A PEGylated Fluorescent Turn-On Sensor
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sensor is not reusable. This sensor might have the potential
for application in the detection for fluoride anion in water
quality monitoring as well as in diagnosis of fluoride poison-
ing. This approach offers some useful insights for realizing
technically simple fluorescent turn-on sensing in the detec-
tion assays for other analytes.
Experimental Section
Materials and reagents: Methoxypolyethylene glycol amine (average mo-
lecular weight 2000, PEG2000), fluorescein isothiocyanate, imidazole,
tert-butyldiphenylchlorosilane, sodium salts of anion (FÀ ClÀ, BrÀ, IÀ,
CH3COOÀ, CO32À, HCO3À, PO43À, HPO42À, H2PO4À, NO2À, NO3À, S2À
,
SO42À), 4-(À2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
and n-cetyltrimethylammonium bromide (CTAB) were purchased from
Aldrich. Human urine was from a healthy male. Fetal bovine serum was
supplied by Hangzhou Sijiqing Biological Engineering Materials Co. Ltd.
The purified water used in this study was triple-distilled, which was then
further treated by ion exchange columns and then by a Milli-Q water pu-
rification system. N,N-dimethylformamide (DMF) was dried with CaH2
and vacuum distilled. Methanol and dichloromethane were analytically
pure solvents and distilled before use.
Figure 7. Fluorescence imaging of Hela and L929 cells incubated with the
sensor before (A and C) and after (B and D) being treated with NaF (1ꢁ
10À4 m).
Synthesis of the PEG-FITC: Methoxypolyethylene glycol amine (average
Mw =2000, 0.4 g, 0.2 mmol) and fluorescein isothiocyanate (79 mg,
0.2 mmol) were dissolved in DMF (5 mL) in a flask. The reaction mixture
was stirred under N2 at room temperature overnight; afterwards, the sol-
vent was removed under reduced pressure. The residue was dissolved in
CH2Cl2 (3 mL), and then diethyl ether (30 mL) was added to precipitate
the product. The mixture solution was centrifuged (10000 rev sÀ1, 8 min).
And the product was obtained as a red solid (442 mg, 92%).
Fluorescence microscope images of both Hela and L929
live cells loaded with the sensor for 2 h at 378C show no flu-
orescence intracellularly, as shown in Figure 7A and C. The
control experiment on cells without the sensor gives no fluo-
rescence either under the same exposure condition. In con-
trast, the intracellular fluorescence for the sensor-stained
cells exposed to 1ꢁ10À4 m of fluoride for 15 min at 378C
gives rise to green fluorescence, as shown in Figure 7B and
D. This result indicates that the sensor can be utilized to
track fluoride level changes inside living cells.
Synthesis of the sensor (PEG-FITC-Si): PEG-FITC (430 mg, 0.18 mmol),
imidazole (34 mg, 0.5 mmol) were dissolved in CH2Cl2 (5 mL) in a flask,
the mixture was stirred and cooled in an ice-bath under N2. Then, tert-bu-
tyldiphenylchlorosilane (138 mg, 0.5 mmol) dissolved in CH2Cl2 (5 mL)
was added into the above solution dropwise. After 1 h, the ice bath was
removed, and the solution was stirred at room temperature for another
18 h. After that, the solution was filtered and CH2Cl2 (30 mL) was added
into the filtrate, which was then washed with deionized water. The com-
bined organic phase was dried in anhydrous MgSO4. Then the solvent
was evaporated under vacuum and the residue was purified by silica-gel
column chromatography by using methanol as an eluent. The product
was obtained as a yellow solid (252 mg, 48.8%).
Conclusion
Cytotoxicity: The cell line, L929 (murine aneuploid fibro-sarcoma cell)
was incubated in RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS) at 378C with 5% CO2. The cytotoxicity of the
sensor against L929 cells was assessed by MTT assay according to ISO
10993-5.
We have successfully constructed a robust fluorescent turn-
on sensing system for use in totally aqueous media, which
features a simple synthesis, and the responsive, sensitive,
and selective detection of fluoride anions. The PEGylation
renders the sensor with good water solubility, cell membrane
permeability, as well as very low cytotoxicity. The incorpora-
tion of tert-butyldiphenylsilyl groups onto fluorescein can ef-
fectively quench the fluorescence of the sensor, whereas
upon addition of fluoride anions, the fluoride-mediated
cleavage of tert-butyldiphenylsilyl groups proceeds selective-
ly and quickly, resulting in the recovery of strong fluores-
cence of fluorescein moiety. This sensor exhibits the lowest
limit of detection among those reported fluorescent fluoride
sensors. Furthermore, it can be used for detecting fluoride
levels in some real samples such as running water, human
urine, and serum; it also can be used for imaging fluoride
anions inside living cells. Since the sensor is based on an ir-
reversible reaction, this sensor exhibits a low detection
limit; however, the disadvantage of this strategy is that the
Cell incubation and imaging: Two cell lines, Hela (human cervical cancer
cell) and L929 (murine aneuploid fibrosarcoma cell), were incubated in
RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS,
Invitrogen). One day before imaging, cells were passed and plated on
30 mm glass culture dishes. Before the experiments, cells were washed
with RPMI 1640, incubated in RPMI 1640 medium containing the sensor
by use of the sensor stock solution (final sensor concentration
0.2 mgmLÀ1) at 378C under 5% CO2 for 2 hours, washed with PBS, and
then treated with a drop of 0.4% trypan blue solution to quench the fluo-
rescence of the sensor adsorbed on the outer cell membrane, so that the
observed fluorescence emission comes from the intracellular sensor.
After that, the cells were washed three times with PBS and then imaged
on an Olympus IX71 inverted fluorescence microscope equipped with a
DP72 color CCD (blue light excitation).
Experiments to assess fluoride uptake were performed as follows: the
cells stained with the sensor were incubated in the HEPES balanced
saline with of sodium fluoride (1ꢁ10À4 m) for 15 min, and washed with
HEPES balanced saline for three times prior to cell imaging.
Chem. Eur. J. 2013, 19, 936 – 942
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
941