Red AIE-Active Fluorescent Probes with Tunable Organelle-Specific Targeting

Article abstract of DOI:10.1002/adfm.201909268

Cell staining is a fascinating research area where monitoring and visualizing different cell organelles can be done using fluorescence techniques. However, the design and synthesis of organelle-targeting fluorophores is still a challenge for several speci

Full text of DOI:10.1002/adfm.201909268

Full PaPer
www.afm-journal.de
Red AIE-Active Fluorescent Probes with Tunable Organelle-
Specific Targeting
Parvej Alam, Wei He, Nelson L. C. Leung, Chao Ma, Ryan T. K. Kwok, Jacky W. Y. Lam,
Herman H. Y. Sung, Ian D. Williams, Kam Sing Wong, and Ben Zhong Tang*
1. Introduction
Cell staining is a fascinating research area where monitoring and visualizing
Cells are fundamental building blocks for
living organisms.[1,2] Indeed, the human
body is made of trillions of cells each with
their own specific functionality.[3] Each cell
is made up of cellular organelles, such
as the plasma membrane, mitochondria,
lysosomes, Golgi apparatus, and endo-
plasmic reticulum (ER).[4] Though it has
long been known that these structures
are vital for cellular operation and conse-
quently, the whole body, further study has
revealed more detailed understanding of
their significance pertaining to specific
biochemical processes and pathways.[5–7]
Due to their nature and size, the study of
organelles, their health, and their func-
tion can be technically difficult. For a long
time, there was limited tools to study such
important structures.[8] With the develop-
ment of fluorescent bioprobes, researchers
became able to visualize the once invisible
organelles revealing important and signifi-
different cell organelles can be done using fluorescence techniques. However,
the design and synthesis of organelle-targeting fluorophores is still a challenge
for several specific organelles. Herein, a platform for synthesizing efficient
red-emitting aggregation-induced emission luminogens (AIEgens) with donor–
acceptor characteristics is reported. The core molecule can be easily functional-
ized in order to modulate organelle targeting. The three synthesized AIEgens
exhibit quantum yields of up to 39.3% and two-photon absorption cross-
section values of up to 162 GM. The two zwitterionic AIEgens, CDPP-3SO3
and CDPP-4SO3, with the sulfonate function group, are successfully utilized
for specific one-photon and two-photon imaging of the endoplasmic reticulum
(ER) in live human cells. Substituting the zwitterionic nature with a singly posi-
tive charge group, one-photon and two-photon imaging of CDPP-BzBr shows
mitochondrial specificity, indicating the importance of the zwitterionic group
for ER-targeting. Owing to the good in vitro photostability, cell viability, and
high efficiency, these red dyes serve as a good potential candidate for specific
organelle targeting, as well as illustrate how such a platform can easily aid in
the study of structure–property relationships for designing such probes.
cant insight.[9,10] Development of the bioprobes has resulted in
organelle specific staining agents allowing researchers to study
the targeted organelle.[11–15] The visualization techniques reveal
the location and morphology of the organelles, and thus enable
the study and monitoring of changes that reveal important bio-
logical states or dysfunction.[16] In particular, super-resolution
microscopy is able to provide unprecedented clarity into the
morphology of these structures.[17–19]
Dr. P. Alam, W. He, Dr. N. L. C. Leung, Dr. R. T. K. Kwok, Dr. J. W. Y. Lam,
Dr. H. H. Y. Sung, Prof. I. D. Williams, Prof. B. Z. Tang
Department of Chemistry
Hong Kong Branch of Chinese National Engineering Research Center
for Tissue Restoration and Reconstruction
Department of Chemical and Biological Engineering
The Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong 999077, China
E-mail: tangbenz@ust.hk
C. Ma, Prof. K. S. Wong
Department of Physics
The Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong 999077, China
Fluorescent probes have greatly aided the study of cellular
structures and biochemical processes, but due to the hydro-
phobic π-conjugated backbone of conventional dyes, many of
these systems suffer aggregation-caused quenching (ACQ), a
reduction in emission efficiency when the molecules interact
with each other.[20,21] The ACQ phenomenon can be particu-
larly egregious when the conventional dyes are used for bio-
imaging applications due to the highly polar nature of the
aqueous media in cells, easily leading the dye molecules to
aggregate together in even slightly higher concentrations. Thus,
typical cell staining dyes are used in nanomolar concentra-
tions to circumvent such issues. However, at such concentra-
tions, quenching due to photobleaching becomes much more
pronounced. Though researchers have come up with a variety
of ingenious methods to overcome the issue of ACQ, there
has been much work successfully incorporating molecules
Prof. B. Z. Tang
Center for Aggregation-Induced Emission
SCUT-HKUST Joint Research Institute
State Key Laboratory of Luminescent Materials and Devices
South China University of Technology
Guangzhou 510640, China
W. He, Dr. R. T. K. Kwok, Prof. B. Z. Tang
HKUST-Shenzhen Research Institute
No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen
518057, China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.201909268.
DOI: 10.1002/adfm.201909268
©
Adv. Funct. Mater. 2020, 1909268
1909268 (1 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
Figure 1. Schematic representation of specific endoplasmic reticulum (ER) targeting by zwitterionic AIEgens. Choline phosphate cytidylyltransferase
(CCT), a membrane-bound enzyme, is found on the ER and is a key enzyme involved in phosphatidylcholine synthesis.
exhibiting aggregation-induced emission (AIE) for a multitude
of applications, including cell staining, in vivo imaging, and
theranostics.[22–29] The concept of AIE describes a phenomenon
whereby emission is observed when the luminogen aggregates
with other molecules. Typically, this luminogen is originally
nonemissive or weakly emissive due to molecular motions
which allow the molecule to decay nonradiatively. Upon
aggregation or binding to molecules, such molecular motions
become restricted, forcing the molecule to decay via radiative
pathways, turning on emission.[30]
that the zwitterionic nature of the pyridinium-sulfonate group
seems to allow ER targeting, with the singly positive charged
molecule only targeting the mitochondria. The selectivity
towards the ER could be due to an abundance of the phospho-
choline cytidylyltransferase (CCT), a key enzyme in regulating
membrane phospholipid synthesis, on the ER membrane.[46–48]
As the α-helix (domain M) of CCT contains multiple positively
charged amino acids and selectively binds to anionic membrane
surfaces, we hypothesized that the zwitterionic probes might
bind to such locations due to their oppositely matching charges
via electrostatic interactions (Figure 1).[49,50]
From our previous structure–property relationship research
regarding AIE systems for cell imaging studies, we have
observed a number of molecular designs that allow for spe-
cific organelle targeting and staining: small hydrophobic sys-
tems are drawn to lipid droplets; hydrophobic molecules with
long alkyl chains target the phospholipid bilayer; positively
charged compounds stain the mitochondria due to the orga-
nelle’s membrane potential.[11,31–33] However, there have been
limited examples of molecules that are capable of staining the
ER.[34–40] The ER is a large membrane-bound compartment
spread throughout the cytoplasm of eukaryotic cells, which is
composed of one completely continuous membrane bilayer and
has a single continuous lumen. The ER plays a key role in cel-
lular metabolism, protein synthesis, and the transport of inter-
mediates and signaling molecules.[34,41–43] Characterization of
the ER structure in living cells is challenging due to a wide 3D
interconnected network of flattened, membrane-enclosed sacks
or tube-like cisterns and tubules with different thicknesses.[44,45]
Herein we designed a simple platform using an AIE-active
molecule that facilitates the rational design and synthesis of bio-
probes. One main feature of the core molecule is its donor–π–
pyridine design, since the pyridine molecule allows researchers
to easily control functionalization and thus, functionality. Upon
functionalization, the pyridine becomes a pyridinium strength-
ening the donor–acceptor properties of the molecule bathochro-
mically shifting emission to deep-red wavelengths, ideal for bio-
imaging applications, with acceptable to great quantum yields.
The ease of modification also simplifies studies regarding struc-
ture–property relationships. In our case, we were able to observe
2. Results and Discussion
2.1. Molecular Design, Synthesis, and Characterization
The synthesis of fluorescent probes with long emission
wavelength >550 nm can be achived by connecting electron-
accepting (A) units with electron-donating (D) units via
π-bridge(s), i.e., D–π–A organic systems. In our strategy, an
electron-donating group, the propeller shaped triphenylamine
(TPA) segment, was chosen as a D unit; an AIE active core, the
α-cyanostilbene, as a π-bridge; and an electron-accepting unit,
the pyridinium, as the A unit. The core molecule (Z)-4-(4-(1-
cyano-2-(4-(diphenylamino)phenyl)vinyl)phenyl)pyridin-1-ium
(CDPP) was synthesized by a two-step reaction: i) a Knoevenagel
condensation between 4-(diphenylamino)benzaldehyde and
2-(4-bromophenyl)acetonitrile, followed by ii) a Suzuki coupling
with 4-pyridine boronic acid, yielding yellow powder with a total
yield of 73% (Scheme S1, Supporting Information).[51] Finally,
the targeted fluorophores CDPP-3SO3, CDPP-4SO3, and CDPP-
BzBr, were synthesized by simple reactions between CDPP and
1,3-propane sultone, 1,4-butane sultone, and 4-bromobenzyl
bromide, respectively (Figure 2A; Scheme S1, Supporting Infor-
mation).[51–53] The chemical structures of all the synthesized
compounds were characterized by standard spectroscopic tech-
1
niques such as H NMR, 13C NMR, and high-resolution mass
spectroscopy (HRMS) (Figures S1–S5, Supporting Information).
©
Adv. Funct. Mater. 2020, 1909268
1909268 (2 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
A
B
1.2
0.9
0.6
0.3
0.0
CDPP-3SO
CDPP-BzBr
CDPP-4SO
3
3
R=
CDPP-3SO3 CDPP-4SO3
CDPP-BzBr
500
600
700
800
φ = 39.3 %
φ = 5.3 %
φ = 8.3 %
Wavelength (nm)
2.0E5
1.5E5
1.0E5
5.0E4
0.0
30
C
D
CDPP-3SO
CDPP-4SO
f
(vol %)
CDPP-3SO
3
w
3
90
80
70
60
50
40
30
20
10
0
25
20
15
10
5
3
CDPP-BzBr
0
500
600
700
800
0
20
40
60
80
100
Wavelength (nm)
Water fractions (f )
w
E
F
G
0%
90%
0%
0%
90%
90%
Figure 2. A) Molecular structures of CDPP derivatives and photographs of all three compounds under 365 nm UV excitation, including their solid-state
quantum yield (Φ). B) Fluorescence spectra of CDPP-3SO3, CDPP-4SO3, and CDPP-BzBr in solid state. C) PL spectra of CDPP-3SO3 in DMSO/water
mixtures with different water fractions (fw); concentration = 10 × 10−6 m. D) Plots of PL maximum and relative PL intensity (αAIE = I/I0) versus the com-
position of the DMSO/water mixture of CDPP-3SO3, CDPP-4SO3, and CDPP-BzBr, where I0 was the PL intensity at fw = 0%; concentration = 10 × 10−6
m, λex = 480 nm. The fluorescence photograph at fw = 0% and 90% and the corresponding SEM image of the nanoaggregates collected at fw = 90% of
E) CDPP-3SO3, F) CDPP-4SO3, and G) CDPP-BzBr; scale bar: 1 µm. The photographs were taken under 365 nm UV irradiation from a hand-held UV
lamp. [CDPP = (Z)-4-(4-(1-cyano-2-(4-diphenylamino)phenyl)vinyl)phenyl)pyridin-1-ium].
2.2. Optical Properties
The absorption spectra of CDPP-3SO3, CDPP-4SO3, and
CDPP-BzBr showed similar absorption maxima at ≈480 nm
(Figure S6A, Supporting Information). The emission spectra
of CDPP-3SO3, CDPP-4SO3, and CDPP-BzBr were found
in the range of 585 to 620 nm in DMSO (Figure S6B, Sup-
porting Information). The solid-state efficiency of CDPP-3SO3,
CDPP-4SO3, and CDPP-BzBr was found to be 39.3%, 5.3%,
The compounds in general exhibited poor solubility in many
polar solvents and were not visibly soluble in nonpolar sol-
vents. Optical properties, absorption and emission spectra,
of CDPP-3SO3, CDPP-4SO3, and CDPP-BzBr were investi-
gated in dimethyl sulfoxide (DMSO) at room temperature.
©
Adv. Funct. Mater. 2020, 1909268
1909268 (3 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
and 8.3%, respectively (Figure 2A). The solid-state emission of
CDPP-3SO3 and CDPP-4SO3 showed similar emission maxima
at ≈620 nm however CDPP-BzBr showed red shifted emission
at 665 nm compare to CDPP-3SO3 and CDPP-4SO3 (Figure 2B).
It is evident that the AIE molecules are highly affected by
the rigidity of their environment. Comparing CDPP-3SO3 and
CDPP-4SO3, the butyl linker of the latter may increase dis-
order resulting in a less rigid environment reducing solid state
quantum yield. From the crystal packing of CDPP-3SO3, we see
the existence of π–π stacking interactions. These interactions
likely exist for CDPP-BzBr as evidenced by the red shifting
of its solid-state emission. The degree of red-shifting likely
indicates the strength of the π–π interaction which ultimately
results in lowering the solid state QY.
Furthermore, the emission behavior of CDPP-3SO3, CDPP-
4SO3, and CDPP-BzBr were studied in DMSO/water solvent
mixtures to evaluate their aggregation properties. For CDPP-
3SO3, increasing the water fraction in DMSO/water mixtures
from 0% to 60% resulted in no visible changes in the photo-
luminescence (PL) intensity. When fw ≥ 70%, emission inten-
sity became enhanced alongside a bathochromic shift (from
620 to 640 nm), signaling the formation of nanoaggregates.
The maximum PL intensity was observed at fw = 90% and αAIE
or I/I0 was found to be ≈14. Similar AIE behavior was observed
for CDPP-4SO3 and CDPP-BzBr in the presence of different
DMSO/water mixtures and their αAIE was found to be ≈17 and
30, respectively (Figure 2C,D; Figures S7 and S8, Supporting
Information). Additionally, scanning electron microscopy
(SEM) was performed to validate aggregate formation when
fw = 90% (Figure 2E–G). SEM revealed that the CDPP-3SO3
and CDPP-BzBr formed spherical shaped nanoaggregates.
CDPP-4SO3, however, formed wire-shaped nanoaggregates
(Figure 2F). Fluorescence decay measurements of CDPP-3SO3,
CDPP-4SO3, and CDPP-BzBr nanoaggregates at fw = 90%
revealed that their lifetimes were 2.1, 2.0, and 2.0 ns, respec-
tively (Figure S9, Supporting Information).
Red, one of the frequently adopted probes for ER-staining.
Both CDPP-3SO3 and CDPP-4SO3 can efficiently stain the cell
within 1 h at 1 × 10−6 m. The fluorescent area matches very well
with those of ER-Tracker Red with a high Pearson’s correlation
factor at 0.85 for CDPP-3SO3, and 0.86 for CDPP-4SO3, indi-
cating that these AIEgens can selectively target ER (Figure 4;
Figure S10, Supporting Information).
In addition, Mito-Tracker Deep Red was employed to inves-
tigate the subcellular localization pattern of CDPP-3SO3 and
CDPP-4SO3, with the resultant images clearly indicating that
their staining region only partially overlapped with Mito-
Tracker Deep Red. The Pearson’s correlation values between
Mito-Tracker Deep Red and CDPP-3SO3 was 0.55 and 0.57 for
CDPP-4SO3, suggesting that these AIEgens did not stain mito-
chondria in HeLa. Moreover, we were also able to successfully
verify the ER staining property of CDPP-3SO3 when employed
to stain 143B cells (Figure S11, Supporting Information).
To investigate the importance of the zwitterionic functional
group, we designed the molecule CDPP-BzBr (Figure 2A),
which contains only one positive charge. This was easy to do as
the core molecule can be simply modified by other functional
groups. This would allow us to investigate how modifications
could affect organelle-targeting properties. Upon staining the
cell, we observed that CDPP-BzBr targeted the mitochondria
instead of the ER. The staining properties of CDPP-BzBr were
verified by co-staining with Mito-Tracker Deep Red (Figure 5). It
showed excellent overlap colocalized pattern with Mito-Tracker
Deep Red in HeLa cells with a high Pearson’s correlation factor
of 0.89. This key difference suggests that the zwitterionic prop-
erty of the functional group is critical for ER staining.
It should be noted that a major component of the phospho-
lipid bilayer of the different membranes of mammalian cells is
phosphatidylcholine (PC), a zwitterionic phospholipid. PC has
oppositely matching charges to CDPP-3SO3 and CDPP-4SO3,
and in theory they would create ideal matching electrostatic
interactions. As is clearly seen in the cell staining (Figure 4;
Figure S10, Supporting Information), no visible emission from
the zwitterionic probes is observed on the different membranes,
only selectively targeting the ER. A major function of the ER is
glyercophospholipid synthesis, such as PC, via the enzyme CCT
located on the ER membrane. The CCT enzyme binds to ani-
onic regions of the ER membrane via electrostatic interactions
with its positively charged α-helical M domain (Figure 1). The
zwitterionic nature of the probes may find it more favorable
to target such locales, as they also have oppositely matching
charges. We are currently investigating with more detail the
mechanism behind the ER targeting of the probes.
2.3. Single X-Ray Crystallography
One of the synthesized compounds, CDPP-3SO3 was success-
fully characterized by single crystal X-ray diffraction (SXRD)
(Figure 3A). The propeller conformation of TPA showed dihe-
dral angles of 54.1°, 68.1°, and 74.2° among the three phenyl
rings. The crystal packing showed various CH···π, π···π,
and CH/π···O interactions, which were measured to be in
the range of 2.72–2.89, 3.39–3.87, and 2.32–3.12 Å, respectively
(Figure 3B,C), with a centroid-to-centroid distance of 3.87 Å
(Figure 3B). It is speculated that the propeller conformation
allows for molecular motions which lead to nonradiative decay
pathways in the solution state. However, the short interactions
suppress molecular motions inhibiting nonradiative decay and
open new radiative channels in the aggregate/solid state.
2.4.1. Two-Photon Imaging
The synthesized CDPP derivatives were anticipated to have
strong two-photon absorption (2PA) because of their strong
electron donating and withdrawing groups.[54] The 2PA
measurements of these compounds were carried out using
two-photon excited fluorescence (TPEF) technique with a fem-
tosecond pulsed laser source, and comparative TPEF intensity
at fw = 90% were measured using Rhodamine B as the standard.
TPEF intensities were scanned between 820 and 1000 nm at an
2.4. Cell Imaging Studies
The specific ER targeting ability of CDPP-3SO3 and CDPP-4SO3
was demonstrated by co-staining HeLa cells with ER-Tracker
©
Adv. Funct. Mater. 2020, 1909268
1909268 (4 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
A
B
2.83
3.39
2.72
2.72
3.39
2.89
2.89
2.83
3.87
C
3.12
2.65
2.63
2.32
2.58
2.63
3.12
2.34
Figure 3. Single crystal structure of CDPP-3SO3, A) thermal ellipsoid (50%) plot and packing of showing B) CH···π and π···π interactions, and C) CH···O
and π···O interactions of CDPP-3SO3.
interval of 30 nm and respective δ2PA values were calculated. The
highest δ2PA value of 162 GM was calculated for CDPP-4SO3 at
820 nm. The δ2PA values of the two other molecules, CDPP-
3SO3 and CDPP-BzBr, were also calculated and found to be 122
and 71 GM at 820 and 970 nm, respectively (Figure 6A). The
obtained 2PA values are much higher than most of fluorescence
proteins such as EGFP (39 GM).[55] Hence CDPP derivatives
may be utilized as good two-photon imaging probes.
parallel, with continuous excitation and sequential scanning
with a confocal microscope. The result showed that the emis-
sion intensity of CDPP-3SO3 and CDPP-4SO3 decreased by
about 25% within 80 irradiation scans. In contrast, the fluores-
cence loss of ER-Tracker Red was around 50% upon irradiation
under the same conditions, demonstrating the superior photo-
stability of CDPP-3SO3 and CDPP-4SO3 to that of ER-Tracker
Red (Figure 6B). CDPP-BzBr showed similar photostability
as Mito-Tracker Red (Figure S12, Supporting Information).
Moreover, two-photon cell imaging was conducted. As seen
The photostability of CDPP-3SO3, CDPP-4SO3, CDPP-
BzBr, ER-Tracker Red, and Mito-Tracker Red were assessed in
©
Adv. Funct. Mater. 2020, 1909268
1909268 (5 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
Figure 4. Confocal microscopy imaging of HeLa cells labeled with CDPP-3SO3 (1 × 10−6 m) and its colocalization with ER-Tracker Red (1 × 10−6 m)
(R = 0.85) (top) and Mito-Tracker Deep Red (250 × 10−9 m) (R = 0.55) (bottom); scale bar = 5 µm.
1
in Figure 6C,D and Figures S13 and S14 in the Supporting
Information, sufficient signals were obtained for CDPP-3SO3,
CDPP-4SO3, and CDPP-BzBr under both one-photon and two-
photon excitation, indicating all three AIEgens were suitable for
two-photon imaging as well as one-photon imaging.
CDPP-BzBr showed the best O2 generation efficiency with a
high consumption rate of ABDA in 2 min (95.86 nmol). Fur-
thermore, the photodynamic therapy (PDT) effect of CDPP-
BzBr was investigated. At higher concentrations (10 × 10−6 m or
above), it showed excellent anticancer properties, while at lower
concentrations (5 × 10−6 m or below), it showed good biocom-
patibility even under white light irradiation. This demonstrated
that CDPP-BzBr can be applied as PDT reagents in higher con-
centrations (Figure S16B, Supporting Information), as well as
long-term tracking fluorescent probes at lower concentrations.
In addition, we have also studied IC50 values with phototoxicity
to HeLa and Cos-7 cells and their IC50 values were calculated to
be 13.4 × 10−6 and 19.3 × 10−6 m, respectively (Figure S16C,D,
Supporting Information).
2.4.2. Cytotoxicity and Phototoxicity
The cytotoxicity of CDPP series AIEgens were evaluated by the
method of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) assay. As shown in Figure S15 in the Sup-
porting Information, CDPP series AIEgens only showed sig-
nificant toxicity to both COS-7 and HeLa cells when staining
concentration was at 40 × 10−6 m. At the working concentration
for cell imaging of 1 × 10−6 m, the dyes exhibited minimal tox-
icity, suggesting that the CDPP AIEgens are suitable for long-
term tracking imaging.
3. Conclusion
Reactive oxygen species (ROS) generation abilities of CDPP
derivatives were also investigated by using 9,10-anthracenediyl-
bis(methylene)dimalonic acid (ABDA) as the singlet oxygen
probe (1O2) (Figure S16A, Supporting Information). All CDPP
derivatives showed good 1O2 generation efficiency at 10 × 10−6 m,
Herein, red emissive fluorescent probes were rationally
designed and synthesized. All these probes exhibited AIE
activity with emission maximum up to 665 nm. The solid-
state efficiency of CDPP-3SO3 was excellent and found to be
39.3%. Additionally, all these molecules showed excellent
Figure 5. Confocal microscopy imaging of HeLa cells labeled with CDPP-BzBr (10 × 10−6 m) and its colocalization with Mito-Tracker Deep Red
(250 × 10−9 m); Pearson’s coefficient (R) = 0.89, scale bar = 10 µm.
©
Adv. Funct. Mater. 2020, 1909268
1909268 (6 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
Figure 6. A) Two-photon absorption cross-section of CDPP-3SO3, CDPP-4SO3, and CDPP-BzBr; condition: DMSO/water (1:9), concentration:
100 × 10−6 m. B) Signal change in HeLa cells stained with ER-Tracker Red, CDPP-3SO3, and CDPP-4SO3 upon continuous scanning by laser light
(λex = 488 nm for CDPP-3SO3 and CDPP-4SO3; λex = 560 nm for ER-Tracker Red). Confocal images of HeLa cells stained with CDPP-3SO3 (1 × 10−6 m),
λex = 480 nm for C) one-photon microscope (1PM) and 820 nm for D) two-photon microscope (2PM); scale bar = 25 µm.
DMSO were procured from the Merck Company. All the molecules
synthesized were purified by column chromatography and recrystallized
photostability with some displaying two-photon absorption
cross-sections of up to 162 GM. These dyes were found to be
good candidates for cell imaging because of high cell biocom-
patibility, high selectivity, high brightness, low background, and
using double layer solution diffusion with dichloromethane/hexane or
dichloromethane/ethyl acetate and fully characterized by 1H NMR, 13C
1
NMR, and HRMS. H and 13C NMR spectra were recorded on a Bruker
excellent photostability. The zwitterionic AIEgens, CDPP-3SO3
and CDPP-4SO3 were successful in imaging the ER in various
live cell lines, such as HeLa cells and 143B cells. In contrast,
the singly positive charged CDPP-BzBr could only stain the
mitochondria. The design strategy demonstrates an easy metho-
dology to develop a variety of different organelle specific effi-
cient red emitters as a platform to study the structure–property
relationships regarding such bioimaging probes.
AV 400 Spectrometer at 400 and 100 MHz in CDCl3 and d6-DMSO,
respectively. Tetramethylsilane (TMS) was used as the internal
standard. High-resolution mass spectra were recorded on a GCT
premier CAB048 mass spectrometer operating in MALDI-TOF mode.
The photoluminescence spectra were measured on a PerkinElmer LS
55 spectrophotometer and Horiba Fluoromax 4 spectrofluorometer.
Quantum yields of the solids were recorded on Hamamatsu, C13534
at room temperature with a calibrated integrating sphere system.
The lifetime was measured on an Edinburgh FLSP 920 fluorescence
spectrophotometer equipped with a Xenon arc lamp (Xe900) and a
microsecond flash-lamp (uF900). Single crystal data was collected
on
monochromated Cu Kα radiation (λ = 1.54178 Å).
Synthesis of Compound CDPBr: 4-(N,N-Diphenylamino)
a Bruker Smart APEXII CCD diffractometer using graphite
4. Experimental Section
Materials
and
Instruments:
4-(diphenylamino)benzaldehyde,
benzaldehyde (5.0 g, 18.30 mmol) was dissolved in 100 mL ethanol,
4-bromophenylacetonitrile (4.28 g, 21.96 mmol), and NaOH (0.88 g,
21.96 mmol) were added, and then stirred at room temperature for 3 h.
After the reaction completed, the product was purified by recrystallization
from EtOH to give the compound CDPBr as a yellow solid. This crude
product was purified by silica-gel column chromatography using
2-(4-bromophenyl)acetonitrile, pyridin-4-ylboronic acid, 4-bromo benzyl
bromide, 1 3-propane sultone, 1 4-butane sultone, sodium hydroxide,
potassium carbonate, and tetrakis(triphenylphosphine)palladium(0)
were purchased from Sigma-Aldrich. All the spectroscopic grade
solvents such as acetonitrile (ACN), ethanol (EtOH), tetrahydrofuran
(THF), dichloromethane (DCM), ethyl acetate (EA), hexane, and
©
Adv. Funct. Mater. 2020, 1909268
1909268 (7 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
DCM/ethyl acetate (v/v = 1:1) as eluent to furnish a yellow solid as
product. Yield: 85%.
(101 MHz, CDCl3) δ 154.77, 150.13, 145.47, 143.88, 143.34, 138.57,
132.10, 131.91, 131.22, 130.65, 130.37, 128.96, 127.90, 126.00, 125.31,
124.68, 124.17, 124.10, 123.75, 119.37, 117.61, 104.07, 62.12. HRMS,
m/z: ([M]+), calculated: 618.1539, found: 618.1553.
Two-Photon Absorption Cross-Sections Measurement: A two-photon
excitation fluorescence cross-section was measured by two-photon
excitation fluorescence method using rhodamine B as a reference. The
excitation source for two-photon excitation was a femtosecond optical
parametric amplifier (Coherent OPerA Solo) pumped by an amplified
Ti:sapphire system (Coherent Legend Elite system) and then detected
with a spectrometer (Acton SpectraPro-500i) coupled to a CCD. The
TPA cross-section (δ) of the sample was calculated at each wavelength
according to the following formula[56]
CDPBr: 1H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) δ
7.76 (d, J = 2.6 Hz, 2H), 7.57–7.48 (m, 4H), 7.40 (s, 1H), 7.37–7.28
(m, 4H), 7.20–7.10 (m, 6H), 7.05 (d, J = 5.2 Hz, 2H). 13C NMR
(101 MHz, CDCl3) 13C NMR (101 MHz, CDCl3) δ 149.58, 145.86, 141.39,
133.43, 131.48, 130.16, 128.99, 126.53, 125.37, 125.19, 123.92, 121.97,
120.04, 117.81, 105.72., HRMS, m/z: ([M]+), calculated: 450.0732, found:
450.0714.
Synthesis of Compound CDPP: Compound CDPBr (1.0 g, 2.22 mmol),
(4-hydroxylphenyl)boronic acid (0.33 mg, 2.66 mmol), potassium
carbonate (3.07 g, 22.21 mmol), and Pd(PPh3)4 (25.41 mg, 0.01 mmol)
were added in 60 mL THF and 9 mL water into a 250 mL two-necked
round bottom flask which was equipped with a condenser under
nitrogen. The mixture was heated to reflux overnight with stirring. After
cooling to room temperature, the mixture was extracted with DCM for
three times. The organic phase was collected, washed with water, and
then dried over anhydrous sodium sulfate. After solvent evaporation, the
crude product was purified by silica-gel column chromatography using
DCM/ethyl acetate (v/v = 2:3) as eluent to furnish an orange solid as
product. Yield: 75%.
φref cref ηref
F
δ = δref
(1)
φ
c
η Fref
where δ is the cross-sectional value, c is the concentration of the
solution, n is the refractive index of the solution, F is the two-photon
excited fluorescence integral intensity of the light emitted at the
excitation wavelength, and φ is the fluorescence quantum yield.
Cell Imaging and Confocal Colocalization: HeLa cells and 143B
cells were cultured in the Dulbecco’s Modified Eagle Medium
(DMEM) containing 10% fetal bovine serum (FBS) and antibiotics
(100 units mL−1 penicillin and 100 mg mL−1 streptomycin) in a 5%
CO2 humidity incubator at 37 °C. After cells were incubated with CDPP
derivatives and the commercial probes at 37 °C for 30 min, the medium
was removed and the cells were rinsed with phosphate buffer saline
(PBS) three times and then imaged under a confocal microscope (ZEISS,
LSM710). The excitation was 488 nm for CDPP derivatives, 560 nm for
ER-Tracker Red, and 633 nm for Mito-Tracker Deep Red.
Cytotoxicity Study in the Dark and under Light Irradiation: Cells were
seeded in 96-well plates (Costar, IL, U.S.A.) at a density of 5000 cells per
well. After overnight culturing, the medium in each well was replaced by
100 µL of fresh medium containing different concentrations of AIEgens.
After 24 h incubation, 10 µL of MTT solution (5 mg mL−1 in PBS) was
added into each well and incubated for 4 h. After a further 30 min of
incubation under light irradiation, in which another array of plates in the
dark was used as the control, 100 µL of DMSO was added to each well
and then vibrated for 15 min. The absorption of each well at 595 nm
was recorded via a plate reader (PerkinElmer Victor3TM). Each trial was
performed with 6 wells parallel.
CDPP: 1H NMR (400 MHz, CDCl3) δ 8.67 (d, J = 5.3 Hz, 2H), 7.79
(dd, J = 15.4, 8.7 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.4 Hz,
1H), 7.57 (m, 4H), 7.36–7.24 (m, 5H), 7.21–7.01 (m, 8H), 6.82 (d, J =
8.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 149.76, 149.61, 149.11,
146.57, 145.85, 145.80, 143.70, 141.54, 137.98, 137.40, 135.20, 133.72,
130.66, 130.25, 129.07, 128.99, 128.93, 127.05, 126.90, 125.69, 125.45,
125.19, 125.16, 125.00, 123.93, 123.85, 120.86, 120.76, 120.13, 120.03,
119.59, 117.96, 108.89, 105.96. HRMS, m/z: ([M]+), calculated: 449.1892,
found: 449.1884.
General Synthesis of CDPP Sulfonate: To an oven dried round bottom
flask sealed with rubber stopper, CDPP (1.6 mmol) and sultones
(1,3 propane sultone/1,4 butane sultone, 1.6 mmol) were added,
followed by the addition of dry acetonitrile (10 mL). The reaction mixture
was refluxed at 95 °C for 6–8 h. Then, the mixture evaporated under
reduced pressure to afford the crude product which was further purified
by column chromatography using MeOH/DCM mixture as an eluent to
furnish a red solid as product. Yield: 75–80%.
CDPP-3SO3: 1H NMR (400 MHz, DMSO) δ 9.11 (d, J = 6.8 Hz, 2H), 8.56
(d, J = 6.8 Hz, 2H), 8.21 (d, J = 8.6 Hz, 2H), 8.13 (s, 1H), 7.96 (d, J = 8.6 Hz,
2H), 7.91 (d, J = 8.9 Hz, 3H), 7.41 (t, J = 7.8 Hz, 3H), 7.19 (dd, J = 16.2,
7.6 Hz, 5H), 6.96 (d, J = 8.8 Hz, 2H), 4.72 (t, J = 6.8 Hz, 2H), 2.45 (t, J =
8.2 Hz, 2H), 2.29–2.23 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 154.76,
150.14, 145.61, 143.93, 143.28, 138.52, 132.34, 130.59, 128.94, 127.79,
126.05, 125.30, 124.78, 124.23, 124.04, 119.53, 117.51, 104.48, 59.84, 29.46,
20.73. HRMS, m/z: ([M+H]+), calculated: 572.2008, found: 572.2015.
Photostability: The cells were imaged using a confocal microscope (Zeiss
Laser Scanning Confocal Microscope; LSM7 DUO) and analyzed using ZEN
2009 software (Carl Zeiss). The concentrations of all the three probes were
1 × 10−6 m. CDPP-3SO3 and CDPP-4SO3 were excited at 488 nm for one-
photon imaging (2% laser power). ER-Tracker Red was excited at 560 nm for
one-photon imaging (2% laser power). The scanning speed was 0.93 s per
scan, and the repeated image scans were taken 80 times. The first scan of
CDPP-3SO3, CDPP-4SO3, and ER-Tracker Red was set to 100%, followed by
which the pixel intensity values were averaged and plotted against the scan
number. The resulting curve represents the bleaching rate.
1
CDPP-4SO3: H NMR (400 MHz, DMSO) δ 9.12 (d, J = 6.9 Hz, 2H),
8.58 (d, J = 6.9 Hz, 2H), 8.22 (d, J = 8.7 Hz, 2H), 8.14 (s, 1H), 7.94
(dd, J = 18.7, 8.8 Hz, 3H), 7.41 (t, J = 7.9 Hz, 3H), 7.25–7.12 (m, 5H),
6.96 (d, J = 8.9 Hz, 2H), 4.61 (t, J = 7.2 Hz, 2H), 2.51–2.41 (m, 2H),
2.05 (dd, J = 14.6, 7.6 Hz, 2H), 1.60 (dt, J = 15.3, 7.6 Hz, 2H). 13C
NMR (101 MHz, CDCl3) δ 154.76, 150.11, 145.47, 143.99, 143.31,
138.44, 132.26, 130.55, 128.90, 127.81, 125.99, 125.26, 124.67, 124.11,
124.02, 119.38, 117.58, 104.14, 58.45, 46.02, 26.21, 17.67. HRMS, m/z:
([M+H]+), calculated: 586.2164, found: 586.2190.
[CCDC 1963718 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.]
Synthesis of CDPP-BzBr: Into a 50 mL two-necked round bottom flask
equipped with a condenser was dissolved CDPP (1000 mg, 2.22 mmol)
in 200 mL acetonitrile. 4-bromo benzyl bromide (662 mg, 2.67 mmol)
was then added and the mixture was heated to reflux for 8 h. After
cooling to room temperature, the mixture was poured into diethyl ether.
The dark red precipitates formed were filtered by suction filtration.
Further, the crude product which was further purified by column
chromatography using MeOH/DCM mixture as an eluent to furnish a
red solid as product. Yield: 80%.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
CDPP-BzBr: 1H NMR (400 MHz, CDCl3) δ 9.13 (d, J = 5.3 Hz, 2H),
8.21 (d, J = 5.7 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H), 7.73 (dd, J = 8.4,
4.2 Hz, 4H), 7.52 (s, 1H), 7.45 (s, 4H), 7.24 (dd, J = 14.6, 6.7 Hz, 4H),
7.07 (d, J = 7.0 Hz, 6H), 6.93 (d, J = 8.8 Hz, 2H), 5.84 (s, 2H). 13C NMR
Acknowledgements
P.A. and W.H. contributed equally to this work. This work was partially
supported by the National Natural Science Foundation of China
©
Adv. Funct. Mater. 2020, 1909268
1909268 (8 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
(Grant No. 21788102), the Research Grants Council of Hong Kong
(16308016, 16308116, C6009-17G, and AoE/P-02/12), the Innovation
and Technology Commission (ITC-CNERC14SC01 and ITCPD/17-9), and
the Science and Technology Plan of Shenzhen (JCYJ20180507183832744
and JCYJ20160229205601482).
[25] H. Wang, E. Zhao, J. W. Y. Lam, B. Z. Tang, Mater. Today 2015, 18,
365.
[26] J. Qian, B. Z. Tang, Chem 2017, 3, 56.
[27] Q. Wan, Q. Huang, M. Liu, D. Xu, H. Huang, X. Zhang, Y. Wei,
Appl. Mater. Today 2017, 9, 145.
[28] F. Hu, S. Xu, B. Liu, Adv. Mater. 2018, 30, 1801350.
[29] S. Wang, F. Hu, Y. Pan, L. G. Ng, B. Liu, Adv. Funct. Mater. 2019, 29,
1902717.
[30] P. Alam, N. L. C. Leung, H. Su, Z. Qiu, R. T. K. Kwok, J. W. Y. Lam,
B. Z. Tang, Chem. - Eur. J. 2017, 23, 14911.
Conflict of Interest
The authors declare no conflict of interest.
[31] Y. Tatenaka, H. Kato, M. Ishiyama, K. Sasamoto, M. Shiga,
H. Nishitoh, Y. Ueno, Biochemistry 2019, 58, 499.
[32] H. Shi, J. Liu, J. Geng, B. Z. Tang, B. Liu, J. Am. Chem. Soc. 2012,
134, 9569.
Keywords
[33] C. Zhang, S. Jin, K. Yang, X. Xue, Z. Li, Y. Jiang, W.-Q. Chen, L. Dai,
G. Zou, X.-J. Liang, ACS Appl. Mater. Interfaces 2014, 6, 8971.
[34] J. M. Meinig, L. Fu, B. R. Peterson, Angew. Chem., Int. Ed. 2015, 54,
9696.
[35] S. Zheng, C. Huang, X. Zhao, Y. Zhang, S. Liu, Q. Zhu, Spectrochim.
Acta, Part A 2018, 189, 231.
aggregation-induced emission, bioimaging, endoplasmic reticulum,
zwitterionic
Received: November 6, 2019
Revised: December 12, 2019
Published online:
[36] L. Shi, X. Gao, W. Yuan, L. Xu, H. Deng, C. Wu, J. Yang, X. Jin,
C. Zhang, X. Zhu, Small 2018, 14, 1800223.
[37] C.-J. Zhang, X. Cai, S. Xu, R. Zhan, W. Jien, B. Liu, Chem. Commun.
2017, 53, 10792.
[1] J. D. Marth, Nat. Cell Biol. 2008, 10, 1015.
[2] K. Mesa, Yale J. Biol. Med. 2015, 88, 100.
[38] S. Hussain, W. Du, M. Zhang, B. Fang, G. Zhang, R. Su, K. Nan,
Q. Zhang, X. Tian, Y. Tian, Y. Chen, J. Mater. Chem. B 2018, 6, 1943.
[39] B. Dong, Y. Lu, N. Zhang, W. Song, W. Lin, Anal. Chem. 2019, 91, 5513.
[40] W. Song, B. Dong, Y. Lu, X. Kong, A. H. Mehmood, W. Lin, Anal.
Methods 2019, 11, 4450.
[41] M. Wang, R. J. Kaufman, Nat. Rev. Cancer 2014, 14, 581.
[42] D. Naon, M. Zaninello, M. Giacomello, T. Varanita, F. Grespi,
S. Lakshminaranayan, A. Serafini, M. Semenzato, S. Herkenne,
M. I. Hernández-Alvarez, A. Zorzano, D. De Stefani, G. W. Dorn,
L. Scorrano, Proc. Natl. Acad. Sci. USA 2016, 113, 11249.
[43] W. Lin, B. Popko, Nat. Neurosci. 2009, 12, 379.
[44] P. Strzyz, Nat. Rev. Mol. Cell Biol. 2019, 20, 453.
[45] W. Li, J. Yang, L. Luo, M. Jiang, B. Qin, H. Yin, C. Zhu, X. Yuan,
J. Zhang, Z. Luo, Y. Du, Q. Li, Y. Lou, Y. Qiu, J. You, Nat. Commun.
2019, 10, 3349.
[3] R. A. Weinberg, Sci. Am. 1996, 275, 62.
[4] C. P. Satori, M. M. Henderson, E. A. Krautkramer, V. Kostal,
M. D. Distefano, E. A. Arriaga, Chem. Rev. 2013, 113, 5699.
[5] K. W. Osteryoung, J. Nunnari, Science 2003, 302, 1698.
[6] A. H. Futerman, G. van Meer, Nat. Rev. Mol. Cell Biol. 2004, 5, 554.
[7] B. Alberts, D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. C. Raff,
K. Roberts, P. Walter, Essential Cell Biology, Garland Science, New
York 2014.
[8] H. F. Steedman, Q. J. Microsc. Sci. 1950, 91, 477.
[9] A. S. Klymchenko, Acc. Chem. Res. 2017, 50, 366.
[10] H. Zhu, J. Fan, J. Du, X. Peng, Acc. Chem. Res. 2016, 49, 2115.
[11] C.-J. Zhang, Q. Hu, G. Feng, R. Zhang, Y. Yuan, X. Lu, B. Liu, Chem.
Sci. 2015, 6, 4580.
[12] F. Xue, P. Wei, X. Ge, Y. Zhong, C. Cao, D. Yu, T. Yi, Dyes Pigm.
2018, 156, 285.
[46] F. Gibellini, T. K. Smith, IUBMB Life 2010, 62, spc1.
[47] R. B. Cornell, I. C. Northwood, Trends Biochem. Sci. 2000, 25, 441.
[48] P. Fagone, S. Jackowski, Biochim. Biophys. Acta, Mol. Cell Biol. Lipids
2013, 1831, 523.
[13] C. Gao, Y. Tian, R. Zhang, J. Jing, X. Zhang, Anal. Chem. 2017, 89,
12945.
[14] Y. Li, Y. Wu, J. Chang, M. Chen, R. Liu, F. Li, Chem. Commun. 2013,
49, 11335.
[49] R. B. Cornell, Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2016,
1861, 847.
[15] M. H. Teiten, L. Bezdetnaya, P. Morlière, R. Santus, F. Guillemin,
Br. J. Cancer 2003, 88, 146.
[50] S. G. Taneva, J. M. C. Lee, R. B. Cornell, Biochim. Biophys. Acta,
Biomembr. 2012, 1818, 1173.
[51] C. Y. Y. Yu, H. Xu, S. Ji, R. T. K. Kwok, J. W. Y. Lam, X. Li, S. Krishnan,
D. Ding, B. Z. Tang, Adv. Mater. 2017, 29, 1606167.
[52] C. DeBruler, B. Hu, J. Moss, J. Luo, T. L. Liu, ACS Energy Lett. 2018,
3, 663.
[53] A. Hassner, D. Birnbaum, L. M. Loew, J. Org. Chem. 1984, 49, 2546.
[54] Z. Zheng, T. Zhang, H. Liu, Y. Chen, R. T. K. Kwok, C. Ma, P. Zhang,
H. H. Y. Sung, I. D. Williams, J. W. Y. Lam, K. S. Wong, B. Z. Tang,
ACS Nano 2018, 12, 8145.
[55] M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, A. Rebane,
Nat. Methods 2011, 8, 393.
[16] F. Hu, B. Liu, Org. Biomol. Chem. 2016, 14, 9931.
[17] S. J. Sahl, S. W. Hell, S. Jakobs, Nat. Rev. Mol. Cell Biol. 2017, 18, 685.
[18] G. Vicidomini, P. Bianchini, A. Diaspro, Nat. Methods 2018, 15, 173.
[19] L. Schermelleh, A. Ferrand, T. Huser, C. Eggeling, M. Sauer,
O. Biehlmaier, G. P. C. Drummen, Nat. Cell Biol. 2019, 21, 72.
[20] P. Alam, N. L. C. Leung, Y. Cheng, H. Zhang, J. Liu, W. Wu,
R. T. K. Kwok, J. W. Y. Lam, H. H. Y. Sung, I. D. Williams, B. Z. Tang,
Angew. Chem. 2019, 131, 4584.
[21] H. Nie, K. Hu, Y. Cai, Q. Peng, Z. Zhao, R. Hu, J. Chen, S.-J. Su,
A. Qin, B. Z. Tang, Mater. Chem. Front. 2017, 1, 1125.
[22] Z. He, C. Ke, B. Z. Tang, ACS Omega 2018, 3, 3267.
[23] Z. Zhao, B. He, B. Z. Tang, Chem. Sci. 2015, 6, 5347.
[24] Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2011, 40, 5361.
[56] Q. Zhang, X. Tian, H. Zhou, J. Wu, Y. Tian, Materials 2017, 10, 223.
©
Adv. Funct. Mater. 2020, 1909268
1909268 (9 of 9)
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim