A ratiometric fluorescent pH probe based on aggregation-induced emission enhancement and its application in live-cell imaging

Article abstract of DOI:10.1039/c1jm12098k

A ratiometric fluorescent pH probe, 4-carboxylaniline-5- chlorosalicylaldehyde Schiff base (1) was synthesized via a facile reaction and used for pH sensing in live cells. While exhibiting weak fluorescence when dispersed in solution, 1 displayed aggregat

Full text of DOI:10.1039/c1jm12098k

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links <
C
Journal of
Materials Chemistry
Cite this: J. Mater. Chem., 2011, 21, 13470
www.rsc.org/materials
PAPER
A ratiometric fluorescent pH probe based on aggregation-induced emission
enhancement and its application in live-cell imaging†
a
a
b
c
a
Panshu Song, Xiaotong Chen, Yu Xiang, Lei Huang, Zhaojuan Zhou, Ruirui Wei and Aijun Tong
a
a
*
Received 12th May 2011, Accepted 20th June 2011
DOI: 10.1039/c1jm12098k
A ratiometric fluorescent pH probe, 4-carboxylaniline-5-chlorosalicylaldehyde Schiff base (1) was
synthesized via a facile reaction and used for pH sensing in live cells. While exhibiting weak fluorescence
when dispersed in solution, 1 displayed aggregation-induced emission enhancement (AIEE)
characteristics in its aggregate/solid state, as a result of the restriction of free intramolecular rotation of
a C–N bond and the non-planar configuration in the aggregate/solid state. The integration of hydroxyl
and carboxyl groups provided 1 with a ratiometric fluorescent response to pH based on AIEE and the
pH-dependent spectral characteristics of compound 1, with proton dissociation constants pKa1 and
pKa2 of 4.8 ꢀ 0.1 and 7.4 ꢀ 0.1, respectively. The probe exhibited a significant fluorescence color change
from orange to green with an intensity ratio (I 516 nm/ I 559 nm) enhanced when the pH increased from 5.0
to 7.0 in aqueous solution. Confocal fluorescence imaging of intracellular pH through ratiometric
response was successfully achieved in live HepG2 cells. The results demonstrate that probe 1 is a good
candidate for monitoring pH fluctuations in live cells with good selectivity, stability, and excellent
membrane permeability.
In recent years, fluorophores displaying aggregation-induced
emission enhancement (AIEE) have provided a powerful plat-
form for the development of novel chemical and biological
sensors.5 It is well known that many organic fluorescent materials
exhibit very strong fluorescence in dilute solution, however their
emission intensities dramatically decrease with increasing
concentrations and formation of aggregates. This aggregation-
caused quenching (ACQ) effect mainly results from strong
intermolecular p–p stacking interactions and non-radiative
decay.6 In contrast, AIEE fluorophores, as first reported by Tang
and Zhu et al. in 2001,7 are weakly fluorescent when dispersed in
solution, but exhibit intense fluorescence in an aggregate or solid
state.8 The restriction of the intramolecular rotation (RIR)
process and the non-planar configuration in their aggregate state
which experiences little p–p stacking interactions are regarded as
the main causes for the AIEE effect.9 Hence, a design principle of
AIEE molecules is to have two or more conjugated/aromatic
moieties connected by rotatable single bond(s), which result in
nonradiative decay in the solution state, but an enhanced emis-
sion in the aggregate state when rotation is restricted.10 On the
basis of this principle, a series of salicylaldehyde azine derivatives
were previously studied in our group, which exhibited AIEE
characteristics by restriction of intramolecular rotation of a N–N
single bond when changed from solution to aggregate state.11
Although AIEE compounds bearing pH sensitive groups have
been shown to demonstrate a pH-dependent fluorescence
response, there is still no such dye reported with a ratiometric
fluorescence response in near-neutral pH ranges.12
Introduction
The development of optical sensors for intracellular pH moni-
toring has attracted great attention since fluctuations in intra-
cellular pH play an essential role in many cellular processes such
as cell growth, enzyme activity and ion transport,1 and variations
in intracellular pH is a common phenotype of cancer cells.2 In
particular, fluorescent pH probes with ratiometric properties are
highly preferred for pH monitoring in complex samples because
of their visible fluorescence color change and better resistance to
variations of sensor concentration and external environment.3 By
combining the use of a fluorescent pH probe and an appropriate
imaging instrument, fluorescent imaging of pH provides
a promising method for studying pH fluctuations in live cells.
However, only a limited number of ratiometric pH probes have
been reported to be practical for intracellular fluorescent
imaging, and the synthetic procedures for these compounds are
often complicated.4
aDepartment of Chemistry, Tsinghua University, Beijing, 100084, PR
China. E-mail: tongaj@mail.tsinghua.edu.cn; Fax: +86-10-62787682;
Tel: +86-10-62787682
bDepartment of Chemistry, University of Illinois at Urbana-Champaign,
Urbana, IL, 61801, USA
cCenter of Biomedical Analysis, Tsinghua University, Beijing, 100084, PR
China
† Electronic supplementary information (ESI) available: Scheme S1 and
Fig. S1–S8. CCDC reference number 780526. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c1jm12098k
13470 | J. Mater. Chem., 2011, 21, 13470–13475
This journal is ª The Royal Society of Chemistry 2011

View Article Online
In this study, an AIEE-active fluorophore, 4-carboxylaniline-
5-chlorosalicylaldehyde Schiff base (1), was facilely synthesized
for sensing pH variations in aqueous solution and in live cells.
This compound showed a ratiometric fluorescent response over
the pH range 5.0 to 7.0 with a fluorescence color change from
orange to green, by the switching of its aggregate and solution
state fluorescence (Scheme 1). Demonstrating good stability,
membrane permeability, and selectivity over metal ions,
compound 1 was successfully applied to intracellular pH
measurement in human hepatocellular liver carcinoma cell line
(HepG2 cells) by confocal microscopy.
conjugated/aromatic moieties connected by a rotatable single
bond (C8–N1), indicating that 1 had the potential of aggrega-
tion-induced emission enhancement (AIEE) feature resulting
from the restriction of free intramolecular rotation in its aggre-
gate/solid state.11,13 In addition, it was found that the carboxyl
and two phenyl groups of the molecule are diorientational due to
the rotation of a C11–C14 single bond which gives a statistical
mirror symmetry to meet the requirement of the C2/m space
group. A dihedral angle of 49.9ꢁ for the [C10-C11-C12] and [O2-
C14-O3] was observed, owing to the propeller-like structure of
the carboxyl groups. Furthermore, the inter-plane distances
ꢀ
between adjacent molecules were as long as 4.32 A (see Fig. S3. †)
due to the non-planar configuration of compound 1, which
hindered any closer packing. In this case, the concentration-
quenching effect caused by strong intermolecular p–p stacking
interactions might be weakened in such structures.14
Results and discussion
Single crystal structure
Single crystal of compound 1 was grown from ethanol solution
and characterized by X-ray crystallography. As shown in Fig. 1,
the crystal structure of 1 contained an intramolecular hydrogen
bond in the salicylaldimine moiety, which ensured two
AIEE characteristics
Compound 1 was soluble in some common organic solvents,
such as ethanol, chloroform, THF, and DMF. However, water
was a poor solvent. Herein, the AIEE feature of 1 was investi-
gated in ethanol/water (from 9 : 1 to 1 : 9, v/v) buffered with
50 mM sodium phosphate at pH 4.42 (Fig. 2). In a good solvent
Scheme 1 Proposed deprotonation processes of compound 1.
Fig. 2 (a) Absorption spectra of 1 (90 mM) in ‘‘solution’’ (ethanol/water,
9 : 1, v/v) and ‘‘aggregate’’ (ethanol/water, 1 : 9, v/v) states, containing
50 mM sodium phosphate at pH 4.42; (b) effect of ethanol volume
fraction on the AIEE fluorescence intensity measured at 559 nm (peaks in
emission spectra). Insert: fluorescence excitation/emission spectra of 1 in
‘‘solution’’ and ‘‘aggregate’’ states. Excitation was performed at 381 nm.
Fig. 1 Crystal structure of compound 1. Left: ORTEP drawing with
35% probability ellipsoids. Right: A packing view along the a direction;
the open bonds showed one orientation of the phenyl and carboxyl
groups, and the solid bonds showed the other orientation.
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 13470–13475 | 13471

View Article Online
Fig. 3 SEM image for solids of 1 obtained from a suspension in 1 : 9
ethanol/water (v/v), buffered by 50 mM sodium phosphate at pH 4.42.
of 9 : 1 ethanol/water, 1 was well dispersed in its ‘‘solution’’ state
with a structured absorption spectra, and displayed weak fluo-
rescence emission (FF ¼ 0.001). However, when 1 was dispersed
in 1 : 9 ethanol/water, a leveling-off in the visible region of its
absorption spectra (commonly observed in nanoaggregate
suspensions15) strongly suggested the formation of a poorly-
soluble ‘‘aggregate’’ state, and an intense fluorescence enhance-
ment was observed (FF ¼ 0.12) with a similar emission wave-
length as observed in its solid state (powder and crystal)
fluorescence spectra (see Fig. S4 †). The blue-shift observed in the
absorption spectrum and the bathochromic shift in its emission
spectrum were attributed to the formation of an H-aggregate in
the aggregate/solid state of 1 with respect to its isolated chro-
mophore, which was supported by the face-to-face arrangement
in its single crystal structure.16 The AIEE effect of compound 1
could be explained by the blocking of nonradiative intra-
molecular rotation decay of excited molecules, as well as by
formation of a non-planar configuration in its aggregate/solid
state which weakened the p–p stacking interaction. As illus-
trated in Fig. 2b, the AIEE effect of 1 occurred at 20% volume
fraction of ethanol. A lower ethanol volume fraction was found
to enhance the AIEE fluorescence intensity, which was well
consistent with the fact that more aggregates would be formed in
poorer solvents. Besides absorption and fluorescence spectra, the
formation of aggregates of 1 was also directly observed using
scanning electron microscopy (SEM): particle size distributions
with 100–200 nm were detected when the solids of 1 were
obtained from the solvent mixtures with 90% buffered water
(pH 4.42) (see Fig. 3); while no particle was observed when the
volume fraction of ethanol was above 20%.
Fig. 4 (a) pH-dependence of the emission spectra of 1 (60 mM) in 50 mM
phosphate buffer, with arrows indicating the change of fluorescence
intensities with pH increase. From acidic to basic conditions: pH 3.43, 3.70,
4.03, 4.40, 4.82, 5.05, 5.22, 5.63, 6.00, 6.28, 6.62, 6.97, 7.20, 7.40, 7.71, 8.06,
8.52, 9.01, and 9.56; (b) ratiometric calibration curve of I 516 /I 559 (intensity
at 516 nm vs. intensity at 559 nm). Excitation was performed at 353 nm.
demonstrated in Fig. 4, the neutral form of 1 tended to aggregate at
acidic pH and exhibited AIEE with a fluorescence maximum at
559 nm. The AIEE effect was weakened as pH increased from 3.43
to 5.63, when the carboxyl group was deprotonated (pKa1 ¼ 4.8 ꢀ
0.1, Fig. S5b.†) and the anionic form of 1 became well-dispersed in
aqueous solution. As pH was further increased from 5.63 to 9.56,
the hydroxyl group of compound 1 was deprotonated (pKa2 ¼ 7.4
ꢀ 0.1, Fig. S5c.†), electron delocalization from the phenolate to the
carbon of the imine group was enhanced due to the negative charge
on the oxygen in the phenolate group. Intramolecular charge
transfer (ICT) was thus enhanced as a result of the expansion of the
conjugated system and the stronger electron-donating ability of the
phenolate group.17 Therefore, a new emission peak for the dianion
form of 1 appeared at 516 nm in a higher pH solution. In partic-
ular, the fluorescence color changed from orange to green with an
intensity ratio (I516 nm / I559 nm) enhancement observed over the pH
range of 5.0 to 7.0, which is ideal for accurate near-neutral pH
measurements.
pH-dependent optical properties
In buffer solution at pH 3.95, 1 (10 mM) exhibited absorption
bands at 258 and 290 nm respectively (Fig. S5a. †). With pH
values increased to 9.52, a new absorption band at 390 nm was
observed, with absorbance enhanced in the 250–275 nm region
and decreased in the 280–350 nm region through two pseudo-
isosbestic points located at 276 and 353 nm.
Testifying the stability and selectivity
The pH-dependent fluorescence response of 1 was also investi-
gated. A ratiometric fluorescence pattern was clearly observed
when the solution of 1 (60 mM) was excited at 353 nm. As
The photostability of probe 1 was tested by measuring the fluo-
rescent response over 30 min under constant excitation. Fig. S6.†
13472 | J. Mater. Chem., 2011, 21, 13470–13475
This journal is ª The Royal Society of Chemistry 2011

View Article Online
showed the time-course fluorescence intensity of 1 (60 mM) in
aqueous solution at pH 6.00 and 7.00 at 37 ꢁC. The fluorescence
intensity at 516 and 559 nm remained stable over the time range
nigericin (5 mg mLꢂ1) to equilibrate the intracellular and extra-
cellular pH, which is a standard approach that has previously
been used to calibrate other pH measurements in vitro.3c,19 As
shown in Fig. 6 (a–f), 1 was well-distributed within live cells
(bright-field transmission image confirmed the viability of the
cells,2d,20 see Fig. 6 (j)), indicating excellent membrane perme-
ability. The dye-loaded cells exhibited weak fluorescence in both
channel I (490–535 nm) and channel II (540–585 nm) in PBS at
pH 5.00; whereas the fluorescence brightness of channel I was
obviously enhanced as the pH was increased. The ratiometric
fluorescence images were shown in Fig. 6 (g–i), which demon-
strated that 1 could readily reveal near-neutral pH variations in
HepG2 cells by a ratiometric response. The mean fluorescent
intensity (MFI) and fluorescence ratio of channel I and channel
II were acquired using commercial software and are displayed in
Fig. 6 (k) (fluorescence images of dye-loaded cells in PBS at pH
tested. Furthermore, the fluorescence ratio (I516 nm/I559
)
nm
exhibiꢁted little variation when the temperature changed from 20
to 40 C (Fig. S7. †). These experiments indicated that 1 could
steadily respond to pH values without interference from photo-
bleaching and variation in temperature.
Considering that a number of salicylaldehyde Schiff base
derivatives can chelate di- or tri-valent metal ions in solution,18
the selectivity of 1 to H+ over metal ions was investigated by
competition experiments. The fluorescence intensity of 1 dis-
played moderate variation in the absence or presence of an excess
of Li+, K+, Zn2+, Mg2+, Ca2+, Fe3+, Cu2+, Mn2+ and Cd2+ ions at
pH 5.98 and pH 6.80. However, as shown in Fig. 5, no significant
changes in the intensity ratio (I516 nm / I559 nm) were observed,
which indicated that the interference from these metal ions on
ratiometric fluorescence pH measurements were negligible.
With satisfied selectivity over metal ions, we realized 1 could be
utilized as an excellent ratiometric fluorescent pH indicator for
real water samples (Table S1†).
Detection of H+ in live cells by confocal microscopy
To further demonstrate the application of the probe, we used 1
for fluorescence imaging of pH fluctuations in live cells. In this
study, HepG2 cells were incubated with 1 (60 mM) and then with
Fig. 6 Confocal fluorescence images of H+ in HepG2 cells after incu-
bation with 1 (60 mM) and nigericin at 37 ꢁC (lex ¼ 405 nm). Left:
collected at channel I (490–535 nm) at pH 5.00 (a), 6.30 (b), and 7.00 (c);
middle: collected at channel II (540–585 nm) at pH 5.00 (d), 6.30 (e), and
7.00 (f); and right (g–i): ratiometric fluorescence images generated from
channel I and channel II; (j) bright-field transmission image of (c). (k)
Data of mean fluorescent intensity (MFI) (gray bar) and fluorescence
ratio of channel I and channel II (black bar) at various pH of 5.00, 5.50,
6.00, 6.30, 6.60, 7.00.
Fig. 5 Ratio of fluorescence intensity at 516 nm and 559 nm of 1 (60 mM)
in the absence or presence of 300 mM Zn2+, Fe3+, Cu2+, Mn2+, Cd2+, 5 mM
Mg2+, Ca2+, and 10 mM Li+, K+ ions in phosphate buffer solution at pH
5.98 (a) and 6.80 (b). 1: H+, 2: Li+, 3: K+, 4: Zn2+, 5: Mg2+, 6: Ca2+, 7: Fe3+
,
8: Cu2+, 9: Mn2+, 10: Cd2+. Excitation was performed at 353 nm.
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 13470–13475 | 13473

View Article Online
5.50, 6.00, and 6.60 are shown in Fig. S8.†). These results have
established that probe 1 is a good candidate for monitoring pH
fluctuations in live cells.
mentioned, all of the measurements were operated at room
temperature and repeated at least once.
Synthesis of compound 1
Conclusion
In a 50 mL flask, 3 mmol of 5-chlorosalicylaldehyde and 3 mmol
of 4-carboxyaniline were dissolved in 25 mL of ethanol, and
stirred at room temperature overnight. The resulting precipitate
was filtered and washed three times with 15 mL of cold ethanol.
After drying, 1 was obtained as an orange solid in a 62% yield
In conclusion, a ratiometric fluorescent pH indicator 4-carbox-
ylaniline-5-chlorosalicylaldehyde Schiff base (1) was synthesized
via a facile method and used to monitor pH fluctuations both in
aqueous solution and in live cells. Compound 1 displayed strong
aggregation-induced emission enhancement (AIEE) fluorescence
in its aggregate/solid state, resulting from the restriction of free
intramolecular rotation of a C–N single bond and its non-planar
configuration in such a state. A significant fluorescence color
1
(Scheme S1 †). H NMR (300 MHz, DMSO-d6, 25 ꢁC, TMS)
d (ppm) ¼ 7.02 (d, J1 ¼ 8.6 Hz, J2 ¼ 2.8 Hz, H), 7.46 (d, J ¼ 8.2
Hz, H), 7.48 (d, J ¼ 8.6 Hz, 2H), 7.78 (d, J ¼ 2.8 Hz, H), 8.01 (d,
J ¼ 8.2 Hz, 2H), 8.96 (s, H); 13C NMR (300 MHz, DMSO-d6, 25
ꢁC, TMS) d (ppm) ¼ 119.3, 121.2, 122.1, 123.3, 129.6, 131.3,
131.4, 133.8, 152.6, 159.4, 163.6, 167.4; ESI mass spectrometry:
m/z 274.2 ([M ꢂ H]ꢂ); Mꢂ calcd 274.0; elemental analysis calcd
(%) for C14H10ClNO3: C 61.09, H 3.67, Cl 12.72, N 5.09, O 17.43;
found: C 60.91, H 3.69, Cl 12.81, N 5.14, O 17.45.
change from orange to green with intensity ratio (I516 nm/ I559 nm
)
enhancement was observed over a pH range from 5.0 to 7.0. The
new probe displays several advantageous properties including
ratiometric fluorescence over a near-neutral pH response range,
high tolerance to metal ions, and excellent stability, all of which
were favourable for intracellular pH imaging. Application of 1
for pH imaging in live HepG2 cells was successfully achieved,
proving that 1 can reveal intracellular pH fluctuations via
a ratiometric response.
Crystal structure determination of compound 1
Crystal data. C14H10ClNO3, M ¼ 275.68, monoclinic, a ¼
ꢁ
ꢀ
ꢁ
ꢀ
ꢀ
6.177(2) A, b ¼ 7.001(3) A, c ¼ 28.415(10) A, a ¼ 90.00 , b ¼
ꢁ
ꢀ
94.40(3) , g ¼ 90.00 , V ¼ 1225.1(8) A3, T ¼ 295 ꢀ 2 K, space
group C2/m (No. 12), Z ¼ 4, 1863 reflections measured, 1244
unique reflections (Rint ¼ 0.0912) which were used in all calcu-
lations. R1 ¼ 0.0808, wR2 ¼ 0.1405, CCDC: 780526.†
Experimental section
Chemicals
Analytical grade absolute ethanol and deionized water (distilled)
were used throughout the experiment as solvents. All materials
for synthesis were purchased from Alfa Aesar Co. and used
without further purification. Nigericin (sodium salt) was bought
from Fermentek Ltd. HepG2 cells were kindly provided by Prof.
J.M. Lin’s research group at Tsinghua University. Solutions of
metal ions were prepared from the corresponding nitrate salts
(analytical grade), except for KCl and MnCl2. Phosphate buffer
solutions (50 mM) at various pH values were prepared under
adjustment by a pH meter using different ratios of phosphoric
acid, sodium dihydrogen phosphate, disodium hydrogen phos-
phate and sodium hydroxide.
Spectral measurements
A stock solution of 1 (3 mM) was prepared in ethanol.
Absorption and fluorescence measurements were performed by
addition of a proper amount of stock solutions to buffered water
or buffered water/ethanol of different pH and ethanol fractions
(totally 3 mL) in a 1 cm quartz cell. After mixing, the solutions
were allowed to stand at ambient conditions for 5 min, and then
absorption or fluorescence spectra were recorded.
Determination of pKa from absorption titration
For determination of pKa, 10 mM of 1 was prepared in aqueous
solution at various pH levels for absorption titration. At this
concentration, 1 was in the ‘‘solution’’ state with structured
absorption spectra. The proton dissociation constants pKa1 and
pKa2 of 1 were calculated from the results of a nonlinear
regression of the absorbance at 290 nm and 390 nm, as previously
described.4b, 21
Instruments
Absorption spectra were measured on a JASCO V-550 UV-vis
spectrophotometer. Fluorescence spectra measurements were
performed on a JASCO FP-6500 spectrofluorimeter equipped
with a xenon discharge lamp, using 1 cm quartz cells. All pH
measurements were made with a METTLER TOLEDO 320 pH
meter (Shanghai, China). NMR spectra were recorded using
a JOEL JNM-ECA300 spectrometer operated at 300 MHz. ESI-
MS spectra were obtained on a HP 1100 LC–MS spectrometer.
X-ray crystal data collection was controlled by XSCANS
program. Computations were constructed using the SHELXTL
NT ver. 5.10 program package on an IBM PC 586 computer.
Dynamic light scattering (DLS) experiments were conducted
using a ZETA3000HS (3 nm–3 mm) light scattering instrument.
Scanning electron microscopy (SEM) observations were per-
formed on a KYKY-2000 instrument. Fluorescent images were
taken using a LMS 710 confocal laser scanning microscope (Carl
Zeiss Co., Ltd.) with an objective lens (ꢃ40). Unless otherwise
pH-dependent intracellular fluorescence imaging
Human hepatocellular liver carcinoma cell line (HepG2 cells)
were incubated in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS),
penicillin (100 units mLꢂ1), and streptomycin (100 mg mLꢂ1).
Cultures were maintained at 37 ꢁC under a humidified atmo-
sphere containing 5% CO2. About 1.0 ꢃ 105 HepG2 cells in
growth medium (2 mL) were seeded on a 35 mm-diameter round
glass petri dish and incubated overnight at 37 ꢁC under 5% CO2.
The medium was then removed. The cells were incubated with
13474 | J. Mater. Chem., 2011, 21, 13470–13475
This journal is ª The Royal Society of Chemistry 2011

View Article Online
B. Z. Tang, J. Am. Chem. Soc., 2011, 133, 660; (f) J. P. Xu,
Z. G. Song, Y. Fang, J. Mei, L. Jia, A. J. Qin, J. Z. Sun, J. Ji and
B. Z. Tang, Analyst, 2010, 135, 3002.
6 (a) H. Y. Li, Z. G. Chi, B. J. Xu, X. Q. Zhang, X. F. Li, S. W. Liu,
Y. Zhang and J. R. Xu, J. Mater. Chem., 2011, 21, 3760; (b)
M. Wang, G. X. Zhang, D. Q. Zhang, D. B. Zhu and B. Z. Tang,
a solution of 1 (60 mM) in medium (2 mL) for 10 min under the
same conditions. After that, the medium was removed and the
cells were treated with nigericin (5 mg mLꢂ1) in PBS (2 mL) for
further 10 min. Before imaging, the dye loaded cells were rinsed
three times and incubated with PBS buffer (50 mM) at various
pH values (pH ¼ 5.0, 5.5, 6.0, 6.3, 6.6, 7.0) for 10 min,
respectively.
€
J. Mater. Chem., 2010, 20, 1858; (c) A. Dreuw, J. Ploner,
L. Lorenz, J. Wachtveitl, J. E. Djanhan, J. Bruning, T. Metz,
€
M. Bolte and M. U. Schmidt, Angew. Chem., Int. Ed., 2005, 44, 7783.
7 J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok,
X. Zhan, Y. Q. Liu, D. B. Zhu and B. Z. Tang, Chem. Commun., 2001,
1740.
8 (a) L. H. Chan, R. H. Lee, C. F. Hsieh, H. C. Yeh and C. T. Chen, J.
Am. Chem. Soc., 2002, 124, 6469; (b) S. J. Lim, B. K. An, S. D. Jung,
M. A. Chung and S. Y. Park, Angew. Chem., Int. Ed., 2004, 46, 6346;
(c) Z. J. Zhao, S. M. Chen, X. Y. Shen, F. S. Mahtab, Y. Yu, P. Lu,
J. W. Y. Lam, H. S. Kwoka and B. Z. Tang, Chem. Commun., 2010,
46, 686; (d) X. Feng, B. Tong, J. B. Shen, J. B. Shi, T. Y. Han,
L. Chen, J. G. Zhi, P. Lu, Y. G. Ma and Y. P. Dong, J. Phys.
Chem. B, 2010, 114, 16731.
Cells were imaged with a LMS 710 confocal laser scanning
microscope equipped with a Chamlide TC system (Live Cell
Instrument, Inc.). The excitation wavelength was 405 nm.
Emission signals from 490–535 nm and 540–585 nm were
collected and denoted channel I and channel II, respectively, and
images were analysed using Image-Pro Plus software. The
intracellular fluorescence intensities and fluorescence ratio of
channel I and channel II were also determined, using a total of 10
cells at each pH tested.
9 (a) Y. Liu, Y. Tang, N. N. Barashkov, I. S. Irgibaeva, J. W. Y. Lam,
R. Hu, D. Birimzhanova, Y. Yu and B. Z. Tang, J. Am. Chem. Soc.,
2010, 132, 13951; (b) J. Wang, J. Mei, W. Z. Yuan, P. Lu, A. J. Qin,
J. Z. Sun, Y. G. Ma and B. Z. Tang, J. Mater. Chem., 2011, 21, 4056;
(c) Z. J. Ning, Z. Chen, Q. Zhang, Y. L. Yan, S. X. Qian, Y. Cao and
H. Tian, Adv. Funct. Mater., 2007, 17, 3799.
10 (a) C. J. Bhongale and C. S. Hsu, Angew. Chem., Int. Ed., 2006, 45,
1404; (b) K. Kokado and Y. Chujo, J. Org. Chem., 2011, 76, 316;
(c) J. Chen, H. Peng, C. C. W. Law, Y. Dong, J. W. Y. Lam,
I. D. Williams and B. Z. Tang, Macromolecules, 2003, 36, 4319.
11 W. X. Tang, Y. Xiang and A. J. Tong, J. Org. Chem., 2009, 74, 2163.
12 (a) Y. Dong, J. W. Y. Lam, A. Qin, Z. Li, J. Liu, J. Sun, Y. Dong and
B. Z. Tang, Chem. Phys. Lett., 2007, 446, 124; (b) Z. Li, Y. Q. Dong,
J. W. Y. Lam, J. X. Sun, A. J. Qin, M. Haeussler, Y. P. Dong,
H. H. Y. Sung, I. D. Williams, H. S. Kwok and B. Z. Tang, Adv.
Funct. Mater., 2009, 19, 905.
Calculation of quantum yield
Quantum yields were calculated using quinine sulfate in 0.1 M
H2SO4 (excitation at 366 nm) as a standard (F ¼ 0.55) for
solution phase samples. For solid phase samples, an integral
sphere was applied with BaSO4 white plates as a standard
(F ¼ 1.0).11
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (NSFC, Nos. 20875054 and
90813014). The authors thank Dr H. F. Li and Prof. J. M. Lin for
kindly providing us the cells. We also thank Prof. R. J. Wang for
the analysis of X-ray crystal data.
13 X. T. Chen, Y. Xiang, N. Li, P. S. Song and A. J. Tong, Analyst, 2010,
135, 1098.
14 (a) D. E. Janzen, M. W. Burand, P. C. Ewbank, T. M. Pappenfus,
ꢁ
H. Higuchi, D. A. Filho, V. G. Young, J. L. Bredas and
K. R. Mann, J. Am. Chem. Soc., 2004, 126, 15295; (b) Z. J. Ning,
Y. C. Zhou, Q. Zhang, D. G. Ma, J. J. Zhang and H. Tian, J.
Photochem. Photobiol., A, 2007, 192, 8; (c) Y. Q. Dong,
J. W. Y. Lam, A. J. Qin, Z. Li, J. Z. Sun, H. H.-Y. Sung,
I. D. Williamsa and B. Z. Tang, Chem. Commun., 2007, 40.
15 (a) B. Z. Tang, Y. Geng, J. W. Y. Lam, B. Li, X. Jing, X. Wang,
F. Wang, A. Pakhomov and X. X. Zhang, Chem. Mater., 1999, 11,
1581; (b) S. Kamino, Y. Horio, S. Komeda, K. Minoura,
H. Ichikawa, J. Horigome, A. Tatsumi, S. Kaji, T. Yamaguchi,
Y. Usami, S. Hirota, S. Enomoto and Y. Fujita, Chem. Commun.,
2010, 46, 9013.
16 (a) D. Oelkrug, A. Tompert, J. Gierschner, H. Egelhaaf, M. Hanack,
M. Hohloch and E. Steinhuber, J. Phys. Chem. B, 1998, 102, 1902; (b)
B. K. An, S. K. Kwon, S. D. Jung and S. Y. Park, J. Am. Chem. Soc.,
2002, 124, 14410; (c) Y. F. Zhao, H. Z. Gao, Y. Fan, T. L. Zhou,
Z. M. Su, Y. Liu and Y. Wang, Adv. Mater., 2009, 21, 3165; (d)
Y. Qian, S. Y. Li, G. Q. Zhang, Q. Wang, S. Q. Wang, H. J. Xu,
C. Z. Li, Y. Li and G. Q. Yang, J. Phys. Chem. B, 2007, 111, 5861.
17 (a) A. Pross, L. Radom and R. Taft, J. Org. Chem., 1980, 45, 818; (b)
Z. Rappoport, The Chemistry of Phenols, John Wiley & Sons,
Chichester, 2003; (c) Y. Fan, Y. M. Zhu, F. R. Dai, L. Y. Zhang
and Z. N. Chen, Dalton Trans., 2007, 3885; (d) Z. Q. Guo, P. Zhao,
W. H. Zhu, X. M. Huang, Y. S. Xie and H. Tian, J. Phys. Chem.
C, 2008, 112, 7047.
18 (a) L. K. Charkoudian, D. M. Pham and K. J. Franz, J. Am. Chem.
Soc., 2006, 128, 12424; (b) N. Li, Y. Xiang and A. J. Tong, Chem.
Commun., 2010, 46, 3363.
19 (a) J. A. Thomas, R. N. Buchsbaum, A. Zimniak and E. Racker,
Biochemistry, 1979, 18, 2210; (b) J. Llopis, J. M. McCaffery,
A. Miyawaki, M. G. Farquhar and R. Y. Tsien, Proc. Natl. Acad.
Sci. U. S. A., 1998, 95, 6803.
20 W. S. Zhang, B. Tang, X. Liu, Y. Y. Liu, K. H. Xu, J. P. Ma,
L. L. Tong and G. W. Yang, Analyst, 2009, 134, 367.
21 E. J. Billo, Excel for Chemists, in A Comprehensive Guide, Wiley-
VCH, New York, 1997, pp. 303.
References
1 (a) R. A. Gottlieb and A. Dosanjh, Proc. Natl. Acad. Sci. U. S. A.,
1996, 93, 3587; (b) K. Xu and A. M. Klibanov, J. Am. Chem. Soc.,
1996, 118, 9815; (c) R. M. Zaguilln and R. J. Gillies, Cell. Physiol.
Biochem., 1996, 6, 169; (d) B. Tang, F. B. Yu, P. Li, L. L. Tong,
X. Duan, T. Xie and X. Wang, J. Am. Chem. Soc., 2009, 131, 3016.
2 (a) M. Stubbs, P. M. J. McSheehy, J. R. Griffiths and C. L. Bashford,
Mol. Med. Today, 2000, 6, 15; (b) J. Srivastava, D. L. Barber and
M. P. Jacobson, Physiology, 2007, 22, 30; (c) J. Y. Han and
K. Burgess, Chem. Rev., 2010, 5, 2709.
3 (a) Y. Xiang and A. J. Tong, Chem. Lett., 2005, 34, 926; (b) X. Y. Peng,
J. J. Du, J. L. Fan, J. Y. Wang, Y. K. Wu, J. Z. Zhao, S. G. Sun and
T. Xu, J. Am. Chem. Soc., 2007, 129, 1500; (c) J. Y. Han, A. Loudet,
R. Barhoumi, R. C. Burghardt and K. Burgess, J. Am. Chem. Soc.,
2009, 131, 1642; (d) L. Xue, C. Liu and H. Jiang, Chem. Commun.,
2009, 1061; (e) Z. X. Han, X. B. Zhang, Z. Li, Y. J. Gong, X. Y. Wu,
Z. Jin, C. M. He, L. X. Jian, J. Zhang, G. L. Shen and R. Q. Yu,
Anal. Chem., 2010, 82, 3108.
4 (a) N. N. Ratchford, S. Bangsaruntip, X. M. Sun, K. Welsher and
H. J. Dai, J. Am. Chem. Soc., 2007, 129, 2448; (b) S. Yao,
K. J. S. Hales and K. D. Belfield, Org. Lett., 2007, 9, 5645; (c)
M. T. Chen, L. M. Gomez, F. N. Ishikawa, P. T. Vernier, C. Zhou
and M. A. Gundersen, Nanotechnology, 2009, 20, 295101; (d)
X. J. Jiang, P. C. Lo, S. L. Yeung, W. P. Fong and D. K. P. Ng,
Chem. Commun., 2010, 46, 3188.
5 (a) Y. N. Hong, J. W. Y. Lama and B. Z. Tang, Chem. Commun.,
2009, 4332; (b) M. Wang, X. G. Gu, G. X. Zhang, D. Q. Zhang
and D. B. Zhu, Anal. Chem., 2009, 81, 4444; (c) Q. Chen, N. Bian,
C. Cao, X. L. Qiu, A. D. Qi and B. H. Han, Chem. Commun., 2010,
46, 4067; (d) Y. Liu, Y. Yu, J. W. Y. Lam, Y. N. Hong, M. Faisal,
W. Z. Yuan and B. Z. Tang, Chem.–Eur. J., 2010, 16, 8433; (e)
Y. Liu, C. M. Deng, L. Tang, A. J. Qin, R. R. Hu, J. Z. Sun and
This journal is ª The Royal Society of Chemistry 2011
J. Mater. Chem., 2011, 21, 13470–13475 | 13475