A NIR fluorescent probe for the detection of fluoride ions and its application in in vivo bioimaging

Article abstract of DOI:10.1039/C6TB03193E

A novel near-infrared (NIR) fluorescent probe for the detection of fluoride ions has been developed, which is based on the F-triggered cleavage reaction of the Si-O bond. This probe exhibits excellent selectivity and sensitivity towards fluoride ions. The results of bioimaging experiments with HepG2 cells and mice show that the fluoride probe is available for visualizing exogenous fluoride ions in vitro and in vivo.

Full text of DOI:10.1039/C6TB03193E

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A NIR fluorescent probe for the detection of fluoride ions and its
application for in vivo bioimaging
Qiuyun Yang,a Chunman Jia,*ab Qing Chen,a Wei Du,a Yile Wang,a Qi Zhang*ab
Received 00th January 20xx,
Accepted 00th January 20xx
A novel near-infrared (NIR) fluorescence probe for the detection of fluoride ions has been developed, which is based on
the F-triggered cleavage reaction of the Si-O bond. This probe exhibits excellent selectivity and sensitivity towards fluoride
ions. The results of bioimaging experiments with HepG2 cells and mice show that the fluoride probe is available for
visualizing exogenous fluoride ions in vitro and in vivo.
DOI: 10.1039/x0xx00000x
www.rsc.org/
probes, those that rely on NIR fluorescence possess unique
advantages of
a
weaker fluorescence background, lower
Introduction
photodamage, intense photon penetration through deep tissues
and light-scattering properties. For these reasons, recent research
has been devoted to the development of NIR fluorescent probes
that capable of detection and sensing of fluoride ions.30 In these
protocols, the classic Si–O or B-Se bond cleavage and H-F bond
interaction were highly effective in response to fluoride ions,
allowing the versatility of fluorescent probes with diverse NIR
signaling units (e.g. DCP,31 BODIPY,32-33 phenanthroimidazole–
cyanine,34 and TDP35). However, all such NIR probes have not been
employed to successfully image of fluoride ions in vivo.
To address this issue, we proposed a hemicyanine skeleton 1 as
the fluorescent report motif, which possesses NIR spectroscopic
features, good photostability, and excellent water solubility. By
incorporating the trigger moiety 4-(bromomethyl)phenoxy)(tert-
butyl)diphenylsilane into this stable hemicyanine skeleton, we
developed a turn-on NIR fluorescence probe 3 depending on the
The fluoride ion, the smallest and most electronegative anion, is
considered to be a micronutrient for human health that prevents
dental cavities and promotes healthy bone growth.1-3 Neverthless,
the toxicity of fluoride ions has also been paid much attention, since
a high intake of fluoride ions could exert a great influence on the
progression of distinct pathophysiological states and even lead to a
diseased state.4-5 To achieve the complete elucidation of fluoride
ion toxicity, the sensitive, specific, and accurate detection of its
distribution and dynamic fluctuation in biological systems is highly
desirable. Among the various analytical methods that are available,
fluorescence sensing is regarded as an ideal technology due to its
advantages of operational simplicity and high specificity and
sensitivity. Despite remarkable advances in the development of
numerous fluorescent probes of fluoride ions,6-15 there are still
some drawbacks in practical applications. For example, some
frequently used absorption and emission wavelengths are within
the ultraviolet-visible (UV-Vis) light range, which introduces
practical problems such as limited tissue penetration, high
rapid cleavage reaction of
a Si-O bond (Scheme 1). Further
experiments disclosed that it is suitable for the real-time detection
in vivo bioimaging of exogenous fluoride ions.
photodamage, and
a low signal-to-noise ratio due to auto-
fluorescence interference. Additionally, most reported fluorescence
fluoride probes can only be applied in pure organic solvents to
detect organic tetrabutylammonium fluoride rather than inorganic
fluoride salts. Hence, the design of a practical fluorescence probe
that responds to fluoride ions, which can be detectable in living
cells and in vivo, is still a challenge that is essential to resolve.
Over the past decade, near-infrared (NIR) fluorescent probes have
become promising modalities for the bioimaging of various
biologically important species, including reactive oxygen species
(ROS)16-18 and reactive nitrogen species (RNS),19-20 amino acids,21
metal ions,22-24 anions,25-26 enzymes27 and intracellular pH
changes.28-29 Compared to most other conventional fluorescent
OH
O
R
R
TBDPSO
TBDPSO
O
Br
O
F-
2
N
1
I
N
I
K2CO3, ACN
O
1a: R = H
1b: R = Cl
1c: R = F
3a: R = H
3b: R = Cl
3c: R = F
Scheme 1 The synthesis method of probe 3.
Experimental section
Materials and Instruments
All experimental reagents and materials were purchased from
commercial suppliers, and dehydration acetonitrile was dried over
CaH2. Deionized water was used throughout the experiment
process. UV-Vis spectrophotometer (UV-1800, Shimadzu) with 1.0
cm quartz cell was used to record the absorbance spectra. NMR
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spectra were recorded on a Bruker AVANCEII 400 MHz NMR (s, 2H), 1.73 (s, 6H). 13C NMR (100 MHz, CD3OD) δ 174.81, 173.95,
spectrometer using TMS as the internal standard and CDCl3 or 163.71, 158.50, 144.44, 142.02, 141.40, 140.37, 130.94, 129.73,
CD3OD as the solvent. Chemical shifts were reported in ppm from 125.86, 123.29, 123.02, 121.95, 116.17, 115.80, 111.48, 103.55,
the solvent resonance as the internal standard (CDCl3: δH = 7.26 99.90, 50.15, 31.45, 29.45, 28.71, 25.24, 22.02. HRMS (ESI, m/z)
ppm, δC = 77.16 ppm; CD3OD: δH = 3.31 ppm, δC = 49.00 ppm). Thin Calcd for [C26H25IClNO2−I−]: 418.1574, found: 418.1573.
layer chromatography (TLC) analysis was performed on silicagel
1c: (yield 49.9%) 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 13.4 Hz,
plates (GF254, 0.20-0.25 mm). Column chromatography was
1H), 7.35 (s, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.26 (d, J = 0.9 Hz, 1H),
conducted over a silica gel (200-300, mesh size) purchased from
7.06 (t, J = 7.4 Hz, 1H), 6.96 (d, J = 10.2 Hz, 1H), 6.85 (d, J = 7.8 Hz,
Qingdao Ocean Chemicals or neutral aluminium oxide (200-300,
1H), 6.77 (d, J = 6.9 Hz, 1H), 5.60 (d, J = 13.4 Hz, 1H), 3.37 (s, 3H),
mesh size) obtained from Shanghai Macklin Biochemical Co., Ltd.
2.70 (t, J = 6.0 Hz, 2H), 2.63 (t, J = 6.5 Hz, 2H), 1.95 – 1.87 (m, 2H),
The fluorescence spectra were recorded on
a
F97pro
1.67 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 164.88, 148.39, 142.17 (d,
fluorospectrophotometer, both of the excitation and emission slit
widths were setting at 10.0 nm. High resolution mass spectrum
(HRMS) was determined on an LCMS-IT TOF (LC30A, Shimadzu).
HPLC experiments were carried out using a C18 column (Shoaex, 4.6
mmID×250 mmL) with a LC1620A HPLC system. The mobile phase
methanol was HPLC grade. Fluorescence imaging in HepG2 cells
used a laser confocal microscope (Leica TCS SP8 CARS) and in vivo
obtained by small animal living imaging system IVIS Lumina Series III
(Perkin Elmer).
1JF –C = 245.8 Hz), 141.80, 139.75, 130.59, 127.56, 127.15, 121.43,
2
118.04, 113.37, 113.16, 109.19, 108.46, 103.89 (d, JF –C = 17.1 Hz),
101.78, 99.58, 91.05, 53.42, 39.60, 39.29, 38.39, 37.88, 35.12, 32.53.
19F NMR (376 MHz, CDCl3) δ -134.89. HRMS (ESI, m/z) Calcd for
[C26H25IFNO2−I−]: 402.1869, found: 402.1861.
Synthesis of compounds 3a-c
Compound 2 were synthesized according to the reported method.37
To a stirred solution of compound 1 (0.3 mM) in acetonitrile were
added K2CO3 (62.2 mg, 0.45 mM) and compound 2 (127.6 mg, 0.30
mM). The reaction mixture was stirred at 50°C for 5 h under
nitrogen atmosphere. The solvent was evaporated under reduced
pressure, and the crude products 3 were purified by neutral
aluminium oxide column chromatography using CH2Cl2/0-20%
methanol as eluent.
General procedures of spectra detection
The stock solutions of 3a-c (0.2 mM) were prepared in dimethyl
sulfoxide (DMSO). Analytes (10 mM) were prepared in deionized
water. (analytes: CH3COONa∙3H2O, NaHCO3, NaNO3, NaF, Na2CO3,
(CH3CH2CH2CH2)4N+F-, (CH3CH2CH2CH2)4N+Cl-, (CH3CH2CH2CH2)4N+Br-,
(CH3CH2CH2CH2)4N+I-, NaHS, NaSCN, Na2SO4, NaH2PO4∙2H2O,
Na2S2O3∙5H2O, FeCl2, KCl, MgSO4, MnCl2, Ni(NO3)2, Zn(NO3)2, AlCl3,
NaCl, CuCl2, Ca(NO3)2). 3a-c (10 µM, 200 µL) was placed in a 5 mL
centrifugal tube, and then being diluted to 4 mL with DMSO (2.6
mL) and PBS (0.01 M, pH=7.4) (1.2 mL). Finally, the analytes were
added according to the demands in the experiment process, and
fluorescence emission spetra and UV-Vis absorbance spectra were
recorded.
3a: (yield 55.0%) 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 14.8 Hz,
1H), 7.69 (s, 2H), 7.67 (s, 2H), 7.48 (d, J = 3.9 Hz, 2H), 7.45 (d, J = 7.4
Hz, 1H), 7.42 – 7.39 (m, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 7.35 (s, 2H),
7.33 (s, 2H), 7.31 (s, 1H), 7.24 (s, 1H), 7.22 (s, 1H), 7.09 (dd, J = 11.5,
5.4 Hz, 3H), 6.79 (s, 1H), 6.77 (s, 1H), 6.64 (d, J = 14.9 Hz, 1H), 5.16
(s, 2H), 4.14 (s, 3H), 2.79 (s, 2H), 2.70 (s, 2H), 1.91 (s, 2H), 1.72 (s,
6H), 1.07 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 177.58, 162.16,
161.59, 155.94, 154.34, 145.76, 142.28, 141.48, 135.51, 133.74,
132.71, 130.02, 129.30, 128.92, 128.05, 127.84, 127.50, 127.28,
122.25, 120.00, 115.99, 115.28, 113.41, 112.90, 104.56, 102.01,
70.79, 50.47, 33.71, 29.71, 29.26, 28.17, 26.51, 24.38, 20.39, 19.47.
HRMS (ESI, m/z) Calcd for [C49H50INO3Si−I−]: 728.3554, found:
728.3499.
Synthesis and characterization of 1 and 3
Synthesis of compounds 1a-c
Compounds 1a-c were synthesized according to previous report.36
A
mixture of compound 6 (2.0 mmol) and K2CO3 (276 mg, 2.0 mmol) in
acetonitrile (5 mL) was stirred for 20 min at room temperature
under nitrogen atmosphere. Then a solution of compound 5 (610
mg, 1.0 mmol) in acetonitrile (5 mL) was added to the above
mixture via a syringe, and the reaction mixture was heated at 50 °C
for 4 h. Finally the solvent was evaporated under reduced pressure,
and the crude product was purified by column chromatography
(CH2Cl2/0-30% methanol) on silica gel, affording the desired
compound 1a-c as a blue-green solid.
3b: (yield 49.9%) 1H NMR (400 MHz, CDCl3) δ8.54 (d, J = 15.0 Hz,
1H), 7.66 (s, 2H), 7.64 (s, 2H), 7.46 (t, J = 8.7 Hz, 3H), 7.37 (s, 1H),
7.35 (s, 2H), 7.33 (s, 1H), 7.31 (s, 1H), 7.29 (s, 2H), 7.27 (s, 1H), 7.24
(s, 1H), 7.22 (s, 1H), 7.08 (s, 1H), 7.00 (s, 1H), 6.77 (s, 1H), 6.75 (s,
1H), 6.54 (d, J = 15.0 Hz, 1H), 5.17 (s, 2H), 4.06 (s, 3H), 2.71 (s, 2H),
2.66 (s, 2H), 1.85 (s, 2H), 1.71 (s, 6H), 1.05 (s, 9H). 13C NMR (100
MHz, CDCl3) δ 178.01, 160.56, 156.80, 155.67, 152.59, 145.74,
142.09, 141.50, 135.38, 132.62, 131.64, 129.89, 129.20, 128.74,
128.45, 127.84, 127.71, 127.52, 122.38, 120.65, 119.85, 115.81,
115.16, 112.96, 105.03, 101.60, 71.59, 50.68, 33.98, 29.19, 27.87,
26.42, 24.30, 20.15, 19.36. HRMS (ESI, m/z) Calcd for
[C49H49IClNO3Si−I−]: 762.3170, found: 762.3170.
1
1a: (yield 57.4%) H NMR (400 MHz, CD3OD) δ 8.39 (d, J = 13.7 Hz,
1H), 7.48 (s, 2H), 7.42 – 7.35 (m, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.22
(d, J = 7.4 Hz, 2H), 6.70 (d, J = 7.4 Hz, 1H), 6.53 (s, 1H), 6.01 (d, J =
13.8 Hz, 1H), 3.57 (s, 3H), 2.69 (s, 2H), 2.62 (s, 2H), 1.87 (s, 2H), 1.72
(s, 6H). 13C NMR (100 MHz, CD3OD) δ 176.04, 173.33, 163.66,
158.71, 144.48, 141.86, 140.81, 140.76, 130.99, 129.68, 125.61,
123.24, 122.62, 122.46, 116.21, 115.68, 111.26, 103.61, 99.43,
49.96,31.33, 29.36, 28.78, 25.22, 22.02. HRMS (ESI, m/z) Calcd for
[C26H26INO2−I−]: 384.1958, found: 384.1928.
3c: (yield 53.3%) 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 14.9 Hz,
1H), 7.69 (d, J = 1.3 Hz, 2H), 7.67 (d, J = 1.5 Hz, 2H), 7.45 (d, J = 5.4
Hz, 2H), 7.43 (s, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 7.35 (d, J = 2.0 Hz, 2H),
7.33 (d, J = 1.2 Hz, 2H), 7.31 (d, J = 1.4 Hz, 1H), 7.23 (s, 1H), 7.18 (s,
1H), 7.16 (s, 1H), 6.87 (dd, J = 8.1, 5.3 Hz, 2H), 6.78 (s, 1H), 6.76 (s,
1H), 6.55 (d, J = 14.9 Hz, 1H), 5.01 (s, 2H), 4.04 (s, 3H), 2.75 (s, 2H),
2.70 (s, 2H), 1.89 (s, 2H), 1.72 (s, 6H), 1.07 (s, 9H). 13C NMR (100
1
1b: (yield 48.5%) H NMR (400 MHz, CD3OD) δ 8.42 (d, J = 14.1 Hz,
1H), 7.48 (d, J = 6.7 Hz, 2H), 7.39 (t, J = 7.7 Hz, 1H), 7.33 (d, J = 8.8
Hz, 1H), 7.28 – 7.20 (m, 2H), 6.72 (d, J = 8.7 Hz, 1H), 6.56 (s, 1H),
6.05 (d, J = 14.1 Hz, 1H), 3.59 (s, 3H), 2.70 (s, 2H), 2.63 (s, 2H), 1.88
1
MHz, CDCl3) δ 177.93,161.02, 156.03, 150.20 (d, JF –C = 245.9 Hz),
150.41, 150.28, 150.12, 148.97, 145.81, 142.38, 141.56, 135.54,
2 | J. Name., 2012, 00, 1-3
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2
132.75, 132.30, 130.05, 129.37, 128.85, 127.87, 127.66 (d, JF –C
=
mL) solution for 10 min, then washed with PBS three times. The
3
36.1 Hz), 122.38, 120.04, 115.33, 115.13 (d, JF –C = 8.1 Hz), 113.08, cells were stained with Hoechst for 10 min, and then washed with
105.08, 103.11, 72.08, 50.69, 34.23, 29.76, 29.39, 28.06, 26.55, PBS three times. Finally the cells were fixed by paraformaldehyde
24.50, 20.32, 19.51. 19F NMR (376 MHz, CDCl3): δ-134.93. HRMS (4%) 10 min at room temperature. Fluorescence imaging were then
(ESI, m/z) Calcd for [C49H49IFNO3Si−I−]: 746.3466, found: 746.3461.
carried out after washing the cells with PBS which was conducted
using a laser confocal microscope by exciting at 690 nm and
emission at about 750 nm.
Detection limit
The detection limit was calculated on the basis of the fluorescence
Living animal imaging
titration.38 The fluorescence emission spectrum of 3a-c (10 µM)
were measured 10 times in a PBS/DMSO= 3 : 7 (v/v, 0.01 M, pH= All animal operations were in accordance with institutional animal
7.4) solution, and the standard deviation of blank measurement use and care regulations approved by the Jiangsu University
was achieved. To gain the slope, the ratio of the emission intensity laboratory animal centre. The study was performed with the
at 730-740 nm was plotted against the concentration of NaF. The Guidelines for the care and use of research animals. First, 3c (20
detection limit was calculated using the following equation:
µM, 192 µL) was injected into nude mouse bodies through the tail
vein as a control experiment. Second, 3c (20 µM, 192 µL) was
injected into nude mouse bodies through the tail vein with a certain
concentration of sodium fluoride solution by intraperitoneal
injection.The amounts of sodium fluoride used were 28 mgF/kg, 12
mgF/kg, and 2 mgF/kg. The fluorescence distributions in the mice
were recorded using a small animal living imaging system after gas
anaesthesia at 15 min, 2 h, and 4 h.
Detection limit = 3 σ/k,
Where σ is the standard deviation of blank measurement, and k is
the slope between the ratio of fluorescence emission intensity
versus NaF.
Photostability
The photostability of probe 3a-c were investigated in DMSO/PBS =
7 : 3 (v/v, 0.01 M, pH = 7.4) solution, which placed in closed box
with an UV lamp (254 nm) 25 cm away from the testing solutions.39
Recorded their fluorescence intensity and UV absorption at
different time.
Results and Discussion
Our probe design focused on 7-substituted 1 that differ in their pKa
of the phenol group but have similar steric demands during binding.
As shown in Scheme 1, hemicyanines 1a were obtained according
to the method reported by Lin et al.40 Compounds 1b and 1c
(Scheme 1), the chloro- and fluoro-substituted 1a, respectively,
were synthesized as controls to investigate the role of the halogen
group in the modulation of the photophysical profiles. As the
HPLC experiments
HPLC experiments were carried out using a C18 column (Shoaex, 4.6
mmID × 250 mmL) with a LC1620A HPLC system. The mobile phase
was a methanol. The flow rate was 0.8 mL/min and detection at 600
nm. The detection solutions of 1a-c (0.2 mM), 3a-c (0.2 mM) and
NaF (0.0, 0.5, 1.0, 1.5 equiv., respectively) in a PBS/DMSO = 3:7 (v/v,
0.01 M, pH = 7.4) solution at room temperature.
response site for the fluoride ion,
a silyl group of tert-
butyldiphenylsilyl (TBDPS) was attached to 1a-c to prepare probes
3a-c. We first investigated the absorption and emission profiles of
compounds 1a−c. As shown in Fig. 1(A), the solutions of 1a-c (10
μM, PBS/DMSO = 3 : 7 (v/v, 0.01 M, pH = 7.4)) showed maximum
absorptions at 707, 717 and 708 nm, respectively. The fluorescence
intensities of 1a-c gradually stabilized at pH 4.9, 5.4 and 6.4 (Fig.
1(B)), respectively. Their pKa values were calculated according to
the Henderson−Hasselbach-type mass action equation. The pKa
value of 1c (4.1) was lower than those of 1a (5.5) and 1b (4.4) (Table
1), which indicates that it could be reasonably applied to biological
systems, and the application of 1c in physiological conditions is
relatively extensive. The UV-Vis absorptions of 1a-c are all stable
and change negligibly when the pH is increased to 7.4 (Fig. S5, S10,
S15). Thus, 3a-c operates well in the physiological pH region, and
pH 7.4 was chosen as the experimental condition.
Cytotoxicity Assay
The cytotoxicity of probe 3a-c was determined by MTT assays.18
HepG2 cells were incubated in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10% FBS (fetal bovine serum) in an
atmosphere of 5% CO2 and 95% air at 37 °C. The cells were placed
in
a 96-well plate and incubated for 24 h upon different
concentrations of probe 3c of 0.0, 5.0, 10.0, 20.0, 40.0, 60.0 and
80.0 μM, respectively. Finally, the MTT assay followed. The
cytotoxic effect (VR) of probe 3c was assessed using the following
equation:
VR = A/A0 × 100%,
Where A and A0 are the absorbance of the experimental group and
control group using a microplate reader at 490 nm, respectively.
Probe 3a and 3b were also evaluated under the same condition.
Living cell culture and imaging
HepG2 Cells were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10% FBS (fetal bovine serum). Before
imaging, the cells were seeded in 12-well flat-bottomed plates in an
atmosphere of 5% CO2, 95% air at 37°C, and seeding at a density of
about 80% - 90%. Immediately before the experiments, the cells
were washed twice with PBS buffer. Subsequently, the cells were
incubated with 3a-c (20 µM) in PBS (containing 5% DMSO as a
cosolvent) for 1h at 37 °C, and then washed with PBS three times.
For the experiment group, the cells were added a NaF (100 µM, 2
Fig. 1 (A) The UV-Vis absorption of 1a-c (10 µM). (B) The fluorescence
emission of 1a-c (10 μM). Inset: thecorrelation between fluorescence
emission intensity and pH (2.4-8.4). All data were measured in PBS/DMSO =
3 : 7 (v/v, 0.01 M, pH = 7.4).
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Table 1 Photophysical Data of 1a-c
Table 2 Photophysical Data of 3a-c
λabs(nm)a λem(nm)b εmax(104 M−1 cm−1)c
DL(µΜ)d
0.116
0.061
0.152
λabs(nm)a λem(nm)b εmax (104 M−1 cm−1
Φd
pKa
c
)
e
3a
3b
3c
611
609
612
731
739
733
4.70
4.19
4.76
1a
1b
1c
707
717
708
732
740
736
3.82
6.85
0.13
0.36
0.41
5.5
4.5
4.1
aMaximal absorption. bMaximal emission wavelength after reacting with
12.24
NaF. Molar extinction coefficient. dDetection limit.42 Concentrations of 3a-c
c
aMaximal absorption. bMaximal emission wavelength. cMolar extinction
coefficient. dRelative fluorescence quantum yield estimated using
indocyanine green (ICG, Φ= 0.13 in DMSO) as a fluorescence standard.41
eCalculated using Henderson−Hasselbach-type mass action equation
(log[(Fmax−F)/(F−Fmin)]=pKa−pH).40 Concentrations of 1a-c are 10 μM.
Measured in PBS/DMSO = 3 : 7 (v/v, 0.01 M, pH = 7.4). λex = 690 nm.
are 10 μM. Measured in PBS/DMSO = 3 : 7 (v/v, 0.01 M, pH 7.4). λex= 690
nm.
Spectroscopic studies of 3a-c towards fluoride ions
To evaluate the fluoride ion sensing ability of the system, the UV-Vis
absorption and fluorescence spectra of 3a-c (10 μM) were
recordedin the presence of F- in a PBS/DMSO = 3:7 (v/v, 0.01 M, pH
= 7.4) solution at room temperature. 3c shows a major absorption
peak at 612 nm (εmax = 4.76×104 M−1 cm−1) (Table 2). When NaF (0.0,
1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 equiv.) was
continually added to the solution, the absorbance at 612 nm
gradually decreased and a new peak appeared at 708 nm with two
isosbestic points at 479 nm and 650 nm, which demonstrates the
formation of a new species. The maximum absorption peak showed
a 96 nm wavelength bathochromic shift, and the colour of the
solution turned from blue to green. As expected, the absorption of
the new species coincided with that of 1c (Fig. 2(A)), indicating that
the cleavage of the Si-O bond was triggered by fluoride ions.
Fig. 3 (A) Fluorescence responses of 3a-c for NaF (3.0 equiv.). (B) Time-
dependent fluorescence intensity changes of 3a-c (10 μM) upon addition of
NaF (2.0 equiv.). Measured in a PBS/DMSO = 3 : 7 (v/v, 0.01 M, pH = 7.4)
solution. λex = 690 nm.
To investigate the effect of H, Cl and F substituents on the
fluorescence properties, their fluorescence emission were
measured at the same conditions (PBS/DMSO = 3 : 7 (v/v, 0.01 M,
pH = 7.4)). As shown in Fig. 3(A), 3a-c all experience clear
fluorescence intensity changes after NaF (3.0 equiv.) is added.
Subsequently, we analysed the time-dependent fluorescence
intensity changes of 3a-c (10 μM) after the addition of NaF (2.0
equiv.). From the trends of the three lines, we found that the
response of 3c is more rapid than those of 3a-b. The fluorescence
intensities of 3c and 3b tended to stabilize at approximately 20 min,
whereas the stabilization of 3a was longer. The fluorescence
intensity and response rate of 3c are superior to those of the other
two.
Under the same condition, 3a shows an absorption band centred
at 611 nm (εmax = 4.70 × 104 M−1 cm−1) (Table 2), The incremental
addition of NaF led to a progressive decrease of the absorption at
611 nm, while a new absorbance band appeared at approximately
707 nm and its absorption increased, similar to the case for 1a. Two
clear isosbestic points were observed at 485 nm and 665 nm (Fig.
S8). The UV-Vis absorption spectra of 3b similarly exhibited
maximum absorption at 609 nm (εmax = 4.19×104 M−1 cm−1). Upon
the addition of NaF (from 0 to 12 equiv.), the absorption at 609 nm
decreased noticeably and a 108 nm bathochromic shift of the
absorption peak to 717 nm occurred. Two clear isosbestic points at
484 nm and 656 nm were observed, accompanied by a solution
colour change from blue to green (Table 2, Fig. S13). 3c (96 nm) and
3b (108 nm) have approximately twice the wavelength shift of 3a
(56 nm), so they have clearer colour changes upon the addition of
NaF to solutions 3a-b.
Selectivity and tolerance of 3a-c for NaF over other
analytes
For the further evaluation of the fluoride-sensing ability of the
system, the selectivity and tolerance of 3a-c for NaF over other
analytes were also tested. As shown in Fig. 4(A), only NaF and TBAF
demonstrated strong fluorescence emissions at 733 nm, while the
-
2-
other analytes (H2PO42-; Ac-; SO4 ; S2O32-; HS-; SCN-; Cl-; Br-; I-; CO3
;NO3 ; HCO3 ; Cu2+; Zn2+; Mn2+; Ni2+; Mg2+; Fe2+; Ca2+; Al3+; K+; Na+)
(3.0 equiv.) show almost no fluorescence response for 3c (10 µM).
This observation clearly demonstrates the high selectivity of 3c
towards F- over other analytes. Evidently, the presence of these
analytes did not have a remarkable influence on the fluorescence
response process of NaF to 3c (Fig. 4(B)). This indicates that 3c has a
great tolerance for F- over other analytes. Although the responses
of 3a and 3b to F- exhibit slight decreases, they still have similar
fluorescent properties and changes (Fig. S6, S7, S11, S12). This
result indicates that the system is an excellent sensitive sensor for
recognizing fluoride ions over other analytes.
-
-
Fig. 2 (A) The UV-Vis absorptions of 1c (10 µM), 3c (10 µM) and 3c (10 µM) +
NaF (3.0 equiv.), measured in PBS/DMSO = 3 : 7 (v/v, 0.01 M, pH = 7.4). Inset:
the colour of 3c and 3c+NaF. (B) UV titration of 3c (10 uM) upon addition of
NaF in a PBS/DMSO = 3 : 7 (v/v, 0.01 M, pH =7.4) solution. Each spectrum
was recorded at 10 min after the addition of NaF (0.0 - 12.0 equiv.).
4 | J. Name., 2012, 00, 1-3
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that the probe 3a-c are all exhibited good photostability, which is a
critical factor for the long-time tracking and bioimaging in vivo.
In order to investigate the correlation between the concentration
of NaF and the fluorescence intensity, a fluorescence titration
experiment was then conducted. As shown
Mechanism studies
To confirm the sensing mechanism, HPLC and ESI-MS methods were
then carried out. We firstly detected the HPLC peaks of 3c (0.2
mM)+ NaF (0.0 equiv. (Fig. 5a), 0.5 equiv. (Fig. 5b), 1.0 equiv. (Fig.
5c), respectively), 1c (0.2 mM) (Fig. 5d), and solvent system (blank
system, Fig. 5e). As shown in Fig. 5, after 3c reacted with different
equivalents of NaF, the peak at 23.385 min decreased, and a new
peak at 5.300 min appeared, with a retention time that coincided
exactly with that of 1c. By contrast, 3a (Fig. S17) and 3b (Fig. S18)
also show the identical results. Therefore, it is logical to expect that
upon the addition of fluoride ions, the fluorescence colour change
from blue to green reflects the extent of the fluoride-induced Si-O
bond cleavage. It is noteworthy that in the HRMS spectra of 3c with
NaF (Fig. S32), a new peak was detected at m/z = 402.1874, which
correspond to the product 1c. These results strongly supports the
supposition that the fluoride ions-triggered cleavage reaction
causes the release of 1c.
Fig. 4 (A) Fluorescence response of 3c (10 μM) to various analytes (3.0
-
2-
equiv.). Other analytes (H2PO42-; Ac-; SO4 ; S2O32-; HS-; SCN-; Cl-; Br-; I-; CO3
;
-
-
NO3 ; HCO3 ; Cu2+; Zn2+; Mn2+; Ni2+; Mg2+; Fe2+; Ca2+; Al3+; K+; Na+). (B)
Fluorescence intensity of 3c (10 μM) in the presence of NaF (3.0 equiv.) with
other analytes (10.0 equiv.). Horizontal axis: (1) F-; (2) F- + SCN-; (3) F- + HS-;
(4) F- + S2O32-; (5) F- + SO42-; (6) F- + Ac-; (7) F- + H2PO42-; (8) F- + HCO3- ; (9) F- +
-
NO3 ; (10) F- + I-; (11) F-+ Br-; (12) F- + Cl-; (13) F- + CO32-; (14) F- + Cu2+; (15) F- +
Zn2+; (16) F- + Mn2+; (17) F- + Ni2+; (18) F- + Mg2+; (19) F- + Fe2+; (20) F- + Ca2+;
(21) F- + Al3+; (22) F- + K+; (23) F- + Na+. (C) Fluorescence titration studies of 3c
(10 µM) upon addition of NaF (0.0 - 11.0 equiv.) at room temperature. Inset:
Linear correlation between the fluorescence intensity and the concentration
of NaF. Measured in a PBS/DMSO = 3: 7 (v/v, 0.01 M, pH = 7.4) solution, 10
min after the addition of NaF.
in Fig. 4(C), the fluorescence intensity is clearly increasing with the
addition of NaF. As the concentration of NaF increases to 100 µM
(10.0 equiv.), the fluorescence intensity reaches its maximum level,
an approximately 37-fold enhancement. The fluorescence
responses of 3a and 3b to NaF achieved their maximum levels at
approximately 120 µM (Fig. S9) and 110 µM (Fig. S14),
approximately 27-fold and 28-fold enhancements, respectively.
From the inset diagrams of the three probes, the fluorescence
intensity has an excellent linear correlation with the concentration
of NaF in the range of 0 to 100 µM (probe 3a, Fig. S9), 0 to 70 µM
(probe 3b, Fig. S14), 0 to 70 µM (probe 3c, Fig. S16). The detection
limits were calculated to be 0.152, 0.116 and 0.061 µM (S/N = 3)
(Table 2), which indicates that the probe is highly sensitive and may
be suitable for studies of NaF in living systems.
Fig. 5 HPLC chromatograms analysis, a) 3c (0.2 mM). b) 3c (0.2 mM)+NaF
(0.5 equiv.). c) 3c (0.2 mM)+NaF (1.0 equiv.). d) 1c (0.2 mM). e) DMSO/PBS=
7: 3. Conditions: incubation for 10 min in a PBS/DMSO = 3 : 7 (v/v, 0.01 M,
pH=7.4) solution at room temperature.
Living cell application
Based on the excellent sensing ability of probes 3a-c to sodium
fluoride in water-containing solutions, we continued to explore
whether probe 3 can operate in living HepG2 cells, and confocal
fluorescence microscope images were investigated. In the control
test (Fig. 6A), we cultivated cells with 3c (20 μM) for 1 h at 37 °C,
and then the cell nuclei were staining with Hoechst for 10 min,
displaying a blue colour by emission from the blue channel (Fig. 6a).
The cells incubated with 3c did not exhibite notable fluorescence,
which indicates that none of the substances are responsive to
3c.Meanwhile, the experimental group (Fig. 6B) was further
incubated with sodium fluoride (100 μM) for another 10 min. As
expected, the probe 3c led to the enhancement of red fluorescence
when the living cells loaded with 3c were further treated with
sodium fluoride, in good agreement with the fluorescence turn-on
Photostability of probe 3a-c
The high photostability of fluorescence probes is highly desirable to
perform long-time in vivo. Therefore the photostability of probe 3a-
c was evaluated by time-course fluorescence and UV absorption
measurements upon sustained illumination. After exposured to UV
lamp (254 nm) for different time, the fluorescence intensity of 3a-c
are almost no change. Simultaneously, the UV absorption only show
a slight decrease (Fig. S2, S3, S4). From above results, we can know
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Fig. 7 Fluorescence images of 3c in mouse. a) Mice injected with probe 3c
(20 μM). b) Mice injected with 3c (20 μM) and 2 mgF/Kg sodium fluoride
recorded at 15 min. c) Mice injected 3c (20 μM) with 2 mgF/Kg sodium
fluoride recorded at 2 h. d) Mice injected 3c (20 μM) with 2 mgF/Kg sodium
fluoride recorded at 4 h.
profile in the solution. 3a (Fig. S22) and 3b (Fig. S23) are similar to
3c. These results imply that probes 3a-c are cell membrane
permeable and responsive to fluoride ions in living cells. In addition,
the cytotoxicity of probe 3a-c in living cells were also tested via MTT
assay. The results indicate that the cells have low cytotoxicity upon
respective treatment with 3a-c (20 μM) for 24 h (Fig. S19, S20, S21).
This illustrate that 3a-c possessed good biocompatibility and was
suitable for biological system.
fluoride ions even at the level of 2 mgF/Kg body weight (Fig. 7, b-d).
In order to observe the distribution of probe 3c in the body, we
analysed the anatomy in mice. From Fig. S22, we can clearly
observe that probe 3c is mainly concentrated in the liver and
spleen, but not in the heart, lung or kidney.
blue channel
red channel
merge
Conclusions
In summary, we have designed and synthesized three probes 3a-c
based on hemicyanine through introducing three different
substituents (F-, Cl-, and H-) at the ortho of the hydroxyl. We
conducted pH effect, UV-titration and fluorescence titration,
selective and competitive experiments. Their excitation wavelength
is 690 nm and their emission wavelengths are 730-740 nm, which
all belong to the NIR region. Compared with the calculated data for
the UV absorption and fluorescen emission spectra, we confirmed
that the fluorescence properties of 3c have more advantages than
those of the other two probes, including a faster reaction rate and
stronger fluorescence intensity. HPLC and HRMS analysis verified
the mechanism is based on the F-triggered rapid cleavage of Si-O
bond. The high specificity of probes 3a-c for fluoride ions permits
accurate detection in living biosystems. Further research of NIR
probes to detect other speciesis underway in our laboratory, the
results of which will be reported in due course.
A
B
Fig. 6 Confocal microscope images of 3c in HepG2 cells. A: control. B: Image
of the cells incubated with 3c (20 µM) for 1 h, and further incubated with
NaF (100 µM) for 10 min. (a) emissions from the blue channel. (b) emissions
from the red channel. (c) Merge.
Acknowledgements
Living animal application
This work has been financially supported by the National Natural
Science Foundation of China (21562018, 21261008, 21166008);
Natural Science Foundation of Hainan Province (20162019) for the
financial assistance.
The prominent features of probe 3 include NIR absorption and
emission wavelengths, excellent selectivity for fluoride ions and
tolerance for other analytes, good cell membrane permeability and
low detection limit for sodium fluoride. These desirable attributes
prompted us to further investigate the suitability of the probe for
detecting fluoride ions in living animals. 3c has more advantages
than the other two and was chosen to conduct living animal
imaging. First, the mice were injected with probe 3c (20 μM) as a
control group (Fig. 7a), and then another group was treated with
sodium fluoride 28 mgF/Kg (Fig. S24), 12 mgF/Kg (Fig. S25), or 2
mgF/Kg (Fig. 7) as an experiment group.The mice were anesthetized
and imaged using an IVIS Lumina Series III in vivo imaging system.
As shown in Fig. 7, the mice injected with both NaF and probe 3c
exhibit that the fluorescence intensity in the mice is time-
dependent, with a maximum fluorescence intensity at 2 h, but
when injected probe 3c without sodium fluoride, they display no
fluorescence (Fig. 7a). The previous studies reported that the
minimum value of the human lethal fluoride doseis approximate 3
mg/Kg for children.43 Gratifyingly, our probe 3c could be utilized as
a n i m a g i n g p r o b e f o r v i s u a l i z i n g e x o g e n o u s
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A near-infrared fluorescence probe has been developed, which is
available for visualizing exogenous fluoride ions in vitro and in vivo.