An aggregation-induced phosphorescent emission-active iridium(III) complex for fluoride anion imaging in living cells

Article abstract of DOI:10.1016/j.jorganchem.2020.121644

Fluoride anion plays a crucial role in bone and tooth growth. However, overdose of fluoride anion can also lead to many chronic diseases and there is no specific medicine for fluorosis so far. Hence, the early detection and accurate estimation of fluoride

Full text of DOI:10.1016/j.jorganchem.2020.121644

Journal of Organometallic Chemistry 932 (2021) 121644
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
An aggregation-induced phosphorescent emission-active iridium(III)
complex for fluoride anion imaging in living cells
Zejing Chena,b,‡, Jiayang Jiangb,‡, Weili Zhaob, Xiaoming Hua, Mingjuan Xieb, Feiyang Lib,
Shujuan Liub,∗∗, Qiang Zhaob,∗
a Jiangxi Key Laboratory for Nanobiomaterials, Institute of Advanced Materials (IAM), East China Jiaotong University (ECJTU), 808 Shuanggang East Main
Street, Nanchang 330013, P. R. China
b Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Nanjing
University of Posts and Telecommunications (NJUPT), 9 Wenyuan Road, Nanjing 210023, P. R. China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluoride anion plays a crucial role in bone and tooth growth. However, overdose of fluoride anion can
also lead to many chronic diseases and there is no specific medicine for fluorosis so far. Hence, the early
detection and accurate estimation of fluoride anion intake are important. In this work, an aggregation-
induced phosphorescent emission (AIPE)-active iridium(III) complex as fluoride anion probe (Ir-AF) has
been developed by incorporating N-(4-hydroxylphenyl)-rhodol protected by tert-butyldiphenylsilyl onto
cyclometalating ligand. As Ir-AF reacts with fluoride anion, the nonradiative transitions of Ir-AF would
be promoted by enhanced photoinduced electron transfer effect and intramolecular rotations, leading to
its phosphorescence quenching dramatically. Thus, Ir-AF has been used for fluoride anion measurement
in the aqueous solution by ratiometric readout. Furthermore, with the utilization of the long-lived phos-
phorescence from Ir-AF, fluoride anion sensing in living cells with high signal-to-noise ratio has been
achieved by time-resolved photoluminescence imaging. This is the first report of AIPE-active probe for
fluoride anion.
Received 12 September 2020
Revised 25 October 2020
Accepted 1 December 2020
Available online 2 December 2020
Keywords:
Iridium(III) complex
Aggregation-induced phosphorescent
emission
Fluoride anion
Probe
Time-resolved photoluminescence imaging
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
the early detection and accurate estimation of fluoride anion are of
great significance.
Fluorine is an essential trace element for human body. Owing
to its strong electronegativity, fluorine is almost in valence state of
negative one in the nature, such as fluoride anion (F−), which is
considered as the largest source to daily fluorine intake [1]. Fluo-
ride anion can promote bone growth and is closely related to the
osteoporosis and odontopathy [2,3]. In the past, the addition of flu-
oride anion to water to ensure fluorine intake is a common prac-
tice [4,5]. And it is also added to toothpastes to reduce the den-
tal cavities and strengthen the resistance of teeth to acid corro-
sion [6,7]. However, studies have shown that overintake of fluoride
anion can lead to not only mottled enamel and skeletal fluorosis
but acute gastric, nephrotoxic changes, brain and immune system
damage [8-11]. As there is no specific medicine for fluorosis so far,
To date, atomic absorption spectrometery, ion chromatogra-
phy, 19 F nuclear magnetic resonance, inductively coupled plasma
mass spectrometery, reverse phase high performance liquid chro-
matography, molecular absorption spectrometery are available ap-
proaches to detect fluoride anion [12-15]. In addition, lumines-
cence detection and imaging have attracted more and more atten-
tion because of their good selectivity, high sensitivity, superb spa-
tial resolution and relatively low cost [16-20]. By a non-invasive
way, luminescence detection and imaging could be used to detect
the target analytes in micromole or even nanomolar levels in intact
living samples in real time, which is in favor of biological processes
study. Consequently, they usually require appropriate probes.
As candidates for luminescent probes, phosphorescent
transition-metal complexes (PTMCs), especially iridium(III) sys-
tem have many advantages, such as high luminescence quantum
yields, excellent photostability, tunable emission, various sensitive
charge-transfer states [21,22]. Moreover, they possess long-lived
phosphorescence which could be easily screened out from com-
plicated environments with short-lived background fluorescence
or autofluorescence from biological samples via time-resolved
∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: iamsjliu@njupt.edu.cn (S. Liu), iamqzhao@njupt.edu.cn (Q.
Zhao).
‡
Chen and Jiang contributed equally to this work
https://doi.org/10.1016/j.jorganchem.2020.121644
0022-328X/© 2020 Elsevier B.V. All rights reserved.

Z. Chen, J. Jiang, W. Zhao et al.
Journal of Organometallic Chemistry 932 (2021) 121644
Fig. 1. Chemical structure of Ir-AF and schematic illustration for detecting fluoride anion.
photoluminescence imaging (TRPI) technique, like photolumi-
nescence lifetime imaging microscopy (PLIM) and time-gated
photoluminescence imaging (TGPI), thus achieving reliable opti-
cal detection [23]. In the past years, lots of luminescent probes
based on PTMCs have been reported [24-27]. Our research group
have also developed a series of iridium(III) complex bioprobes,
including fluoride anion probes [28-31]. However, the propensity
of PTMCs to aggregate in poor solvent sometimes may cause
severe triplet-triplet annihilation (TTA) which often occurs in the
solid state, leading to aggregation-caused quenching (ACQ). On the
contrary, aggregation-induced emission (AIE) that is an unusual
photophysical phenomenon of luminescent materials could break
the limitations that the materials are not suitable for luminescent
application in the solid state or at a high concentration [32,33].
In 2008, our group first conducted an in-depth research about the
aggregation-induced phosphorescent emission (AIPE) mechanism
based on two octahedral cyclometalated Ir(III) complexes [34].
Then, there has been a rising interest in developing AIPE-active
PTMCs and exploring their applications [35-43].
visible absorption spectra and emission spectra were conducted
with a Shimadzu UV-3600 spectrophotometer and Edinburgh FL
920 spectrophotometer, respectively. Besides, an integrating sphere
was employed to collect data of absolute quantum yields in N2
atmosphere. Luminescence lifetime of compounds were measured
through a semiconductor laser unit on Olympus Edinburgh LFS-
920 spectrometer. Time-resolved photoluminescence imaging ex-
periments were conducted on an Olympus FV1000 laser scanning
confocal microscope and the lifetime data was processed by correl-
ative and professional software provided by Becker & Hickl GmbH
Company. Moreover, cell flow cytometer analysis was carried out
with FlowSight imaging flow cytometer. The details of experimen-
tal section were shown in the supplementary data.
3. Results and discussion
3.1. Synthesis and characterization
The chemical structure of iridium(III) complex Ir-AF is shown
in Scheme 1, which was confirmed by NMR and mass spec-
tra. Briefly, the skeleton of cyclometalating HC^N ligand (4-((tert-
butyldiphenylsilyl)oxy)-N-(4-(pyridin-2-yl)phenyl)aniline) was pre-
pared in high yield via a Buchwald–Hartwig coupling reaction
of aryl trifluoromethanesulfonate with aniline derivatives [45].
Then, Ir-AF was synthesized according to the typical three-step
method for cationic iridium(III) complex [46]: the cyclometalated
iridium(III)-chlorobridged dimers (C^N)2Ir(μ-Cl)2Ir(C^N)2 were pre-
pared by refluxing iridium trichloride with cyclometalating HC^N
ligand, which was treated with 1,10-phenanthroline (N^N ligand)
without further purification, followed by counterion exchange re-
action from chloride ion to hexafluorophosphate anion. The de-
tailed synthetic routes and characterization data for complex Ir-AF
and corresponding intermediates are presented in the supplemen-
tary data.
In our recent study, N-(4-hydroxylphenyl)-rhodol moiety
grafted onto the cyclometalating ligand have been found to facil-
itate the nonradiative transitions of phosphorescent iridium(III)
complexes by the photoinduced electron transfer (PET) effect and
intramolecular rotations [44]. We speculated that introducing moi-
eties which could restrain PET effect imposed by the electron-rich
hydroxyphenyl group or alleviate intramolecular rotations may
recover the efficient phosphorescence of the related iridium(III)
complexes. In this work, an cationic iridium(III) complexes (Ir-AF)
containing tert-butyldiphenylsilyl (TBDPS) moieties on the oxygen
atom of the N-(4-hydroxylphenyl)-rhodol has been designed and
synthesized (Fig. 1). As Ir-AF possesses the characteristics of AIPE,
it has been used for fluoride anion measurement in the aqueous
solution by ratiometric readout. Importantly, utilizing the long-
lived phosphoresncence of Ir-AF, accurate fluoride anion sensing
in living cells was achieved by time-resolved photoluminescence
imaging. To the best of our knowledge, this is the first report of
AIPE-active probe for fluoride anion.
3.2. Photophysical properties
The photophysical properties of Ir-AF have been studied by
UV-vis absorption and photoluminescence spectra in different sol-
vents, and the photophysical data are summarized in Table S1
and S2. As depicted in Fig. 2a, the strong absorption bands be-
low about 310 nm are assigned to 1π–π∗ transitions of the cy-
clometalating ligands. The moderately intense absorption bands
over about 310–450 nm are attributed to the spin-allowed sin-
glet metal-to-ligand charge-transfer (1MLCT) and ligand-to-ligand
charge-transfer (1LLCT) transitions. The extremely weak absorption
bands above about 450 nm are originated from the mixed 1MLCT
2. Experimental section
All the raw materials and reagents were purchased from com-
mercial suppliers and used without further purification except
some were noted. All solvents were purified and dried under re-
flux over CaH2 and used freshly. All aqueous solutions were pre-
pared with deionized water. The structure of Ir-AF was charac-
terized by 1H NMR and autoflex matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry. The UV-
2

Z. Chen, J. Jiang, W. Zhao et al.
Journal of Organometallic Chemistry 932 (2021) 121644
Fig. 2. Absorption spectra (a) and emission spectra (b) of Ir-AF in dichloromethane, acetonitrile, dimethylsulfoxide, water (contain 10% dimethylsulfoxide) or solid state,
respectively.
and spin-forbidden triplet metal-to-ligand charge-transfer (3MLCT)
dominantly AIPE-active in aqueous solution, and the hypoxic effect
accompanying with aggregate formation could be ignored in the
air. Additionally, the photobleaching experiments in which the so-
lution is under the illumination of 405 nm laser source have been
carried out (Fig. 3f). Compared with the gradually decreasing in-
tensities of commercial dyes (Hoechst and Lyso-Tracker Red), the
phosphorescence intensity at 622 nm did not show obvious de-
crease within 20 min, suggesting that Ir-AF has a good photosta-
bility and its AIPE properties are steady.
transitions [47]. It is worth noting that the absorption spectra pro-
files of Ir-AF in three kinds of good solvents (dichloromethane, ace-
tonitrile and dimethylsulfoxide) are similar, while the absorption
in aqueous solution shows a little weaker, which may be arised
from the slight aggregation of Ir-AF in poor solvent. The photolu-
minescence spectra at ambient temperature were recorded in the
air (Fig. 2b). Upon photoexcitation at 405 nm, the emission of Ir-
AF could hardly be observed in three good solvents. In contrast,
Ir-AF displayed an intense band with the peak around 622 nm
(ꢀ = 0.045) in aqueous solution (contain 10% dimethylsulfoxide).
This featureless and broad band, which could be assigned to 3MLCT
transitions, is similar to the emission band of Ir-AF in the solid
state (ꢀ = 0.012), indicating that the phosphorescence of Ir-AF is
probably induced by aggregation.
3.4. Mechanism of fluoride anion detection
Considering that tert-butyldiphenylsilyl (TBDPS) moieties on the
oxygen atom of the phenol are often used as the candidate recog-
nition sites for fluoride anion, the response of Ir-AF toward fluo-
ride anion was evaluated preliminarily. The product extracted from
the reaction mixtures of iridium(III) complex and fluoride anion
has been studied by matrix-assisted laser desorption ionization
time-of-flight mass spectrometry. As shown in Fig. 4, the molecular
weight of products is 478 less than that of Ir-AF, which were con-
sistent with the expectation that the desilylation process occurred
in the reaction. The main products possessing phenol moieties gen-
erated in the above reactions were also verified by 1H NMR spectra
(Fig. S2). Therefore, the significantly reduced phosphorescence can
be attributed to PET process, which is consistent with the previous
reports [28,29].
3.3. Aggregation-induced phosphorescent emission
The AIPE characteristics of Ir-AF have been investigated by
emission spectra in a dimethylsulfoxide/water mixed solvent sys-
tem with different water fractions at room temperature. As shown
in the Fig. 3a, no emission band of Ir-AF was observed in dimethyl-
sulfoxide solution, and the emission intensity nearly kept constant
even when the water fraction was less than 40%. Nevertheless, the
phosphorescence intensity at around 622 nm was dramatically en-
hanced to maximum when the water fraction increases from 40%
to 50% (Fig. 3b). Dynamic light scanning (DLS) and Transmission
electron microscopy (TEM) analyses revealed an average diameter
of 72 nm for Ir-AF dispersed in above mixed solution (Fig. 3c).
These results implied that the emission of Ir-AF is indeed induced
along with aggregate formation. Notably, at the water fraction of
above 50%, the phosphorescence intensity gradually decreases as
water content rose. This phenomenon may be explained by TTA,
which was further proved by significantly increased effective di-
ameter and polydispersity index measurements in solution with
increasing water content (Fig. S1).
3.5. Fluoride anion sensing properties
The phosphorescence intensity and lifetime response of Ir-AF
to fluoride anion in dimethylsulfoxide/water (V/V = 1/9) mixed so-
lution has been studied in detail by spectrofluorometric titrations.
As shown in Fig. 5a, a gradual dose dependent phosphorescence
diminishment was observed with the sequential addition of fluo-
ride anion. When fluoride anion concentration reached 5 μM, the
phosphorescence intensity at 622 nm dropped to half compared
with those in the absence of fluoride anion. Meanwhile, the phos-
phorescence lifetime decreased from 213 ns to 168 ns (Fig. 5b).
Upon addition of 20 μM fluoride anion, the emission bands nearly
disappeared and the quenching efficiencies (defined by the equa-
tion η = (I0-I)/I0, I0 and I denote the phosphorescence intensity be-
fore and after the addition of fluoride anion, respectively.) reached
97%. The phosphorescence lifetime shortened to 113 ns accord-
ingly. Concomitantly, the phosphorescence intensity ratio (I/I0) var-
ied with an approximate linear relationship in fluoride anion con-
centration range of 0−8 μM (Fig. 5c). The related detection limita-
tion is calculated to be (2.6 × 10−2 μM), suggesting the sensitivity
To further confirm the AIPE behavior of Ir-AF and explore
whether the potential hypoxic effect formed in aggregation is the
other factor for promoting emission of Ir-AF, the emission spectra
in three good solvents in the absence and presence of O2 were in-
vestigated. As described in the Fig. 3d, the emission band of Ir-AF
in six conditions could be hardly detectable, which indicated the
emission of Ir-AF cannot be activated in good solvents even though
in the hypoxic environment. In general, the phosphorescence from
iridium(III) complexes could be quenched by oxygen molecule and
are susceptible to oxygen content in the environment, as depicted
in Fig. 3e. Thus, the results mean that the emission of Ir-AF is
3

Z. Chen, J. Jiang, W. Zhao et al.
Journal of Organometallic Chemistry 932 (2021) 121644
Fig. 3. (a) Emission spectra of Ir-AF (10 μM) in dimethylsulfoxide solution added with different fraction of water (λex = 405 nm). (b) Plot of I622 nm as a function of H2O
fraction. The insert pictures were Ir-AF in dimethylsulfoxide solution (left) and in dimethylsulfoxide /water (v/v=1:1) solution (right). (c) DLS analysis of Ir-AF in aqueous
solution. (d) Emission spectra of Ir-AF (10 μM) in dichloromethane, acetonitrile and dimethylsulfoxide saturated with air or N2, respectively. (e) Emission spectra of Ir-AF (10
μM) in aqueous solution saturated with air or N2, respectively. (f) Continuously monitoring luminescence intensities of Ir-AF at 622 nm, Hoechst at 460 nm and Lyso-Tracker
Red at 590 nm in aqueous solution within 20 min, respectively. Excitation wavelength was 405 nm.
Fig. 5. Response of Ir-AF to fluoride anion. (a) Phosphorescence response of Ir-AF
(10 μM) to different amounts of fluoride anion (0-20 μM) in aqueous solution. (b)
The plot of phosphorescence intensity ratio (I/I0) against the fluoride anion concen-
tration. (c) Phosphorescence decays of Ir-AF in different amounts of fluoride anion,
monitored at 622 nm. (d) Emission intensity of Ir-AF (10 μM) in aqueous solution
containing other anions in the absence or presence of fluoride anion within 200 s.
Excitation wavelength was 405 nm.
Fig. 4. MALDI-TOF-MS spectra of Ir-AF (a) and the MALDI-TOF-MS spectra of Ir-AF
incubated with excessive fluoride anion (b) for 5 min.
is qualified for detecting trace amounts of fluoride anion in the liv-
ing cells.
To further justify the selectivity of Ir-AF to fluoride anion, sev-
eral anions were selected as interference. As shown in the Fig. 5d,
these anions hardly affected the phosphorescence intensity of Ir-
AF, and the intensity changed remarkably only in presence of fluo-
ride anion. These phenomenon are attributed to the good stabil-
ity of Ir-AF and unique silicon-fluoride anion interactions. Addi-
tionally, the time-dependent intensity record illustrated that the
phosphorescence intensity at 622 nm reached a plateau within 120
s after treatment with 20 μM fluoride anion, meaning that the
progress of Ir-AF reacting with fluoride anion proceeded fast in
aqueous solution (Fig. S3).
4

Z. Chen, J. Jiang, W. Zhao et al.
Journal of Organometallic Chemistry 932 (2021) 121644
3.6. Fluoride anion imaging in living cells
The biocompatibility of Ir-AF for Hela cells, A549 cells and
HepG2 cells was estimated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyltetrazolium bromide (MTT) assay, respectively (Fig. S4).
When these cells were incubated with Ir-AF of 20 μM (a concen-
tration higher than that used in cell imaging) for 24 h, the cell
viabilities of three kinds of cells all kept above 70%, demonstrat-
ing that Ir-AF hardly has cytotoxicity, which can ensure its further
applications in living cells.
Subsequently, the cell imaging experiments were conducted by
confocal laser scanning microscopy. The luminescence signals from
550–650 nm were collected. As shown in Fig. S5a, the obvious lu-
minescence could be observed from three kinds of cells, which in-
dicated Ir-AF could enter into the living cells nonspecifically and
formed the aggregates in living cells. Interestingly, the emission
peak recorded by Lambda scanning model shifted from 625 to 560
nm. This may be because the polarity of intracellular environment
is different from of that of aqueous solution that pulled down the
levels of the 3MLCT of Ir-AF (Fig. S5b). Furthermore, after illumi-
nation of build-in 405 nm laser source with the power of 100 μW
for 60 s, the phosphorescence intensity from cells remains almost
no change, stating that Ir-AF is a photo-stable imaging reagents
(Fig. S6). Besides, the cells co-stained with Ir-AF and commercial
nuclear dye Hoechst 33342 have been also investigated by time-
resolved photoluminescence imaging. As shown in Fig. S7, upon
excitation at 405 nm, the luminescence collected from 430–470
nm was located in the cell nucleus and the average photolumines-
cence lifetime was about 5 ns, while the luminescence collected
from 550–650 nm distributed in the cell cytoplasm and the aver-
age photoluminescence lifetime reached around 150 ns. When 50
ns,
100 ns or 150 ns delay was exerted, only luminescence sig-
nals from the cell cytoplasm could be collected. These results illus-
trated that Ir-AF mainly gathered in cell cytoplasm and the long-
lived phosphorescence of Ir-AF can be easily distinguished from
the short-lived fluorescence of commercial fluorescent dye or back-
ground fluorescence via TRPI.
Based on the good comprehensive performance of Ir-AF, the
fluoride anion imaging and sensing in live cells were conducted.
After pretreated with Ir-AF (5 μM), Hela cells were subsequently
incubated with PBS or different concentrations of fluoride anion
for 30 min. The CLSM and TRPI images under different conditions
were shown in Fig. 6 and S8. In the control group of PBS, the lumi-
nescence intensity collected from 550–650 nm was intensive and
the average lifetime was about 150 ns. As the used fluoride anion
increased from 0 to 20 μM, the recorded average phosphorescence
intensity reduced from 2675 to 389 counts, and the average life-
time shortened from 149 to 75 ns. Accordingly, the signal intensity
decreased markedly in the TGPI images which is recorded after a
few dozens of nanoseconds delay. Meanwhile, similar quantitative
analysis of phosphorescence intensity variation by flow cytometry
was also obtained (Fig. S9). These results demonstrated that Ir-AF
could function as a reliable probe for detecting intracellular fluo-
ride anion.
Fig. 6. Confocal laser scanning images (CLSM) (a), photoluminescence lifetime im-
ages (PLIM) (b) and time-gated photoluminescence images (TGPI) (c) of living Hela
cells labeled with Ir-AF were subsequently treated with PBS, different amounts of
fluoride anion (5 μM, 10 μM and 20 μM), respectively. The orange channels in
CLSM images were acquired by collecting the photoluminescence from 550 to 650
nm and photoluminescence was collected through longpass filter (≥ 550 nm) for
PLIM and TGPI images. Excitation wavelength was 405 nm.
signal to noise ratio via time-resolved photoluminescence imag-
ing. Next, work will focus on regulating its water-dispersibility and
developing near-infrared-excited probes with aggregation-induced
phosphorescent emission for fluoride anion imaging in vivo.
Declaration of Competing Interest
4. Conclusion
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
In conclusion, a novel cationic iridium(III) complex fluoride
anion probe Ir-AF which exhibits aggregation-induced phospho-
rescent emission was designed and synthesized. Distinctively, its
working mechanism is based on the synergistic effect including en-
hancement of PET process and attenuation of AIPE effect. Owing
to its fine photostability, high sensitivity, excellent selectivity, good
biocompatibility, and long emission lifetime, Ir-AF has been used
for fluoride anion imaging and sensing in living cells with high
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21501098 and 21671108), Science and Tech-
nology Research Project of Jiangxi Provincial Department of
5

Z. Chen, J. Jiang, W. Zhao et al.
Journal of Organometallic Chemistry 932 (2021) 121644
Education (GJJ190350), Natural Science Foundation of Jiangxi
Province (20202BAB214012) and the open research fund of Key
Laboratory for Organic Electronics and Information Displays.
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Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jorganchem.2020.
121644.
Appendix A. Supplementary data
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