetate–hexane, 1 : 10) to give compound
3 as a pink solid. Yield 78%, 388.6 mg. Melting point
1
265–266 °C. H NMR (CD2Cl2): δ = 8.24 (d, J = 8.7 Hz, 2H),
7.87 (s, 1H), 7.83 (d, J = 16.8 Hz, 1H), 7.76 (d, J = 9 Hz, 2H),
7.52–7.61 (m, 5H), 7.43 (d, J = 16.5 Hz, 1H), 7.04 (d, J =
4.5 Hz, 1H), 7.00 (d, J = 4.8 Hz, 1H), 6.90 (d, J = 3.9 Hz, 1H),
6.58 (d, J = 2.1 Hz, 1H). 13C NMR (CD2Cl2): δ = 155.6, 148.1,
148.0, 145.0, 142.7, 142.6, 137.4, 135.2, 134.2, 132.5, 130.95,
130.90, 130.3, 128.8, 128.4, 124.5, 123.0, 118.5, 117.8. MS
(EI): m/z (%) = 415 (100.0), 414 (30.1), 349 (8.4), 347 (10.4),
291 (5.1), 174 (4.8); HRMS (EI): calcd for C23H16BF2N3O4
415.1304; found: 415.1303.
Synthesis of compound 4. Iron powder (803.5 mg,
14.4 mmol) and water (4 mL) were added to a solution of com-
pound 3 (373.6 mg, 0.9 mmol) in methanol (12 mL). It was then
treated with HCl in methanol (6 mL, 0.5 mol L−1). The reaction
mixture was heated at 80 °C for 6 h. The reaction mixture was
cooled to room temperature, and concentrated at reduced
pressure. The crude product was purified by column chromato-
graphy (ethyl acetate–hexane, 1 : 3) to give a blue solid. Yield
80%, 277.3 mg). Melting point 238–239 °C. 1H NMR
(CD2Cl2): δ = 7.69 (s, 1H), 7.48–7.57 (m, 6H), 7.47 (d, J =
Determination of the binding stoichiometry and the
apparent dissociation constants for the binding of
Hg(II) to MS1
The binding stoichiometry of MS1–Hg2+ complexes was deter-
mined from a Job plot.11 The fluorescence intensity at 650 nm
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 5410–5416 | 5415

View Online
was plotted against the molar fraction of MS1 with a total con-
centration of the sensor and Hg2+ ion of 8.0 μM. The molar frac-
tion at maximum emission intensity represents the binding
stoichiometry of the MS1–Hg2+ complexes. The maximum
emission intensity was reached at a molar fraction of 0.5 (In
Fig. 4). This result indicates that chemosensor MS1 forms a 1 : 1
complex with Hg2+. The apparent association constant (Ka) of
MS1–Hg2+ complexes was determined by the Benesi-Hildebrand
eqn (1)12,13
Acknowledgements
We gratefully acknowledge the financial support of the National
Science Council (ROC) and National Chiao Tung University.
Notes and references
1 (a) T. Takeuchi, N. Morikawa, H. Matsumoto and Y. Shiraishi, Acta
Neuropathol., 1962, 2, 40–57; (b) M. Harada, Crit. Rev. Toxicol., 1995,
25, 1–24.
2 K. Leopold, M. Foulkes and P. Worsfold, Anal. Chim. Acta, 2010, 663,
127–138.
3 (a) Y. Gao, S. De Galan, A. De Brauwere, W. Baeyens and
M. Leermakers, Talanta, 2010, 82, 1919–1923; (b) M. J. da Silva,
A. P. S. Paim, M. F. Pimentel, M. L. Cervera and M. de la Guardia, Anal.
Chim. Acta, 2010, 667, 43–48.
4 (a) F. Moreno, T. Garcia-Barrera and J. L. Gomez-Ariza, Analyst, 2010,
135, 2700–2705; (b) M. V. B. Krishna, K. Chandrasekaran and
D. Karunasagar, Talanta, 2010, 81, 462–472; (c) W. R. L. Cairns,
M. Ranaldo, R. Hennebelle, C. Turetta, G. Capodaglio, C. F. Ferrari,
A. Dommergue, P. Cescon and C. Barbante, Anal. Chim. Acta, 2008,
622, 62–69.
1=ðF ꢀ F0Þ ¼ 1=fKa ꢁ ðFmax ꢀ F0Þ ꢁ ½Hg2þꢂg
þ 1=ðFmax ꢀ F0Þ;
ð1Þ
where F is the fluorescence intensity at 650 nm at any given
Hg2+ concentration, F0 is the fluorescence intensity at 650 nm
in the absence of Hg2+, and Fmax is the maxima fluorescence
intensity at 650 nm in the presence of Hg2+ in solution.
The association constant Ka was evaluated graphically by plot-
ting 1/(F − F0) against 1/[Hg2+]. Data were linearly fitted
according to eqn (1) and the Ka value was obtained from the
slope and intercept of the line.
5 X. Chai, X. Chang, Z. Hu, Q. He, Z. Tu and Z. Li, Talanta, 2010, 82,
1791–1796.
6 (a) M. H. Lee, S. W. Lee, S. H. Kim, C. Kang and J. S. Kim, Org. Lett.,
2009, 11, 2101–2104; (b) J. Fan, K. Guo, X. Peng, J. Du, J. Wang,
S. Sun and H. Li, Sens. Actuators, B, 2009, 142, 191–196; (c) V. Bhalla,
R. Tejpal, M. J. Kumar and A. Sethi, Inorg. Chem., 2009, 48, 11677–
11684; (d) K. G. Vaswani and M. D. Keranen, Inorg. Chem., 2009, 48,
5797–5800; (e) N. Wanichacheva, M. Siriprumpoonthum, A. Kamkaew
and K. Grudpan, Tetrahedron Lett., 2009, 50, 1783–1786; (f) J. Du,
J. Fan, X. Peng, P. Sun, J. Wang, H. Li and S. Sun, Org. Lett., 2010, 12,
476–479; (g) W. Shi, S. Sun, X. Li and H. Ma, Inorg. Chem., 2010, 49,
1206–1210; (h) S. Atilgan, T. Ozdemir and E. U. Akkaya, Org. Lett.,
2010, 12, 4792–4795; (i) O. A. Bozdemir, R. Guliyev, O. Buyukcakir,
S. Selcuk, S. Kolemen, G. Gulseren, T. Nalbantoglu, H. Boyaci and
E. U. Akkaya, J. Am. Chem. Soc., 2010, 132, 8029–8036;
( j) B. N. Ahamed and P. Ghosh, Dalton Trans., 2011, 40, 12540–12547;
(k) F. Wang, S.-W. Nam, Z. Guo, S. Park and J. Yoon, Sens. Actuators, B,
2012, 161, 948–953; (l) X. Ma, J. Wang, Q. Shan, Z. Tan, G. Wei,
D. Wei and Y. Du, Org. Lett., 2012, 14, 820–823; (m) X. Jiang, C. Wong,
P. Lo and D. K. P. Ng, Dalton Trans., 2012, 41, 1801–1807;
(n) M. Vedamalai and S. Wu, Eur. J. Org. Chem., 2012 (6), 1158–1163;
(o) J. Dessingou, K. Tabbasum, A. Mitra, V. K. Hinge and C. P. Rao,
J. Org. Chem., 2012, 77, 1406–1413; (p) B. Bag and B. Biswal, Org.
Biomol. Chem., 2012, 10, 2733–2738.
Cell culture
The cell line HeLa was provided by the Food Industry Research
and Development Institute (Taiwan). The HeLa cells were grown
in DMEM (Dulbecco’s modified Eagle’s medium) supplemented
with 10% FBS (fetal bovine serum) at 37 °C and 5% CO2. Cells
were plated on 14 mm glass coverslips and allowed to adhere for
24 hours.
Fluorescence imaging
Experiments to assess Hg2+ uptake were performed in PBS with
2 μM Hg(ClO4)2. Treat the cells with 4 μL of 1 mM metal
ions (final concentration: 2 μM) dissolved in sterilized PBS
(pH 7.4) and incubated for 30 min at 37 °C. The treated cells
was washed PBS (3 × 2 mL) to remove remaining metal ions.
Culture media (2 mL) was added to the cell culture, which was
treated with a 10 mM solution of chemosensor MS1 (4 μL; final
concentration: 2 μM) dissolved in DMSO. The samples were
incubated at 37 °C for 30 min. The culture media was removed,
and the treated cells were washed with PBS (3 × 2 mL) before
observation. Fluorescence imaging was performed with a ZEISS
Axio Scope A1 Fluorescence Microscope. Cells loaded with
MS1 were excited at 545 nm using a lamp (Hg 50 W). Emission
Filter was 570 nm.
7 D. S. McClure, J. Chem. Phys., 1952, 20, 682–686.
8 (a) W. Qin, T. Rohand, W. Dehaen, J. N. Clifford, K. Driesen,
D. Beljonne, B. Van Averbeke, M. Van der Auweraer and N. Boens,
J. Phys. Chem. A, 2007, 111, 8588–8597; (b) A. Loudet and K. Burgess,
Chem. Rev., 2007, 107, 4891–4932.
9 M. Kaupp, O. L. Malkina, V. G. Malkin and P. Pyykko, Chem.–Eur. J.,
1998, 4, 118–126.
10 R. P. Brinnas and C. Bruckner, Tetrahedron, 2002, 58, 4375–4381.
11 A. Senthilvelan, I. Ho, K. Chang, G. Lee, Y. Liu and W. Chung, Chem.–
Eur. J., 2009, 15, 6152–6160.
12 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71, 2703–
2707.
13 R. B. Singh, S. Mahanta and N. Guchhait, J. Mol. Struct., 2010, 963,
92–97.
5416 | Org. Biomol. Chem., 2012, 10, 5410–5416
This journal is © The Royal Society of Chemistry 2012