A highly selective and sensitive fluorescent turn-on sensor for Hg 2+ and its application in live cell imaging

Article abstract of DOI:10.1039/b902912e

A boron-dipyrromethene (BODIPY) derivative containing a tridentate diaza-oxa ligand (8H-BDP) was synthesized as a fluorescent turn-on chemosensor for Hg²⁺ with high sensitivity (detection limit ≤2 ppb), a rapid response time (≤5 seconds) and specific selectivity over other cations under physiological conditions and in live cells according to the confocal fluorescence microscopy experiment. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b902912e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A highly selective and sensitive fluorescent turn-on sensor for Hg2+ and
its application in live cell imaging†
Hua Lu,a Liqin Xiong,b Hanzhuang Liu,a Mengxiao Yu,b Zhen Shen,*a Fuyou Li*b and Xiaozeng You*a
Received 11th February 2009, Accepted 6th April 2009
First published as an Advance Article on the web 29th April 2009
DOI: 10.1039/b902912e
A boron–dipyrromethene (BODIPY) derivative containing a tridentate diaza-oxa ligand (8H-BDP) was
synthesized as a fluorescent turn-on chemosensor for Hg2+ with high sensitivity (detection limit ≤2 ppb),
a rapid response time (≤5 seconds) and specific selectivity over other cations under physiological
conditions and in live cells according to the confocal fluorescence microscopy experiment.
hydroxyphenyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-di-
Introduction
aza-s-indacene (8H-BDP) (Scheme 1) as a highly selective and sen-
sitive fluorescence turn-on chemosensor for Hg2+. Furthermore,
confocal fluorescence microscopy experiments demonstrated that
8H-BDP could be used to monitor Hg2+ in living cells.
The concern for the worldwide spread of mercury pollution and its
damage to human health and the environment has stimulated the
development of efficient, selective and sensitive methods for mon-
itoring Hg2+ in environmental and biological systems.1 A number
of highly selective and sensitive fluorescent chemosensors have
been extensively exploited for the detection of Hg2+ in solution,
however, only a few examples of in vivo monitoring of Hg2+ have
been reported,2 including two fluorescein chemosensors bearing a
thio-crown moiety,2d –2e several rhodamine-based fluorescent turn-
on probes,2f –2k and a ratiometric fluorescent probe based on a
BODIPY-rhodamine system.2l Several limitations such as low
water solubility, poor selectivity toward other metal ions, and
weak sensitivity in physiological conditions prevent the sensor
molecules being applicable in biological systems. Moreover, Hg2+
often causes a turn-off fluorescence response due to the heavy
atom or spin-orbit coupling effect. Therefore, the exploration of
new fluorescent turn-on probes for analyzing Hg2+ in vivo with
appropriate selectivity, high sensitivity, rapid response, and good
water solubility remains a challenge.
Owing to their remarkable photophysical properties, such as
high molar extinction coefficients (usually >80 000 L (mol·cm)-1),
high fluorescence quantum yields (commonly Uf > 0.5), excellent
photostability, easy structural modification and appropriate redox
potential,3 recently, boron–dipyrromethene (BODIPY) derivatives
have been widely employed for detecting K+,4 Cd2+,5 CN-,6 ClO-,7
Zn2+,8and Hg2+.9 However, the lipophilicity of the BODIPY
moiety limited their applications in biotechnology.10 Herein,
we report a rational design, relying on the “receptor–spacer–
fluorophore” approach,11 toward the development of fluores-
cence sensors for Hg2+ in aqueous media as well as in living
cells. With a hydrophilic diaza-oxa tridentate ligand based on
Hpyramol (2-N-(2-pyridylmethyl)amino-phenol)12 as a receptor,
we designed and synthesized 8-(3-N-(2-pyridylmethyl)amino-4-
Scheme 1 Synthetic procedures for the preparation of 8H-BDP.
Scheme 1 outlines the synthesis of 8H-BDP. The intermediate 2
was prepared through the TFA catalyzed condensation reaction of
3-nitro-4-hydroxybenzaldehyde with 2,4-dimethylpyrrole accord-
ing to a published procedure.3a Reduction of 2 with Fe–HCl gave
another compound 3, which was reacted with picolinaldehyde
to afford 8H-BDP in 19% yield. The structure of 8H-BDP was
confirmed by 1H NMR, MS spectra and single-crystal X-ray
diffraction analysis.
Results and discussion
aState Key Laboratory of Coordination Chemistry, Nanjing National
Laboratory of Microstructures, Nanjing University, Nanjing, 210093, PR
China. E-mail: zshen@nju.edu.cn, youxz@nju.edu.cn
Structure of 8H-BDP
bDepartment of Chemistry & Laboratory of Advanced Materials, Fudan
University, Shanghai, 200433, PR China. E-mail: fyli@fudan.edu.cn
The molecular structure of 8H-BDP is shown in Fig. 1. Similar
to the previously reported structures of alkyl substituted
BODIPYs,13 the indacene plane in 8H-BDP was nearly planar
since the deviation from the mean plane was 0.0581 A. The meso
(or 8)-phenyl ring was virtually orthogonal to the indacene plane
† Electronic supplementary information (ESI) available: Supplementary
data, 1H NMR spectra and X-ray analysis of 8H-BDP. CCDC reference
number 671844. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/b902912e
˚
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Fig. 1 ORTEP view of the molecular structure of 8H-BDP at 30%
probability. The solvent molecule was omitted for clarity.
with the torsion angle being 87.5◦. The pyridyl ring was tilted to
the meso-phenyl plane with a torsion angle of 80.2◦. The neigh-
boring 8H-BDP molecules were linked through intermolecular
O1-H ◊ ◊ ◊ N4 hydrogen bonds to form a one-dimensional chain
along the b axis (Fig. S1, ESI†). There were no strong p–p
interactions within the chain.
Fig. 2 (a) Fluorescence responses of 5 mM of 8H-BDP upon addition of
50 mM of different metal ions and 5 mM of Hg2+. (b) Fluorescence responses
of the addition of 5 mM Hg2+ to 5 mM of 8H-BDP in the presence of the
appropriate metal ions (200 mM for Mg2+, Na+, Ca2+ and Zn2+; 50 mM for
Cd2+, Co2+, Fe3+, Mn2+, Ni2+, Ag+, Al3+, Fe2+ and Pb2+; 5 mM for Cu2+).
Spectra were acquired in a DMSO–HEPES buffer (50 mM HEPES, 50 mM
KNO3, 1 : 99, v/v, pH = 7.2) solution, lex = 470 nm.
Hg2+ recognition in aqueous solution
As expected, 8H-BDP in DMSO–HEPES buffer (1 : 99, v/v,
pH = 7.2) solution showed a very weak fluorescence with a
quantum yield (U0) of 0.002, which was attributed to the efficient
photoinduced electron transfer (PET) quenching process from
the electron-donating Hpyramol receptor to the excited BDP
fluorophore.14 Upon addition of Hg2+, the fluorescence intensity
increased remarkably and a fluorescence enhancement factor at
509 nm of approximately 27-fold was estimated. Its fluorescence
intensity was almost not influenced by the addition of 10 eq of
Zn2+, Cu2+, Cd2+, Co2+, Fe3+, Mn2+, Ni2+, Mg2+, Na+, Al3+, Ca2+,
and Fe2+, respectively. A small influence by the addition of 10 eq
of Pb2+ (I/I0 = 2.4) and 10 eq of Ag+ (I/I0 = 3.6) was observed
(Fig. 2a). To evaluate the utility of 8H-BDP as a Hg2+-selective
fluorescent sensor, ion interference experiments were carried out.
As shown in Fig. 2b, its Hg2+ response was not interfered in the
background containing the appropriate metal ions. These facts
suggested that 8H-BDP could recognize Hg2+ ions with high
selectivity under physiological conditions.
The sensitivity of 8H-BDP to Hg2+ was further investigated
by fluorescence spectroscopy. Upon addition of the Hg2+ ion at
different concentrations in a DMSO–HEPES buffer (1 : 99, v/v,
pH = 7.2) solution, as shown in Fig. 3, the fluorescence intensity
of 8H-BDP increased gradually with a virtually unchanged peak
position, typical of PET.15 Interestingly, before reaching the
stoichiometric point, the observed fluorescent intensity was pro-
Fig. 3 Fluorescence response of 5 mM of 8H-BDP upon addition of Hg2+
.
The amounts of Hg2+employed are 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35,
0.4, 0.45, 0.5, 0.6, 0.7, and 1.0 equiv., respectively. Spectra were acquired
in a DMSO–HEPES buffer (50 mM HEPES, 50 mM KNO3, 1 : 99, v/v,
pH = 7.2) solution. lex = 470 nm.
portional to the concentration of Hg2+ ion; subsequent addition of
Hg2+ (>0.5 eq.) caused an unchanged behaviour in the fluorescence
intensity, indicating that the Hg2+ sensor had a 1 : 2 stoichiometry16
(Fig. 3). The association constant logKs was determined to be
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18.2 0.1 from a nonlinear least-square analysis of fluorescence
intensity versus the concentration of Hg2+ (Fig. S2, ESI†).
For a fluorescent molecular sensor to be practically used, the de-
tection limit is an important factor. When 8H-BDP was employed
at 0.5 mM in a DMSO–HEPES buffer (50 mM HEPES, 50 mM
KNO3, 1 : 999, v/v, pH = 7.2) solution, Hg2+ could be detected at
least down to 2 ppb, which is the U.S. Environmental Protection
Agency’s limit for drinking water.17 Under these conditions, the
fluorescent intensity of 8H-BDP was nearly proportional to the
concentration of Hg2+ added (Fig. 4). Furthermore, the interaction
of 8H-BDP with Hg2+ was completed in a few seconds (Fig. S3,
ESI†), therefore 8H-BDP could be used for the real-time and real-
space analysis of Hg2+ ions in cells and organisms.
Fig. 5 Confocal fluorescence and brightfield images of HeLa cells. (a)
Cells incubated with 50 mM of 8H-BDP for 10 min at 25 ◦C. (b) Cells
supplemented with 50 mM of Hg2+ in the growth media for 1 h at 37 ◦C
and then stained with 50 mM of 8H-BDP for 10 min at 25 ◦C. (c) Brightfield
image of cells showed in panel b. The overlay of panels b and c is shown in
d (lex = 488 nm).
Fig. 4 Fluorescence response of 8H-BDP (0.5 mM) to Hg2+ in a
DMSO–HEPES buffer (50 mM HEPES, 50mM KNO3, 1 : 999, v/v, pH =
7.2) solution. lex = 470 nm. Inserted figure: titration curve of I509nm vs.
Hg2+ concentration.
Intracellular imaging of Hg2+
Then, we evaluated the practical applicability of 8H-BDP as a
Hg2+ probe to operate within living cells. Cells supplemented with
50 mM of HgCl2 for 1 h at 37 ◦C and then incubated with 50 mM
of 8H-BDP for 10 min at 25 ◦C, showed a significant fluorescence
emission from the intracellular region (Fig. 5b), as determined by
laser scanning confocal microscopy (using an Olympus FluoView
FV1000). In the control experiment, staining HeLa cells with
only 50 mM 8H-BDP or 50 mM Hg2+ under the same conditions
showed weak intracellular fluorescence (Fig. 5a and Fig. S4, ESI†).
The marked increase observed in intracellular fluorescence was
attributed to the interaction between 8H-BDP and Hg2+. Further
brightfield measurements after treatment with both Hg2+ and
8H-BDP confirmed that the cells were viable throughout the
imaging experiments (Fig. 5c). As shown in Fig 5d, the overlay of
fluorescence and brightfield images revealed that the fluorescence
signals were localized in the perinuclear region of the cytosol,
Fig. 6 Z-scan image of the live HeLa◦cells supplemented with 50 mM of
Hg2+ in the growth media for 1 h at 37 C and then stained with 50 mM of
8H-BDP for 10 min at 25 ◦C.
which was confirmed by the high signal-to-noise ratio (I2/I1
>
200) between the cytoplasm (region 2) and the background (region
1) (Fig. S5, ESI†).
cell-permeable and could respond to variations in intracellular
Hg2+.
In addition, large signal ratios (I2/I3 >20) between the cy-
toplasm (region 2) and nucleus (region 3) mean weak nuclear
uptake and exclusive staining in the cytoplasm for 8H-BDP.
This was also confirmed by Z-scan luminescence imaging of
HeLa cells stained with both 8H-BDP and Hg2+ (Fig. 6 and
Fig. S6, ESI†). The above results indicated that 8H-BDP was
In summary, we have demonstrated a boron–dipyrromethene
(BDP) derivative 8H-BDP with a diaza-oxa tridentate ligand of
Hpyramol as a fluorescence turn-on chemosensor for Hg2+ in
aqueous media. This chemosensor showed a very high sensitivity
(detection limit ≤2 ppb), a rapid response time (≤5 seconds),
and high selectivity for Hg2+ over other metal cations. Such
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significantly enhanced fluorescence of 8H-BDP upon addition of
Hg2+ is probably attributed to the formation of a 2 : 1 complex of
8H-BDP–Hg2+ in which the PET quenching process is efficiently
suppressed by coordination with the Hg2+ ion. Moreover, confocal
fluorescence microscopy experiments have established the utility
of 8H-BDP in monitoring Hg2+ within living cells, indicating its
potential application for studying the effect of Hg2+ in biological
systems.
56.52◦, R1 = 0.0673, Rw2 = 0.1892, GOF = 0.930, residual electron
-3
˚
density between 0.883 and -0.685 e A .†
Cell culture and fluorescence bioimaging
The HeLa cell line was provided by the Institute of Biochemistry
and Cell Biology, SIBS, CAS (China). Cells were grown in MEM
(modified Eagle’s medium) supplemented with 10% FBS (fetal
◦
bovine serum) at 37 C and 5% CO2. Cells (5 ¥ 108 per L) were
plated on 14 mm glass coverslips and allowed to adhere for 24 h.
Experiments to assess Hg2+ uptake were performed over 1 h in the
same medium supplemented with 50 mM Hg(ClO4)2.
Experimental section
Materials and methods
Immediately before the experiments, cells were washed with
PBS buffer and then incubated with 50 mm of 8H-BDP in PBS
for 10 min at 25 ◦C. Cell imaging was then carried out after
washing the cells with PBS. Confocal fluorescence imaging was
performed with an Olympus FV1000 laser scanning fluorescence
microscope and a 60¥ oil-immersion objective lens. Cells incubated
with 8H-BDP were excited at 488 nm using a multi-line argon laser.
Emission was collected from 500 to 540 nm.
All reagents were obtained from commercial suppliers and used
without further purification unless otherwise indicated. All air-
and moisture-sensitive reactions were carried out under nitro-
gen atmosphere in oven-dried glassware. Dichloromethane was
distilled over calcium hydride. Triethylamine was obtained by
simple distillation. Elemental analyses for C, H and N were
performed on a Perkin-Elmer 240C elemental analyzer. NMR
spectra were recorded on a Bruker DRX500 spectrometer and
referenced to the residual proton signals of the solvent. Mass
spectra were measured with a Bruker Daltonics Autoflex IITM
MALDI TOF spectrometer. Luminescence spectra were measured
on an Edinburgh LFS-920 fluorescence spectrophotometer and
an Aminco Bowman 2 Luminescene spectrophotometer. The
fluorescence quantum yield was calculated using Rhodamine 6G
(U = 0.88 in EtOH) as a reference.
Synthesis
8-(3-Nitro-4-hydroxyphenyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-
bora-3a,4a-diaza-s-indacene (2). 2,4-Dimethylpyrrole (380 mg,
4 mmol) and 3-nitro-4-hydroxybenzaldehyde (334 mg, 2 mmol)
were dissolved in dry CH2Cl2 (150 mL) under nitrogen. One drop
of trifluoroacetic acid (TFA) was added, and the solution was
stirred for 4 h at ambient temperature in the dark (until TLC
indicated complete consumption of the aldehyde). 2,3-Dichloro-
5,6-dicyanoquinone (DDQ, 442 mg, 2 mmol) was added, and the
mixture was stirred for an additional 30 min. The reaction mixture
was then treated with triethylamine (3 mL) for 5 min. Boron
trifluoride etherate (3 mL) was added and the mixture was stirred
for another 40 min, the dark brown solution was washed with
water (3 ¥ 20 mL) and brine (30 mL), dried over anhydrous mag-
nesium sulfate, and concentrated at reduced pressure. The crude
product was purified by silica-gel flash column chromatography
(10% EtOAc–petroleum ether) and recrystallization from CHCl3–
hexane yielded 2 as red crystals (150 mg, yield 19%). Mp >200 ◦C.
IR (KBr pellet, cm-1): 3447, 2924, 1634, 1548, 1512, 1465, 1415,
1382, 1311, 1192, 1156, 1088, 937. lmax (CHCl3)/nm 479 (e/dm3
mol-1 cm-1 14 800) and 507 (61 700). 1H NMR (500 MHz, CDCl3)
10.73 (s, 1 H), 8.13 (s, 1 H), 7.55 (d, 1 H, J = 5 Hz), 7.36 (d, 1 H, J =
8.5 Hz), 6.05 (s, 2 H), 2.59 (s, 6 H), 1.49 (s, 6 H). MS (MALDI-
TOF): m/z 385.210 [M]+. Anal. Calcd for C19H18BF2N3O3; C,
59.25; H, 4.71; N, 10.91; found C, 59.38; H, 4.63; N, 10.93%.
Stock solutions of the metal ions (1 mM) and Hg2+ (10 mM,
1 mM, 0.1 mM) were prepared in deionized water. A stock solution
of 8H-BDP (0.5 mM) was prepared in DMSO. The solution of 8H-
BDP was then diluted to 5 mM with HEPES buffer solution (1 :
99, v/v, pH 7.2). In the titration experiments, a 2 mL solution
of 8H-BDP (5 mM, 0.5 mM) was poured into a quartz optical
cell of 1 cm optical path length each time, and the Hg2+ stock
solution was added into the quartz optical cell gradually by using
a micro-pipette. Spectral data were recorded immediately after
the addition. In selectivity experiments, the test samples were
prepared by placing the appropriate amounts of metal ion stock
solution into 2 mL solution of 8H-BDP (5 mM). For fluorescence
measurements, excitation was provided at 470 nm, and emission
was collected from 500 to 580 nm.
X-Ray structure determination
The X-ray diffraction data were collected on a Bruker Smart
Apex CCD diffractometer with graphite monochromated Mo Ka
˚
8-(3-Amino-4-hydroxyphenyl)-4,4-difluoro-1,3,5,7-tetramethyl-
radiation (l = 0.71073 A) using the w-2q scan mode. The data
4-bora-3a,4a-diaza-s-indacene (3). Compound
2 (150 mg,
were corrected for Lorenz and polarization effects. The structure
was solved by direct methods and refined on F2 by full-matrix
least-squares methods using SHELXTL-2000. All calculations
and molecular graphics were carried out on a computer using
the SHELX-2000 program package and Diamond.
0.39 mmol) was dissolved in 5 mL of methanol. H2O (3 mL) and
Fe (300 mg, 5.4 mmol) were added and the reaction mixture was
heated to reflux. Hydrochloric acid in a methanol solution (2 mL,
0.6 mol L-1) was added dropwise . The solution was refluxed for
2 h until TLC monitoring indicated complete consumption of the
starting material. After cooling to room temperature, filtration
and concentration at reduced pressure, the crude product was
purified by silica-gel flash column chromatography (20% EtOAc–
petroleum ether) and recrystallization from CHCl3–hexane yielded
3 as red crystals (105 mg, yield 75%). Mp >200 ◦C. IR (KBr pellet,
8H-BDP·CH3OH. C26H29BF2N4O2; a red block-like crystal
of the approximate dimensions 0.23 ¥ 0.21 ¥ 0.20 mm3 was
˚
measured. Monoclinic, space group P21/c, a = 14.6725(14) A,
◦
˚
˚
b = 10.8383(10) A, c = 18.7883(13) A, b= 127.732(5) , V =
3
-3
˚
2363.0(4) A , Z = 4, F(000) = 1008, r = 1.396 Mgm , 2qmax
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cm-1): 3430, 2923, 1620, 1544, 1507, 1469, 1406, 1382, 1302, 1197,
1158, 1085, 983. lmax (CHCl3)/nm 474 (e/dm3 mol-1 cm-1 12 900)
and 502 (56 700). 1H NMR (500 MHz, CDCl3) 6.84 (d, 1 H, J =
9 Hz), 6.66 (s, 1 H), 6.56 (d, 1 H, J = 7.5 Hz), 5.99 (s, 2 H), 2.56 (s, 6
H), 1.54 (s, 6 H). MS (MALDI-TOF): m/z 355.148 [M]+, 336.157
[M - F]+ Anal. Calcd for C19H20BF2N3O; C, 64.25; H, 5.68; N,
11.83; found C, 64.43; H, 5.62; N, 11.72%.
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8-(3-N-(2-Pyridylmethyl)amino-4-hydroxyphenyl)-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene
(8H-BDP).
Compound 3 (105 mg, 0.3 mmol), picolinaldehyde (32 mg,
0.3 mmol) and one drop of acetic acid in MeOH (10 mL) were
refluxed for 24 h. After cooling to room temperature, NaBH4
(30 mg, 0.8 mmol) was slowly added and the reaction mixture
was refluxed continuously for another 12 h. After removing the
volatiles under reduced pressure, water (20 mL) was added to the
resulting residue. The pH value of the mixture was adjusted to
ca. 7 by adding glacial acetic acid, and the mixture was extracted
with EtOAc (3 ¥15 mL), dried over anhydrous magnesium
sulfate, and concentrated at reduced pressure. The crude product
was purified by silica-gel flash column chromatography (40%
EtOAc–petroleum ether) and recrystallization from CH3OH–
hexane yielded 8H-BDP as red crystals (25 mg, yield 18.7%). Mp
◦
>200 C. IR (KBr pellet, cm-1): 3428, 2924, 1601, 1546, 1510,
1470, 1407, 1384, 1307, 1197, 1157, 1085, 978. lmax (CH3OH)/nm
469 (e/dm3 mol-1 cm-1 13 500) and 507 (52 100). 1H NMR
(500 MHz, CD3OD) 8.39 (d, 1 H, J = 4.5 Hz), 7.70 (m, 1 H), 7.43
(d, 1H, J = 6 Hz), 7.21 (m, 1 H), 6.79 (d, 1 H, J = 9 Hz), 6.33 (d,
1 H, J = 9 Hz), 6.24 (s, 1 H), 5.93 (s, 2 H), 5.46 (s, 2 H), 4.44 (s, 2
H), 2.40 (s, 6 H), 1.33 (s, 6 H). MS (MALDI-TOF): m/z 446.387
[M]+, 427.399 [M - F]+. Anal. Calcd for C25H25BF2N4O; C, 67.28;
H, 5.65; N, 12.55; found C, 67.15; H, 5.51; N, 12.38.
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Acknowledgements
We are thankful for financial support from the National
Basic Research Program of China (Nos. 2006CB806104 and
2007CB925103), NSFC (Nos. 20875043 and 20775017), and the
Shanghai Leading Academic Discipline Project (B108).
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2558 | Org. Biomol. Chem., 2009, 7, 2554–2558
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