Exploiting aggregation induced emission and twisted intramolecular charge transfer in a BODIPY dye for selective sensing of fluoride in aqueous medium and living cells

Article abstract of DOI:10.1016/j.jphotochem.2018.03.020

A novel red fluorescent probe based on boron dipyrromethene (BODIPY) was successfully designed and synthesized, consisting of electron acceptor 1,3,5,7-tetramethyl-8-phenyl-BODIPY (BDP) and electron donor triphenylamine (TPA) units using Suzuki cross-coup

Full text of DOI:10.1016/j.jphotochem.2018.03.020

Accepted Manuscript
Title: Exploiting Aggregation Induced Emission and Twisted
Intramolecular Charge Transfer in a BODIPY Dye for
Selective Sensing of Fluoride in Aqueous Medium and Living
Cells
Authors: Weilong Che, Guangfu Li, Jianxu Zhang, Yun Geng,
Zhigang Xie, Dongxia Zhu, Zhongmin Su
PII:
S1010-6030(17)31700-8
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.03.020
JPC 11189
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
21-11-2017
12-3-2018
13-3-2018
Please cite this article as: Weilong Che, Guangfu Li, Jianxu Zhang, Yun Geng, Zhigang
Xie, Dongxia Zhu, Zhongmin Su, Exploiting Aggregation Induced Emission and
Twisted Intramolecular Charge Transfer in a BODIPY Dye for Selective Sensing
of Fluoride in Aqueous Medium and Living Cells, Journal of Photochemistry and
Photobiology A: Chemistry https://doi.org/10.1016/j.jphotochem.2018.03.020
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Exploiting Aggregation Induced Emission and Twisted Intramolecular
Charge Transfer in a BODIPY Dye for Selective Sensing of Fluoride in
Aqueous Medium and Living Cells
Weilong Che a, Guangfu Li a, Jianxu Zhang b, Yun Geng a, Zhigang Xie b, Dongxia Zhu a,*, Zhongmin Su
a,*
a Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department
of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024,
P.R. China
b
State key laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry
Chinese Academy of Sciences, Changchun, 130022, P. R. China
*Corresponding authors
E-mail addresses: zhudx047@nenu.edu.cn (D-X. Zhu.), zmsu@nenu.edu.cn (Z-M. Su.)
Highlights
 A novel BODIPY-based fluorescent probe TPA-BDP has been successfully designed and
synthesized. The D-A-D structure and twisted molecular conformation make TPA-BDP
have the AIE and TICT properties simultaneously. This new probe features high sensitivity
and excellent selectivity to fluoride with a detection limit of 0.73 μM in aqueous medium.
TPA-BDP is cell-permeable and can effectively respond to F- as a suitable fluorescence
probe in living cells with very low cytotoxicity.
Abstract
A novel red fluorescent probe based on boron dipyrromethene (BODIPY) was successfully designed

and synthesized, consisting of electron acceptor 1,3,5,7-tetramethyl-8-phenyl-BODIPY (BDP) and
electron donor triphenylamine (TPA) units using Suzuki cross-coupling methods. TPA-BDP exploits
the advantages of both aggregation-induced emission (AIE) and twisted intramolecular charge transfer
(TICT). An X-ray crystal structure of TPA-BDP reveals a network of C–H···F intermolecular
interactions which can contribute to rigidifying the molecular conformation. Thereby, aggregation
restricts the intramolecular rotation associated with the donor moiety, inducing a great enhancement in
the emission efficiency. This new dye features high sensitivity and excellent selectivity to fluoride ions
with a detection limit of 0.73 μM in aqueous medium. The sensing mechanism was systematically
investigated by 1H NMR, 19F NMR, 11B NMR and mass spectrometry. It is proposed that fluoride reacts
in a nucleophilic displacement reaction at the BDP core which disrupts the structure of TPA-BDP,
thereby causing the red fluorescence of TPA-BDP quenched. It is noteworthy that TPA-BDP has been
utilized for the fluorescence imaging and fluoride ions detection in living cells with very low
cytotoxicity.
Keywords
Aggregation-induced emission; TICT; Fluorescent probe; BODIPY; DFT; Cell image
1. Introduction
Traditional organic luminophores usually emit efficiently in solutions whereas their emission is
weakened or even totally quenched upon the formation of molecules aggregates, which facilitate
nonradiative pathways and exciton interactions [1-4]. Obviously, aggregation caused quenching (ACQ)
is a harmful photophysical effect for light emission and for many practical applications. A very different
photophysical property associated with luminophore aggregation is aggregation-induced emission
(AIE), first reported by Ben Zhong Tang’s group in 2001 [5]. AIE arises as a result of restricted

intramolecular rotations, and is the opposite of ACQ. Since then, AIE materials have attracted
considerable attention for potential applications in fields such as the fluorescent detection of ions [6-8].
Recently, various AIE-active materials have been developed for fluorescent sensing [9-13] and
bioimaging [14-17]. Previously, we demonstrated an AIE-active ionic iridium complex as a probe for
−
ClO4 with high sensitivity and selectivity in bioimaging [18].
Fluoride anions (F−) play an essential role in human health and fluorescent methods to detect F−
can provide both quantitative and qualitative information [19, 20]. For examples, viable F− detection
has been developed at low energy consumption [21-24], low cost and with the capability for
intracellular fluoride detection [25]. It is well known that fluoride has strong affinity toward water.
Consequently, few of the reported systems tolerate aqueous conditions [26, 27]. Based on the strong
Lewis acid optical chemosensors can be successfully used in water to detect fluoride (e.g., zirconium，
lanthanide or organoboron compounds) [28]. And, until now, many fluorescent probes are limited in
biosystems due to their ACQ property [29, 30]. Therefore, the development of a fluorescent probe with
AIE property to detect F− in biosystems is an ongoing challenge.
Boron dipyrromethene (BODIPY) dyes are organoboron compounds. It is well-known to be
effective in energy-transfer processes, due to their excellent optical properties, such as sharp and intense
absorption with relatively long excited-state lifetimes, high fluorescence quantum efficiencies and high
molar absorption coefficients [31-34]. Thus, they are frequently used in light-harvesting molecules, as
materials for hybrid inorganic/organic LEDs [35] and as probes and labels for biomolecules [36-39]. In
2014, Rurack et al. reported three BODIPY-amidothiourea probes can be used for quantitative detection
of inorganic F− ions in aqueous solution with a remarkable detection limit due to a photoinduced
electron-transfer (PET) process [40]. By using this probe, a simple and user-friendly test strip was
fabricated that can be applied to determine the F− ion content in drinking water. Many BODIPY-based
dyes emit red light, which is an attractive attribute in the development of efficient bioimaging systems.
However, the applications of most BODIPY dyes to living cells are severely restricted due to one or
more of the following disadvantages: high background noise, difficulty of preparation, small Stokes

shifts (less than 40 nm), and aggregation-caused quenching (ACQ).
Insert Scheme 1
Scheme 1. Synthesis of the target TPA-BDP and Ph-BDP.
Highly luminescent BODIPY derivatives with AIE performance are rare [41, 42]. Notably, Hu et
al. obtained a series of BODIPY derivatives with AIE and twisted intramolecular charge transfer (TICT)
properties by introducing an electron donor triphenylamine unit at 8 position [43]. Increasing the “D-A”
effect in fluorophores can result in a red shift in their UV-vis absorption and emission spectra, increase
the Stokes shift and enlarge the geometrical relaxation [44]. Based on these considerations, we now
report a new AIE-active fluorophore containing 1,3,5,7-tetramethyl-8-phenyl-BODIPY (BDP) as the
electron acceptor (A) and triphenylamine (TPA) as the electron donor (D) (TPA-BDP, Scheme 1). In
our design, we introduced the TPA donor to the conventional 1,3,5,7-tetramethyl-8-phenyl-BODIPY at
the 2 and 6 positions, to form a symmetrical donor (D)-acceptor (A)-donor (D) structure. This
functionalization is clearly distinct from the BODIPY derivatives of Hu et al [43]. Due to the donor
(D)-acceptor (A)-donor (D) structure of TPA-BDP, it exhibits both AIE and TICT properties. Based on
this unusual combination of properties, TPA-BDP selectively and sensitively detects F− among a series
of anions. Moreover, TPA-BDP is shown to determine F− in biosystems with very low cytotoxicity.
2. Experimental section
2.1 Materials and instruments
1H NMR and 19F NMR spectra were recorded at 25 ºC on a Varian 500 MHz spectrometer. Absorption
spectra were measured with a Shimadzu UV-3100 spectrophotometer. Photoluminescence spectra were
collected on a Shimadzu RF-5301PC spectrophotometer and Maya 2000Pro optical fiber
spectrophotometer. PL efficiencies were measured with an integrating sphere (C-701, Labsphere
Inc.), with a 365 nm Ocean Optics LLS-LED as the excitation source, and the laser was
introduced into the sphere through the optical fiber. Transmission electron microscopy (TEM) of the

sample were performed using a TECNAI F20 microscope. The samples were prepared by placing
microdrops of the solution on a holey carbon copper grid. Electrospray ionization and time-of-flight
analyzer (ESI-TOF) mass spectra were recorded by both high-resolution electrospray ionization
time-of-flight mass spectrometry equipped with high performance liquid chromatography (Agilent
1200HPLC-micrOTOFESI-TOF-MS, and C18 columns was applied). Elemental analysis was obtained
using a Flash EA1112 analyser. Fluorescence microscopy images were taken on Nikon Eclipse Ti-S
inverted fluorescence microscope.
2.2 Synthesis
2.2.1 Synthesis of TPA-BDP
1,3,5,7-tetramethyl-8-phenyl-BODIPY
(4,4-Difluoro-8-phenyl-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)
and
1
(4,4-Difluoro-8-phenyl-2,6-diiodo-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)
were
synthesized by using literature procedures [45-47]. TPA-BDP was synthesized from
1 as
follows. A mixture of
1 (576 mg, 1.0 mmol), (4-(diphenylamino)phenyl)boronic acid (700 mg,
2.4 mmol) and Pd(PPh3)4 (60 mg, 0.05 mmol) was dissolved in THF (40 mL) and CH3OH (10
mL) under nitrogen atmosphere. Aqueous potassium carbonate solution (2 M, 10 mL) was added
to the reaction solution and stirred at 80 °C for 24 h. After cooling to room temperature, the
reaction mixture was extracted by dichloromethane (3 × 50 mL). The combined organic layer
was washed with water and dried over Na2SO4. After removal of the solvent, the crude product
was purified by column chromatography on silica gel (eluent: ethyl acetate /petroleum ether (1:1,
1
v/v) to obtain TPA-BDP (664 mg, 82% yield) as a red solid. H NMR (500 MHz, CDCl3), δ
(ppm): 7.48 (t, J = 8.0 Hz, 3H), 7.36 (d, J = 8.0 Hz, 2H), 7.28 (t, J = 8.0 Hz, 8H), 7.12 (d, J = 7.5
Hz, 8H), 7.07 (d, J = 8.5 Hz, 4H), 7.02 (t, J = 7.5 Hz, 8H), 2.53 (s, 6H), 1.34 (s, 6H). 13C NMR
(125 MHz, CDCl3), δ (ppm): 12.88, 13.49, 123.01, 124.54, 127.43, 128.12, 129.00, 129.22,
129.29, 130.87, 131.35, 135.55, 146.73, 147.66. ESI-MS: Calcd for C55H45BF2N4 (M+), m/z
810.37; found 810.33. Anal. Calcd. for C55H45BF2N4: C 81.48, H 5.59, N 6.91. Found C 81.47,

H 5.57, N 6.94. Crystals for X-ray analysis were grown by slow evaporation of a solution of
TPA-BDP in a mixture of CH2Cl2 and CH3OH.
2.2.2 Synthesis of Ph-BDP
Ph-BDP
,
was
synthesized
by
using
the
above
procedure
except
that
(4-(diphenylamino)phenyl)boronic acid was replaced by phenylboronic acid. Ph-BDP was
1
obtained as a red solid (362 mg, 76% yield). H NMR (500 MHz, CDCl3), δ (ppm): 7.50 (t, J =
7.5 Hz, 2H), 7.47 (t, J = 6.0 Hz, 1H), 7.39 (d, J = 6.0 Hz, 3H), 7.36 (d, J = 8.5 Hz, 3H), 7.29 (t, J
= 6.5 Hz, 2H), 7.16 (t, J = 7.5 Hz, 4H), 2.54 (s, 6H), 1.31 (s, 6H). 13C NMR (125 MHz, CDCl3),
δ (ppm): 12.73, 13.39, 127.07, 128.06, 128.31, 129.04, 129.24, 130.19. ESI-MS: Calcd for
C31H27BF2N2 (M+), m/z 476.22; found 499.21 (M+Na)+. Anal. Calcd. for C31H27BF2N2: C 78.16,
H 5.71, N 5.88. Found C 78.17, H 5.69, N 5.89.
2.3 X-ray crystallography
For the crystal structure of TPA-BDP, the data collection was performed on a Bruker Smart Apex II
CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71069 Å) at room
temperature. All absorption corrections were performed by using the SADABS program. The crystal
structure was solved by direct methods of SHELXTL-97 [48] and refined by full-matrix least-squares
techniques using SHELXTL-97 within WINGX. All hydrogen atoms of the aromatic rings were
included in the structure factor calculation at idealized positions by using a riding model. The detailed
crystallographic data and structure refinement parameters are summarized in Table S1. Further details of
the crystal structure determination have been deposited to the Cambridge Crystallographic Data Centre
as supplementary publication. CCDC 1522662 (TPA-BDP) contains the supplementary crystallographic
data for this paper.
2.4 Theoretical calculations
The electronic structures of the ground states of TPA-BDP were investigated by performing
density functional theory (DFT) calculations at the B3LYP level. The 6-31G** basis sets were

employed to optimize the C, H and N atoms. All calculations were performed with the D.01
version of the Gaussian 09 program package [49].
2.5 UV-vis and fluorescence (FL) spectra
The stock solution of TPA-BDP was prepared in different solvents (10 μM) for
solvatochromism experiments. In AIE tests, the stock solution of TPA-BDP was prepared in
CH3CN (10 μM) and deionized water was added with different water fractions (0-90 vol%).
For selectivity measurements, all the anions were used as their tetrabutylammonium salts. The
test samples were prepared in CH3CN:H2O (200 μM, 1:4 v/v). Equal volumes of different anions
stock solution were added to TPA-BDP solutions (20 μM). The concentrations of the anions
were 100 µM for TPA-BDP, respectively. Then the absorbance of the solutions of different
compositions were recorded. The absorption and fluorescence measurements of the resulting
solutions were then performed immediately.
For sensitivity measurements, stock solutions of TPA-BDP were prepared in CH3CN:H2O (10
μM, 1:4 v/v). The F− salt was dissolved in CH3CN:H2O (1:4 v/v). The titration experiments were
carried out by taking 10 μM TPA-BDP with the increasing concentration of F−. The absorption
and fluorescence measurements of the resulting solutions were then performed immediately.
For competition measurements, equal concentrations of other anions and F− were simultaneously
added to TPA-BDP solutions. The absorption and fluorescence measurements of the resulting
solutions were then performed immediately.
For calculation of LOD, the FL intensity (value of the emission maxima) was plotted as a
function of F- concentration for determination of the limit of detection (LOD). LOD values were
calculated by standard deviation of the measured value of the emission maxima of a blank
solution for eleven times.
2.6 Cell culture

HeLa cells were propagated to confluence in Dulbecco's Modified Eagle Medium (DMEM)
supplemented with 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 10% FBS, and
maintained at 37 oC in a humidified atmosphere of 5% CO2 for further cell experiments.
2.7 Cell viability analysis
Cells harvested in a logarithmic growth phase were seeded in 96-well plates at a density of
8×103 cells per well and incubated in DMEM for 24 h. The medium was then replaced by 200
μL of DMEM containing predetermined concentrations of TPA-BDP (0, 2.5, 5, 12.5, 25, 37.5
and 50 µg mL-1), and then incubated for 24 h, followed by MTT assays to measure the live cells.
Cell viabilities were determined by reading the absorbance of the plates at 490 nm with a
microplate reader. The cells incubated with DMEM were used as the control. The cell viability
(%) =A sample / A control ×100%.
2.8 Cell imaging
Cells harvested in a logarithmic growth phase were seeded in 6-well plates at a density of
2.5×105 cells/well and incubated in DMEM for 24 h. The medium was then replaced by DMEM
(2 mL) containing TPA-BDP (8.1 µg mL-1) and incubated for 1 h at 37 °C. After that, the cells
were washed with phosphate buffer saline (PBS) three times and then supplemented with 50
−
equiv. of F− (130.5 µg mL-1), Ac− (159 µg mL-1), Cl− (139 µg mL-1) and PF6 (193.5 µg mL-1) at
37 °C for another 1 h, respectively. Later, the cells were washed with PBS and observed using
an inverted fluorescence microscope. The cell samples incubated with only TPA-BDP (8.1 µg
−
mL-1), only F− (130.5 µg mL-1), only Ac− (159 µg mL-1), only Cl− (139 µg mL-1) and only PF6
(193.5 µg mL-1) were also measured as the controls.
3 Results and discussion
3.1 Synthesis and characterization
The syntheses of target TPA-BDP and the model analog Ph-BDP are shown in Scheme 1. In the first
step, 1,3,5,7-tetramethyl-8-phenyl-BODIPY was synthesized by using literature procedures [45, 46] and

then treated with iodine and iodic acid to produce BDP intermediate 1 [47]. Palladium-mediated Suzuki
cross-coupling reaction between 1 and 4-(diphenylamino)phenylboronic acid or 4-phenylboronic acid
gave TPA-BDP or Ph-BDP, respectively. The identity of the products was established by NMR, mass
spectra and elemental analysis. Additionally the single-crystal X-ray structure of TPA-BDP was
obtained (Fig. 1 and Table S1). The torsion angles between the phenyl rings of triphenylamine in
TPA-BDP are 34.4 °, 44.7°, 49.4°, 30.8°, 44.5° and 57.5° (Fig. 1a). It is shown that three kinds of
C–H···F intermolecular interactions exist in the molecular packing of crystalline TPA-BDP (Fig. 1b).
The network of C–H···F intermolecular interactions is expected to contribute to rigidifying the
molecular conformation when the molecule aggregates.
Insert Fig. 1
Fig. 1 (a) The molecular structure of TPA-BDP in the crystal. The H atoms and solvent molecules are
omitted for clarity. (b) The molecular packing in the crystal structure of TPA-BDP showing a network
of intermolecular C–H···F interactions. (Colour code for heteroatoms: N blue; F green; B brown).
3.2 Photophysical properties
When photoexcited in acetonitrile, 1,3,5,7-tetramethyl-8-phenyl-BODIPY (BDP) emits green light (Fig.
S5c) with high fluorescence quantum yield (ΦF = 92%, Scheme 1). The related model compound
Ph-BDP with phenyl rings at the 2, 6 and 8 positions emits with a much lower fluorescence quantum
yield about 50.4% in solution. In marked contrast, under the same conditions TPA-BDP emits
extremely weak red light with ΦF = 0.3% as the rotational movement of the phenyl rings of the TPA
units nonradiatively deactivates its excited state to a large extent [50]. In order to study the electron
transfer process of TPA-BDP, UV-vis absorption spectra, emission spectra and corresponding
excitation spectra of TPA and BDP have been investigated, respectively (Fig. S5-S6). TPA shows a
longer fluorescence lifetime (3.54 ns) compared to BDP (1.54 ns) and TPA-BDP (1.48 ns). Moreover,
the overlap between the absorption spectra of BDP and the emission spectra of TPA indicates the
presence of electron transfer process from TPA to BDP (Fig. S7). The fluorescence emission of
TPA-BDP in different solvents was also investigated (Fig. 2). Dual emission peaks derived from

locally-excited (LE) and TICT states at 412 nm (Fig. 2, insert) and 598 nm, respectively, were
simultaneously observed in a single spectrum in nonpolar hexane solvent upon excitation at 365
nm. Since the dual emission features of TPA-BDP in solution was observed, thus, the excitation
wavelength corresponding to the short-wavelength emission was selected for photophysical
measurement to make sure both emission peaks can be observed (Fig. S8). With increasing
solvent polarity, the short-wavelength emission band is nearly no shift, so it is assigned to a LE
process from BDP (Fig. 2, insert). The long-wavelength emission gradually shifts from λmax 598
to 668 nm and the fluorescence quantum yield decreases dramatically from 47.9% (hexane) to
∼
0.3% (acetonitrile) (Fig. 2). The color shift with solvent polarity is caused by an intramolecular
charge transfer (ICT) process. The value of fluorescence quantum yield should be significantly
smaller in polar vs nonpolar solvents, since the propensity of TICT state formation is larger in
more polar solvents [51]. While electronic excitation generally planarizes the system and thus
increases the charge-delocalization interactions, discrepancies have been found for some D-A
systems, which undergo torsional motion about the D-A bond and lead to a charge-separated
twisted ICT (TICT) state [51]. TPA-BDP has a twisted conformation due to the three-bladed
propeller-like structure of the triphenylamine units and the twisted conformation will be
stabilized in polar solvents, which consequently red-shift the emission spectra. In addition to the
bathochromic shift, the emission intensity is decreased as the TICT state is affected by various
nonradiative quenching processes [52]. There is precedent that the emission in the TICT state
can be modulated by AIE with a simple physical process in aqueous media [43].
Insert Fig. 2

Fig. 2 The fluorescence spectra of TPA-BDP (10 μM) in different solvents in long-wavelength band at
room temperature. Inset: The fluorescence spectra of TPA-BDP (10 μM) in different solvents in
short-wavelength band at room temperature, excited at 365 nm.
To investigate the AIE property, the fluorescence spectra of TPA-BDP in CH3CN/water mixtures
with different fractions of water (fw) are shown in Fig. 3. TPA-BDP is fluorescent in CH3CN (10 µM)
upon excitation at 365 nm. Dual emission peaks derived from LE and TICT states at 412 and 668 nm,
respectively. The corresponding absorption spectrum is shown in Fig. S9. TPA-BDP in CH3CN solution
displayed three absorption peaks centered at 301, 432 and 541 nm. The data are summarised in Table 1.
In polar solvent CH3CN, the emission around λmax 668 nm of the TPA-BDP is from the TICT state that
dominates its fluorescence spectra. Addition of large amounts of poor solvents such as water into the
solution of the TPA-BDP induces the molecules to aggregate, which boosts their TICT emission
through the AIE process. The emission intensity at 668 nm increased dramatically with increasing the
water content, indicating that TPA-BDP is AIE-active (Fig. 3a). However, the fluorescence intensity
decreased when the water fraction was higher than 60% (Fig. 3b). This result can be interpreted as the
simultaneous formation of crystalline particles and amorphous particles when the fw > 60% [53]. The
emission of TPA-BDP in the CH3CN/water mixture (fw = 60%) was similar to the solid-state emission
of TPA-BDP located at λmax 665 nm (Fig. S10). Transmission electron microscopy (TEM) experiments
showed that the TPA-BDP is uniformly dispersed in the CH3CN solution, while nanoaggregates are
formed in CH3CN/water mixtures with 60% water fraction (Fig. S11). Thereby, aggregation restricts the
intramolecular rotation associated with the donor moiety, inducing a great enhancement in the emission
efficiency (Fig. S12).
Insert Fig. 3
Fig. 3 (a) The fluorescence spectra of TPA-BDP (10 μM) in CH3CN/water mixtures with different
water fractions (0–90% v/v) at room temperature, excited at 365 nm; (b) Emission peak intensity of
TPA-BDP (10 μM) in CH3CN/water mixtures with different fws.

Insert Table 1
Table 1 Detailed photophysical data of TPA-BDP in CH3CN solution (10 µmol).
In contrast, Ph-BDP exhibited significant ACQ as expected (Fig. S13). Ph-BDP showed strong
fluorescence in CH3CN solution as the methyl groups exert steric hindrance on the rotational motion of
the phenyl rings at the 2 and 6 positions, thus effectively reducing the energy loss from nonradiative
decay. Its emission intensity is gradually decreased with increasing water content. This confirms that the
AIE property of TPA-BDP originate from the intramolecular rotation associated with the TPA units.
3.3 DFT calculations
To gain further understanding the photophysical and AIE properties, the geometry of TPA-BDP
was optimized in dilution dichloromethane solution with reference to the X-ray structural data
using density functional theory (DFT) methods. The data are given in Tables S2 and S3. Table
S2 shows that the major molecular orbitals involved in the main absorption transitions are
HOMO-2, HOMO and LUMO, which are exhibited in Fig. S14. The 540 nm absorption band is
attributed to electron transfers from the TPA to BDP unit for S1 excitation, which is inseparable
from the donating and accepting electron ability of TPA and BDP, respectively. The absorption
band at 432 nm shows a π→π* transition on the BDP unit. The corresponding calculated
emission data listed in Table S3 indicate the dual emission peaks derived from locally-excited
(LE) and TICT states.
According to the Huang-Rhys (HR) factor equation, namely Si = (ωiDi2)/2ћ, we calculated
the HR factors for TPA-BDP (Fig. S15). Since HR factor Si is calculated in terms of the
displacement in the normal mode coordinate Di, it can characterize the geometrical modification
resulting from vibrations when a molecule changes from one electronic state to another, which is
important for the internal conversion rate. It is clearly seen from Fig. S15 that most of the large
HR factors distribute in the low frequency region, which indicates that molecular rotational
vibrations make important contributions to the internal conversion, since rotational vibrations

mostly appear for the low frequency modes. It can be further proved by the vibration analysis of
the modes at 114.67 and 127.04 cm-1 with the larger HR factors in Fig. S16. It was shown that
both the vibrations are mainly derived from the rotational vibration of the three phenyl rings of
TPA and the four methyl groups. These rotations are generally easy to restrict by the molecular
packing or aggregation, and thus the nonradiative decay is reduced and luminescence is
enhanced in the aggregated state.
3.4 Detection of F−
With its primary AIE property, we investigated the ability of TPA-BDP for anion detection in
CH3CN:H2O (1:4 v/v) (10 μM) at room temperature. The solutions of anions (F−, Cl−, Br−, I−,
−
2−
−
−
Ac−, HSO4 , HPO4 , PF6 , ClO4 , CN−) were prepared from their tetrabutylammonium salts.
Free TPA-BDP exhibited high fluorescence intensity in the solution at λmax 668 nm (Fig. 4a).
There is no significant change in the emission spectrum for addition of 10 equiv. of other anions
except for F−. It is interesting to observe that the fluorescent intensity significantly decreased at
668 nm in the presence of F−, which is about 200 times lower than that of the blank solution of
TPA-BDP. This means that the TICT emission might be disrupted by interaction of TPA-BDP
specifically with F−. Its luminescence nearly disappeared in the presence of F− but showed only
little change for other anions, indicating that TPA-BDP is highly selective for detecting F−.
Insert Fig. 4
Fig. 4 (a) The fluorescence spectra of TPA-BDP (10 μM) in the presence of different anions (1 - blank,
−
2−
−
−
2 - F−, 3 - Ac−, 4 - Br−, 5 - Cl−, 6 - ClO4 , 7 - HPO4 , 8 - HSO4 , 9 - I−, 10 - PF6 , 11 - CN−)(10 equiv.)
in CH3CN:H2O (1:4 v/v), excited at 365 nm. (b) UV-Vis absorption spectra of TPA-BDP (10 μM) in
the presence of different anions (10 equiv.) in CH3CN:H2O (1:4 v/v) solution at room temperature.
To further study the TPA-BDP sensing behavior, its absorption spectra changes upon addition of
−
various anions were investigated. The solutions with 10 equiv. of anions (F−, Cl−, Br−, I−, Ac−, HSO4 ,
2−
−
−
HPO4 , PF6 , ClO4 , CN−) were added to CH3CN:H2O (1:4 v/v) solutions of TPA-BDP (10 µM) at

room temperature. Evident changes of the absorption spectrum induced by F− were observed, whereas
the other anions gave only a negligible change in the spectrum (Fig. 4b). Upon addition of F−, the
original absorption band of TPA-BDP at 540 nm sharply decreased and slightly red-shifted to 551 nm.
The band at 432 nm disappeared, implying new species with less conjugation were formed. This is
consistent with the fluorescence spectra (Fig. 4a), further proving that TPA-BDP is suitable for selective
detection of F−.
Insert Fig. 5
Fig. 5 (a) UV-Vis absorption spectra of TPA-BDP (10 μM) upon addition of different amounts of F−
anion (0, 1, 3, 5, 7, 9, 10, 20, 30, 40, 50, 60, 80, 100 µM) in CH3CN:H2O (1:4 v/v) solution; (b) The
corresponding fluorescence spectra at 668 nm, excited at 365 nm.
In order to evaluate the sensing properties of TPA-BDP, UV-vis spectrophotometric
titrations were carried out by adding standard solutions of F− to the CH3CN:H2O (1:4 v/v)
solutions of TPA-BDP (10 μM). With the addition of an increasing concentration of F−, the
absorption band at 540 nm decreased gradually and slightly red-shifted to 551 nm (Fig. 5a). And
the F− sensing property was further investigated through detailed fluorescent titration studies.
The emission at 668 nm decreased gradually until nearly disappearing (Fig. 5b), in agreement
with the absorption response. These data suggest that the structure of TPA-BDP and
consequently the TICT emission is disrupted by the addition of F−.
Insert Fig. 6
Fig. 6 Plot of the emission intensity of TPA-BDP system in the presence of different anions: TPA-BDP
(10 µM) + F− (100 µM) + other anions (100 µM), where other anions = (1 - Ac−, 2 - Br−, 3 - Cl−, 4 -
−
2−
−
−
ClO4 , 5 - HPO4 ,6 - HSO4 , 7 - I−, 8 - PF6 , 9 - CN−) in CH3CN:H2O (1:4 v/v) solution, excited at 365
nm.

It is important to note that TPA-BDP retained the sensing response to F− even in the
presence of other anions. TPA-BDP in CH3CN:H2O (1:4 v/v) solutions (10 μM) were treated
−
2−
−
with 10 equiv. of F− in the presence of 10 equiv. of Cl−, Br−, I−, Ac−, HSO4 , HPO4 , PF6 ,
−
ClO4 , CN−. Even in the presence of these anions, the fluorescence intensities of TPA-BDP with
F− were quenched immediately (Fig. 6). This indicates that the effect of competitive anions on
TPA-BDP detection of F− is slight. This confirms that TPA-BDP selectively interacts with F−,
implying that these anions' affinity with TPA-BDP are not comparable with F− in aqueous
conditions. The limit of detection (LOD) of F− is as low as 0.73 μM (0.191 mg/L) which is
determined from a plot of normalized fluorescence intensity as a function of the concentration of
added F− (Fig. S19) [54]. This value is much lower than the maximum contaminant level (MCL)
defined by the U.S. Environmental Protection Agency (EPA) for fluoride in drinking water (2
mg/L) [55]. Therefore, TPA-BDP shows a highly sensitive and selective detection of F− in the
presence of different anions, which is sufficient to detect F− pollution in Eco-water.
Insert Scheme 2
Scheme 2 A possible scheme for the reaction between TPA-BDP and F−.
3.5 Mechanism study
To understand the highly selective fluorescence quenching of TPA-BDP upon addition of F−, 1H
19
NMR, F NMR, 11B NMR and MS analyses were conducted. It seems that nucleophilic
displacement favors happening at the hybridized boron center, and fluoride is likely to disrupt
the ring by complexing with the central boron atom [56]. So the quenching response of
TPA-BDP with F− can be ascribed to nucleophilic displacement of the sp3 hybridized boron
atom in the center of BDP, this proposed mechanism is illustrated in Scheme 2. As shown in Fig.
1
7, a singlet at 7.22 ppm in the H NMR spectrum assigned to the N-H proton was present after

addition of tetrabutylammonium fluoride (TBAF) to TPA-BDP. The singlet disappeared on
adding one drop of D2O, consistent with an exchangeable N-H signal. Similarly, there were
obvious changes in the 19F NMR spectra upon addition of TBAF (Fig. 8).
1-Iodo-3-(trifluoromethyl)benzene was added as an internal standard. A new peak at −153.50
−
ppm in the 19F NMR spectra is the same as [(n-Bu)4N]+BF4 , which proves the formation of the
−
11
BF4 anion in the solution. Furthermore, corresponding B NMR spectra was also investigated
(Fig. S20). Upon addition of F− anions, the triplet signal at 1.0 ppm shifted to the 0.8 ppm,
which can be assigned to a partially coordinated BF3 unit [57]. The new singlet peak at -1.0 ppm
−
−
is corresponding to free BF4 anions. Moreover, the negative-ion mass spectrum shows that BF4
anions are generated in the solution after the addition of TBAF (Fig. S21). The peak at m/z
810.33 in mass spectrum is correspond to TPA-BDP (Fig. S22a). When adding TBAF in the
solution, the peak at m/z 810.33 disappears and a new peak at m/z 764.81 appears (Fig. S22b).
The new peak corresponds to TPA-DP, coming from the damaged structure of TPA-BDP by the
nucleophilic reaction between F− and TPA-BDP. Furthermore, mass spectrometry experiments
1
and H NMR of BDP
(
Ph-BDP) and BDP
(
Ph-BDP) with F− in CH3CN/CDCl3 have also been
1
performed, as shown in Fig. S23-S26. Mass spectrometry and H NMR results showed that the
BF2 unit bridging two pyrroles are partially detached when added with F− [57]. The detection of
F− anions cannot be carried out with both BDP and Ph-BDP compounds due to their native
ACQ properties in aqueous media. Therefore, the fluorescence quenching mechanism of
TPA-BDP response to F− is identified as: (1) nucleophilic addition of F−; (2) detachment of the
stable difluoroboron bridges; (3) breaking the B-N bond and yielding new B-F and N-H bonds.
The D-A-D structure of TPA-BDP and the TICT emission are disrupted, resulting in the
quenching of the fluorescence of TPA-BDP in the presence of F−.
Insert Fig. 7

Fig. 7 1H NMR spectra of TPA-BDP
,
TPA-BDP + TBAF and TPA-BDP + TBAF + D2O in
CDCl3.
Insert Fig. 8
−
Fig. 8 19F NMR spectra of TPA-BDP + TBAF, TPA-BDP, [(n-Bu)4N]+BF4 and TBAF in
CDCl3.
3.6 Cellular imaging
The cytotoxicity of TPA-BDP was investigated by studying the metabolic viability of HeLa
cells after incubation with different concentrations of the TPA-BDP. As shown in Fig. 9, no
significant change for the cell viability was observed even at a concentration of 50 µg mL-1 of
TPA-BDP after 24 h which indicates that TPA-BDP is biocompatible with living cells. This
suggests that TPA-BDP has a very low cytotoxicity and enables its application as a fluorescent
probe for imaging in live cells.
Insert Fig. 9
Fig. 9 Cell viabilities of HeLa cells after incubation with different concentrations of TPA-BDP
(0, 2.5, 5, 12.5, 25, 37.5 and 50 µg mL-1) for 24 h.
The fluorescence imaging of F− detection using TPA-BDP was studied in living cells using
scanning confocal microscopy (Fig. 10). Human HeLa cells were incubated with TPA-BDP (8.1
µg mL-1) in Dulbecco's Modified Eagle Medium (DMEM) for 1 h at 37 °C. Another sample of
HeLa cells was incubated with TPA-BDP (8.1 µg mL-1) for 1 h at 37 °C and then supplemented
with F− (130.5 µg mL-1) at 37 °C for another 1 h, and washed with phosphate buffer saline (PBS)
to remove the remaining compound. Bright intracellular fluorescence was observed with
TPA-BDP (Fig. 10a) whereas no intracellular fluorescence can be seen after addition of F− (Fig.

10c). The marked quenching in intracellular fluorescence suggests that TPA-BDP can permeate
the cell membrane and respond to the presence of F− in living cells. As shown in Fig. 10b and
10d, the bright-field measurements suggest that the cells are viable upon treatment with both
TPA-BDP and F− throughout the imaging experiments. The contrast experiments were carried
out to ensure that only the F− is responsible for the fluorescence quenching (Fig. S27-S29). After
−
addition of Ac−, Cl− and PF6 to HeLa cells under the same experimental conditions, no
intracellular fluorescence quenching were observed. Thus, these are promising initial
proof-of-concept results and TPA-BDP has been identified as a potential probe for F− in living
cells.
Insert Fig. 10
Fig. 10 (a) Fluorescence and (b) bright-field images of HeLa cells cultured in the presence of TPA-BDP
(8.1 µg mL-1) in DMEM buffer at 37 oC for 1 h; (c) fluorescence and (d) bright-field images of the HeLa
o
cells cultured in the presence of TPA-BDP (8.1 µg mL-1) in DMEM buffer at 37 C for 1 h and TBAF
(130.5 µg mL-1) in DMEM buffer at 37 oC for an additional 1 h. The scale bar is 50 µm.
4. Conclusions
In summary, a novel BODIPY-based probe TPA-BDP has been designed and synthesized using
triphenylamine and 1,3,5,7-tetramethyl-8-phenyl-BODIPY as the electron donating and accepting units,
respectively. The D-A-D structure and twisted molecular conformation endow TPA-BDP with both AIE
and TICT properties simultaneously, as observed in photophysical experiments and supported by DFT
calculations. Obvious fluorescence quenching is observed upon the addition of F− to the solution of
TPA-BDP in CH3CN:H2O (1:4 v/v). The system shows a high selectivity for detection of F− with
negligible influence from other anions. The very low limit of detection of F− by TPA-BDP [0.73 μM
(0.191 mg/L)] indicates that this dye can detect F− in Eco-water. The fluorescence quenching of
TPA-BDP can be explained as that a nucleophilic reaction between F− and TPA-BDP at the BF2 bridges
damages the molecular structure and disrupts the TICT emission. Cellular imaging experiments indicate

that TPA-BDP has a very low cytotoxicity and could be utilized as a biocompatible fluorescent probe
for HeLa cells. From these results we can conclude that specifically functionalized BODIPY derivatives
with AIE properties are promising candidates in the development of novel probes for F− in biosystems.
Acknowledgments
The work was funded by NSFC (No.51473028), the key scientific and technological project of Jilin
province (20150204011GX, 20160307016GX), the development and reform commission of Jilin
province (20160058). Martin R. Bryce (Durham University, UK) is thanked for helpful discussions.
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Figure Captions
Scheme 1. Synthesis of the target TPA-BDP and Ph-BDP.
Fig. 1 (a) The molecular structure of TPA-BDP in the crystal. The H atoms and solvent molecules are
omitted for clarity. (b) The molecular packing in the crystal structure of TPA-BDP showing a network
of intermolecular C–H···F interactions. (Colour code for heteroatoms: N blue; F green; B brown).
Fig. 2 The fluorescence spectra of TPA-BDP (10 μM) in different solvents in long-wavelength band at
room temperature. Inset: The fluorescence spectra of TPA-BDP (10 μM) in different solvents in
short-wavelength band at room temperature, excited at 365 nm.
Fig. 3 (a) The fluorescence spectra of TPA-BDP (10 μM) in CH3CN/water mixtures with different
water fractions (0–90% v/v) at room temperature, excited at 365 nm; (b) Emission peak intensity of
TPA-BDP (10 μM) in CH3CN/water mixtures with different fws.
Table 1 Detailed photophysical data of TPA-BDP in CH3CN solution (10 µmol).
Fig. 4 (a) The fluorescence spectra of TPA-BDP (10 μM) in the presence of different anions (1 - blank,
−
2−
−
−
2 - F−, 3 - Ac−, 4 - Br−, 5 - Cl−, 6 - ClO4 , 7 - HPO4 , 8 - HSO4 , 9 - I−, 10 - PF6 , 11 - CN−)(10 equiv.)
in CH3CN:H2O (1:4 v/v), excited at 365 nm. (b) UV-Vis absorption spectra of TPA-BDP (10 μM) in the
presence of different anions (10 equiv.) in CH3CN:H2O (1:4 v/v) solution at room temperature.
Fig. 5 (a) UV-Vis absorption spectra of TPA-BDP (10 μM) upon addition of different amounts of F−
anion (0, 1, 3, 5, 7, 9, 10, 20, 30, 40, 50, 60, 80, 100 µM) in CH3CN:H2O (1:4 v/v) solution; (b) The
corresponding fluorescence spectra at 668 nm, excited at 365 nm.
Fig. 6 Plot of the emission intensity of TPA-BDP system in the presence of different anions: TPA-BDP
(10 µM) + F− (100 µM) + other anions (100 µM), where other anions = (1 - Ac−, 2 - Br−, 3 - Cl−, 4 -

−
2−
−
−
ClO4 , 5 - HPO4 ,6 - HSO4 , 7 - I−, 8 - PF6 , 9 - CN−) in CH3CN:H2O (1:4 v/v) solution, excited at 365
nm.
Scheme 2. A possible scheme for the reaction between TPA-BDP and F−.
Fig. 7 1H NMR spectra of TPA-BDP, TPA-BDP + TBAF and TPA-BDP + TBAF + D2O in CDCl3.
−
Fig. 8 19F NMR spectra of TPA-BDP + TBAF, TPA-BDP, [(n-Bu)4N]+BF4 and TBAF in CDCl3.
Fig. 9 Cell viabilities of HeLa cells after incubation with different concentrations of TPA-BDP (0, 2.5,
5, 12.5, 25, 37.5 and 50 µg mL-1) for 24 h.
Fig. 10 (a) Fluorescence and (b) bright-field images of HeLa cells cultured in the presence of TPA-BDP
(8.1 µg mL-1) in DMEM buffer at 37 oC for 1 h; (c) fluorescence and (d) bright-field images of the HeLa
o
cells cultured in the presence of TPA-BDP (8.1 µg mL-1) in DMEM buffer at 37 C for 1 h and TBAF
(130.5 µg mL-1) in DMEM buffer at 37 oC for an additional 1 h. The scale bar is 50 µm.

Scheme 1. Synthesis of the target TPA-BDP and Ph-BDP.
Fig. 1 (a) The molecular structure of TPA-BDP in the crystal. The H atoms and
solvent molecules are omitted for clarity. (b) The molecular packing in the crystal
structure of TPA-BDP shows a network of intermolecular C–H···F interactions.
(Colour code for heteroatoms: N blue; F green; B brown).

Fig. 2 The fluorescence spectra of TPA-BDP (10 μM) in different solvents in
long-wavelength band at room temperature. Inset: The fluorescence spectra of
TPA-BDP (10 μM) in different solvents in short-wavelength band at room
temperature, excited at 365 nm.
Fig. 3 (a) The fluorescence spectra of TPA-BDP (10 μM) in CH3CN/water mixtures
with different water fractions (0–90% v/v) at room temperature, excited at 365 nm;
(b) Emission peak intensity of TPA-BDP (10 μM) in CH3CN/water mixtures with
different fws.

Table 1 Detailed photophysical data of TPA-BDP in CH3CN solution (10 µmol).
λabs
(nm)
λem
(nm)
ΦF
(％)
τ
(ns)
kr
knr
(×107 s-1) (×107 s-1)
301, 432,
541
412,
668
2.4,
0.3
1.45,
1.48
1.66,
0.20
67.31,
63.36
TPA-BDP
The radiative kr and non-radiative knr values in neat film were calculated according to
the equations: kr = Φ/τ and knr = (1 - Φ)/τ, from the quantum yields ΦF and the
lifetime τ values.
Fig. 4 (a) The fluorescence spectra of TPA-BDP (10 μM) in the presence of different
−
2−
−
anions (1 - blank, 2 - F−, 3 - Ac−, 4 - Br−, 5 - Cl−, 6 - ClO4 , 7 - HPO4 , 8 - HSO4 , 9 -
−
I−, 10 - PF6 , 11 - CN−)(10 equiv.) in CH3CN:H2O (1:4 v/v), excited at 365 nm. (b)
UV-Vis absorption spectra of TPA-BDP (10 μM) in the presence of different anions
(10 equiv.) in CH3CN:H2O (1:4 v/v) solution at room temperature.