A series of triphenylamine-based two-photon absorbing materials with AIE property for biological imaging

Article abstract of DOI:10.1039/c4tb00464g

A specific series of D-π-A (1A-3A) and D-π-A (1B-3B) structural chromophores with various electron donors and π-conjugated bridges were designed, synthesized, and fully characterized. Their crystal structures were determined by single crystal X-ray diffraction analysis. The one/two-photon absorption properties of the chromophores have been successfully tuned by using different electron donors and π-bridges. Interestingly, it was found that chromophore 3B shows quite weak fluorescence in pure DMSO, while a significant AIE (aggregation-induced emission) effect is observed in water-DMSO (v/v 90%) mixtures with a sharp increase in fluorescence intensity of about 11 times. Furthermore, chromophore 3B shows strong two-photon excited fluorescence (TPEF) and has a large 2PA action cross-section (394 GM). The results of live-cell imaging experiments show that 3B can be effectively used as a bio-imaging probe in two-photon fluorescence microscopy towards HepG2 cells in vitro. The TPEF images of 3B not only exhibit cellular cytosol uptake but also exhibit actin regulatory protein uptake which are different from OPEF images.

Full text of DOI:10.1039/c4tb00464g

Journal of
Materials Chemistry B
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PAPER
A series of triphenylamine-based two-photon
absorbing materials with AIE property for biological
Cite this: J. Mater. Chem. B, 2014, 2,
imaging†
5430
Yanqiu Liu, Ming Kong, Qiong Zhang, Zhiwen Zhang, Hongping Zhou, Shengyi Zhang,
*
*
Shengli Li, Jieying Wu and Yupeng Tian
A specific series of D–p–A (1A–3A) and D–p–A (1B–3B) structural chromophores with various electron
donors and p-conjugated bridges were designed, synthesized, and fully characterized. Their crystal
structures were determined by single crystal X-ray diffraction analysis. The one/two-photon absorption
properties of the chromophores have been successfully tuned by using different electron donors and p-
bridges. Interestingly, it was found that chromophore 3B shows quite weak fluorescence in pure DMSO,
while a significant AIE (aggregation-induced emission) effect is observed in water–DMSO (v/v 90%)
mixtures with a sharp increase in fluorescence intensity of about 11 times. Furthermore, chromophore
3B shows strong two-photon excited fluorescence (TPEF) and has a large 2PA action cross-section (394
GM). The results of live-cell imaging experiments show that 3B can be effectively used as a bio-imaging
probe in two-photon fluorescence microscopy towards HepG2 cells in vitro. The TPEF images of 3B not
only exhibit cellular cytosol uptake but also exhibit actin regulatory protein uptake which are different
from OPEF images.
Received 24th March 2014
Accepted 8th May 2014
DOI: 10.1039/c4tb00464g
www.rsc.org/MaterialsB
(Fs, where F is the uorescence quantum yield) is highly
desired. It is necessary that 2PA chromophores are water soluble
Introduction
or dispersable and remain highly uorescent in aqueous media.
However, highly efficient 2PA uorescence has suffered the
defect of low uorescence efficiency in aqueous media because
most 2PA molecules are hydrophobic and their uorescence
quantum yields are considerably reduced in water, by self-
aggregation, which generally leads to uorescence quenching.18
To overcome this limitation, a special molecular design for a
2PA chromophore is required not only to ensure a large two-
photon activity, but also, more importantly, to overcome uo-
rescence quenching at high concentration or in a biological
aqueous environment, which is generally observed for common
organic uorophores. Recently, some novel 2PA chromophores
were synthesized both with large two-photon activity and high
uorescence quantum yields in aggregation. Prasad and co-
workers had synthesized a chromophore with aggregation-
enhanced uorescence and two-photon absorption in nano-
aggregates due to the hindering of molecular internal rotation.19
Tang discovered luminogenic molecules and polymers of AIE
and 2PA based on tetraphenylethene.20 Recently, our group has
reported several series of compounds based on isophorone21 or
triphenylamine22 with good AIE property.
Organic molecules with large two-photon absorption (2PA) draw
great interest in the eld of materials science due to their various
applications including optical limiting,1–3 up-converted lasing,4
3D optical data storage,5 micro-fabrication,6 bio-imaging,7 and
photodynamic therapy.8 With signicant potential applications,
a large amount of compounds with large 2PA cross-sections (s)
and good processabilities have been developed. The molecular
design strategies include symmetrical donor–acceptor–donor (D–
A–D), donor–p-bridge–acceptor (D–p–A), and donor–p-bridge–
donor (D–p–A) structures.9–16 These studies revealed that the
intramolecular charge transfer (ICT) from the donor groups to
the p-bridge can enhance the 2PA cross-sections. Other factors
such as the strength of the donor and acceptor, the character of
the conjugated bridge, the planarity of chromophores, and the
dimensionality of the charge-transfer network17 can also enhance
the 2PA cross-section.
For bio-photonic applications, a good 2PA labeling agent
with a high 2PA cross-section and large 2PA action cross-section
Department of Chemistry, Key Laboratory of Functional Inorganic Materials Chemistry
of Anhui Province, Anhui University, Hefei 230039, P. R. China. E-mail: jywu1957@
163.com; yptian@ahu.edu.cn
Triphenylamine has been widely used in opto- and electro-
active materials due to its good electron-donating and trans-
porting capabilities, as well as its special propeller starburst
molecular structure.23,24 Recently, 2PA materials with triphe-
nylamine as the electron donor have aroused great interest and
† Electronic supplementary information (ESI) available: CIF les giving X-ray
crystallographic data. CCDC 1006493, 1006514 and 1006515. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c4tb00464g
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1
spectrometer (KBr discs) in the 4000–400 cmꢀ1 region. H and
13C NMR spectra were recorded on a 400 and 100 MHz NMR
instrument using (CD3)2SO or (CD3)2CO as a solvent. Chemical
shis were reported in parts per million (ppm) relative to
internal TMS (0 ppm) and coupling constants in Hz. Splitting
patterns were described as singlet (s), doublet (d), triplet (t),
quartet (q), or multiplet (m). MALDI-TOF mass spectra were
recorded on a time-of-ight (TOF) mass spectrometer using a
337 nm nitrogen laser with alpha-cyano-4-hydroxycinnamic
acid as the matrix.
Optical measurements
The one-photon absorption (OPA) spectra were obtained on a
UV-265 spectrophotometer. The one-photon excited uores-
cence (OPEF) spectra measurements were performed using a
Hitachi F-7000 uorescence spectrophotometer. OPA and OPEF
of all chromophores were measured in ve organic solvents of
different polarities with the concentration of 1.0 ꢁ 10ꢀ5 mol
Lꢀ1. The quartz cuvettes used are of 1 cm path length. The
uorescence quantum yields (F) were determined by using
coumarin 307 as the reference according to the literature
method.31 Quantum yields were corrected as follows:
Scheme 1 Synthesis routes to chromophores 1A–3A and 1B–3B.
ꢀ
ꢁ
2
Arhs Ds
2
Fs ¼ Fr
Ashs Dr
become the focus of intensive research in this eld.25,26 Ethyoxyl
was also attached to the triphenylamine group to enhance its
electron-donating ability and improve solubility of the
compounds. Moreover, cyano-substituted compounds show
good optical and electrical properties due to their high electron
affinities. Molecules including an electron-withdrawing cyano
group on the central p-bridge exhibit strong uorescence and
higher 2PA cross-section values.27,28 Some cyano-substituted
compounds have been reported to show unique enhanced
emission rather than uorescence quenching in the solid
state,29,30 which enlightened us to design novel functional
molecules with both AIE and 2PA properties. In the present
study, we report synthesis and optical properties (linear and
nonlinear) of six novel type D–p–A (1A–3A) and D–p–A (1B–3B)
chromophores (Scheme 1) based on cyano-substituted triphe-
nylamine with derivatives. The correlation between molecular
structures and spectral properties is discussed by quantum-
chemical calculation and X-ray crystallography. Interestingly, a
simple modication on the p-bridge of chromophore 3B results
in good AIE characteristics together with fascinating 2PA
properties compared with other related chromophores, sug-
gesting the suitability of 3B for high-contrast bio-imaging
application.
where the s and r indices designate the sample and reference
samples, respectively, A is the absorbance at lexc, h is the
average refractive index of the appropriate solution, and D is the
integrated area under the corrected emission spectrum.32
For time-resolved uorescence measurements, the uores-
cence signals were collimated and focused onto the entrance
slit of a monochromator with the output plane equipped with a
photomultiplier tube (HORIBA HuoroMax-4P). The decays were
analyzed by ‘least-squares’. The quality of the exponential ts
was evaluated by the goodness of t (c2).
Two-photon absorption (2PA) cross-sections (s) of the
samples were obtained using the two-photon excited uores-
cence (TPEF) method33 at femtosecond laser pulse and a
Ti:sapphire system (680–1080 nm, 80 MHz, 140 fs, Chameleon
II) as the light source. The sample was dissolved in benzene
solvent at a concentration of 1.0 ꢁ 10ꢀ3 mol Lꢀ1. The intensities
of TPEF spectra of the reference and the sample were deter-
mined at their excitation wavelength. Thus, s of samples was
determined by the following equation:
FFref Cref nref
s ¼ sref
F
ref FCn
where the ref subscripts stand for the reference molecule (here
uorescein in the aqueous NaOH solution (1 mol Lꢀ1) at a
concentration of 1.0 ꢁ 10ꢀ3 mol Lꢀ1 was used as the reference).
s is the 2PA cross-section value, c is the concentration of the
solution, n is the refractive index of the solution, F is the TPEF
Experimental section
General procedures
All chemicals are commercially available and used as obtained. integral intensities of the solution emitted at the exciting
The solvents were puried by conventional methods before use. wavelength, and F is the uorescence quantum yield. The sref
IR spectra were recorded with a Nicolet FT-IR NEXUS 870 value of reference was taken from the literature.34
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DMSO) were added to obtain nal concentrations of 0, 10, 20,
40, 60, and 80 mM. The treated cells were incubated for 24 h at
37 C and under 5% CO2. Subsequently, the cells were treated
Computational studies
Density functional theory (DFT) calculations on all the
compounds were carried out in vacuo for a better understanding
of the charge transfer state. Optimizations were carried out with
B3LYP[LANL2DZ] without any symmetry restraint, and the time-
dependent density functional theory (TD-DFT) {B3LYP
[LANL2DZ]} calculations were performed on the optimized
structure.35 All calculations, including optimizations and TD-
DFT, were performed using G03 soware.36 Geometry optimi-
zation of the singlet ground state and the TD-DFT calculation of
the lowest 25 singlet–singlet excitation energies were calculated
with a basis set composed of 6-31 G* for C, H, N, and O atoms
were downloaded from the EMSL basis set library.
ꢂ
with 5 mg mLꢀ1 MTT (40 mL per well) and incubated for an
additional 4 h (37 ꢂC, 5% CO2). Then, DMEM was removed, the
formazan crystals were dissolved in DMSO (150 mL per well),
and the absorbance at 490 nm was recorded. The cell viability
(%) was calculated according to the following equation: cell
viability % ¼ OD490(sample)/OD490(control) ꢁ 100, where
OD490(sample) represents the optical density of the wells treated
with various concentrations of the compounds and
OD490(control) represents that of the wells treated with DMEM +
10% FCS. Three independent trials were conducted, and the
averages and standard deviations are reported. The reported
percent cell survival values are relative to untreated control
cells.
X-ray structural determinations
Single crystals of the compounds used in X-ray determination
were obtained by slow evaporation of their mother liquors. X-ray
diffraction data of 1A–3A and 3B were collected on a Bruker
Smart 100 CCD area detector diffractometer. Both of the radi-
Synthesis
The chromophores 1A and 1B were prepared as reported in the
literature.38
˚
ation sources were Mo Ka (l ¼ 0.71073 A). Empirical absorption
correction was applied to the data. The structures were solved
by direct methods and rened by full-matrix least-squares
methods on F2. All the nonhydrogen atoms were located from
the trial structure and then rened anisotropically with
SHELXTL using the full matrix least-squares procedure.37 The
hydrogen atom positions were geometrically idealized and
generated in idealized positions and xed displacement
parameters.†
N-(4-(4-Nitrostyryl)phenyl)-4-ethoxy-N-(4ethoxyphenyl)benzen-
amine (2A). Diethyl(4-nitrophenyl)methyphosphonate (M1) (2.73
g, 10 mmol), t-BuOK (2.24 g, 20 mmol), 4-(bis(4-ethoxyphenyl)
amino)benzaldehyde (M2) (3.61 g, 10 mmol), and a modicum of
18-crown-6 were placed into a dry mortar. The mixture was milled
vigorously for about 20 min and aer completion of the reaction
(monitored by TLC), the mixture was dissolved in 150 mL CH2Cl2
and washed with Di-water. The organic layer was separated, dried
over MgSO4, ltered and the solvent was removed in vacuo. The
product was puried by recrystallization from anhydrous ethanol
to get 2.41 g dark red crystals. Yield: 51%. IR (KBr, cmꢀ1): 2978,
2924, 1580, 1504, 1476, 1391, 1334, 1239, 954, 834. 1H-NMR (400
MHz, (CD3)2SO) d (ppm): 8.20 (d, J ¼ 8.80 Hz, 2H), 7.79 (d, J ¼ 8.80
Hz, 2H), 7.46 (m, 3H), 7.19 (d, J ¼ 16.0 Hz, 1H), 7.08 (d, J ¼ 8.80
Hz, 4H), 6.92 (d, J ¼ 8.80 Hz, 4H), 6.83 (d, J ¼ 8.80 Hz, 2H), 4.04
(q, J ¼ 6.80 Hz, 4H), 1.37 (t, J ¼ 6.80 Hz, 6H). 13C-NMR (100 MHz,
(CD3)2SO) d (ppm): 156.98, 150.51, 145.90, 140.86, 134.17, 128.14,
127.46, 123.97, 119.79, 116.27, 64.24, 15.16. MALDI-TOF calcd for
C30H28N2O4, 480.21; found, 480.25.
N-(4-(4-Aminostyryl)phenyl)-4-ethoxy-N-(4-ethoxyphenyl)ben-
zenamine (2B). 2A (0.96 g, 2 mmol) was dissolved in ethanol
(25 mL) and added into a round-bottom ask equipped with a
magnetic stirrer. The mixture is then heated to 80 ꢂC. 0.06 g Pd/
C catalyst was added into the preceding reaction system and a
solution of ethanol (25 mL) containing 85% hydrazine hydrate
(5 mL) was added dropwise over 0.5 h. Aer the reaction
completed, the solvent was removed under reduced pressure.
The mixture was washed with water and extracted with ethyl
acetate. The organic phase was dried over MgSO4, ltered and
Cell image
HepG2 cells were seeded in 6 well plates at a density of 2 ꢁ 105
cells per well and grown for 96 h. For live cell imaging cell
cultures were incubated with the complexes (10% PBS : 90% cell
media) at a concentration of 40 mM and maintained at 37 ꢂC in
an atmosphere of 5% CO2 and 95% air for incubation times
ranging for 2 h. The cells were then washed with PBS (3 ꢁ 3 mL
per well) and 3 mL of PBS was added to each well. The cells were
imaged using confocal laser scanning microscopy and water
immersion lenses. An excitation energy of 720 nm was used and
the uorescence emission was measured at 495–582 nm.
HepG2 cells were luminescently imaged on a Zeiss LSM 710
META upright confocal laser scanning microscope using
magnication 40ꢁ and 100ꢁ water-dipping lenses for mono-
layer cultures. Image data acquisition and processing was per-
formed using Zeiss LSM Image Browser, Zeiss LSM Image
Expert and Image J.
Cytotoxicity assays in cells
To ascertain the cytotoxic effect of the treatment of all the concentrated to provide light red oil. The obtained oil is then
compounds over a 24 h period, the 5-dimethylthiazol-2-yl-2,5- puried by silica gel column chromatography using a mixture of
diphenyl-tetrazolium bromide (MTT) assay was performed. petroleum ether (b.p. 60–90 ꢂC)–ethyl acetate (10 : 1 by volume)
HepG2 cells were trypsinized and plated to ꢃ70% conuence in to get 0.55 g yellow powder. Yield: 60%. IR (KBr, cmꢀ1): 3452,
96-well plates 24 h before treatment. Prior to the treatment of 3374, 3028, 2979, 2925, 1607, 1503, 1476, 1392, 1283, 1238, 1046,
compounds, the DMEM was removed and replaced with fresh 962, 827. 1H-NMR (400 MHz, (CD3)2SO), d (ppm): 7.31 (d, J ¼ 8.8
DMEM, and aliquots of the compound stock solutions (500 mM Hz, 2H), 7.21 (d, J ¼ 8.4 Hz, 2H), 6.97 (d, J ¼ 8.8 Hz, 4H), 6.88 (t,
5432 | J. Mater. Chem. B, 2014, 2, 5430–5440
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5H), 6.82 (d, J ¼ 13.2 Hz, 1H), 6.73 (d, J ¼ 8.8 Hz, 2H), 6.54 (d, J ¼
8.4 Hz, 2H), 5.22 (s, 2H), 3.99 (q, J ¼ 6.9 Hz, 4H), 1.32 (t, J ¼ 6.8
Hz, 6H). 13C-NMR (100 MHz, (CD3)2SO) d (ppm): 154.83, 148.24,
146.92, 140.03, 130.21, 127.16, 126.52, 126.30, 125.16, 122.56,
119.84, 115.34, 113.90, 63.13, 14.68. MALDI-TOF calcd for
C30H30N2O2, 450.23; found, 450.28.
The crystal structure descriptions of 1A–3A and 3B
The crystal structures of the chromophores are shown in Fig. 1–
3 and S1–S3.† The crystal data collection and renement
parameters are listed in Table S1.† The selected bond distances
and angles are given in Table S2.† The structure of chromo-
phore 1A has been reported previously,39 which is is listed here
for the comparison.
The ORTEP diagram of chromophore 2A with the atom
numbering scheme is depicted in Fig. 1a. As shown in this
gure, the central nitrogen (N2) and its three bonded carbon
atoms are in distorted pyramidal geometry of the bond angles in
the range of 117.2–120.6ꢂ. The dihedral angle between the two
benzene rings P1 and P2 is 4.37ꢂ, which is much smaller than
that of 1A (29.59ꢂ), revealing that the planarity of 2A is much
better than that of 1A by introducing the ethyoxyl chain to the
triphenylamine moiety. Furthermore, the linkage between the
(Z)-3-(4-(Bis(4-ethoxyphenyl)amino)phenyl)-2-(4-nitro-phenyl)-
acrylonitrile (3A). M2 (1.80 g, 5 mmol), ethanol (150 mL), p-
nitrobenzylcyanide (0.81 g, 5 mmol), and piperidine (0.25 mL)
were added into a one-neck ask. The mixture was reuxed at
80 ꢂC for 4 h (monitored by TLC). Aer cooling to room
temperature, the red crystal which had formed was ltered off
and washed with ethanol three times to get 2.00 g of red crystal.
1
Yield: 80%. H-NMR (400 MHz, (CD3)2CO) d (ppm): 8.32 (d, J ¼
8.80 Hz, 2H), 7.99 (m, 3H), 7.91 (d, J ¼ 8.80 Hz, 2H), 7.20 (d, J ¼
9.2 Hz, 4H), 6.98 (d, J ¼ 8.80 Hz, 4H), 6.83 (d, J ¼ 8.40 Hz, 2H),
4.07 (q, J ¼ 7.2 Hz, 4H), 1.38 (t, J ¼ 8.80 Hz, 6H). 13C-NMR (100
MHz, (CD3)2CO) d (ppm): 157.88, 152.95, 148.03, 146.17, 142.68,
139.56, 132.55, 129.07, 126.99, 125.05, 124.72, 118.98, 117.58,
116.48, 103.89, 64.30, 15.12. MALDI-TOF calcd for C31H27N3O4,
505.20; found, 505.28.
˚
rings is quite conjugated with bond lengths of 1.498(6) A (C23–
˚
˚
C22), 1.280(5) A (C22]C21) and 1.465(5) A (C21–C20). The
bond-length alternation (BLA, the difference between the
average lengths of carbon–carbon single and double bonds)40
˚
across the p-bridge of 2A is 0.20 A, which is much smaller than
(Z)-3-(4-(Bis(4-ethoxyphenyl)amino)phenyl)-2-(4-amino-phenyl)-
acrylonitrile (3B). A solution of ethanol (150 mL) with 3A (1.10 g,
2 mmol) was added into a ask equipped with a magnetic
stirrer, and then SnCl2$2H2O (2.49 g, 11 mmol) was added and
the mixture was reuxed for 0.5 h. The resulting mixture was
neutralized with saturated NaHCO3 to weak basicity, diluted
with water (about 50 mL), and extracted with dichloromethane.
The combined organic phase was dried over Na2SO4, ltered
and concentrated to give red oil. The obtained oil was then
puried by silica gel column chromatography using a mixture of
petroleum ether (b.p. 60–90 ꢂC)–ethyl acetate (10 : 1 by volume)
to get a 0.56 g yellow solid. Yield: 54%. 1H-NMR (400 MHz,
(CD3)2SO), d (ppm): 7.69 (d, J ¼ 8.80 Hz, 2H), 7.50 (s, 1H), 7.36
(d, J ¼ 7.35 Hz, 2H), 7.10 (d, J ¼ 8.80 Hz, 4H), 6.94 (d, J ¼ 9.20 Hz,
4H), 6.72 (d, J ¼ 8.80 Hz, 2H), 6.62 (d, J ¼ 8.80 Hz, 2H), 5.51 (s,
2H), 4.01 (q, J ¼ 6.80 Hz, 4H), 1.33 (t, J ¼ 7.20 Hz, 6H). 13C-NMR
(100 MHz, (CD3)2SO) d (ppm): 155.77, 149.57, 145.35, 138.76,
136.68, 129.90, 127.59, 126.23, 125.06, 121.42, 118.94, 117.07,
115.55, 113.93, 106.16, 63.20, 14.64. MALDI-TOF calcd for
˚
that of 1A (1.27 A). These structural features suggest that
chromophore 2A possesses higher delocalization within the
molecule and more charge-transfer features of the ground state
in the solid state compared with 1A.
The unit cell of chromophore 3A contains two molecules as
shown in Fig. 2a, while the unit cell of chromophore 3B
C
31H29N3O2, 475.23; found, 475.30.
Fig. 1 (a) ORTEP diagram of 2A, hydrogen atoms are omitted for
clarity. (b) The side elevation of 2A.
Results and discussion
Structural studies
The synthesis routes to the targeted chromophores (1A–3A and
1B–3B) are presented in Scheme 1. The molecular structures are
changed from D–p–A to D–p–A by restoring NO2 to NH2. These
six chromophores and their intermediates were prepared via the
Knoevenagel and Witting-Horner reactions in high yield. All the
chromophores are soluble in common organic solvents, such as
benzene, ethyl acetate, ethanol, acetonitrile, and N,N-dime-
thylformamide (DMF). They were fully characterized by 1H and
13C-NMR, mass spectrometry, and elemental analysis. The
structures of 1A–3A and 3B were also characterized by single
crystal X-ray diffraction analysis.
Fig. 2 (a) ORTEP diagram of 3A, hydrogen atoms are omitted for
clarity. (b) The side elevation of 3A.
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the other molecule (Fig. 3d). This is a common feature of AIE
active molecules.41,42 Beside C–H/p interaction, there also
exists C–H/N and N–H/O hydrogen bonds within the aggre-
gate structure. The various inter-molecular interactions help
rigidify the conformation and lock the intramolecular rotations
of the phenyl rings. As a result, the excited-state energy
consumed by intramolecular rotation is greatly reduced, thus
enabling the molecule to emit intensely in the solid state.
Hence, the structural characters of 3B, such as a distorted
molecular plane, steric hindrance of the cyano segment in the
case of p–p stack, and the smaller value of BLA, make the
chromophore a highly promising candidate for AIE and two-
photon absorption materials.
Linear absorption and one-photon excited uorescence
(OPEF)
The photophysical data of chromophores 1A–3A and 1B–3B
in ve different solvents (c ¼ 1 ꢁ 10ꢀ5 mol Lꢀ1) are shown in
Table S3.†
Absorption properties. The absorption spectra of chromo-
phores are shown in Fig. S4.† Two major absorption bands can
be observed: the band at about 305 nm is attributed to the p–p*
transition of the triphenylamine moiety, which remains almost
unchanged. While the other band located at about 380–460 nm
with stronger intensity is assigned as the intramolecular charge-
transfer (ICT) absorption band. This band shows regular red-
shi from chromophore 1A(B) to 3A(B), as shown in Fig. 4. This
is attributed to the introduction of ethoxyl and cyano groups
which increases the electron-donating strength of the end
group and extends the conjugation length of the system.
Fig. 3 (a) ORTEP diagram of 3B, hydrogen atoms are omitted for
clarity. (b) The side elevation of 3B. (c) One-dimensional chain of 3B
showing the C–N/H (violet) and C–O/H (blue) bonds along the b-
axis. (d) Two-dimensional layer structure of 3B showing the C–H/p
stacking (red) along the a-axis. Hydrogen atoms except H2A, H2B,
H13A and H23 are omitted for clarity.
contains one molecule (Fig. 3a). Similar to 2A, around the
central nitrogen, three phenyl ring planes are arranged in a
pyramidal geometry. The dihedral angles between P1 and P2
(13.58ꢂ for 3A and 12.63ꢂ for 3B) are greater than that of 2A.
Obviously, the cyano group plays an important role in twisting
the main structure in comparison with 2A. However, a direct
comparison of the linkage between phenyl rings clearly indi-
cates that the conjugation degree of the chromophores 3A and
3B seems to be extended by the cyano group. Notice that the
˚
˚
value of BLA across the p-bridge (0.11 A for 3A and 0.12 A for 3B)
˚
is smaller than that of 2A (0.20 A). It is crucial that BLA has been
established as a useful parameter in relation to the NLO
response.40
What is more, the cyano group also inuences their stack
structure. In the case of chromophores 1A and 2A, the mole-
cules are xed into centrosymmetric antiparallel dimers by N–
O/H hydrogen bonding into a one-dimensional chain along
axis a (Fig. S1c and S2†). Different from them, chromophores 3A
and 3B are xed into centrosymmetric antiparallel dimers by
weak C–N/H stacking interactions into a one-dimensional
chain along axis b (Fig. S3† and 3c). Meanwhile, from further
inspection of the crystal structure of 3B, the 2D layer structure is
formed through, however, multiple C–H/p hydrogen bonds
˚
Fig. 4 Linear absorption spectra of six chromophores (1.0 ꢁ 10ꢀ5 mol
with a distance of 2.861 A between the hydrogen atom in one
molecule and the p electron cloud of the planar phenyl ring in
L
ꢀ1) in ethyl acetate.
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Table 1 Selected experimental and computed optical data for chromophores 1A–3A and 1B–3B
e
Compd
1A
Transitions (S. no.)
OIa (Tc)b
Ec (eV)
Cal. lmaxd (nm)
Obs. lmax (nm)
f f
Character
1
2
1
2
1
1
2
1
2
1
102(Hꢀ1) / 104(L)
103(H) / 108(L+4)
126(Hꢀ1) / 128(L)
127(H) / 132(L+4)
133(H) / 135(L+1)
96(H) / 100(L+3)
96(H) / 98(L+1)
120(H) / 122(L+1)
120(H) / 125(L+4)
125(H) / 127(L+1)
3.2869
4.0296
2.7525
4.0483
2.6702
4.1425
3.6687
3.4673
4.0808
3.1295
377.2
307.7
450.4
306.3
464.3
299.3
337.9
357.6
303.8
396.2
426.3
303.4
446.7
304.5
463.6
302.1
352.4
372.2
303.4
405.5
0.7868
0.2042
0.6268
0.2063
0.5896
0.0405
0.0331
0.0294
0.0428
0.0221
p / p*ICT
p / p*
p / p*ICT
p / p*
p / p*ICT
p / p*
p / p*ICT
p / p*ICT
p / p*
2A
3A
1B
2B
3B
p / p*ICT
a
Orbitals involved in the excitations. b Transition coefficients. c Excitation energies (eV). d Calculate peak position of the longest absorption band.
e
Observed peak position of the longest absorption band. f Oscillator strength.
Additionally, the introduction of the cyano group also leads to that the optical properties of the chromophores strongly
the disappearance of the short wavelength band of 3A(B). depend on the length of the p-bridge and the nature of
Quantum chemical calculations. TD-DFT computational peripheral substituting donor groups relying on the theoretical
studies were performed to establish the electronic structure of calculation. The results of 1A–3A are similar to those of 1B–3B
the chromophores and aid in assignment of optical transitions. with satisfying agreement with experimental results.
The energy and composition of ICT are listed in Table 1 and the
spatial plots of selected TD-DFT frontier molecular orbitals are
One-photon excited uorescence (OPEF)
shown in Fig. 5 and Fig. S5.† The HOMOs (H) of all chromo-
phores are localized on the main conjugated framework
between the triphenylamine moiety and aniline (or nitroben-
zene), and the LUMOs (L) are mainly located on the styryl-
benzenamine (or nitro-styrylbenzene) units.
Representative one-photon induced emission spectra of 2B in
different solvents are shown in Fig. 6, and the others are
included in the ESI (Fig. S6†). These chromophores (especially
1B–3B) show regular red-shi as the solvent polarity increases,
indicating that this charge separation increases in the excited
state, resulting in a larger dipole moment than that in the
ground state, which explains the sensitivity of the emission
spectra of these chromophores to solvent polarity.43 It is noticed
that these chromophores exhibit much remarkable bath-
ochromic shi in ethanol compared to other solvents, indi-
cating that hydrogen bond interactions may occur between the
chromophores and ethanol.
As shown in Fig. 7a, chromophores 1A–3A and 1B–3B in
benzene all show obvious bathochromic shi from 1A(B) to
3A(B), respectively. They are all in agreement with the order of
the extension of the p-system: 4-ethoxy-N-(4-ethoxy-phenyl)-N-
phenyl aniline > triphenylamine. Meanwhile, note that 1B–3B
exhibit much stronger uorescence intensity compared with
that of 1A–3A by changing from nitro into amino. In addition, it
As shown in Fig. 5, two transitions of 1B and 2B resemble
those observed in the experimental linear absorption spectra.
The low-energy band originating from H / L+1 transition is
assigned to the intramolecular charge transfer (ICT) transition
(aniline / triphenylamine moiety), while the high-energy band
is assigned to the H / L+2 (1B) or H / L+5 (2B) transition
(ICT) and mixed with the p–p* transition of triphenylamine. In
the case of 3B, the transition at ca. 396 nm originating from the
H / L+1 transition is assigned as the ICT transition (triphenyl-
amine / p-bridge). Additionally, the energy gap between the H
and L of 3B (0.62 eV) is much smaller than that of 1B (3.44 eV)
and 2B (3.33 eV), which is caused by the introduction of a cyano
group in the p-bridge, which may make the electron transfer
easy in conjugated systems. This may be the condition for 3B
bearing stronger two-photon absorbing activity. It is obvious
Fig. 6 The one-photon excited fluorescence spectra of 2B in different
Fig. 5 Molecular orbital energy diagram of 1B–3B.
solvents.
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s
F ¼
(2)
s0
Except for the tunable uorescence and increased FL life-
time, the exciton–plasmon interaction and energy transfer of 3B
also result in dramatical optimization on NLO activity, such as
2PA and TPEF.
Aggregation-induced enhanced emission
To investigate the AIE attribute of 3B, different amounts of
water (a poor solvent for 3B) were added to the pure DMSO
solution by dening the water fractions (fw) of 0–99% and then
monitored the photoluminescence (PL) change with the exci-
tation wavelength of 503 nm, respectively. Fig. 8a shows that the
emission of 3B is dramatically weakened with a gradual addi-
tion of water into DMSO and the emission band is bath-
ochromically shied when fw # 50%. The light emission is
invigorated from fw z 60% and reaches its maximum value at
90% water content, which is 11-fold higher than that in pure
DMSO solution. Meanwhile, the emission maximum is gradu-
ally red-shied to 556 nm when fw reaches 99%. It is presum-
able that molecules of 3B may cluster together to form random,
amorphous aggregates in the mixture with low water fractions
(0–50%). When the water fraction becomes higher, the chro-
mophores may agglomerate in an ordered fashion to form
crystal-like aggregates.45 This phenomenon is also probably
caused by the change of solvent polarity with addition of water
at low water fractions, and then the water can interact with
solute molecules immediately, which would weaken the emis-
sion gradually.46 As can be seen in the inset of Fig. 8a, aer
reaching a maximum intensity at 90% water content, the PL
Fig. 7 The one-photon excited fluorescence spectra of 1A–3A, 1B–
3B (a) and 2B–3B (b) in benzene.
can be seen from Fig. 7b that the uorescence wavelength of 3B
is evidently red-shied by 2B and the intensity becomes much
weaker, which is due to the introduction of the cyano group.
Meanwhile, the time-resolved uorescence measurements
were performed and the detailed data of the uorescence decay
curves are listed in Table S3.† The experimental errors are
estimated to be ꢄ11% from sample concentrations and
instruments. The lifetimes of 1B–3B in the solution were
obtained by monitoring the monomer emission. The decay
behaviors of 1B–3B were of double-exponential manner
obtained by monitoring the monomer emission. As shown in
Table S3,† the weighted mean lifetime of 3B (0.97 ns) in ethyl
acetate is longer than those of 1B (0.51 ns) and 2B (0.73 ns). This
may be attributed to the larger delocalization, leading to a larger
molecular stabilization effect for the excited state.
Additionally, the uorescence lifetime of 3B in ethyl acetate
was calculated by multiplying the corresponding quantum yield
with natural lifetime, which could be easily calculated from the
known Strickler–Berg equation (eqn (1)).44 A calculated uo-
rescence lifetime of 1.02 ns was obtained, which is in excellent
agreement with the experimental value (0.97 ns) and further
validate the experimental time-resolved technique.
ð
ꢂ ꢃ
ð
I ~n d~n
1
3ð~nÞ
~n
2
n
¼ 2:88 ꢁ 10ꢀ9
d~n
(1)
ð
ꢂ ꢃ
s0
I ~n ~nꢀ3d~n
in which n is the refractive index, I is the uorescence emission,
3 is the extinction coefficient, and ~n is the wavenumber. The
natural radiative lifetime s0 and the uorescence lifetime s are
related through the quantum yield F by
Fig. 8 PL spectra (a) and absorption spectra (b) of 3B in DMSO–water
mixtures with different water fraction (fw). The inset depicts the
changes of PL peak intensity with different water fractions.
5436 | J. Mater. Chem. B, 2014, 2, 5430–5440
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Journal of Materials Chemistry B
intensity decreases with increasing water content. This
TPEF spectra of chromophores 2B and 3B in ethyl acetate
phenomenon was oen observed in some compounds with AIE pumped by femtosecond laser pulse at 300 mw at different
properties, but the reasons remain unclear. There are two excitation wavelengths are presented in Fig. S7 (le).† Fig. S7†
possible explanations for this phenomenon: (1) aer the (right) shows logarithmic plots of the uorescence integral
aggregation, only the molecules on the surface of the nano- versus pumped power with a slope of 1.93 and 1.97 when the
particles emit light and contribute to the uorescence intensity input laser power is increasing, suggesting a two-photon exci-
upon excitation, leading to a decrease in uorescence intensity. tation mechanism. As no linear absorption was observed in the
However, the restriction of intramolecular rotation in the range from 680–1080 nm, the emission excited by 700 nm laser
aggregation state can enhance light emission. The net outcome wavelength can be attributed to the TPEF mechanism. The TPEF
of these antagonistic processes depends on which process plays bands located at 500 nm for 2B and 550 nm for 3B, which are
a predominant role in affecting the uorescence behavior of the evidently red-shied by 52 and 23 nm compared with those of
aggregated molecules.47 (2) When water is added, the solute OPEF in ethyl acetate due to the reabsorption effect. In addition,
molecules can aggregate into two kinds of nano-particle a red-shi of 3B compared with 2B is observed, revealing that
suspensions: crystal particles and amorphous particles. The the nature of the p-bridge plays an important role in TPEF. It is
former one results in an enhancement in the PL intensity, while reasonable that the cyano group substituted on the ethylenic
the latter leads to a reduction in intensity.48
bond increased the electron density of the p-bridge (shown in
The absorption spectra of 3B in the DMSO–water mixtures DFT calculations), favoring the ICT between the core and the
(c ¼ 1 ꢁ 10ꢀ5 mol Lꢀ1) are shown in Fig. 8b. The spectral two end groups.
proles are signicantly changed when fw > 60%, respectively.
The 2PA cross-sections (s) of chromophores 2B and 3B were
The intensity of absorption peaks of compound 3B positioned measured in the wide wavelength range from 680–1050 nm.
at 400 nm gradually decreases with the increasing water Fig. 10 (le) indicates that in the measured region, the 2PA
content, while the new peak located at about 490 nm emerges cross-section s value of chromophore 3B is much enhanced
aerward, indicating the formation of nanoscopic aggregates of compared with that of 2B. Furthermore, two-photon action
3B. The representative scanning electron microscopy (SEM) cross-sections (Fs) are shown in Fig. 10 (right), the maximum
image in Fig. 9a shows the formation of nano-aggregates of 3B by values are 527 GM for 2B and 394 GM for 3B, respectively. Notice
the addition of the 90% fraction of water; the image indicates that that though the OPEF intensity of 3B is much lower than that of
the AIE dots are well dispersed and have a needle-like arrange- 2B, the two-photon action absorption cross-section of 3B is only
ment with a mean size of about 200 nm. Fig. 9b and c show slightly smaller than that of 2B. On account of the extended p-
uorescence images of 3B in the DMSO solution, nanoparticle system and enhanced intra-molecular charge transfer (ICT)
suspension (90% water content) and powder under UV light.
from the triphenylamine to the cyano group, the result proves
To quantitatively evaluate the AIE effect of 3B, the quantum that the strategy of increasing the ICT effect is an effective
efficiency of the solid powder was measured and the value is approach for enhancing the 2PA cross-section of the molecule.
7.13%, measured by using an integrating sphere, which is Therefore, the value of the 2PA cross-section to the functional
higher than that of 3B in the solution (F < 0.4%) and manifest material can be modulated by simple modication on the p-
its AIE feature.
bridge. Signicantly, by comparing 3B with 2B, it not only shows
larger s and Fs values, but also exhibits higher PL intensity at
90% water content due to the AIE property, which spurred us
further to explore its potential application in biological
imaging.49
Two-photon excited uorescence (TPEF)
The two-photon excited uorescence (TPEF) spectra of chro-
mophores 2B and 3B were recorded at their maximum excita-
tion wavelength with a pulse duration of 140 fs at 300 mW
(milliwatt). The TPEF spectral data of 2B and 3B are described
in Table S3,† which were measured in ethyl acetate (c ¼ 1 ꢁ
10ꢀ3 mol Lꢀ1).
Two-photon microscopy biological imaging application of 3B
Considering the comprehensive merits of the chromophores,
we singled out 3B for biological imaging application research.
Fig. 9 (a) SEM image of 3B in the water–DMSO mixture with 90%
water fraction at concentration of 1 ꢁ 10ꢀ5 M, (b) solid powder of 3B Fig. 10 Two-photon absorption cross-sections (left) and two-photon
and (c) PL emission photos of 1 ꢁ 10ꢀ5 M in the water–DMSO mixtures action absorption cross-sections (right) (s, 1 GM ¼ 10ꢀ50 cm4 s per
with 0% and 90% water concentration under 365 nm UV light photon per molecule) of chromophores 2B and 3B in ethyl acetate
illumination.
versus excitation wavelengths of an identical energy of 0.30 W.
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To analyze the potential of 3B as an antitumor agent, its cyto- and clearly emerged from cellular cytoplasm (Fig. 11d and e).
toxicity was measured toward the human hepatoma cancer cells We presume that the luminescence from punctuate bright dots
(HepG2). The tested chromophore was dissolved in DMSO and outside the nuclei region is because of the aggregation of the
then serially diluted in complete culture medium. It is note- complex inside the lysosome. This result demonstrates that the
worthy that the live cells were stained with 3B; the cells were observed lysosome uptake may be due to the presence of the
active for 24 h during the incubation time (Fig. S8†). The results intact 3B.
clearly indicate that HepG2 cells incubated with a concentration
of 10 mm of 3B remained 90% viable aer 24 h of feeding time,
demonstrating the superior biocompatibility of 3B.
Conclusion
To evaluate the internalisation of 3B in living cells, one- and
A series of triphenylamine derivatives with D–p–A (1A–3A) or D–
two-photon uorescence microscopy micrographs were
p–A (1B–3B) conguration have been synthesised by a conve-
obtained from HepG2 incubated with 3B, owing to its relatively
nient method. Their photophysical properties can be tuned by
simple modication of electron donor and p-bridge groups,
correlating both experimentally and theoretically. Interestingly,
the introduction of a cyano-group in chromophore 3B results in
lower OPEF intensity but stronger intensity of TPEF than that of
corresponding 2B. Signicantly, 3B emits intensely upon
aggregated formation in a water–DMSO mixture, demonstrating
a typical AIE feature. Therefore a simple modication on the p-
lower toxicity toward live cells and two-photon behavior
together with the AIE property. A bright-eld image (Fig. 11a) of
each cell was taken immediately prior to imaging. To minimize
the side effects of organic solvent toward live cells, 3B was
dissolved in DMSO at high concentration and diluted with
phosphate buffer solution to a working concentration. OPEF
images (Fig. 11b) of each live cell were successfully taken which
clearly display the cytoplasm structure. The TPEF images of 3B
bridge of chromophore 3B results in excellent 2PA and AIE
(Fig. 11c) exhibit similar cellular cytosol uptake at the excitation
properties which pave the way for its biophotonic application.
wavelength of 700 nm. It shows the remarkable stability of
Our initial investigation demonstrates that 3B is successfully
applied to a two-photon uorescent probe for labeling the
uorescence from cellular cytosol as we switch one photon
excitation to two-photon excitation. It is worth noting that the
cytoplasm and actin regulatory protein in live cells. Finally, it
TPEF images of 3B not only exhibit cellular cytosol uptake but
can be said that the research results open a new way to search
for applicable new TPA materials.
also exhibit actin regulatory protein staining. As most of
currently commercial available cellular actin probes are
membrane impermeable, highly toxic and require pre-xation,
therefore, 3B might be able to have potential as a more safe Acknowledgements
uorescence probe for cellular cytosol and actin in the two-
This work was supported by a grant for the National Natural
photon excitation range.50 Our preliminary confocal microscopy
Science Foundation of China (51372003, 21271004, 21275006
and 21271003), the Natural Science Foundation of Anhui Prov-
studies reveal 3B with function as a luminescent cellular cytosol
probe for HepG2 cells being successfully taken up by live cells
ince (1208085MB22, 1308085MB24), the Natural Science Foun-
dation of Education Committee of Anhui Province
(KJ2012A024), the Ministry of Education Funded Projects Focus
on returned overseas scholar And the Foundation for
Outstanding Youth of the Department of Science and Tech-
nology of Anhui Province.
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