A highly selective colorimetric and ratiometric fluorescent chemodosimeter for imaging fluoride ions in living cells

Article abstract of DOI:10.1039/c1cc11308a

A simple but highly selective colorimetric and ratiometric fluorescent chemodosimeter was designed and synthesized to detect fluoride ions (F ^-) in aqueous solution and living cells by virtue of the strong affinity of F^- toward silic

Full text of DOI:10.1039/c1cc11308a

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COMMUNICATION
A highly selective colorimetric and ratiometric fluorescent
chemodosimeter for imaging fluoride ions in living cellsw
Baocun Zhu,a Fang Yuan,a Rongxia Li,a Yamin Li,b Qin Wei,c Zhenmin Ma,*a Bin Du*a
and Xiaoling Zhang*b
Received 5th March 2011, Accepted 12th May 2011
DOI: 10.1039/c1cc11308a
A simple but highly selective colorimetric and ratiometric
fluorescent chemodosimeter was designed and synthesized to
detect fluoride ions (FÀ) in aqueous solution and living cells by
virtue of the strong affinity of FÀ toward silicon.
coumarin needs to be excited by UV light, which poses potential
drawbacks to the bioimaging. These above-mentioned difficulties
should be overcome when designing FÀ indicators for chemical
and biological applications.
During the preparation of our manuscript, two studies on
colorimetric and ratiometric fluorescent indicators have been
reported.7 In both papers, the recognition properties of
indicators for FÀ were investigated in detail in organic
solvents. Although two indicators can work in the mixture
of water and organic solvents, twenty percent water content
greatly demolished their excellent performances owing to the
strong solvation of FÀ. So, they have potential difficulties in
the determination of FÀ in living matrices.
Fluoride as a useful additive is frequently found in toothpaste,
pharmaceutical agents, and even drinking water owing to
its important roles in preventing dental caries and in the
treatment of osteoporosis.1 However, excessive ingestion of
fluoride has been proven to cause some diseases and cancers.2
For example, sodium fluoride (NaF) can affect a number of
essential cell signaling components and disturb normal cellular
metabolism at a higher concentration.3 Thus, the quantitative
detection of fluoride is of growing importance in chemical and
biological systems.
Herein, we address these challenges by designing an amphi-
pathic fluorescent chemodosimeter 1 (Scheme 1, 1) employing
an internal charge transfer (ICT) mechanism for the ratio-
metric determination of FÀ in buffered aqueous solution and
living systems. Previous studies have demonstrated that the
subtle equilibrium between hydrophilicity and lipophilicity
of indicators is favourable for both cell permeability and
intracellular fluorescence imaging.8 So, in the design of a
chemodosimeter, we chose the hydrophilic benzothiazolium
hemicyanine dye as the fluorophore because of its desirable
spectral performances, excellent ICT structure and non-
toxicity,9 and a lipophilic tert-butyldiphenylsilyl (TBDPS)
moiety as the specific reaction site for FÀ.2c,5a,10 We expected
that the reaction of chemodosimeter 1 with FÀ would trigger
the cleavage of the Si–O bond and release the longer wave-
length fluorescence of compound 2. The reaction mechanism
of chemodosimeter 1 and FÀ is shown in Scheme 1.
Currently, considerable attention has been paid to fluores-
cent indicators for the determination of fluoride ions (FÀ) due
to their apparent advantages over other methods, such as high
sensitivity, operational simplicity, and bioimaging analysis
in vivo, even in single living cells.1b,4 Despite advances in the
development of new reagents, there are still some limitations
in the quantitative detection and bioimaging.5 First, most
fluoroionophores can only be operated in organic solvents to
detect tetrabutylammonium fluoride rather than inorganic
fluoride salts. Second, most of the current fluorescent indicators
respond to FÀ with the changes only in fluorescence intensity,
which are prone to be disturbed in the quantitative detection by
many factors, such as instrumental efficiency, environmental
conditions, and probe distribution.6 Third, as far as we know,
only a fluorescent reagent for the imaging of FÀ in living
matrices has been reported for such reagents must meet various
requirements including water solubility, cell permeability, non-
toxicity, etc.2c But this fluorescent reagent derived from
a School of Resources and Environment, University of Jinan, Jinan
250022, China. E-mail: mazhmujn@163.com, dubin61@gmail.com;
Fax: +86 531-82765969; Tel: +86 531-82769235
b Key Laboratory of Cluster Science of Ministry of Education,
Department of Chemistry, School of Science, Beijing Institute of
Technology, Beijing 100081, China. E-mail: zhangxl@bit.edu.cn
c School of Chemistry and Chemical Engineering, University of Jinan,
Jinan 250022, China
w Electronic supplementary information (ESI) available: Synthesis
procedures, additional spectra and experimental details. See DOI:
10.1039/c1cc11308a
Scheme 1 Proposed reaction mechanism of 1 and FÀ.
c
7098 Chem. Commun., 2011, 47, 7098–7100
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Chemodosimeter 1 was easily synthesized in a satisfactory
yield from N-ethyl-2-methyl-benzothiazolium iodide and
4-(tert-butyldiphenylsilyloxy)benzaldehyde, which was prepared
from tert-butylchlorodiphenylsilane and 4-hydroxybenzaldehyde.
Detailed procedures and characterizations are described in ESI.w
In this communication, the determination of FÀ with chemo-
dosimeter 1 was investigated in a mixture of ethanol and water
(3 : 7, v/v) solution containing phosphate buffered saline (PBS)
(20 mM, pH 7.4). Chemodosimeter 1 displays one major
absorption band centered at 407 nm (Fig. S1, ESIw). When
FÀ was gradually added to the solution of 1, the maximum
absorption peak showed a 110 nm red shift with an isosbestic
point at 442 nm and the colour of the solution turned from
slight yellow to orange (Fig. S1 and S2, ESIw). Therefore,
chemodosimeter 1 can serve as a ‘‘naked-eye’’ indicator for
FÀ. In the fluorescence emission spectrum (Fig. 1), chemo-
dosimeter 1 exhibits the maximum emission peak at 500 nm in
the absence of FÀ. Upon addition of different concentrations
of FÀ, the maximum emission peak undergoes a red shift to
558 nm with an isoemission point at 539 nm. Additionally,
there was a good linearity between the fluorescence intensity
ratio, R (F500/F558) and the concentrations of FÀ in the range
of 0.5 to 28 mM with a detection limit of 0.08 mM,11 which
allowed the determination of FÀ by a ratiometric fluorescence
method. Obviously, chemodosimeter 1 is a very promising
indicator for the detection of FÀ in environmental and biological
science because fluoride becomes toxic at a higher concentration
(0.1 mM for drinking water standard and more than 3 mM for
biological systems).2b,5a
We then evaluated the selectivity of chemodosimeter 1
toward FÀ under the same conditions. As expected, nearly
no fluorescence intensity changes were observed in the
presence of CO32À, SO42À, SCNÀ, NO3À, N3À, ClÀ, BrÀ, IÀ,
cysteine (Cys), glutathione (GSH), bovine serum albumin
(BSA), and human serum albumin (HSA) (Fig. 2). Also, the
effects of interference of the above-mentioned analytes on
monitoring FÀ were investigated (Fig. 2). These results
demonstrated that chemodosimeter 1 possesses high selectivity
toward FÀ when present with other anions and biorelevant
analytes.
To confirm the mechanism of chemodosimeter 1 in sensing
FÀ, the reaction of 1 and FÀ was conducted under the same
conditions as described above. The reaction products were
subjected to electrospray ionization mass spectral analysis,
and the peak at m/z 282.09453 [M–I]+ corresponding to
compound 2 was clearly observed (Fig. S7, ESIw). Further-
more, when the pH of the reaction mixture was adjusted to 4
using increasing concentrations of hydrochloric acid, the
colour of solution gradually faded and the maximum absorp-
tion and fluorescence emission peaks were blue-shifted to
412 nm and 512 nm, respectively (Fig. S8, ESIw). The result
showed that compound 2 existed mainly in the deprotonated
form in the above analytical system. Therefore, all these data
indicated that the reaction most likely undergoes the proposed
mechanism as shown in Scheme 1.
Next, to further demonstrate the ability of chemodosimeter
1 to image FÀ in living matrices, we carried out experiments in
live RAW 264.7 macrophage cells. The cells incubated with
chemodosimeter 1 (5 mM) for 5 min showed a clear intra-
cellular fluorescence (Fig. 3b and c). The result revealed that
chemodosimeter 1 can penetrate the cell membrane. And then,
Fig. 1 Fluorescence responses of 1 (5 mM) toward different concen-
trations of FÀ in PBS (20 mM, pH 7.4) solution (ethanol/water = 3 : 7,
v/v). (a) Fluorescence spectra of 1 in the presence of increasing
concentrations of FÀ (final concentration: 0, 0.5, 2, 4, 6, 10, 13, 16,
20, 24, 28, 36, 40, 50, 60 mM); (b) fluorescence intensity ratio
(F500/F558) of 1 versus increasing concentrations of FÀ (final concentration:
0.5, 2, 4, 6, 10, 13, 16, 20, 24, 28 mM). Each spectrum was obtained after
FÀ addition at 25 1C for 50 min.
Fig. 2 Fluorescence responses of 1 (5 mM) toward various analytes in
PBS (20 mM, pH 7.4) solution (ethanol/water = 3 : 7, v/v). Light gray
bars represent the addition of a single analyte including 60 mM anions
(CO32À, SO42À, SCNÀ, NO3À, N3À, ClÀ, BrÀ, IÀ, FÀ), 300 mM Cys,
3 mM GSH, 1 mg mLÀ1 BSA, and 1 mg mLÀ1 HSA. Black bars
represent the subsequent addition of FÀ to the mixture. All data
represent the fluorescence intensity ratio F558/F500. Each spectrum was
obtained after various analytes addition at 25 1C for 50 min.
c
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Technology (2008ZX07422), and National Science and Tech-
nology Major Project (2009ZX07212-003) for financial sup-
port and Hongying Jia in Institute of Chemistry CAS for help
in the bioimaging experiments.
Notes and references
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Fig. 3 Confocal fluorescence images of live RAW 264.7 macrophage
cells: the cells were incubated with chemodosimeter 1 (5 mM) for 5 min;
(a) bright-field transmission image, (b) blue channel at 490 Æ 20 nm,
(c) orange channel at 560 Æ 20 nm, and (d) ratio image generated from
(c) and (b). The above cells after addition of 3 mM sodium fluoride for
another 20 min; (e) bright-field transmission image, (f) blue channel at
490 Æ 20 nm, (g) orange channel at 560 Æ 20 nm, and (h) ratio image
generated from (g) and (f). Incubation was performed at 37 1C under a
humidified atmosphere containing 5% CO2. Scale bar = 20 mm.
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another 20 min (Fig. 3f and g). As expected, distinct changes
of ratio fluorescence responses in living cells were observed
(Fig. 3d and h). The bright-field images (Fig. 3a and e)
confirmed that the cells are viable throughout the bioimaging
experiments. These results demonstrated that chemodosimeter
1 can be used for the ratiometric fluorescence imaging of FÀ in
living cells. Moreover, to evaluate cytotoxicity of chemodosi-
meter 1, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assays in HeLa cells with 5 and
10 mM chemodosimeter 1 for 1 h, respectively. The result
clearly showed that chemodosimeter 1 was of low toxicity to
cultured cells under the experimental conditions at the con-
centration of 5 mM for 25 min (Fig. S5, ESIw).
In conclusion, we have presented the synthesis and proper-
ties of a new ICT-based fluorescent chemodosimeter 1 for FÀ
with benzothiazolium hemicyanine dye as the fluorophore.
The chemodosimeter exhibits high FÀ-selectivity over various
anions and biorelevant analytes, which is ascribed to the
strong affinity of FÀ toward silicon. Additionally, the chemo-
dosimeter can not only serve as a ‘‘naked-eye’’ probe for FÀ,
but also detect FÀ quantitatively by a ratiometric fluorescence
method in buffered aqueous solution. Importantly, the amphi-
pathic chemodosimeter was successfully applied to the ratio-
metric fluorescence imaging of FÀ in living cells. We highlight
the simplicity of the design and synthesis, yet its combined
properties, such as high specificity, longer excitation and
emission wavelength, visual and ratiometric fluorescence de-
termination with suitable sensitivity, desirable response time
and bioimaging in living cells, and anticipate that this chemo-
dosimeter would be of great benefit to biomedical researchers
for investigating the effects of sodium fluoride in biological
systems.
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We thank the National Natural Science Foundation of
China (No. 21075052, 20975012, and 40672158), National
Major Projects on Water Pollution Control and Management
11 See ESIw for a detailed calculation method.
c
7100 Chem. Commun., 2011, 47, 7098–7100
This journal is The Royal Society of Chemistry 2011