1,6-Elimination reaction induced detection of fluoride ions in vitro and in vivo based on a NIR fluorescent probe

Article abstract of DOI:10.1016/j.saa.2019.05.013

Near-Infrared “turn on” type fluorescent probes are attractive and promising in the fields of chemical sensing and bioimaging. Here, a new dicyanomethylene-4H-pyran derivative (DCM-Si) NIR fluorescent probe was designed and synthesized for specific lighting up F^? in living cells and bodies. Si–O bond was used as F^? trigger, and the release of fluorophore (DCM-NH₂) occurred after substituent reaction and subsequent 1,6-elimination. This NIR probe displayed high sensitivity and selectivity for the sensing of F^?, and the detection limit was calculated to be as low as 157 nM. Moreover, the “off-on” fluorescent signal changes can be realized by adding F^? in living cells and zebrafish embryos.

Full text of DOI:10.1016/j.saa.2019.05.013

Accepted Manuscript
1
,6-Elimination reaction induced detection of fluoride ions in
vitro and in vivo based on a NIR fluorescent probe
Aiqing Feng, Yongmei Jia, Liping Huang, Lin Wang, Guohua
Zhou, Sheng Wang, Peilian Liu
PII:
S1386-1425(19)30489-5
https://doi.org/10.1016/j.saa.2019.05.013
SAA 17108
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 22 November 2018
Revised date:
Accepted
date:
23 April 2019
9
May 2019
Please cite this article as: A. Feng, Y. Jia, L. Huang, et al., 1,6-Elimination reaction
induced detection of fluoride ions in vitro and in vivo based on a NIR fluorescent probe,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/
1
0.1016/j.saa.2019.05.013
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ACCEPTED MANUSCRIPT
1
,6-Elimination reaction induced detection of fluoride ions in
vitro and in vivo based on a NIR fluorescent probe
b
a
a
c
a
Aiqing Feng , Yongmei Jia , Liping Huang , Lin Wang , Guohua Zhou ,
a
a*
Sheng Wang , Peilian Liu
a. School of Chemistry and Chemical Engineering, Lingnan Normal University, Zhanjiang
24048, Guangdong, PR China
5
b. Department of Life Science, Luoyang Normal University, Luoyang 471934, PR China
c. Analytical and Testing Center, Jinan University, Guangzhou 510632, PR China
Corresponding Author
Tel.: 86-759-3183176. Fax: 86-759-3183176. E-mail address: liupeilian2010@126.com
1

ACCEPTED MANUSCRIPT
ABSTRACT
Near-Infrared “turn on” type fluorescent probes are attractive and promising in the fields of
chemical sensing and bioimaging. Here, a new dicyanomethylene-4H-pyran derivative
-
(
DCM-Si) NIR fluorescent probe was designed and synthesized for specific lighting up F in
-
living cells and bodies. Si-O bond was used as F trigger, and the release of fluorophore
(
DCM-NH ) occurred after substituent reaction and subsequent 1,6-elimination. This NIR
2
-
probe displayed high sensitivity and selectivity for the sensing of F , and the detection limit
was calculated to be as low as 157 nM. Moreover, the “off-on” fluorescent signal changes can
-
be realized by adding F in living cells and zebrafish embryos.
Keywords: 1,6-elimination; fluoride ions; fluorescent probe; NIR
2

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1.
Introduction
The development and synthesis of fluorescent probes for sensing anions in biologically
and chemically relevant conditions have attracted much attention [1-11]. Fluoride is
considered as a special ion due to its dual natural properties in biological science. Appropriate
intake of fluoride can reduce the risk of dental caries, but the excessive fluoride will bring
some diseases including dental and skeletal fluorosis, and even cause kidney failure [12-16].
Therefore, it is very necessary to explore sensors for accurate monitoring fluoride ions in
drinking water as well as in living species. Although ion-selective electrode and ion
-
chromatography have been considered as the standard lab methods to detect F quantitatively,
the required equipment and facilities are tedious and expensive [17,18]. In recent years, the
-
fluorescent probes for F have attracted more attention because they provide a highly selective,
sensitive and convenient strategy in real sample testing [19-27]. However, most conventional
probes display short emissive wavelength (in the UV or visible range) that greatly limit their
application in living cells and in vivo.
In the past decade, near-infrared (NIR) fluorescent probes have received much attention
because of their unique optical performances including weaker fluorescence background,
lower photobleaching, and strong photon penetration depth [28-33]. Many NIR fluorescent
probes have been designed, prepared, and applied for detecting and imaging various important
analytes (such as proteins and active radicals) in the field of biology [34-38]. Generally, the
-
mechanism of sensing for F can be divided into three interacted types: hydrogen bonding,
boron-fluoride complexation, and Si-O cleavage [39-44]. Although some NIR probes have
-
been reported for the detection of F in aqueous solution or living cells based on
3

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-
fluoride-mediated desilylation strategy, few of them can be applicated for F imaging in vivo
45].
In this paper, a new fluoride-activatable NIR fluorescent probe (DCM-Si) was designed
[
-
and synthesized for the detection of F in vitro and in vivo. As shown in Scheme 1, DCM-Si
containing a Si-O linking erasable group as fluorescence quencher and specific fluoride
-
trigger for minoring F in living cells and zebrafish based on the cleavage of Si-O bond
followed by 1,6-elimination to release DCM-NH . As expected, a “turn on” fluorescent
2
-
emission of DCM-Si located at 665 nm was observed upon adding F , which providing a
strong broad band in the near infrared region. Moreover, further studies proved the DCM-Si
-
with low cytotoxicity was suitable to track F level elevation in living cells and zebrafishes
through fluorescent imaging technology.
2.
Experimental section
2.1. Materials
(
2-Hydroxy-5-methyl-1,3-phenylene) dimethanol (98%), 2-iodylbenzoic acid (97%), ethyl
2
-(triphenylphosphoranylidene) acetate (98%), tert-butylchlorodimethylsilane (TBSCl, 99%),
triethylamine (AR), diisobutyl aluminium hydride (1.5 M in toluene), tert-butoxycarbonyl
anhydride (98%), trifluoroacetic acid (AR), and triphosgene (98%) were purchased from
Energy Chemical, Shanghai, China. Tetrabutylammonium fluoride (1 M in THF),
tetrabutylammonium
chloride
(98%),
tetrabutylammonium
bromide
(99%),
tetrabutylammonium iodide (99%), tetrabutylammonium acetate (97%), tetrabutylammonium
hydrogen sulfate (98%), tetrabutylammonium phosphate (99%), tetrabutylammonium nitrate
(
98%), and tetrabutylammonium hydroxide (1M in MeOH) were purchased from Aladdin
4

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Chemistry Co. Ltd.. All used organic solvents were purchased from Guangzhou Chemical
Reagent Factory and used without further purification.
2.2. Apparatus
UV-Vis spectra were recorded on TECHCOMP spectrophotometer in the range of 320-750
nm with a slit of 2 nm. Fluorescence and excitation spectra were measured using a Hitachi
1
F-7000 fluorescence spectrophotometer with a 150 W xenon lamp as a light source. H NMR
1
3
and C NMR spectra were recorded on a Bruker 400 MHz NMR Spectrometer. HPLC
analyses were performed on Agilent 1260. Cells imaging experiments were carried out on a
Leica TCS SP5 CLSM using 488 nm laser as the excitation light source. The fluorescence
images of zebrafish were afforded by an Olympus IX 71 fluorescence microscope with a
DP72 color CCD.
2.3. Synthesis of the probe
Synthesis of compound 1: To a solution of (2-hydroxy-5-methyl-1,3-phenylene)
dimethanol (1 g, 5.95 mmol) in ethyl acetate (20 mL) was added 2-iodylbenzoic acid (5 g,
7.86 mmol). The mixture solution was stirred under refluxing for 3 h. After filtrating the
1
solid, the filtrate was evaporated to obtain the crude product. Then, the product was purified
by column chromatography on silica gel to obtain the target compound as a white solid (yield:
1
7
3
6.3%). H NMR (400 MHz, CDCl , ppm): 11.47 (s, 1H), 10.23 (s, 2H), 7.78 (s, 2H), 2.40 (s,
3
H).
Synthesis of compound 2: Compound 1 (745 mg, 4.54 mmol) and ethyl
2
-(triphenylphosphoranylidene) acetate (6.3 g, 18.1 mmol) were dissolved in dichloromethane
(
30 mL), and the mixture was stirring under room temperature for 6 h. The organic solvent
5

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was removed by decompression. Then, the crude product was purified by column
1
chromatography on silica gel to afford the compound 2 as a white solid (yield: 86.4%). H
NMR (400 MHz, CDCl , ppm): 8.10-8.20 (d, 2H), 7.35 (s, 2H), 6.45-6.55 (d, 2H), 4.25-4.40
3
1
3
(
m, 4H) 2.32 (s, 1H), 1.30-1.40 (t, 6H), 1.27 (s, 1H). C NMR (101 MHz, CDCl , ppm):
3
167.68, 152.01, 139.47, 130.42, 129.99, 123.01, 119.15, 60.82, 20.52, 14.33.
Synthesis of compound 3: Compound 2 (0.5 g, 4.54 mmol) was dissolved in 30 mL of
dichloromethane, and 10 eq. of triethylamine was added into the solution. Subsequently,
tert-butylchlorodimethylsilane (1.739 g, 7 eq.) was added into the mixture carefully. After
stirring under room temperature for 6 h, the mixture was diluted with a certain amount of
dichloromethane, and washed with DI water three times. The organic phase was first dried by
anhydrous sodium sulfate, and then removed under vacuum to obtain the crude product. The
product was purified by column chromatography on silica gel to afford the compound 3 as a
1
yellow powder (yield: 95.9%). H NMR (400 MHz, CDCl , ppm): 7.85-8.00 (d, 2H), 7.41 (s,
3
2
6
H), 6.32-4.36 (d, 2H), 4.23-4.35 (m, 4H), 2.32 (s, 1H), 1.32-1.35 (t, 6H), 1.11 (s, 9H), 0.12 (s,
1
3
H). C NMR (101 MHz, CDCl , ppm): 166.89, 151.29, 140.03, 131.41, 129.32, 126.99,
3
118.03, 60.49, 25.89, 20.73, 18.50, 14.34, -3.82.
Synthesis of compound 4: Compound 3 (650 mg, 1.55 mmol) was sealed up within the
round bottom flask (100 mL), then tetrahydrofuran (30 mL) was added into the bottom under
the protection of nitrogen gas. After cooling down to -78℃, diisobutyl aluminium hydride
(
20.7 mL) was drop into the reaction bottle slowly in about 45 min, and the mixture was
stirring for 1 h. Then, the temperature was slowly heated up to room temperature, and the
mixture was reacted for further 1 h. 20 mL water was slowly drop into the bottle to quench the
6

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reaction. The solution was extracted with ethyl acetate three times, then the organic phase was
combined and dried by anhydrous sodium sulfate overnight. The purified compound 4 with
1
yellow color was obtained by column chromatography on silica gel (yield: 97.6%). H NMR
(
400 MHz, DMSO-d , ppm): 7.21 (s, 2H), 6.73-6.78 (d, 2H), 6.20-6.26 (m, 2H), 4.10-4.12 (m,
6
1
3
4
1
H), 2.24 (s, 3H), 1.01 (s, 9H), 0.07 (s, 6H). C NMR (101 MHz, DMSO-d , ppm): 147.68,
6
30.95, 130.73, 129.02, 125.92, 124.50, 62.17, 26.44, 20.90, 18.71, -2.99.
Synthesis of compound 5: DCM-NH (68 mg, 0.22 mmol) and triphosgene (194.8 mg,
2
0.66 mmol) were added into the 25 mL of round bottom flask under the protection of nitrogen.
After being dissolved by dichloromethane (9 mL), the solution was cooled to 0 ℃. 3 mL of
dichloromethane containing pyridine (158.2 μL, 1.97 mmol) was added into the bottom slow
down with a syringe. After stirring at 0 ℃for 1 h, the mixture was keeping further 2 h at room
temperature. Dichloromethane was removed by decompression under room temperature. 10
mL dichloromethane solution of compound 4 (220.5 mg, 0.22 mmol) was rapidly into the
bottom under the protection of nitrogen gas. The mixture was stirring under room temperature
overnight. The solvent was removed under vacuum to obtain the crude product. The product
was purified by column chromatography on silica gel to obtain the compound 5 as a yellow
1
solid (yield: 48.3%). H NMR (400 MHz, CDCl , ppm): 8.93-8.95 (m, 1H), 7.74-7.78 (m, 1H),
3
7
4
.46-7.63 (m, 7H), 7.23-7.24 (d, 2H), 6.74-6.99 (m, 6H), 6.20-6.30 (m, 2H), 4.86-4.88 (d, 2H),
1
3
.33-4.35 (d, 2H), 2.31 (s, 3H), 1.08(s, 9H), 0.12 (s, 6H). C NMR (101 MHz, DMSO-d ,
6
ppm): 157.66, 152.87, 152.92, 152.34, 148.41, 140.04, 138.30, 134.61, 131.04, 130.57,
29.73, 129.11, 128.77, 128.25, 128.12, 127.38, 126.99, 126.50, 125.95, 125.85, 122.47,
18.68, 118.58, 117.87, 117.30, 116.90, 115.82, 106.60, 66.40, 64.10, 62.43, 29.72, 26.03,
1
1
7

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20.80, -3.36.
2.4. The procedure for spectral analysis
All absorption and emission spectra of DCM-Si were carried out in DMSO containing 5%
H O. 100 μL of water, 1.88 mL of DMSO, and 20 μL DMSO stock solution of DCM-Si (0.5
2
mM) were added into a 2 mL tube to afford the final concentration (5 μM).
-
-
-
-
-
-
-
4
-
-
Tetrabutylammonium salt including (F , Cl , Br , I , AcO , HSO , H PO , NO , or OH ) was
4
2
3
dissolved in DMSO, and an appropriate volume was added to the sample solution. Then, the
solution was transferred into a 10×10 mm quartz cuvette to conduct the measurement of
absorption and fluorescence spectra.
2.5. DFT calculations
The nature of the ground states (HOMO) and singlet excited states (LUMO) of DCM-Si
and DCM-NH was calculated by quantum chemistry theory. The geometry optimization and
2
energy level for samples were displayed by the method of the density functional theory (DFT)
at the B3LYP level using a suite of Gaussian 09 programs [46,47].
2.6. Cell culture.
Human epithelioid cervical carcinoma cell line (HeLa cells) was used in this study. Cells
were cultured in Dulbecco’s modified medium (DMEM) supplemented with 10% fetal bovine
serum and 1% antibiotics penicillin at 37 °C under 5% CO atmosphere.
2
2.7. Cell viability assay
To explore the cytotoxicity of DCM-Si, the HeLa cells were cultured at 37 °C with 5%
CO and grown for one day. After removal of the medium, cells were cultured with standard
2
-
medium containing various concentrations of F (0, 5, 10, 15, 20, 25, or 30 μM) for further
8

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one day. The cytotoxicity of DCM-Si was evaluated by MTT assay based on ISO 1099. Each
sample was performed in five replicates, and the cell viability was performed by the statistical
mean and standard deviation.
2.8. Cell imaging
The HeLa cells were plated on polylysine-coated cell culture glass slides inside the 30 mm
glass culture dishes and allowed grown to 70% confluency. After washing with DMED
-
medium, cells were cultured with various amount F (0, 50, and 100 μM, respectively) at
3
7 °C under 5% CO for 60 min. Cells were washed with medium again prior to incubate with
2
the medium containing DCM-Si (10 μM) and 0.5% DMSO for another 60 min. Subsequently,
we picked up the glass slides, and washed the cells by PBS buffer for three times. Finally, the
cells were imaged on a Leica TCS SP5 CLSM with a blue laser (λ = 488 nm), and the
ex
fluorescent signal collection wavelength was 620 to 720 nm.
2.9. Zebrafish imaging
Zebrafish embryos of the pharyngula period (48 hpf) were incubated with E3 media (15
mM NaCl, 0.5 mM KCl, 1 mM MgSO , 1 mM CaCl , 0.15 mM KH PO , 0.05 mM Na HPO ,
4
2
2
4
2
4
0
.7 mM NaHCO ; pH 7.5) containing DCM-Si (20 μM) and 0.5% DMSO for 4 h. The
3
zebrafishes were washed with E3 media three times prior to imaging. As for the fluoride ions
-
imaging, two groups were further cultured with 50 and 100 μM of F for another 1h,
respectively.
3.
Result and discussion
3.1 Synthesis of the probe (DCM-Si)
Scheme 2 illustrated the synthetic route of DCM-Si. The two benzyl alcohol groups of
9

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(
2-hydroxy-5-methyl-1,3-phenylene) dimethanol were oxidized to aldehyde groups by adding
the oxidant (2-iodylbenzoic acid) into the reaction system, and the crude product was purified
to form compound 1. Compound 1 was reacted with ethyl 2-(triphenylphosphoranylidene)
acetate via Witting reaction to form compound 2. The phenolic hydroxyl of compound 2 was
allowed to react with TBSCl to afford compound 3. Then, the carboxylate esters were reduced
by diisobuty aluminium hydride to alcohols at low temperature (-78 ℃) to yield compound 4.
Finally, DCM-NH [48] was bonded with compound 4 using triphosgene to obtain the target
2
1
13
probe compound 5 (DCM-Si). Compound 1-5 were well characterized by H and C nuclear
magnetic resonance (Figs. S3-S11).
-
3.2 UV response of DCM-Si toward F
-
The UV spectra of DCM-Si in the presence or absence of F were investigated. As shown
in Fig. S1, the prepared NIR probe DCM-Si has a broad absorption band (centered at 455 nm)
with the range from 350 to 625 nm, which is attributed to the existence of the intramolecular
charge transfer (ICT) in the conjugate system. This peak was dramatically reduced in the
-
presence of F , and a new absorption peak appeared at 516 nm which was almost at the same
-
location for the free DCM-NH . This phenomenon might due to the reaction between F and
2
-
-
-
-
-
4
-
DCM-Si to form DCM-NH . Some similar anions including Cl , Br , I , AcO , HSO , H PO ,
2
2
4
-
-
NO , and OH were added into the solution of DCM-Si, respectively. There was almost no
3
obvious change in the UV absorption curves, proving that the NIR probe can distinguishing
-
the fluoride ion selectively. Furthermore, the response time of DCM-Si toward F was also
studied. The maximum absorption wavelength of DCM-Si was red shift range from 455 nm to
-
5
16 nm with the increasing time exposing to the F in the solution (Fig. 1). As time goes on,
1
0

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the obviously color change from yellow to pink was observed, which provided the
-
colorimetric recognition for F directly by naked eye (Figure 1, inset photo).
-
3.3 Fluorescent response of DCM-Si toward F
-
The fluorescent spectra of DCM-Si (5 μM) toward F in DMSO aqueous solution
water/DMSO = 5/95, v/v) at room temperature (λ = 480 nm) were investigated. As shown
(
ex
in Fig. 2, DCM-Si exhibited the faint emission with the maximum wavelength at 570 nm.
-
Upon adding 20 eq. of F (100 μM) into the DCM-Si solution, the peak located at 570 nm was
dramatically decreased, while a new emission band located at 665 nm was occurred and
continuous to enhance with the increasing time. These results displayed the silicon oxygen
-
bond of DCM-Si was broke successfully in the presence of F , and the 1,6-elimination
reaction was occurred subsequently. The totally process was almost done in 40 min. And we
therefore clearly observed the fluorescence color changes of DCM-Si from dark to red under
the irradiation of ultraviolet lamp (Fig. 2, inset photo).
In addition, the quantitative detection ability of DCM-Si for fluorine ion was evaluated by
-
using the fluorescence titration experiments. After exposing to different concentrations of F
(
0 - 100 μM) with 40 min, the emission intensities of DCM-Si significantly increased and
-
almost reached a balance at the 100 μM of F (Fig. 3). The scatter plot of the relative intensity
-
of DCM-Si at 665 nm versus the concentration of F was investigated in Fig. 3 inset photo.
The fluorescence intensity increased rapidly until the concentration of fluoride reached 50 μM,
-
and became slow afterward. Interestingly, the emission intensity change (F-F ) versus the F
0
2
concentration range from 1 to 40 μM followed a simple linear equation y = 40.77x -36.23 (R
-
=
0.998), and the detection limit (DL) of DCM-Si for F had been calculated as 157 nM by the
1
1

ACCEPTED MANUSCRIPT
equation DL = 3SD / slope, where SD is the standard deviation of the blank signal of DCM-Si
(
n=5) (Fig. 4).
The selectivity of DCM-Si toward F was also evaluated by the fluorescence emission
-
-
-
spectra of the NIR probe in the presence of fluorine ion or some other similar anions (Cl , Br ,
-
-
-
4
-
4
-
3
-
-
I , AcO , HSO , H PO , NO , OH ). As shown in Fig. 5, adding F into the DCM-Si solution
2
could induce significant enhancement of the emission curve, while almost no change was
observed in the presence of the other analytes. Moreover, the selectivity performance of
-
DCM-Si under the coexistence of F and other analytes was also explored. DCM-Si can still
-
make a specific response for F in the mixture of coexisting ions at the same concentration.
All these results therefore proved that the NIR probe based on DCM-Si was selective for
sensing fluoride ions.
-
3.4 Mechanism of DCM-Si responding to F
To investigate the detecting mechanism, the data was collected for the standard DCM-NH2,
-
and DCM-Si in presence or absence of F using the HPLC method. As shown in Fig. 6, upon
-
adding 10 eq. of F , the peak located at 3.909 mins of DCM-Si was disappeared, and a new
peak was observed at 2.801 min which was consistent with the retention time of DCM-NH2.
This result clearly showed that the increasing of the fluorescence intensity was caused by the
-
release of DCM-NH . Before treating with F , the fluorescence of the DCM moiety was
2
quenched through the weaker intramolecular charge transfer (ICT) process to ensure a low
-
fluorescence background in the probe. Upon addition of F , the silicon oxygen bond of
DCM-Si was cleavage to generate a phenolic-hydroxyl containing intermediate which was
chemically labile and underwent a 1,6-elimination spontaneously to release DCM-NH2
1
2

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concomitantly, and thus lead to the fluorescence turn-on process due to the strong ICT. The
-
sensing mechanism of DCM-Si toward F was depicted in Scheme 1. In fact, the identical
absorption spectra of DCM-Si and DCM-NH also proved that the Si-O bond of DCM-Si was
2
-
cracked to release DCM-NH with the treatment of F (Fig. S1).
2
Density functional theory (DFT) calculations were evaluated for the electron density of
DCM-Si and DCM-NH at the B3LYP level through the Gaussian 09 package. As for the NIR
2
probe DCM-Si, the electron density mainly located at the benzene ring linked with siloxane in
its HOMO orbital, and the electron transfer occurred to the benzopyran unit in its lowest
unoccupied molecular orbital (LUMO) (Fig. 7). Compared with the band gap of DCM-Si
(
2.973 ev), the smaller band gap (2.902 ev) of DCM-NH can explain the red shift of
2
maximum absorption (from 455 nm to 516 nm) and fluorescence emission (from 570 nm to
-
6
65 nm) of the probe with the treatment of F (Fig. S1 and Fig. 2).
3.5 Detection ability of DCM-Si in vitro
-
To evaluate the detection ability of the probe toward F within living cells, HeLa cell line
was chosen to study the cytotoxicity of DCM-Si by MTT assay. As shown in Fig. S2, the cell
mortality rate was lower than 10% upon respective treatment with high concentration of
DCM-Si (30 μM) for 24 h, indicating that DCM-Si possessed good biocompatibility, and
-
could be utilized as a NIR “off-on” type probe for sensing F in live cells. In addition, the
HeLa cells were also used for exploring the cell imaging capability of DCM-Si with various
-
concentrations of F by confocal laser scanning microscope (Fig. 8). No obvious signal in red
channel was observed from cells treated with DCM-Si (10 μM) for 60 min (A). In contrast,
the cells displayed NIR fluorescence signal in the same channel upon being precultured with
1
3

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-
F (50 μM) for 60 min and then incubated with DCM-Si (10 μM) for further 60 min (B). The
-
red was enhanced depending on the changes in concentration of the preincubated F (100 μM,
C), indicating that DCM-Si not only had excellent cell membrane permeability, but also could
be activated by the fluorine ions inside cells. Therefore, this novel low toxic NIR fluorescence
-
probe would offer an efficient way for tracking F in biological systems.
3.6 Detection ability of DCM-Si in vivo
Zebrafish was displayed as the animal model to demonstrate that the fluorescence imaging
-
of F in vivo could be tracked by DCM-Si, as the zebrafish possessed many advantages (such
as excellent optical transparency, high fecundity, external fertilization, and so on.).
Zebrafishes were incubated with free DCM-Si (10 μM) as a control group, and another two
groups were stained with DCM-Si (10 μM) for a certain amount of time prior treating with 50
-
and 100 μM F separately. As shown in Fig. 9, there was no fluorescence signal observed in
-
the control experiment treating without F (A). The obviously red fluorescence located at the
-
yolk of embryo was obtained with the addition of F (50 μM, B), and the emission of yolk was
-
increased with the higher concentration of F (100 μM, C). Combined with the behavior of
DCM-Si in vivo, it can be clearly concluded that the Si-O bond of DCM-Si was successful
-
broke by F in living bodies.
4.
Conclusions
In summary, we have reported a novel “off-on” NIR fluorescent probe by utilizing the
inhibition of intramolecular charge transfer (ICT). DCM-Si exhibits almost no fluorescent
-
emission in terms of ICT block from the amino group of DCM-NH . When adding F , the
2
Si-O bond of DCM-Si is substituted to the form of phenolic hydroxyl, and the 1,6-elimination
1
4

ACCEPTED MANUSCRIPT
can subsequently be spontaneously to release DCM-NH concomitantly with the NIR
2
fluorescence (at 665 nm) turn-on process due to the strong ICT from amino group. Notably,
DCM-Si is successfully applied for the quantitative sensing of exogenous fluoride ions in
HeLa cells and zebrafish embryos through the fluorescence imaging.
Acknowledgements
The work was supported by Natural Science Foundation of Guangdong Province
(
2017A030310604;
2018A030307035;
2016A030310362;
2018A030307037;
2014A030310274), Guangdong University Provincial Key Platform and Major Research
Projects: Characteristic Innovation Project (2017KTSCX118), Natural Science Foundation of
Lingnan Normal University (ZL1802), and National Natural Science Foundation of China
(
21878136).
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Figure Captions
Scheme 1. Schematic illustration of DCM-Si based NIR fluorescence probe for detection of
-
F in vitro an in vivo.
Scheme 2. The synthesis method of probe DCM-Si.
-
Fig. 1. Time-dependent absorption spectra of DCM-Si (5 μM) with 100 μM F in aqueous
solution (DMSO/water = 95/5, v/v). Inset: Photos of DCM-Si solution before and after
-
addition of F with the range of time.
-
Fig. 2. Time-dependent emission spectra of DCM-Si (5 μM) with 100 μM F in aqueous
solution (DMSO/water = 95/5, v/v). Inset: Photos of DCM-Si solution before and after
-
addition of F with the range of time under the excitation of 365 nm UV light (left); plot
relative intensity at 665 nm versus time for DCM-Si (5 μM) solution upon addition of 100 μM
-
F (right).
Fig. 3. Emission spectra of DCM-Si (5 μM) in aqueous solution (DMSO/water = 95/5, v/v)
-
upon the addition various concentrations of F (from 0 to 100 μM). Inset: plot relative
-
intensity at 665 nm for DCM-Si (5 μM) solution versus F concentration (0-100 μM).
-
Fig. 4. Linear relationship between the relative intensity at 665 nm (F-F ) and F
0
concentration (1-40 μM).
Fig. 5. Fluorescence emission spectra of DCM-Si (5 µM) in aqueous solution (DMSO/water =
2
2

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-
-
-
-
-
-
4
9
5/5, v/v) with F (100 µM) and free various interfering analytes (Cl , Br , I , AcO , HSO ,
-
-
3
-
-
H PO , NO , and OH , 100 µM), and coexistence of F and various interfering ions including
2
4
-
-
-
-
-
-
-
-
Cl , Br , I , AcO , HSO , H PO , NO , and OH .
4
2
4
3
Fig. 6. HPLC chromatogram analysis of DCM-NH (5 µM), DCM-Si (5 µM), and DCM-Si (5
2
-
µM) + F (100 µM). Solvent ratio was acetonitrile/water = 90/10.
Fig. 7. Frontier molecular orbital profiles of DCM-Si and DCM-NH based on DFT
2
(
B3LYP/6-31G*) calculations.
Fig. 8. Confocal microscopy images of DCM-Si (10 μM) in HeLa cells. The cells were
incubated with only DCM-Si (10 μM) for 60 min as control (A). The cells were incubated
-
with 50 μM (B), or 100 μM (C) F for 60 min, and further incubated with DCM-Si (10 μM)
for 60 min as experimental groups.
Fig. 9. Fluorescence microscope images of DCM-Si (10 μM) in zebrafish. The zebrafishes
were incubated with only DCM-Si (10 μM) for 60 min as control (A). The zebrafishes were
-
incubated with 50 μM (B), or 100 μM (C) F for 60 min, and further incubated with DCM-Si
(
10 μM) for 60 min as experimental groups.
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Scheme 1
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Scheme 2
Figure 1
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Figure 2
Figure 3
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2
1
1
000
500
000
Equation
y = a + b
Adj. R-Squa 0.99798
Value
Intercept -36.2334
Standard Err
12.56091
0.57995
B
B
Slope
40.7672
5
00
0
0
10
20
30
40
50
-
Concentration of F / M
Figure 4
Figure 5
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-
DCM-Si + F
DCM-NH2
DCM-Si
2
4
6
8
10
Time / min
Figure 6
Figure 7
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Figure 8
Figure 9
2
9