Aggregation-induced emission and biological application of tetraphenylethene luminogens

Article abstract of DOI:10.1071/CH11170

Tetraphenylethene derivatives [Ph(PhCH=CHPhR)C=C(PhCH=CHPhR)Ph, R=H, CN, NO₂, NPh₂] with green, yellow-green, and orange emission colours were designed and synthesized. These molecules are practically non-emissive in their dilute sol

Full text of DOI:10.1071/CH11170

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2011, 64, 1203–1210
Aggregation-Induced Emission and Biological Application
of Tetraphenylethene Luminogens
A
A
A
Yuning Hong, Jacky Wing Yip Lam, Sijie Chen,
A B C
, ,
and Ben Zhong Tang
A
Department of Chemistry, Nano Science and Technology Program,
Bioengineering Graduate Program, The Hong Kong University of Science
and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Department of Polymer Science and Engineering, Institute of Biomedical
Macromolecules, Zhejiang University, Hangzhou 310027, China.
B
C
Corresponding author. Email: tangbenz@ust.hk
Tetraphenylethene derivatives [Ph(PhCH¼CHPhR)C¼C(PhCH¼CHPhR)Ph, R¼H, CN, NO2, NPh2] with green, yellow-
green, and orange emission colours were designed and synthesized. These molecules are practically non-emissive in their
dilute solutions but emit intensely as nanoaggregates in poor solvents, demonstrating a novel phenomenon of aggregation-
induced emission. Their blended films with poly(methyl methacrylate) also display bright emissions. Restriction of
intramolecular motion in the condensed phase may be responsible for such unusual behaviour. Multilayer electrolumi-
nescence devices with a configuration of indium tin oxide/N,N0-di(1-naphthyl)-N,N0-diphenylbenzidine/emitter/tris
(8-hydroxyquinolinolato)aluminum (Alq3)/LiF/Al were constructed, which gave green light with a maximum luminance
and current efficiency of 12930 cd cmꢀ2 and 3.04 cd Aꢀ1 respectively. The tetraphenylethenes can serve as excellent cell
staining agents for selectively illuminating the cytoplasm and vesicles of living cells.
Manuscript received: 29 April 2011.
Manuscript accepted: 21 May 2011.
Published online: 27 July 2011.
Introduction
has limited the scope of their applications and it is therefore
desirable to develop luminophores that can overcome this
‘aggregation-caused emission quenching’ problem.
The development of fluorescent materials is of critical impor-
tance to biological science and biotechnology because it offers a
direct visualization tool for detecting biological macromolecules
and monitoring biological events.[1] A large variety of materials,
such as organic dyes, inorganic quantum dots (QD), fluorescent
proteins (FP), and polymeric and silica nanoparticles have been
studied as potential candidates for biosensory applications.[2]
Inorganic QD are highly emissive and photostable but inher-
ently cytotoxic because they are typically composed of heavy
metals and chalcogens (e.g. CdSe and PbS).[3] The capping
agents used in the QD preparation, such as trioctylphosphine,
can also be of biological concern. Similarly, the surfactants used
in the fabrication of polymeric and silica nanoparticles, such
as sodium dodecyl sulfate, are harmful to living cells, because
they can stimulate cytolysis even at a concentration as low as
0.0005 %.[4] Recently, FP have attracted much attention because
they can be genetically encoded to the target of interest. The use
of FP, however, requires a complicated transfection process and
their large size may interfere with the conformation and dynam-
ics of the target.[5]
Our group has worked on the development of efficient
organic chromophores and has discovered an unusual phenom-
enon of aggregation-induced emission (AIE): a series of non-
emissive linear and cyclic p-conjugated organic polyenes
including siloles, butadienes, 4H-pyrans, fulvenes, and tetra-
phenylethenes (TPE) are induced to emit efficiently by aggre-
gate formation.[9] Attracted by this fascinating phenomenon,
many groups have worked on AIE materials and done a great
deal of outstanding work.[10] Among these, the TPE system has
drawn much of our interest because TPE are stable, easy to make
and readily functionalized, and exhibit appreciably high quan-
tum yields.[9g,11] Thanks to the research efforts of scientists,
TPE has been functionalized with ionic or polar functional
groups to endow it with good water solubility.[12,13] These
fluorophores have been successfully used as fluorescent probes
for nucleic acid detection, enzymatic activity assay, and metallic
ion tracing.[12,13] Most of these TPE derivatives, however, emit
blue light. To expand their applications as light-emitting mate-
rials and in cell imaging, varied emission colours are highly
desirable. This may be achieved by extension of their electronic
conjugation through attachment of different substituents. In this
work, we report the synthesis of TPE with various substituents
on the peripheral phenyl rings and present their optical proper-
ties (Scheme 1). These new molecules show AIE features and
can be used for the construction of electroluminescence (EL)
In contrast, organic dyes enjoy structural and property diver-
sity and are easy to use.[6] Conventional organic luminophors
often exhibit remarkably bright emission in dilute solutions but
become weakly or even non-emissive when they are dispersed in
aqueous media or aggregated in living cells.[7] The close prox-
imity of the chromophores often induces non-radiative energy
transfer, resulting in self-quenching of the luminescence.[8] This
Ó CSIRO 2011
10.1071/CH11170

RESEARCH FRONT
1204
Y. Hong et al.
Br
TiCl4, Zn
THF
NBS, BPO
CCl4
O
Br
5
6
7
ϪBrPh3Pϩ
R
CHO, t-BuOK, DMF
PPh3, DMF
PϩPh3BrϪ
8
R
R ϭ H (1), CN (2), NO2 (3), N(C6H5)2 (4)
R
Scheme 1. Synthetic route to tetraphenylethene (TPE) derivatives 1–4.
devices. Although they are not soluble in water, they work well
as fluorescent stains for intracellular imaging and can selec-
tively light up the cytoplasm and vesicles inside living HeLa
cells without interference with their proliferation process.
CDCl3) 7.11–7.00 (m, 10H), 6.91 (d, 8H), 2.26 (d, 6H). dC
(75 MHz, CDCl3) 144.8, 141.6, 141.1, 136.6, 132.0, 131.9,
129.0, 128.2, 126.9, 21.9. m/z (FAB) 360.2 [Mþ]; calc. 360.2.
1,2-Bis[4-(bromomethyl)phenyl]-1,2-diphenylethene 7
Experimental
To a mixture of 6 (1.8 g, 5.0 mmol) and NBS (1.7 g,
10.0 mmol) in CCl4 was added catalytic amount of BPO at room
temperature. The mixture was stirred and heated to reflux for
8 h. After filtration and solvent evaporation, the product was
purified by silica gel chromatography using DCM/hexane
(1:4 v/v) as eluent. Compound 7 was isolated as pale yellow
solid in 43 % yield. dH (300 MHz, CDCl3) 7.14–7.07 (m, 10H),
7.02–6.96 (m, 8H), 4.41 (d, 4H). dC (75 MHz, CDCl3) 144.4,
143.9, 141.5, 136.6, 132.3, 132.0, 129.2, 128.5, 127.4, 34.3. m/z
(FAB) 518.0 [Mþ]; calc. 518.2.
Materials and Instruments
THF (Labscan) was purified by simple distillation from sodium
benzophenone ketyl undernitrogen immediately before use. Zinc
dust, titanium(IV) chloride, N-bromosuccinimide (NBS), benzoyl
peroxide (BPO), triphenylphosphine (PPh3), benzaldehyde,
4-cyanobenzaldehyde, carbon tetrachloride, 4-nitrobenzaldehyde,
triphenylamine, N,N-dimethylformamide (DMF), dichloro-
methane (DCM), dimethylsulfoxide (DMSO), and other reagents
were all purchased from Aldrich and used as received.
1H and 13C NMR spectra were measured on a Bruker ARX
300 spectrometer in CDCl3 using tetramethylsilane (TMS;
d ¼ 0 ppm) as the internal standard. Mass spectra were recorded
on a Finnigan TSQ 7000 triple quadrupole spectrometer operat-
ing in fast-atom bombardment (FAB) mode. UV-vis spectra
were measured on a Milton Roy Spectronic 3000 Array spec-
trophotometer. Photoluminescence (PL) spectra were recorded
on a Perkin–Elmer LS 50B spectrofluorometer with a xenon
discharge lamp excitation.
1,2-Bis[4-(2-phenylvinyl)phenyl]-1,2-diphenylethene 1
Triphenylphosphonium salt 8 was prepared from 7 and
triphenylphosphine in DMF at 1008C. After stirring for 24 h,
the solution was poured into large amount of toluene. The white
precipitate was collected and directly used for the next reaction
without purification. Into another round-bottom flask were
added benzaldehyde (0.1 g, 0.94 mmol), 8 (0.1 g, 0.19 mmol),
and a catalytic amount of 18-crown-6 in DMF (8 mL) under
nitrogen. The solution was cooled to 0–58C with an ice bath, into
which potassium t-butoxide (0.056 g, 0.50 mmol) was added.
The mixture was stirred under nitrogen at room temperature for
48 h and poured into a large amount of water. The precipitate
was collected and the crude product was purified on a silica gel
column using hexane as eluent. Yellow solid of 1 was obtained
in 78 % yield. dH (300 MHz, CDCl3) 7.46 (d, 2H), 7.40 (s, 2H),
7.32 (t, 2H), 7.23 (m, 2H), 7.19–7.17 (m, 6H), 7.15–7.10 (m,
6H), 7.06–6.97 (m, 8H), 6.88–6.85 (m, 2H), 6.51–6.49 (m, 2H).
dC (75 MHz, CDCl3) 143.5, 142.6, 140.8, 137.3, 135.3, 131.5,
130.0, 129.5, 128.7, 128.1, 127.6, 127.3, 127.0, 126.5, 125.8. m/z
(FAB) 536.3 [Mþ]; calc. 536.7.
Synthesis
The TPE derivatives 3–6 were prepared according to Scheme 1.
Typical procedures for their synthesis are shown below.
1,2-Bis(4-methylphenyl)-1,2-diphenylethene 6
A suspension of 4-methylbenzophenone (5, 3.6 g, 10.0mmol),
TiCl4 (1.9 g, 10.0 mmol), and Zn dust (1.3 g, 20.0 mmol) in dry
THF (100 mL) was refluxed for 20 h. Afterward, the reaction
mixture was cooled to room temperature and filtered. The
filtrate was evaporated and the crude product was purified on
a silica-gel column using dichloromethane as eluent. Compound
6 was isolated as white solid in 94 % yield. dH (300 MHz,
Compounds 2–4 were prepared by similar procedures
to those described above using appropriate aldehydes.

RESEARCH FRONT
Tetraphenylethene Luminogens
1205
1,2-bis{4-[2-(4-cyanophenyl)vinyl]phenyl}-1,2-diphenylethene
2: Yellow solid; yield 85 %. dH (300 MHz, CDCl3) 7.98–7.82
(m, 4H), 7.67–7.62 (m, 2H), 7.54–7.51 (m, 2H), 7.47–7.44
(m, 2H), 7.17–7.14 (m, 6H), 7.08–7.02 (m, 6H), 6.95–6.91
(m, 4H), 6.65 (d, 2H), 6.48 (d, 2H). dC (75 MHz, CDCl3)
144.7, 144.1, 142.6, 141.6, 135.1, 133.6, 133.2, 132.6, 132.0,
130.3, 128.8, 128.5, 127.4, 127.0, 119.7, 111.0. m/z (FAB)
587.2 [Mþ]; calc. 586.7.
1,2-Bis{4-[2-(4-nitrophenyl)vinyl]phenyl}-1,2-diphenylethene
3: Orange solid; yield 66 %. dH (300 MHz, CDCl3) 8.19
(d, 4H), 8.04 (d, 4H), 7.58 (d, 4H), 7.31–7.26 (m, 4H),
7.19–7.14 (m, 4H), 7.08–7.03 (m, 4H), 6.95–6.91 (m, 2H),
6.70 (d, 2H), 6.53 (d, 2H). dC (75 MHz, CDCl3) 146.7, 144.5,
144.1, 143.7, 143.5, 141.2, 134.5, 133.9, 133.2, 131.6, 129.8,
128.3, 127.0, 126.2, 124.0. m/z (FAB) 627.1 [M þ 1]þ; calc.
627.1.
4-(Diphenylamino)benzaldehyde (reactant for the synthesis
of 4) was prepared by formylation of triphenylamine following
the Vilsmeier–Haack procedure. White solid; yield 67 %. dH
(300 MHz, CDCl3) 9.81 (s, 1H), 7.68 (d, 2H), 7.37–7.32 (m, 4H),
7.19–7.15 (m, 6H), 7.01 (d, 2H). dC (75 MHz, CDCl3) 191.2,
146.9, 132.0, 130.4, 129.8, 127.0, 125.8, 124.9, 120.0.
1,2-Bis{4-[2-(4-diphenylaminophenyl)vinyl]phenyl}-1,2-diphe-
nylethene 4: Yellow solid; yield 50 %. dH (300 MHz, CDCl3)
7.68 (d, 4H), 7.37–7.32 (m, 8H), 7.29–7.23 (m, 4H), 7.19–7.17
(m, 8H), 7.11–7.09 (m, 8H), 7.03–7.00 (m, 12H), 6.96–6.87 (m,
4H), 6.42 (m, 2H). dC (75 MHz, CDCl3) 148.3, 148.0, 144.4,
143.6, 141.3, 136.3, 132.5, 132.0, 130.5, 130.0, 128.5, 128.4,
128.0, 127.5, 126.3, 125.3, 124.2, 123.7, 123.3. m/z (FAB) 872.0
[M þ 1þ]; calc. 872.1.
electroluminescence spectra were obtained with a PR650
spectrophotometer. All measurements were carried out under air
at room temperature without device encapsulation.
Cell Imaging
HeLa cells were grown overnight on a plasma-treated 25-mm
round coverslip mounted onto a 35-mm Petri dish with an
observation window. The living cells were stained with either
5 mM of 2 or 4 (by adding 2 mL of a 5 mM stock solution of 2 or
4 in DMSO to 2 mL culture) for 30 min to 5 h. The cells were
imaged under an upright fluorescence microscope (Olympus
BX41) or an inverted fluorescence microscope (Nikon Eclipse
TE2000-U) using the combination of excitation and emission
filters: excitation wavelength¼ 330–380 nm, diachronic mirror ¼
400 nm. The images of the cells were captured using a computer-
controlled SPOT charged-couple device camera (Spot RT SE 18
Mono).
Results and Discussion
Dye Synthesis
We synthesized four TPE derivatives with different substituents
on the peripheral phenyl rings and designed a multistep reaction
route for their synthesis. TPE derivatives 1–4 were prepared
according to the synthetic route shown in Scheme 1. 1,2-Bis(4-
methylphenyl)-1,2-diphenyl-ethene (6) was first synthesized
from McMurry coupling of 4-methylbenzophenone (5) using
titanium(IV) chloride and zinc dust as catalysts. Bromination of 6
gave 7, whose reaction with triphenylphosphine in DMF gen-
erated triphenylphosphonium salt 8. Wittig reactions of 8 with
the corresponding benzaldehydes in the presence of potassium
tert-butoxide furnished TPEs 1–4 in 50–85 % yields. All the
reactions proceeded smoothly and the desired products were
isolated in satisfactory to high yields. We characterized the
products by NMR and mass spectroscopy and all of them gave
satisfactory analysis data corresponding to their molecular
structures. The lipophilic TPE derivatives are completely solu-
ble in common organic solvents such as acetonitrile, chloro-
form, and THF, slightly soluble in methanol and ethanol, but
insoluble in water.
Preparation of TPE-Doped Poly(methyl methacrylate) Films
The TPE-doped poly(methyl methacrylate) (PMMA) films were
prepared by dissolving PMMA and TPE derivatives (10:1 by
weight) in chloroform and then spin-coating the resultant solu-
tions onto quartz plates.
Device Fabrication
The devices were fabricated on 80-nm indium tin oxide (ITO)-
coated glass with a sheet resistance of 25 O sqꢀ1. Prior to loading
into the pretreatment chamber, the ITO-coated glass was soaked
in detergent and ultrasonicated for 30 min, followed by spraying
with de-ionized water for 10 min, soaking in water and ultra-
sonicated de-ionized water for 30 min, and oven-baking for 1 h.
The cleaned samples were treated with perfluoromethane (CF4)
plasma with a power of 100 W, gas flow of 50 sccm, and pres-
sure of 0.2 Torr (26.6 Pa) for 10 s in the pretreatment chamber.
The samples were transferred to the organic chamber with a base
pressure of 7 ꢁ 10ꢀ7 Torr for the deposition of N,N-bis(1-naph-
thyl)-N,N-diphenylbenzidine (NPB), 2,9-dimethyl-4,7-diphenyl-
1,10-phenanthroline (BCP) and tris(8-hydroxyquinolinolato)-
aluminium (Alq3), which serve as hole-transport, hole-block,
and electron-transport layers respectively. The samples were
then transferred to the metal chamber for cathode deposition,
which was composed of lithium fluoride (LiF) capped with
aluminium (Al). The light-emitting area was 4 mm2. The current
density–voltage characteristics of the devices were measured
with a HP4145B semiconductor parameter analyzer. The for-
ward-direction photons emitted from the devices were detected
by a calibrated UDT PIN-25D silicon photodiode. The lumi-
nance and external quantum efficiencies of the devices were
inferred from the photocurrent of the photodiode. The
Aggregation-induced Emission
All the TPE are practically non-emissive when molecularly
dissolved in good solvents but emit intensely in the aggregate
state. An example of the UV and PL spectra of 1 is given in
Fig. 1.
The acetonitrile solution of 1 emits dimly at ,415 nm when
photoexcited at 370 nm. When 99 % water is added to the
acetonitrile solution, an intense PL signal is recorded at
511 nm under the same measurement conditions. As 1 is
insoluble in water, aggregates must have been formed in
aqueous mixtures with high water contents. The solution is,
however, homogeneous with no precipitates, suggesting that the
aggregates are of nanodimension. The formation of nanoaggre-
gates can be inferred from the level-off tail observed at the
longer wavelength in the UV spectrum in an acetonitrile/water
mixture with a 99 % water fraction. To have a quantitative
picture, we measured its fluorescence quantum yield (FF).
The FF in acetonitrile solution is as low as 0.48 %, revealing
that 1 is a genuinely weak emitter in the solution state. The FF
remains unchanged when up to 50 % water is added (Fig. 2).
Afterward, the value increases swiftly. At 90 % water fraction,
the FF value rises to ,26 %. The AIE effect can be quantified

RESEARCH FRONT
1206
Y. Hong et al.
by the extent of emission enhancement (aAIE),[14] as defined
below:
owing to the change in the packing order of the aggregates from
a crystalline state to an amorphous one.[15] At a ‘lower’ water
ratio, the molecules may have sufficient time to assemble in an
ordered fashion to form more emissive crystalline clusters,
whereas at a ‘higher’ water ratio, the molecules may abruptly
agglomerate in a random way to form less emissive amorphous
powders.
FF;a
FF;s
aAIE
¼
;
ð1Þ
where FF,a and FF,s are the quantum yields in the aggregate and
solution states respectively. From Eqn 1, the aAIE value for 1 was
calculated to be 53.8.
Careful observation reveals that the FF value decreases
slightly when the water content is raised to ,99 %, probably
Similar phenomena are observed in 2–4 (Table 1) and all of
these are AIE-active. The maximum wavelength of emission of
2 in 99 % aqueous mixture is ,10 nm red-shifted from that of 1,
suggestive of its higher conjugation due to the electronic
communication between the polarized cyano groups and the
ethene core. The emission of 3 is observed at an even longer
wavelength. Its FF value in the aggregate state is, however,
five-fold lower than 1, which is in some sense understandable
because nitro groups are known to induce intersystem crossing
and cause non-radiative transition. Among the molecules, 4
shows the smallest aAIE value because its dilute solution shows
comparably stronger emission owing to the contribution from
the triphenylamine unit. Although 4 emits more efficiently when
aggregated in poor solvent, the associated FF value is similar to
those of 1 and 2. These two effects thus mainly contribute to
the lower aAIE value of 4. Fig. 2 shows the photographs of
acetonitrile/water mixtures (1:99 v/v) of 1–3 taken under UV
illumination. Whereas 1 and 2 emit cyan and green light, 3 is
an orange emitter, demonstrating that the optical properties of
TPE can be readily tuned by molecular engineering alteration
of its structure.
Water content
(vol-%)
99
0
ϫ20
To gain insight into the mechanism of the AIE effect, we
doped TPE derivatives 1–4 into PMMA film (10 % by weight),
in which PMMA serves as a solid ‘solvent’ to disperse the TPE
molecules and impede their aggregate formation. Similarly to
their nanoaggregates in the suspensions (Table 1), all the TPE-
doped PMMA films emit bright light on photoexcitation, sug-
gesting that intermolecular interactions play little role in their
photophysical properties and the AIE phenomenon results from
the restriction of intramolecular rotations, which blocks the
non-radiative relaxation processes and hence boosts their PL
efficiency.
300
400
500
600
700
Wavelength [nm]
Fig. 1. UV and photoluminescence (PL) spectra of 1 in pure acetonitrile
(dashed line) and acetonitrile/water mixture (1:99 v/v; solid line); concen-
tration, 1:2.5 mM; excitation wavelength, 370 nm. Arrows pointing left
indicate the curves are UV spectra. Arrows pointing right indicate the curves
are PL spectra. The PL spectrum of 1 in pure acetonitrile is magnified by
20-fold.
30
Nanoscopic
aggregates
1
2
3
24
1
2
3
4
18
12
Isolated
species
6
0
0
20
40
60
80
100
Water fraction [%]
Fig. 2. Dependence of quantum yields of 1–4 on solvent compositions of acetonitrile/water mixtures. Photographs of 1–3 in
acetonitrile/water mixture (1:99 v/v) taken under 365-nm UV illumination; dye concentration, 2.5 mM; excitation wavelength, 370 nm.

RESEARCH FRONT
Tetraphenylethene Luminogens
1207
The AIE feature of the TPE derivatives prompted us to
explore their application in light-emitting diodes. We studied
their EL by fabrication of multilayer devices with a configura-
tion of ITO/NPB (60 nm)/TPE (30 nm)/BCP (20 nm)/Alq3
(20 nm)/LiF (1 nm)/Al (150 nm). As shown in Fig. 3, the
EL spectrum of 2 peaks at ,520 nm. The device is turned on
at ,10 V and radiates brilliantly with a luminance of up to
12930 cd cmꢀ2 at ,18 V. The maximum current efficiency
attained by the device is 3.04 cd Aꢀ1, which is equivalent
to a power efficiency of 0.87 lm Wꢀ1 at a current density of
,45 A mꢀ2 and an applied voltage of ,11 V. Disappointingly,
only weak EL was detected in the devices of other luminogens.
The exact reason, however, is unknown at present.
were prepared in Minimum Essential Medium (Sigma–Aldrich)/
DMSO mixtures and the HeLa cells were imaged using a stan-
dard cell-staining protocol. The living cells were incubated with
2 or 4 (5 mM) for 5 h and then washed three times with phosphate
buffered saline solution. As shown in Fig. 4a and c, the living
cells grow normally even in the presence 2 or 4, indicating that
the TPE pose no toxicity to living cells.
The fluorescent images were taken on an upright fluores-
cence microscope under UV illumination. Although both 2 and 4
are yellowish-green emitters in the aggregate state (cf. Fig. 2),
after entering the cell membrane, their emission colour changes
to blue (Fig. 4b and d). The change in the emission colour can be
discerned from Fig. 4d, where the extracellular nanoaggregates
exhibit strong yellow emission. This phenomenon is commonly
observed in TPE-based stains. It is plausible that the macromo-
lecular crowding environment in the interior of the cell forces
the dye molecules to adopt a more twisted conformation, thus
resulting in emission in the shorter-wavelength region.[16] The
performance of 2 is much better than 4 in terms of emission
intensity. Thus, we used 2 for further investigation.
Cell Imaging
The TPE derivatives are not soluble but form highly emissive
nanoaggregates in aqueous medium, which inspired us to explore
their application as fluorescent stains for living cell imaging. As
2 and 4 possess higher quantum efficiencies in the aggregate
state, we chose them for the study. The nanoaggregates of 2 and 4
Table 1. Optical properties of 1 4 in solution (Soln), aggregate (Aggr), and film states
]
F,a and FF,s, quantum yields in the aggregate and solution states respectively
F
l
ab [nm]D
AggrB
l
em [nm]E
Aggr (FF,a
Film
SolnA
FilmC
Soln (FF,s
)
)
FF,a/FF,s
1
2
3
4
335
334
380
383
359
370
404
390
365
395
443
419
415 (0.48)
418 (0.66)
467 (0.07)
517 (1.85)
511 (25.8)
521 (25.3)
586 (5.13)
525 (27.7)
53.8
38.3
73.3
15.0
476
496
540
502
AIn acetonitrile solutions (2.5 mM).
BIn acetonitrile/water mixtures (1:99 v/v, 2.5 mM).
CBlended with PMMA (poly(methyl methacrylate); 10 % by weight).
DAbsorption maximum.
EEmission maximum with quantum yield (FF, %) given in parentheses. The FF values were determined using quinine sulfate (for 1 and 2), fluorescein (for 3)
and 9,10-diphenylanthracene (for 4); excitation wavelength: 370 nm.
7500
6000
4500
3000
1500
0
3.5
2.8
2.1
1.4
0.7
0.0
(b)
(a)
104
103
102
101
100
10Ϫ1
380
460
540
620
700
Wavelength [nm]
6
8
10
12
14
16
18
0
1400
2800
4200
5600
7000
Voltage [V]
Current density [A mϪ2
]
Fig. 3. (a) Change in current density and luminance with voltage, and (b) current efficiency versus current density curve of a multilayer electroluminescence
(EL) device of 2. Inset in panel (b): EL spectrum of the device. Arrow pointing left indicates the curve is current density and arrow pointing right indicates the
curve is luminance. The circles represent the whole curves.

RESEARCH FRONT
1208
Y. Hong et al.
(a)
(b)
(c)
(d)
Fig. 4. (a and c) Phase contrast and (b and d) fluorescence images of HeLa cells stained with (a and b) 2 and (c and d) 4 for 5 h. Scale
bar ¼ 10 mm; [dye] ¼ 5 mM.
(a)
(b)
(c)
(d)
Fig. 5. (a and c) Phase contrast and (b and d) fluorescent images of HeLa cells stained with 2 for (a and b) 0.5 h and (c and d) 3 h.
Scale bar ¼ 10 mm; [dye] ¼ 5 mM.
Some commercially available fluorescence dyes, such as
CellTracker GreenTM, stain the entire cell; only the cytoplasm
of the cells, however, was stained by the TPE dyes, presumably
owing to their hydrophobic nature. Water-soluble fluorogens
such as CellTracker GreenTM can enter the cells through the
cell membrane. As they are genuinely dissolved in the aqueous
medium, their isolated molecules can enter the nucleus via the
nuclear pores. Therefore, these dyes can label both the cyto-
plasm and the nuclear compartments of the cells. In contrast, the
major route for the nanoaggregates of 2 to enter a living cell is
through endocytosis. During the process, the nanoaggregates are
enclosed by the cell membrane to form small vesicles that can be

RESEARCH FRONT
Tetraphenylethene Luminogens
1209
internalized by the cell. Inside the cell, the aggregates can be
further processed in endosomes and lysosomes and are eventu-
ally released from the cellular organelles. The hydrophobic
nature of the aggregates prevents them from entering the
nucleus. When the nanoaggregates are bound to the biomacro-
molecules in the cytoplasm, they emit more intensely owing
to the additional physical restriction to their intramolecular
rotations.
References
[1] (a) S. M. Borisov, O. S. Wolfbeis, Chem. Rev. 2008, 108, 423.
doi:10.1021/CR068105T
(b) Y. Wang, J. Y. J. Shyy, S. Chien, Annu. Rev. Biomed. Eng. 2008,
10, 1. doi:10.1146/ANNUREV.BIOENG.010308.161731
(c) S. B. VanEngelenburg, A. E. Palmer, Curr. Opin. Chem. Biol. 2008,
12, 60. doi:10.1016/J.CBPA.2008.01.020
(d) D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008, 4,
168. doi:10.1038/NCHEMBIO.69
Close inspection of the cell image reveals some highly
emissive particles in the cytoplasm. These fluorescent spots
may correspond to vesicles (endosomes, lysosomes, etc.) with
large amounts of TPE nanoaggregates. To prove that the dye
aggregates enter the cell through endocytosis, we performed the
cell staining at different time intervals. The fluorescence images
were taken with an inverted fluorescence microscope to have a
better comparison of their brightness. Photographs were
obtained in black and white and pseudocoloured green, which
was the same colour emitted by 2 in the aggregate state.
The HeLa cells were first incubated with 2 for 30 min. As
shown in Fig. 5b, the nanoaggregates of 2 light up the whole
cytoplasm of the cell uniformly. Many dark spots are also
observed. The nanoaggregates of 2 may have entered the cell
membrane but not yet accumulated in the vesicles because of
insufficient incubation time. As expected, the vesicles become
emissive when the incubation time is prolonged to 3 h.
The nanoaggregates of 2 can selectively stain the cytoplasm
of the cells, endowing them with a unique advantage over
commercial CellTracker, which stains the entire cells. In most
cases, it is necessary to use two different fluorogenic dyes to
stain a cell: one to stain DNA in the nucleus, with the other to
stain the cytoplasm surrounding the nucleus. The fluorescent
dyes we developed here are therefore useful staining agents
when combined with a DNA-staining fluorogen. The nano-
aggregates of 2 can specifically accumulate in the vesicles of
the living cells, making them promising for the tracing of the
endocytosis process.
(e) A. Mayer, S. Neuenhofer, Angew. Chem. Int. Ed. Engl. 1994, 33,
1044. doi:10.1002/ANIE.199410441
[2] (a) B. N. G. Giepmans, S. R. Adams, M. h. Ellisman, R. Y. Tsien,
Science 2006, 312, 217. doi:10.1126/SCIENCE.1124618
(b) X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J.
Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, S. Weiss, Science 2005,
307, 538. doi:10.1126/SCIENCE.1104274
(c) S. W. Thomas III, G. D. Joly, T. M. Swager, Chem. Rev. 2007, 107,
1339. doi:10.1021/CR0501339
(d) A. Burns, H. Ow, U. Wiesner, Chem. Soc. Rev. 2006, 35, 1028.
doi:10.1039/B600562B
(e) N. L. Rosi, C. A. Mirkin, Chem. Rev. 2005, 105, 1547. doi:10.1021/
CR030067F
[3] (a) I. L. Medintz, H. T. Uyeda, E. R. Goldman, H. Mattoussi, Nat.
Mater. 2005, 4, 435. doi:10.1038/NMAT1390
(b) J. M. Klostranec, W. C. W. Chan, Adv. Mater. 2006, 18, 1953.
doi:10.1002/ADMA.200500786
[4] (a) F. C. Krebs, S. R. Miller, B. J. Catalone, R. Fichorova, D. Anderson,
D. Malamud, M. K. Howett, B. Wigdahl, Antimicrob. Agents
Chemother. 2002, 46, 2292. doi:10.1128/AAC.46.7.2292-2298.2002
(b) H. Li, Q. Zhou, W. Liu, B. Yan, Y. Zhao, G. Jiang, Sci. China Ser.
Biol. Chem. 2008, 51, 393. doi:10.1007/S11426-008-0057-9
[5] I. Chen, A. Y. Ting, Curr. Opin. Biotechnol. 2005, 16, 35. doi:10.1016/
J.COPBIO.2004.12.003
[6] (a) M. Sameiro, T. Gonc¸alves, Chem. Rev. 2009, 109, 190. doi:10.1021/
CR0783840
(b) A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891. doi:10.1021/
CR078381N
[7] (a) F. h. Quina, E. A. Lissi, Acc. Chem. Res. 2004, 37, 703. doi:10.1021/
AR030152þ
(b) I. Capek, Adv. Colloid Interface Sci. 2002, 97, 91. doi:10.1016/
S0001-8686(01)00049-5
Conclusions
[8] (a) J.-L. Mergny, J.-C. Maurizot, ChemBioChem 2001, 2, 124.
doi:10.1002/1439-7633(20010202)2:2,124::AID-CBIC124.3.0.CO;
2-L
In this work, a series of tetraphenylethene derivatives (1–4) with
variedemission colours were designed and synthesized. Whereas
they are practically non-emissive in dilute solutions, they are
induced to emit intensely by aggregate formation, demonstrating
a novel phenomenon of AIE. Restriction of intramolecular
motions is responsible for this effect. A multilayer EL device
using 2 as emitter was fabricated, which emits green light with a
maximum luminance and current efficiency of 12930 cd mꢀ2 and
3.04 cd Aꢀ1 respectively. The nanoaggregates of 1–4 are highly
emissive and cytocompatible, enabling their use for sensitive and
selective imaging the cytoplasm of living cells. Further synthesis
of red-emitting AIE luminogens and investigation of their
biological application are under way in our laboratories.
(b) J. L. Mergny, Biochemistry 1999, 38, 1573. doi:10.1021/
BI982208R
(c) N. Venkatesan, Y. J. Seo, B. H. Kim, Chem. Soc. Rev. 2008, 37,
648. doi:10.1039/B705468H
[9] (a) J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S.
Kwok, X. Zhan, Y. Liu, D. Zhu, B. Z. Tang, Chem. Commun. 2001,
1740. doi:10.1039/B105159H
(b) J. Chen, C. C. W. Law, J. W. Y. Lam, Y. Dong, S. M. F. Lo,
I. D. Williams, D. Zhu, B. Z. Tang, Chem. Mater. 2003, 15, 1535.
doi:10.1021/CM021715Z
(c) J. Chen, B. Xu, X. Ouyang, B. Z. Tang, Y. Cao, J. Phys. Chem. A
2004, 108, 7522. doi:10.1021/JP048475Q
(d) H. Tong, Y. Dong, M. Haussler, J. W. Y. Lam, H. H. Y. Sung, I. D.
Williams, J. Sun, B. Z. Tang, Chem. Commun. 2006, 1133. doi:10.1039/
B515798F
Acknowledgement
(e) Q. Zeng, Z. Li, Y. Dong, C. Di, A. Qin, Y. Hong, Z. Zhu, C. K. W.
Jim, G. Yu, Q. Li, Z. Li, Y. Liu, J. Qin, B. Z. Tang, Chem. Commun.
2007, 70. doi:10.1039/B613522F
(f) Y. Dong, J. W. Y. Lam, A. Qin, J. Sun, J. Liu, Z. Li, J. Sun, H. H. Y.
Sung, I. D. Williams, H. S. Kwok, B. Z. Tang, Chem. Commun. 2007,
3255. doi:10.1039/B704794K
(g) H. Tong, Y. Hong, Y. Dong, M. Haussler, J. W. Y. Lam, Z. Li,
Z. Guo, Z. Guo, B. Z. Tang, Chem. Commun. 2006, 3705. doi:10.1039/
B608425G
This work was partially supported by the Research Grants Council of Hong
Kong (Project numbers 603509, HKUST13/CRF/08 and HKUST2/CRF/10),
the Innovation and Technology Commission (ITP/008/09NP), the Univer-
sity Grants Committee of Hong Kong (AoE/P-03/08) and the National
Science Foundation of China (20974028). B.Z.T. acknowledges the support
of the Cao Guangbiao Foundation of Zhejiang University and Dr Jiaxin Sun
and Professor Hoi Sing Kwok in the Department of Electronic and Computer
Engineering in Hong Kong University of Science and Technology for the
electroluminescence measurement.

RESEARCH FRONT
1210
Y. Hong et al.
[10] (a) Y. T. Wu, M. Y. Kuo, Y. T. Chang, C. C. Shin, T. C. Wu, C. C. Tai,
T. H. Cheng, W. S. Liu, Angew. Chem. Int. Ed. 2008, 47, 9891.
doi:10.1002/ANIE.200802560
(b) Y. Dong, J. W. Y. Lama, A. Qin, J. Liu, Z. Li, B. Z. Tang, Appl.
Phys. Lett. 2007, 91, 11111. doi:10.1063/1.2753723
[12] (a) Y. Hong, M. Haeussler, J. W. Y. Lam, Z. Li, K. K. Sin, Y. Dong,
H. Tong, J. Liu, A. Qin, R. Renneberg, B. Z. Tang, Chemistry 2008, 14,
6428. doi:10.1002/CHEM.200701723
(b) C. J. Bhongale, C. W. Chang, C. S. Lee, E. W. G. Diau, C. S. Hsu,
J. Phys. Chem. B 2005, 109, 13472. doi:10.1021/JP0502297
(c) Z. Xie, B. Yang, W. Xie, L. Liu, F. Shen, H. Wang, X. Yang,
Z. Wang, Y. Li, M. Hanif, G. Yang, L. Ye, Y. Ma, J. Phys. Chem. B
2006, 110, 20993. doi:10.1021/JP064069Q
(d) C. X. Yuan, X. T. Tao, L. Wang, J. X. Yang, M. h. Jiang, J. Phys.
Chem. C 2009, 113, 6809. doi:10.1021/JP8111167
(e) Z. Yang, Z. Chi, T. Yu, X. Zhang, M. Chen, B. Xu, S. Liu,
Y. Zhang, J. Xu, J. Mater. Chem. 2009, 19, 5541. doi:10.1039/
B902802A
(b) Y. Hong, H. Xiong, J. W. Y. Lam, M. Haessler, J. Liu, Y. Yu,
Y. Zhong, H. H. Y. Sung, I. D. Williams, K. S. Wong, B. Z. Tang,
Chemistry 2010, 16, 1232. doi:10.1002/CHEM.200900778
(c) Y. Hong, C. Feng, Y. Yu, J. Liu, J. W. Y. Lam, K. Q. Luo, B. Z.
Tang, Anal. Chem. 2010, 82, 7035. doi:10.1021/AC1018028
(d) H. Tong, Y. N. Hong, Y. Q. Dong, M. Haussler, J. W. Y. Lam,
Z. Li, Z. F. Guo, Z. H. Guo, B. Z. Tang, Chem. Commun. 2006,
3705. doi:10.1039/B608425G
(f) Z. J. Ning, Z. Chen, Q. Zhang, Y. L. Yan, S. X. Qian, Y. Cao,
H. Tian, Adv. Funct. Mater. 2007, 17, 3799. doi:10.1002/ADFM.
200700649
(e) Y. Yu, Y. Hong, C. Feng, J. Liu, J. W. Y. Lam, M. Faisal,
K. M. Ng, K. Q. Luo, B. Z. Tang, Sci. China Ser. B: Chem. 2009,
52, 15. doi:10.1007/S11426-009-0008-0
(g) G. Qian, B. Dai, M. Luo, D. Yu, J. Zhan, Z. Zhang, M. Dongge,
Z. Y. Wang, Chem. Mater. 2008, 20, 6208. doi:10.1021/CM801911N
(h) W. X. Tang, Y. Xiang, A. J. Tong, J. Org. Chem. 2009, 74, 2163.
doi:10.1021/JO802631M
(i) J. He, B. Xu, F. Chen, H. Xia, K. Li, L. Ye, W. Tian, J. Phys. Chem.
C 2009, 113, 9892. doi:10.1021/JP900205K
(j) E. Quartapelle Procopio, M. Mauro, M. Panigati, D. Donghi,
P. Mercandelli, A. Sironi, G. D’Alfonso, L. De Cola, J. Am. Chem.
Soc. 2010, 132, 14397. doi:10.1021/JA106772V
(f) T. Sanji, K. Shiraishi, M. Tanaka, ACS Appl. Mater. Interfaces
2009, 1, 270. doi:10.1021/AM800224R
[13] M. Wang, G. Zhang, D. Zhang, D. Zhu, B. Z. Tang, J. Mater. Chem.
2010, 20, 1858 and references therein. doi:10.1039/B921610C
[14] Z. Zhao, S. Chen, X. Shen, F. Mahtab, Y. Yu, P. Lu, J. W. Y. Lam,
H. S. Kwok, B. Z. Tang, Chem. Commun. 2010, 46, 686. doi:10.1039/
B915271G
[15] Y. Q. Dong, J. W. Y. Lam, A. Qin, J. X. Sun, J. Z. Liu, Z. Li, J. Z. Sun,
H. H. Y. Sung, I. D. Williams, H. S. Kwok, B. Z. Tang, Chem.
Commun. 2007, 3255. doi:10.1039/B704794K
[11] (a) H. Tong, Y. Hong, Y. Dong, M. Haussler, Z. Li, J. W. Y. Lam,
Z. Li, Y. Dong, H. H. Y. Sung, I. D. Williams, B. Z. Tang, J. Phys.
Chem. B 2007, 111, 11817. doi:10.1021/JP073147M
[16] R. J. Ellis, Curr. Opin. Struct. Biol. 2001, 11, 500. doi:10.1016/S0959-
440X(00)00239-6