A ratiometric fluorescent probe based on FRET for imaging Hg2+ ions in living cells

Article abstract of DOI:10.1002/anie.200803246

(Chemical Equation Presented) Mercury rising: A FRET-based ratiometric probe (see picture; FRET = fluorescence resonance energy transfer) can selectively detect Hg²⁺ ions on the parts-per-billion scale under physiological conditions. The probe undergoes a clear Hg²⁺-induced change in the intensity ratio ofthe two well-separated and equally intense emission bands of dipyrrometheneboron difluoride and rhodamine. Experiments show that the probe can be used in living cells.

Full text of DOI:10.1002/anie.200803246

Angewandte
Chemie
DOI: 10.1002/anie.200803246
Fluorescent Probes
A Ratiometric Fluorescent Probe Based on FRET for Imaging Hg2+
Ions in Living Cells**
Xiaolin Zhang, Yi Xiao,* and Xuhong Qian*
Mercury ions can easily pass through biological membranes
and cause serious damage to the central nervous and
endocrine systems.[1] Therefore, imaging of Hg2+ ions in
living cells is crucial for the elucidation of their biological
effects. Fluorescence spectroscopy has become a powerful
tool for sensing and imaging trace amounts of samples
because of its simplicity and sensitivity.[2] Thus, the develop-
ment of fluorescent Hg2+ probes,[3] particularly those that
have practical application in living cells,[4] has attracted much
attention. Most reported examples of fluorescent sensing of
Hg2+ ions in living cells function by the enhancement of
fluorescence signals. However, as the change in fluorescence
intensity is the only detection signal, factors such as instru-
mental efficiency, environmental conditions, and the probe
concentration can interfere with the signal output.[5] Ratio-
metric sensors can eliminate most or all ambiguities by self-
calibration of two emission bands.[6]
Ratiometric probes can be designed to function following
two mechanisms: intramolecular charge transfer (ICT) and
fluorescence resonance energy transfer (FRET). ICT probes
have been frequently reported and some work well under
physiological conditions. Two aspects which potentially influ-
ence the accuracy of ICT probes are: 1) Binding of the target
ions promotes or inhibits ICT interactions, which results in
remarkable shifts of the sensorsꢀ absorption maxima; but if
multiple excitation wavelengths are used to match the
different excitation maxima, their difference in efficiency
may be a potential origin of inaccuracy. 2) Relatively broad
fluorescence spectra are often observed for ICT fluoro-
phores; in a significant number of cases the broad fluores-
cence spectra before and after binding target ions have a high
degree of overlap (or in an extreme case, a broad spectrum
with high intensity completely covers one with lower inten-
sity), which makes it difficult to accurately determine the ratio
of the two fluorescence peaks. Theoretically, the above
problems can be avoided by using a FRET-based sensor for
which the single excitation wavelength of a donor fluorophore
results in emission of the acceptor at a longer wavelength.[7]
Herein we present a BODIPY-rhodamine (BODIPY=
boron–dipyrromethene) FRET “off–on” system 3 as a
ratiometric and intracellular Hg2+ sensor. A leuco-rhodamine
derivative was chosen as a sensitive and selective chemo-
sensor for Hg2+ ions. This was inspired by Tae and co-workers
as well as other research groups,[8], who used these leuco
derivatives with unconjugated structures as fluorogenic and
chromogenic sensors. A highly efficient ring-opening reaction
induced by Hg2+ generates the long-wavelength rhodamine
fluorophore which can act as the energy acceptor. BODIPY[9]
was chosen as the energy donor because its intense fluores-
cence is insensitive to environmental factors and its fluores-
cence spectrum matches well with the absorption spectrum of
rhodamine. The choice of the connection between the donor
and acceptor was equally important; a rigid and conjugated
phenyl–ethynyl–phenyl spacer, which not only facilitates the
through-bond energy transfer process[7a] but also greatly
simplifies the synthesis of a relatively large molecule, was
identified as an ideal bridge.
Both sensor 3 and ring-opened product 4 were efficiently
synthesized (Scheme 1) and well characterized. An Hg2+-
induced process can change the emission maximum of the
system from 514 nm (the characteristic peak of BODIPY) to
589 nm (the characteristic peak of rhodamine). This wave-
length shift allows the ratiometric detection of Hg2+ ions both
in ethanol/water solution and in living cells. To the best of our
knowledge, this is the first successful intracellular application
of a FRET-based ratiometric Hg2+ sensor.
[*] Dr. X. Zhang, Dr. Y. Xiao
The hydrophilicity of the rhodamine moiety and the
lipophilicity of the BODIPY moiety mean that sensor 3 has
the advantage of proper amphipathicity and can be dissolved
in mixtures of organic solvents and water. Previous stud-
ies[4c,6b] have shown that the subtle equilibrium between
hydrophilicity and lipophilicity is a very important factor for
both cell permeability and intracellular fluorescent imaging.
The UV/Vis spectrum of 3 (ethanol/water 80:20, pH 7.0)
showed only the absorption profile of the donor (BODIPY),
which has a maximum at 501 nm (loge = 4.80). Addition of
Hg2+ ions immediately induced an increase in the absorption
intensity at 560 nm, which corresponds to the absorption of
rhodamine. The absorption stabilized after the amount of
added Hg2+ ions reached 1 equiv (Figure 1) and a significant
color change from green to pink could be observed easily by
eye. This confirms that the addition of Hg2+ ions can promote
State KeyLaboratoryof Fine Chemicals
Dalian Universityof Technology
Zhongshan Road 158, Dalian, 116012 (China)
Fax: (+86)411-8367-3488
E-mail: xiaoyi@chem.dlut.edu.cn
Dr. X. Qian
Laboratoryof Chemical Biology
East China Universityof Science and Technology
Meilong Road 130, Shanghai, 200237 (China)
Fax: (+86)21-64252603
[**] We thank the NSF of China (Nos. 20406004, 20572012, 20536010)
and the Program for Changjiang Scholars and Innovative Research
Team in University(IRT0711) for financial support. We also thank
Dr. Liji Jin and Xueliang Niu (Dalian Universityof Technology) for
help with cell culture experiments.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200803246.
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Communications
Table 1: Spectroscopic data for compounds.
Compd.
labs [nm]
loge [mÀ1 cmÀ1
]
lem [nm]
Ff
501
565
501
565
4.80
3.05
4.90
4.99
514
589
514
589
0.35[a]
3
4
0.03[a]
0.001[a]
0.20[a], 0.26[b]
[a] lex =488 nm. [b] lex =543 nm.
the fluorescence spectrum stopped and the ratio of the
emission intensities at 589 nm and 514 nm (F589/F514) became
constant when the amount of Hg2+ ions added reached
60 parts per billion (ppb; 1 equiv of the sensor, Figure 2b).
The color of the fluorescence clearly changed from green to
organge-red. It was clear that the FRET process was switched
on by Hg2+ ions as excitation of BODIPY at 488 nm resulted
in the emission of rhodamine with a maximum of 589 nm. The
Förster energy transfer efficiency between BODIPY and
rhodamine in compound 4 was calculated to be 99%, and the
calculated R0 value (the distance at which the energy transfer
efficiency is 50%) was 58.9 Š (see the Supporting Informa-
tion), which indicates the occurrence of a highly efficient
Scheme 1. Synthesis of compounds 3 and 4.
the formation of the ring-opened compound 4, which posesses
a high molar extinction coefficient (Table 1).
Excitation of 3 at 488 nm resulted in the emission profile
of BODIPY at 514 nm (Ff = 0.35). A fluorescence titration
with Hg2+ ions was conducted on a solution of 3 (0.3 mm) in
ethanol/water (80:20) at pH 7.0. Upon addition of Hg2+ the
BODIPY emission at 514 nm decreased, and a new emission
band with a maximum at 589 nm (rhodamine) appeared and
gradually increased in intensity (Figure 2a). These changes in
Figure 2. a) Fluorescence titration spectra of 3 (0.3 mm) in C2H5OH/
H2O (8:2) at pH 7.0 upon gradual addition of Hg2+ ions (in 2 ppb
increments); b) Fluorescence intensityratio changes ( F589/F514) of 3
(0.3 mm) in C2H5OH/H2O (8:2) at pH 7.0 upon gradual addition of
Hg2+ ions. lexc =488 nm.
Figure 1. UV/Vis spectra of 3 in C2H5OH/H2O (8:2) at pH 7.0 upon
gradual addition of Hg2+ ions (mole equivalents=0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, and 1.5).
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Chemie
FRET process. This FRET “off–on” sensing system has two
distinct advantages. One is the large shift (100 nm) between
donor excitation and acceptor emission, which rules out any
influence of excitation backscattering effects on fluorescence
detection. The other is the presence of two well-separated
emission bands with comparable intensities, which ensures
accuracy in determining their intensities and ratios.
The new FRET probe showed excellent selectivity toward
Hg2+ ions. In fact, 3 did not give any observable response for
many metal ions such as Mg2+, Na+, Mn2+, Ni+, Pb2+, Al3+,
Zn2+, Ba2+, Ca2+, Cd2+, Co2+, Cr2+, Cu2+, or K+ (Figure 3).
Figure 4. Fluorescence intensityratio ( F589/F514) of 3 (square) and 4
(circle) in C2H5OH/H2O (8:2) vs. pH at 258C. lex =488 nm.
Figure 5. Confocal fluorescence images of MCF-7 (breast cancer) cells
(lex =488 nm; Leica TCS-SP2 confocal fluorescence microscope,
20”objective lens). a) MCF-7 cells incubated with 3 (5 mm) for 30 min
at room temperature, emission measured at (514Æ15) nm. b, c) MCF-
7 cells incubated with 3 (5 mm) and then further incubated with Hg2+
ions (5 mm), emission measured at (514Æ15) nm (b) and
(589Æ15) nm (c).
Figure 3. Fluorescence intensityratio ( F589/F514) of 3 (1 mm) in
C2H5OH/H2O (8:2) at pH 7.0 in the presence of 1 equiv of Mg2+, Na+,
Mn2+, Ni+, Pb2+, Al3+, Zn2+, Ba2+, Ca2+, Cd2+, Co2+, Cr2+, Cu2+, K+, Ag+,
or Hg2+ ions.
cate that 3 can provide ratiometric detection for intracellular
Hg2+ ions. Therefore, it could be a useful molecular probe for
studying biological processes involving Hg2+ ions within living
cells.
Only Ag+ ions had a slight effect with a much longer
equilibrium time (> 40 min) than that of Hg2+ ions
(< 5 min).[8a] A pH titration showed that both 3 and 4 had
stable fluorescence properties over a wide pH span of 4–10
(Figure 4), which suggests that sensor 3 is suitable for
application under physiological conditions.
In summary, we have developed a FRET-based ratiomet-
ric probe 3 that can selectively detect amounts of Hg2+ ions on
the ppb scale under physiological conditions. It exhibits a
clear Hg2+-induced change in the intensity ratio of the two
well-separated and comparably strong emission bands of
BODIPY and rhodamine. The significant changes in the
fluorescence color can be observed by eye. Preliminary
confocal laser scanning microscopy experiments show that 3
has practical application in living cells. Further investigations
on its applications in life science are still underway.
Fluorescence images of MCF-7 cells were observed under
a Leica TCS-SP2 confocal microscope. The double-channel
fluorescence images at (514 Æ 15) and (589 Æ 15) nm are
shown in Figure 5. MCF-7 cells incubated with 3 (5 mm) for
30 minutes at room temperature showed a clear green
intracellular fluorescence, which suggested that 3 was cell
permeable. When cells stained with compound 3 were
incubated with HgCl2 (5 mm) in phosphate-buffered saline
(PBS) for 30 minutes and washed, a partial quenching of the
green fluorescence intensity (Figure 5b) and a remarkable
increase in the red fluorescence intensity (Figure 5c) was
observed. The intensity ratio data were obtained using
commercial software (see Figure S17 in the Supporting
Information). A similar phenomenon can be observed even
if the concentrations of the probe 3 and HgCl2 were further
lowered to 1 mm. The cells remained viable and no apparent
toxicity and side effects were observed throughout the
imaging experiments (about 3–4 h). These experiments indi-
Experimental Section
All chemicals were obtained from commercial suppliers and used
without further purification. Tetrahydrofuran was distilled from
sodium prior to use. Column chromatography was performed on
silica gel (0.035–0.070 mm). 1H NMR and 13C NMR spectra were
measured on a Bruker AV-400 spectrometer using TMS as an internal
standard. Mass spectra were measured on HP 1100 LC-MSD and GC-
Tof MS spectrometers. Fluorescence spectra were measured on a PTI-
700 fluorescence lifetime spectrophotometer. Absorption spectra
were determined on a PGENERAL TU-1901 UV/Vis spectropho-
Angew. Chem. Int. Ed. 2008, 47, 8025 –8029
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Communications
tometer. Fluorescence quantum yields were determined in ethanol/
(q, J = 5.72 Hz, 8H, CH2CH3), 2.55 (s, 6H, CH3), 1.42 (s, 6H, CH3),
1.32 ppm (t, J = 5.72 Hz, 12H, CH2CH3); 13C NMR (100 MHz,
CDCl3): d = 160.6, 158.3, 157.9, 156.2, 155.6, 143.1, 141.2, 139.5,
135.2, 134.3, 132.2, 132.2, 131.6, 131.3, 130.8, 130.2, 130.0, 129.8, 129.5,
128.1, 125.1, 124.8, 123.9, 121.3, 118.1, 114.3, 114.1, 96.6, 91.8, 87.3,
46.1, 14.6, 14.1, 12.7 ppm; UV/Vis (ethanol/water 8:2): lmax (e) = 501
water (8:2) relative to rhodamine 6G (ff = 0.88 in enthanol).[10] The
given quantum yields are averaged from values measured with the
absorption maximum 0.02–0.05.
1: A stirred solution of 4-ethynylaniline (62 mg, 0.53 mmol),
BODIPY (200 mg, 0.44 mmol), CuI (3.5 mg, 0.022 mmol), and
[PdCl2(PPh3)2] (6 mg, 0.044 mmol) in triethylamine (0.5 mL) and
THF (2 mL) was heated for 2 h at 408C under an argon atmosphere.
The solution was cooled and filtered and the resulting solid was
washed with additional CH2Cl2 (5 mL). The filtrate was then
evaporated under reduced pressure. The residual solid was purified
on flash silica gel using dichloromethane as eluent to give 1 (120 mg)
as a solid. Yield: 62%. M.p. > 3008C. Rf = 0.3 (SiO2, CH2Cl2).
1H NMR (400 MHz, CDCl3): d = 7.62 (d, J = 8.0 Hz, 2H, Ar-H),
7.36 (d, J = 8.4 Hz, 2H, Ar-H), 7.25 (d, J = 8.0 Hz, 2H, Ar-H), 6.66 (d,
J = 8.4 Hz, 2H, Ar-H), 6.0 (s, 2H, pyrrole-H), 3.87 (s, 2H, NH2), 2.56
(s, 6H, CH3), 1.44 ppm (s, 6H, CH3); 13C NMR (100 MHz, CDCl3):
d = 155.6, 147.0, 143.1, 141.1, 134.2, 133.1, 132.0, 131.3, 128.1, 124.8,
121.3, 114.8, 112.0, 91.7. 86.7, 14.6 ppm (superposition of two signals);
TOF MS calcd for C27H24N3BF2: 439.2031; found: 439.2040.
(79432), 565 nm (97723); luorescence (ethanol/water 8:2, lexc
=
488 nm): lmax = 589 nm; TOF MS calcd. for C56H53BF2N7O2:
904.4316, found: 904.4559.
Received: July 4, 2008
Published online: September 15, 2008
Keywords: fluorescent probes · FRET · mercury· rhodamine ·
.
sensors
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1
petroleum ether= 1:1). H NMR (400 MHz, CDCl3): d = 7.67 (d, J =
8.0 Hz, 2H, Ar-H), 7.54 (d, J = 6.8 Hz, 2H, Ar-H), 7.32 (d, J = 8.0 Hz,
2H, Ar-H), 7.24 (d, J = 6.8 Hz, 2H, Ar-H), 6.00 (s, 2H, pyrrole-H),
2.57 (s, 6H, CH3), 1.44 ppm (s, 6H, CH3); 13C NMR (100 MHz,
CDCl3): d = 155.8, 142.9, 140.6, 136.9, 135.4, 132.8, 132.3, 131.4, 131.2,
128.3, 125.9, 123.6, 121.9, 121.4, 90.5, 89.6, 14.6 ppm (superposition of
two signals); TOF MS calcd for C28H22N3BF2S: 481.1596; found:
481.1607.
3: A stirred solution of rhodamine B hydrazide[11] (45.6 mg,
0.10 mmol) and compound 2 (50 mg, 0.10 mmol) in DMF was heated
at 508C for 6 h. The resulting solution was cooled and poured into
water (5 mL). The resulting precipitate was filtered and purified on
flash silica gel using dichloromethane /acetone (200:1) as eluent.
Yield: 50 mg, 53%. M.p. 200–2028C. Rf = 0.2 (SiO2; dichloromethane/
1
acetone 200:1). H NMR (400 MHz, CDCl3): d = 8.02 (d, J = 7.2 Hz,
1H, Ar-H), 7.62 (m, 4H, Ar-H), 7.56 (s, 1H, NH), 7.36 (d, J = 7.6 Hz,
2H, Ar-H), 7.29 (m, 3H), 7.16 (d, J = 7.6 Hz, 2H, Ar-H), 6.95 (s, 1H,
NH), 6.48 (d, J = 7.0 Hz, 2H, xanthene-H), 6.45 (s, 2H, xanthene-H),
6.32 (d, J = 7.0 Hz, 2H, xanthene-H), 6.0 (s, 2H, pyrrole-H), 3.35 (q,
J = 5.6 Hz, 8H, CH2CH3), 2.56 (s, 6H, CH3), 1.43 (s, 6H, CH3),
1.16 ppm (t, J = 5.6 Hz, 12H, CH2CH3); 13C NMR (100 MHz, CDCl3):
d = 182.3, 167.2, 155.8, 154.4, 150.1, 149.5, 143.0, 140.9, 138.1, 134.9,
134.4, 132.3, 131.7, 131.3, 129.2, 129.1, 128.2, 127.6, 124.8, 124.2, 124.1,
123.9, 121.4, 120.0, 108.5, 104.3, 98.5, 90.5, 88.6, 67.4, 44.5, 14.6 ppm
(superposition of two signals), 12.6; UV/Vis (ethanol/water 8:2): lmax
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=
488 nm): lmax = 514 nm; TOF MS calcd for C56H55BF2N7O2S
[M+H]+: 938.4121; found: 938.4450.
4: A mixture of compound 3 (30 mg, 0.032 mmol) and HgCl2
(9 mg, 0.033 mmol) was stirred in acetonitrile for 15 min at room
temperature. The solvent was evaporated under reduced pressure and
the crude product was purified by column chromatography on silica
gel using dichloromethane/acetone (100:1). Yield: 28 mg, 97%. M.p.
289–2918C. Rf = 0.1 (SiO2; dichloromethane/acetone 100:1). 1H NMR
(400 MHz, CDCl3): d = 11.73 (s, 1H, NH), 8.39 (d, J = 6.2 Hz, 1H, Ar-
H), 7.87 (d, J = 6.8 Hz, 2H, Ar-H), 7.72 (t, J = 6.2 Hz, 1H, Ar-H), 7.62
(m, 3H), 7.39 (d, J = 6.8 Hz, 2H, Ar-H), 7.27(m, 1H, Ar-H), 7.23 (d,
J = 6.6 Hz, 2H, Ar-H), 7.17(d, J=7.6 Hz, 2H, Ar-H), 6.81 (d, J =
7.6 Hz, 2H, Ar-H), 6.76 (s, 2H, Ar-H), 6.0 (s, 2H, pyrrole-H), 3.58
8028 www.angewandte.org
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