Novel fluorescent pH sensors and a biological probe based on anthracene derivatives with aggregation-induced emission characteristics

Article abstract of DOI:10.1021/la904727t

Three functionalized 9,10-distyrylanthracene (DSA) derivatives, namely, 9,10-bis(4-hydroxystyryl)anthracene (2), 9,10-bis{4-[2-(diethylamino)ethoxy] styryl}anthracene (4), and 9,10-bis{4-[2-(N,N,N-triethylammonium)ethoxy]styryl} anthracene dibromide (5), were synthesized and their fluorescence properties were investigated. The three DSA derivatives possess a typical aggregation-induced emission (AIE) property (i.e., they are nonluminescent in dilute solutions but are efficiently fluorescent as induced by molecular aggregation). Different AIE properties were tuned through molecular structure control. Dye 2 is a phenol-moiety-containing compound, which shows aggregation at pH values smaller than 10, resulting in a high fluorescence intensity. Thus, dye 2 has a pK_a of 9.94. 4 is an amine-containing compound that starts to aggregate at slightly basic conditions, resulting in a pK_a of 6.90. Dye 5 is an ammonium-salt-containing compound. Because it is very soluble in water, this compound has no AIE phenomenon but can interact strongly with protein or DNA to amplify its emission. Therefore, 5 is a fluorescent turn on biological probe for protein and DNA detection and it is also selective, which works for native BSA and ct DNA but not their denatured forms. Therefore, we not only developed a few new compounds showing the AIE phenomena but also controlled the AIE through environmental stimulation and demonstrated that the new AIE molecules are suitable for pH and biomacromolecule sensing.

Full text of DOI:10.1021/la904727t

pubs.acs.org/Langmuir
© 2010 American Chemical Society
Novel Fluorescent pH Sensors and a Biological Probe Based on Anthracene
Derivatives with Aggregation-Induced Emission Characteristics
Hongguang Lu, Bin Xu, Yujie Dong, Feipeng Chen, Yaowen Li, Zaifang Li, Jiating He, Hui Li, and
Wenjing Tian*
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University,
Changchun 130012, PR China
Received October 28, 2009. Revised Manuscript Received January 13, 2010
Three functionalized 9,10-distyrylanthracene (DSA) derivatives, namely, 9,10-bis(4-hydroxystyryl)anthracene
(2), 9,10-bis{4-[2-(diethylamino)ethoxy]styryl}anthracene (4), and 9,10-bis{4-[2-(N,N,N-triethylammonium)ethoxy]-
styryl}anthracene dibromide (5), were synthesized and their fluorescence properties were investigated. The three DSA
derivatives possess a typical aggregation-induced emission (AIE) property (i.e., they are nonluminescent in dilute
solutions but are efficiently fluorescent as induced by molecular aggregation). Different AIE properties were tuned
through molecular structure control. Dye 2 is a phenol-moiety-containing compound, which shows aggregation at pH
values smaller than 10, resulting in a high fluorescence intensity. Thus, dye 2 has a pKa of 9.94. 4 is an amine-containing
compound that starts to aggregate at slightly basic conditions, resulting in a pKa of 6.90. Dye 5 is an ammonium-salt-
containing compound. Because it is very soluble in water, this compound has no AIE phenomenon but can interact
strongly with protein or DNA to amplify its emission. Therefore, 5 is a fluorescent turn “on” biological probe for protein
and DNA detection and it is also selective, which works for native BSA and ct DNA but not their denatured forms.
Therefore, we not only developed a few new compounds showing the AIE phenomena but also controlled the AIE
through environmental stimulation and demonstrated that the new AIE molecules are suitable for pH and
biomacromolecule sensing.
Introduction
ACQ. Since the first AIE-active material, 1-methyl-1,2,3,4,5-
pentaphenylsilole, was reported by Tang’s group,4 many AIE-
active dyes have been developed by various research groups, such
as siloles,5 CN-MBE,6 DPDSB derivatives,7 DPDBF deriva-
tives,8 conjugated polymers,9 and others.10 The AIE dyes are
highly emissive in the solid state and hence are mainly used for the
construction of organic light-emitting diodes. In the past few
years, the application of AIE molecules to building new chemo-/
biosensors has attracted significant scientific interest. Typical AIE
molecules are tetraphenylethylene (TPE) derivatives, which can
Fluorescence (FL) chemo-/biosensors have received a great
deal of attention because of their potential applications in
chemistry, materials science, biology, and medicine.1 The FL-
based technique offers high sensitivity, low background noise,
and wide dynamic ranges.2 When sensors bind with ions or
neutral organic or inorganic molecules, their FL can be
enhanced/quenched and/or hypsochromically/bathochromically-
shifted, thus enabling the visual observation of the analytes.
However, FL dyes tend to aggregate when dispersed in aqueous
media or when bound to the analytes in large quantities. The
aggregation often quenches FL, which results in drastic reduc-
tions in their FL signals. This aggregation-caused quenching
(ACQ) has been a thorny problem in the development of efficient
FL sensing systems, especially in bioassays of trace numbers of
biomolecules.3 Thus, there is a high demand for the development
of simple, stable FL sensors without ACQ.
(4) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.;
Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Chem. Commun. 2001, 1740.
€
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Kwok, H. S.; Dong, Y.; Williams, I. D.; Liu, Y.; Luo, Y.; Shuai, Z.; Zhu, D.; Tang,
B. Chem. Commun. 2005, 3583. (b) Yu, G.; Yin, S.; Liu, Y.; Chen, J.; Xu, X.; Sun, X.;
Ma, D.; Zhan, X.; Peng, Q.; Shuai, Z.; Tang, B.; Zhu, D.; Fang, W.; Luo, Y. J. Am.
Chem. Soc. 2005, 127, 6335.
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124, 14410. (b) Lim, S.-J.; An, B.-K.; Jung, S.-D.; Chung, M.-A.; Park, S. Y. Angew.
Chem., Int. Ed. 2004, 43, 6346. (c) An, B.-K.; Gihm, S. H.; Chung, J. W.; Park, C. R.;
Kwon, S.-K.; Park, S. Y. J. Am. Chem. Soc. 2009, 131, 3950.
Recently, materials with aggregation-induced emission (AIE)
properties have attracted more and more attention because they
offer an efficient path to the solution of this spiny problem of
(7) (a) Xie, Z.; Yang, B.; Cheng, G.; Liu, L.; He, F.; Shen, F.; Ma, Y.; Liu, S.
Chem. Mater. 2005, 17, 1287. (b) Li, Y.; Li, F.; Zhang, H.; Xie, Z.; Xie, W.; Xu, H.; Li,
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*Corresponding author. E-mail: wjtian@jlu.edu.cn. Fax: þ86-431-
85193421.
Williams, I. D.; Sun, J.; Tang, B. Chem. Commun. 2006, 1133. (b) Tong, H.; Dong, Y.;
€
Hong, Y.; Haussler, M.; Lam, J. W. Y.; Sung, H. H.-Y.; Yu, X.; Sun, J.; Williams, I. D.;
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6838 DOI: 10.1021/la904727t
Published on Web 01/29/2010
Langmuir 2010, 26(9), 6838–6844

Lu et al.
Article
be used as FL turn-on bioprobes for protein, DNA detection,11
G-quadruplex formation, and real-time monitoring of DNA
folding.12 Other AIE-active compounds such as silole derivatives
have been applied in the FL turn-on detection of heparin in
serum,13 DNA and label-free fluorescence nuclease assay,14 con-
tinuous on-site label-free ATP fluorometric assay,15 and pH
sensing.16 The AIE dyes used for the FL sensors exhibit the
following advantages: (i) the AIE dyes do not suffer from the
problem of ACQ and hence extend the effective ranges of the
sensors; (ii) most of the sensors based on the AIE feature, in which
the FL intensities are turned on from an initially low level, could
be more sensitive and faster;17 and (iii) in contrast to the tradi-
tional molecular sensors utilizing different photophysical pro-
cesses such as photoinduced electron transfer (PET), photo-
induced charge transfer (PCT), and excimer/exciplex formation,
the AIE-active sensor operates with a new working mechanism
that could offer some new possibilities for species detection. The
addition of analyte could induce the AIE-active sensors to
aggregate or deaggregate, leading to a change in the fluorescence
optical signals and thus accomplishing analyte detection. For
example, Zhang et al. have established a simple AIE-active probe
for in situ, continuous, qualitative adenosine triphosphate (ATP)
detection based on the positively charged quaternary ammonium
modified silole, which can aggregate on the negatively charged
ATP template through charge-charge interaction and emit
strong fluorescence in aqueous solution.15 In addition, they have
recently reported a fluorescence turn-on sensing ensemble for
cyanide in aqueous solution by making use of the AIE feature of
the silole compound.18 However, until now, studies utilizing AIE
dyes as FL sensors have mainly concentrated on TPE derivatives
and silole derivatives, so developing new AIE-active sensory
materials would be very rewarding work.19
AIE-active DSA derivatives; dyes 2 and 4 and water-soluble
cationic salt 5; and explored their utility as pH sensors and
biological probes on the basis of their AIE characteristics. The
FL sensors based on compounds 2 and 4 exhibit well-defined on-
and-off behavior in response to the change in pH. Salt 5 is an
excellent fluorescent turn-on biological probe for protein and
DNA detection and it is also selective, which works for native
BSA and ct DNA but not their denatured forms.
Experimental Section
General. All reagents and starting materials are commercially
available and were used as received. 9,10-Dibromoanthracene
was purchased from Acros and used without further purification.
Dimethyl acetamide (DMAc) and tetrahydrofuran (THF) were
purified by fractional distillation before use as solvents. As shown
in Scheme 1, 1-methoxy-4-vinylbenzene was prepared according
to literature procedures.22 Bovine serum albumin (BSA; >98%,
fraction V) and calf thymus (ct) DNA were purchased as lyophi-
lized crystalline powders from Sigma.
The 1H and 13C NMR spectra were recorded at 298 K on an
AVANCZ 500 spectrometer and a Varian Mercury-300 NMR,
respectively, with tetramethylsilane (TMS) as the standard. Mass
spectra were recorded on an Agilent 1100 LC-MS system.
Elemental analysis was performed with a Flash EA 1112 elemen-
tal analyzer. UV-vis absorption spectra were recorded on a
Lambda-800 spectrophotometer. Fluorescence measurements
were carried out with RF-5301PC. Scanning electron microscope
(SEM) images were obtained with a field-emission scanning
electron microscope (FE-SEM, JEOL JSM-6700F).
UV and FL Spectra. Thestock solutionof4 was 1.0 ꢀ 10-4
M
in acetonitrile. A sample mixture used to measure the UV and FL
spectra was prepared by adding 1 mL of a stock solution to 9 mL
of acetonitrile or water under vigorous stirring at room tempera-
ture. The mixture was stirred for halfanhourprior to recording its
spectrum. Similarly, BSA was dissolved in a pH 7.0 phosphate
buffer solution (0.1 and 1.0 mg/mL), and DNA was dissolved in
deionized water (0.1 and 1.0 mg/mL). The stock solution of 5 was
2.5 ꢀ 10-4 M in water. FL titration was carried out by adding
aliquots of BSA or DNA solution to 0.1 mL of a stock solution of
5, followed by adding an aqueous phosphate buffer (10 mM, pH
7.0) to acquire a 10.0mL solution. The mixturewas stirred for half
an hour prior to recording its spectrum. The relative fluorescence
quantum yield (ΦF) was determined by the standard method
using quinine sulfate in 0.1 N H2SO4 solution as a reference.
Anthracene and its derivatives constitute a very famous class of
fluorophoresthathave beenwidelyused in the development of FL
sensors because of their excellent photoluminescence properties
and chemical stability.20 We have recently reported a class of
9,10-distyrylanthracene (DSA) derivatives with AIE properties.21
The investigation indicates that the restricted intramolecular
rotations between the 9,10-anthylene core and the vinylene
segment are the cause of the AIE phenomenon. These dye
molecules, showing typical AIE characteristics and excellent
photoluminescence properties, could be promising candidates
as FL sensory materials. Herein, we synthesized three new
DNA Cleavage Reaction with the Hydroxyl Radical.
A
total volume of 1 mL of a reaction mixture containing ct DNA
(10.0μg/mL), FeSO4 (14μM), and H2O2 (3.6 mM) was placedina
tube. The mixture was incubated at 37 °C for 30 min and then was
added to a buffer solution of 5 (2.5 ꢀ 10-6 M in 10 mM phosphate
buffer solution, pH 7.0) for FL spectral measurements.
€
(11) (a) Tong, H.; Hong, Y.; Dong, Y.; Haussler, M.; Lam, J. W. Y.; Li, Z.; Guo,
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€
Haussler, M.; Li, Z.; Lam, J. W. Y.; Dong, Y.; Sung, H. H.-Y.; Williams, I. D.; Tang, B.
J. Phys. Chem. B 2007, 111, 11817.
Synthesis of 9,10-Bis(4-methoxystyryl)anthracene (1).
A
€
(12) Hong, Y.; Haussler, M.; Lam, J. W. Y.; Li, Z.; Sin, K. K.; Dong, Y.; Tong,
H.; Liu, J.; Qin, A.; Renneberg, R.; Tang, B. Chem. Eur. J. 2008, 14, 6428.
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2008, 80, 6443.
round-bottomed flask (25 mL) was oven dried and cooled under a
N2 atmosphere. 1-Methoxy-4-vinylbenzene (0.32 g, 2.4 mmol),
9,10-dibromoanthracene (0.34 g, 1 mmol), K3PO4 (0.64 g,
3 mmol), and Pd(OAc)2 were dissolved in 10 mL of dry DMAc.
The reaction mixture was heated to 110 °C in an oil bath and
stirred for 24 h at this temperature. After being cooled to room
temperature, the reaction mixture was poured into water and
extracted with CH2Cl2 (3 ꢀ 40 mL). The combined organic
extracts were washed with brine, dried (Mg2SO4), and concen-
trated to dryness under vacuum. The crude product was purified
by silica gel chromatography (4:1 petroleum ether/CH2Cl2) to
(15) Zhao, M.; Wang, M.; Liu, H.; Liu, D.; Zhang, G.; Zhang, D.; Zhu, D.
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(16) Li, Z.; Dong, Y.; Lam, J. W. Y.; Sun, J.; Qin, A.; Haussler, M.; Dong, Y.;
€
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19, 1.
(17) (a) Mortellaro, M. A.; Nocera, D. G. J. Am. Chem. Soc. 1996, 118, 7414. (b)
McQuade, D. T.; Hegedus, A. H.; Swager, T. M. J. Am. Chem. Soc. 2000, 122, 12389.
(c) Ma, G.; Cheng, Q. Langmuir 2006, 22, 6743.
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(19) (a) Ning, Z.; Chen, Z.; Zhang, Q.; Yan, Y.; Qian, S.; Cao, Y.; Tian, H. Adv.
Funct. Mater. 2007, 17, 3799. (b) Yuan, C.; Tao, X.; Wang, L.; Yang, J.; Jiang, M.
J. Phys. Chem. C 2009, 113, 6809.
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A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515.
(b) Martinez-Manez, R.; Sancenon, F. Chem. Rev. 2003, 103, 4419.
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1
give a yellow solid (0.29 g, 65% yield). H NMR (CDCl3, 500
MHz): δ (TMS) 8.39-8.41 (m, 4H), 7.79 (d, J=16.5 Hz, 2H), 7.63
(d, J=8.5 Hz, 4H), 7.45-7.47 (m, 4H), 7.00 (d, J=8.5 Hz, 4H),
6.88 (d, J=16.5 Hz, 2H), 3.89 (s, 6H). 13C NMR (CDCl3, 75
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Langmuir 2010, 26(9), 6838–6844
DOI: 10.1021/la904727t 6839

Article
Lu et al.
Scheme 1. Synthesis of Functionalized DSA Derivatives 2, 4, and 5 from 1
MHz): δ (TMS) 159.73, 136.90, 132.83, 130.35, 129.73, 127.83,
126.55, 125.10, 123.05, 114.35, 55.45. EI-MS m/e: 443.4
([M þ H]þ, calcd 443.19). Anal. Calcd for C32H26O2: C, 86.85;
H, 5.92. Found: C, 86.71; H, 6.01.
and 0.94 g of 3 (1.50 mmol) in 31 mL of THF/H2O (30:1 v/v).
After being refluxed for 24 h, the mixture was cooled to room
temperature and 3 mL of 37% hydrochloric acid and 50 mL of
water were added. The mixture was extracted with 100 mL of
CHCl3 three times. The organic layers were combined and dried
over Mg2SO4 overnight. After filtration and solvent evaporation,
the crude product was purified by silica gel chromatography (1:1
petroleum ether/CH2Cl2) to give a yellow solid (0.58 g, 63%
yield). 1H NMR (CDCl3,500 MHz): δ (TMS) 8.39-8.41 (m, 4H),
7.76 (d, J=16.5 Hz, 2H), 7.60 (d, J=8.5 Hz, 4H), 7.45-7.47 (m,
4H), 7.00(d, J=8.5 Hz, 4H), 6.85 (d, J=16.5Hz, 2H), 4.14(t, 4H),
2.94 (t, 4H), 2.67 (m, 8H), 1.11 (t, 12H). 13C NMR (CDCl3, 75
MHz): δ (TMS) 158.86, 136.85, 132.77, 130.24, 129.64, 127.77,
126.52, 125.08, 122.90, 114.89, 66.67, 51.76, 47.85, 11.79. EI-MS
m/e: 613.4 ([M þ H]þ, calcd 613.37). Anal. Calcd for
C42H48N2O2: C, 82.31; H, 7.89; N, 4.57. Found: C, 82.29; H,
7.92; N, 4.49.
Synthesis of 9,10-Bis(4-hydroxystyryl)anthracene (2).
Into a 100 mL flask were added 1.33 g of 1 (3.00 mmol) and 30
mL of dichloromethane (DCM). The flask was placed in an
acetone-dry ice bath at -78 °C. A solution of 3.02 g of boron
tribromide (12.0 mmol) in 20 mL of DCM was added carefully to
the mixture with stirring. The resultant mixture was allowed to
warm to room temperature and was stirred overnight. The
reaction product was hydrolyzed by careful shaking with 20 mL
of water. The organic phase was separated and concentrated by a
rotary evaporator. The crude product was purified by silica gel
chromatography (3:1 petroleum ether/acetone) to give a yellow
1
solid (1.03 g, 83% yield). H NMR (DMSO-d6, 500 MHz): δ
(TMS): 9.67 (s, 2H), 8.37-8.39 (m, 4H), 7.86 (d, J=16.5 Hz, 2H),
7.63 (d, J=8.5 Hz, 4H), 7.53-7.55 (m, 4H), 6.85 (d, J=8.5 Hz,
4H), 6.80 (d, J=16.5 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): δ
(TMS): 157.61, 137.03, 132.36, 128.98, 128.09, 128.06, 126.22,
125.31, 121.11, 115.55. EI-MS m/e: 415.2 ([M þ H]þ, calcd
415.16). Anal. Calcd for C30H22O2: C, 86.93; H, 5.35. Found:
C, 86.79; H, 5.46.
Synthesis of 9,10-Bis{4-[2-(N,N,N-triethylammonium)-
ethoxy]styryl}anthracene Dibromide (5). A 100 mL flask with
a magnetic spin bar was charged with 0.11 g of 3 (0.17 mmol)
dissolved in 50 mL of THF. To this solution was added 5 mL of
triethylamine (35.9 mmol). The mixture was heated to reflux and
stirred for 3 days. During this period, 10mL of waterwas added at
several intervals. THF and extra triethylamine were evaporated.
The watersolutionwaswashed with chloroformthree times. After
solvent evaporation, the residue was washed with chloroform and
acetone and then dried overnight in vacuo at 50 °C. The product
Synthesis of 9,10-Bis[4-(2-bromoethoxy)styryl]anthra-
cene (3). Into a 100 mL round-bottomed flask were added 3.31
g of K2CO3 (24.0 mmol), 1.00 g of 2 (2.40 mmol), and 9.02 g of
1,2-dibromoethane (48.0 mol) in 40 mL of dry acetone. The
mixture was heated to reflux and stirred for 24 h. After filtration
and concentration, the crude product was purified by silica gel
chromatography (2:1 petroleum ether/CH2Cl2) to give a yellow
solid (0.79 g, 52% yield). 1H NMR (CDCl3,500 MHz): δ (TMS)
8.38-8.40 (m, 4H), 7.79 (d, J=16.5 Hz, 2H), 7.62 (d, J=8.5 Hz,
4H), 7.46-7.49 (m, 4H), 7.00 (d, J = 8.5 Hz, 4H), 6.86 (d,
J=16.5 Hz, 2H), 4.38 (t, 4H), 3.70 (t, 4H). 13C NMR (CDCl3,
125 MHz): δ (TMS): 158.08, 136.69, 132.73, 130.98, 129.63,
127.91, 126.50, 125.17, 123.41, 115.15, 68.05, 29.02. EI-MS m/e:
629.1 ([M þ H]þ, calcd 629.04); Anal. Calcd for C34H28Br2O2: C,
64.99; H, 4.49. Found: C, 64.81; H, 4.47.
1
was isolated in 48% yield (0.07 g). H NMR (DMSO-d6, 500
MHz): δ (TMS) 8.37-8.39 (m, 4H), 8.00 (d, J=16.5 Hz, 2H), 7.81
(d, J=8.5 Hz, 4H), 7.56-7.58 (m, 4H), 7.10 (d, J=8.5 Hz, 4H),
6.89 (d, J=16.5 Hz, 2H), 4.48-4.50 (t, 4H), 3.72-3.74 (t, 4H),
3.40-3.45(m, 12H), 1.25-1.28(t, 18H). 13C NMR (DMSO-d6, 75
MHz): δ (TMS) 157.40, 136.57, 132.34, 130.46, 128.97, 128.15,
126.23, 125.50, 122.82, 114.95, 61.18, 55.22, 52.97, 7.36. EI-MS m/e:
335.3 ([M - 2Br]2þ, calcd 335.23). Anal. Calcd for C46H58Br2N2O2:
C, 66.50; H, 7.04; N, 3.37. Found: C, 66.28; H, 7.27; N, 3.21.
Results and Discussion
Synthesis and Characterization. The DSA derivatives were
prepared by the synthesis route shown in Scheme 1. DSA
derivative 1 was obtained in all-trans conformations by the Heck
Synthesis of 9,10-Bis{4-[2-(diethylamino)ethoxy]styryl}-
anthracene (4). Into a 100 mL round-bottomed flask were added
1.05 g of K2CO3 (7.50 mmol), 3 mL of diethylamine (30.0 mmol),
6840 DOI: 10.1021/la904727t
Langmuir 2010, 26(9), 6838–6844

Lu et al.
Article
Figure 1. (A) FL spectra of 4 in acetonitrile/water mixtures and (B) dependence of the I/I0 ratios of 4 on the solvent composition of the
acetonitrile/water mixture. The concentration of 4 is 10 μM, and the excitation wavelength is 425 nm.
coupling reaction. The demethylation of 1 by BBr3 followed by
treatment with water gave dihydroxylated DSA derivative 2. The
reactions of 2 with 1,2-dibromoethane in the presence of K2CO3
yielded DSA derivative 3. DSA derivative 4 was synthesized by
the reaction of 3 with diethylamine. The quaternization of 3 by
NEt3 gave salt 5. The structures of the product molecules were
confirmed by 1H NMR, 13C NMR, EI-MS, and elemental
analysis. Dyes 2 and 4 are soluble in common organic solvents
such as acetonitrile (AN), acetone, chloroform, and THF but
insoluble in water. Salts 5 are soluble in water as well as methanol,
dimethyl formamide (DMF), and dimethyl sulfoxide (DMSO).
AIE Effect. 4 is almost nonluminescent in acetonitrile (AN)
with an extremely low fluorescence quantum yield (ΦF) of 0.25%,
and a suspension of 4 in AN/water with a high water fraction
larger than 65% is highly emissive (Figure 1A), suggesting strong
aggregations of 4 in water-rich solvents. As indicated in Figure S1
(Supporting Information), the absorption of 4 obviously de-
creases along with the FL emission increase. The formation of
Figure 2. Emission spectra of 5 (2.5 μM) in a glycerol/water
mixture with 90% glycerol at -78, 0, and 25 °C. Data for 5 in
water (2.5 μM) at 25 °C is shown for comparison.
nanoscopic aggregates of 4 is suggested by the leveling-off tail in
the visible region of the absorption spectrum because of the Mie
effect of the nanoparticles.23 The nanoscopic aggregations of 4
were virtually confirmed by SEM imaging (Figure S2). Nano-
particles with average sizes of ∼100-150 nm are clearly observed.
Thus, 4 is AIE-active.
The light-scattering effect of nanoaggregates in aqueous mix-
tures with different water contents makes the ΦF measurements
inaccurate.10e We thus used the changes in the FL intensity of 4 in
the AN/water mixtures with different water contents to reveal the
AIE characteristics. The I/I0 ratio in the solvent mixtures remains
almost unchanged until up to ∼65% water is added (Figure 1B).
The ratio starts to increase when the dyes begin to cluster together
in the water/AN mixtures with >65% water. When the water
fraction is increased to 90%, the fluorescence intensity is ∼82-fold
higher than in pure AN solution (I0).
Salt 5 is completely soluble in water. The addition of AN, THF,
and methanol to the solution of the sample in water fails to make
the dye molecules aggregate, possibly because of its amphiphilic
nature, which is associated with its binary tetraalkylammonium
moieties.11a The emission in the mixtures is as weak as that in the
pure water solution. However, the increasing viscosity and
decreasing temperature of the solution of 5 can increase its FL
intensity (Figure 2). At room temperature, the FL intensity of a
dilute solution of 5 in a viscous glycerol/water mixture (9:1 v/v) is
much higher than that in pure water. As the solution temperature
is decreased from 25 to -78 °C, the FL intensity of 5 is increased.
These phenomena occur because high viscosity and low tempera-
ture can hamper intramolecular rotation, leading to the closure of
the nonradiative decay channel and thus enhanced FL emission.
The results indicate that the restriction of intramolecular rotation
plays a very important role in inducing the dye to emit, which
coincides with our previous study on the AIE of the DSA
derivatives.21
pH Sensing. Recently, stimuli-sensitive functional materials
have received much attention.24 In particular, fluorescent pH
sensors have extensively been pursued in analytical chemistry,
bioanalytical chemistry, cellular biology, and medicine.25 DSA
derivatives show a distinct phenomenon of AIE: the nonemissive
molecules can be induced to emit efficiently by aggregate forma-
tion, which provides an opportunity to design a novel pH sensor
by making use of the AIE feature.
(24) (a) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.;
Okano, T. Nature (London) 1995, 374, 240. (b) Roy, I.; Gupta, M. N. Chem. Biol. 2003,
10, 1161. (c) Yu, X.; Wang, Z.; Jiang, Y.; Shi, F.; Zhang, X. Adv. Mater. 2005, 17, 1289.
(25) (a) Lee, S.-H.; Kumar, J.; Tripathy, S. K. Langmuir 2000, 16, 10482. (b) Lee,
J. W.; Lee, J.-S.; Chang, Y.-T. Angew. Chem., Int. Ed. 2006, 45, 6485. (c) Uchiyama, S.;
Makino, Y. Chem. Commun. 2009, 2646.
€
(23) Auweter, H.; Haberkorn, H.; Heckmann, W.; Horn, D.; Luddecke, E.;
Rieger, J.; Weiss, H. Angew. Chem., Int. Ed. 1999, 38, 2188.
Langmuir 2010, 26(9), 6838–6844
DOI: 10.1021/la904727t 6841

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Lu et al.
Figure 3. (A) FL spectra of 4 (20 μM) at different pH values in an acetonitrile/water mixture (3:7 v/v). (B) pH dependence of 4 (I/Imax) at
296 K in acetonitrile/water mixtures (3:7 v/v) at different pH values.
As discussed above, dye 4 is nonluminescent in acidic solution
with pH lower than 4. At pH higher than 4, the FL intensity starts
to increase quickly. At a pH of 8.5, the solution emits strongly in
the green spectral window at 510 nm and the FL intensity is
70-fold that at pH 3.0 (Figure 3). Furthermore, the difference in
the fluorescence intensities of 4 at high and low pH can be easily
distinguished by the naked eye under UV light (365 nm) illumina-
tion (inset of Figure 3B). Furthermore, the FL intensity of 4
exhibits a distinct increase when the pH increases from 2 to 12,
and its emission could be quenched when the pH is reduced from
12 to 2. The reversibility experiments demonstrate that the AIE of
compound 4 is reversible when the pH is increased from 2 to 12
and again returns to 2 (Figure S3 in Supporting Information).
These phenomena occur because the structure of4 can be changed
from an isolated species to aggregated clusters in response to the
variation in pH. At low pH, 4 is converted into an ammonium salt
form and thus dissolves in water. The solution is nonemissive. A
similar phenomenon was observed for 5 and has been discussed.
When the pH is higher than 5, the ammonium salt form of 4
started to convert back to its free amine form, which is insoluble
and aggregates in water. Thus, the fluorescence coming from the
aggregation state is turned on. The AIE nature makes 4 pH-
sensitive.
Figure 4. pH dependence of 2 (I/Imax) at 296 K in acetonitrile/
water mixtures (3:7 v/v) of differing pH.
Table 1. Fluorescence Quantum Yield of DSA Derivatives 2, 3, 4, and
5 under Acidic, Neutral, and Basic Conditions
Φ
F (%)a
Different from 4, compound 2 bearing two phenol substituents
behaves in an exactly opposite manner. As shown in Figure 4, the
FL emission of 2 is turned on at low pH values and turned off at
high pH values, which is attributed to deionization and ionization
under acidic and basic conditions, respectively. The phenol
substituents may be converted into the quinone structure under
basic conditions, but compound 2, within 2 weeks, cannot
converted to the quinone form under basic conditions, as sug-
gested by the fact that the absorption spectra of a basic solution of
2 undergo no distinct change within 2 weeks. Figures 3B and 4
illustrate the pH response of sensors 2 and 4 as a function of I/Imax
versus pH, where I is the measured FL emission at that pH and
Imax is the maximum output of the sensor. The pKa can be
estimated where I/Imax is 0.5. The pKa values for 2 and 4 are
9.94and 6.90, respectively, showing the structural influence onthe
pH response. Neutral compound 3 also showed strong aggrega-
tion in water-rich solvent; however, because 3 has no pH-sensitive
moiety, its aggregation state has no significant differences at
various pH values, which can be reflected in its quantum yields
(Table 1).
DSA
pH 2
pH 7
pH 12
2
3
4
5
6.87
12.74
0.14
5.82
11.76
9.12
0.02
10.33
20.40
0.19
0.19
0.20
a Fluorescence quantum yield (ΦF) of DSA derivatives 2, 3, 4,
and 5 (20 μM) at different pH values in an acetonitrile/water mixture
(3:7 v/v).
because of its potential application in biological science and
technology.26 However, the aggregation of dyes causes FL
quenching, which limits the effective ranges of the probes. This
makes it particularly difficult to assay low-abundance biomacro-
molecules. The bioprobe using an AIE dye does not suffer from
ACQ.
Figure 5A shows the FL spectral changes in 5 before and
after the addition of BSA. Water-soluble salt 5 is nonluminescent
in a pH 7.0 phosphate buffer; however, the FL intensity of the
(26) (a) Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2000, 100, 2479. (b)
Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435.
(c) Herland, A.; Inganas, O. Macromol. Rapid Commun. 2007, 28, 1703.
Biological Probing. The study of the detection of biomacro-
molecules by FL probes has received a great deal of attention
€
6842 DOI: 10.1021/la904727t
Langmuir 2010, 26(9), 6838–6844

Lu et al.
Article
Figure 5. (A) Change in the FL spectrum of 5 (2.5 μM) with the addition of BSA to an aqueous phosphate buffer (pH 7.0). (B) Plot of FL
intensity vs BSA concentration.
Figure 7. Plot of FL intensity of 5 (2.5 μM) in an aqueous
phosphate buffer (pH 7.0) vs ct DNA concentration.
Figure 6. Effect of BSA (100 μg/mL) and/or CTAB (0.6 mg/mL)
on the FL spectrum of a buffer solution of 5 (2.5 μM).
It is well known that Fenton’s reagent can generate HO from
buffer solution of 5 increased gradually when BSA was intro-
duced. For instance, the FL intensity of 5 (2.5 μM) at 510 nm is
enhanced by 50-fold after the concentration of BSA reached
500 μg/mL (Figure 5B). The detection limit can reach 500 ng/mL
for BSA. Meanwhile, as indicated in Figure 6, the FL of the
ensemble of 5 (2.5 μM) and BSA (100 μg/mL) is rather strong;
however, it is dramatically weakened after cetyltrimethyl-
ammonium bromide (CTAB) is added to the BSA solution
of 5. The probe is thus folding-structure-sensitive because the
surfactant molecules of CTAB can unfold the folding structure
of BSA chains and destroy the native hydrophobic regions of
the protein.27
In addition to protein probing, salt 5 is also a sensitive FL
bioprobe for DNA detection. The emission of 5 (2.5 μM) is turned
on when ct DNA is added to its phosphate buffer solution
(pH 7.0, Figure S5 in Supporting Information). The FL intensity
of 5 was enhanced 15-fold after the concentration of ct DNA
reached 10 μg/mL (Figure 7). Actually, the FL difference in the
solution of 5 before and after the addition of ct DNA can be
distinguished bythe naked eye asdisplayed intheinset ofFigure 7.
Moreover, the detection limit can reach 100 ng/mL for ct DNA.
3
Fe2þ and H2O2 and HO can cut DNA into different sequence
3
fragments and even single bases.28 The cleavage process of DNA
by HO can be followed with salt 5. As indicated in Figure 8, the
3
FL of the ensemble of 5 (2.5 μM in 10 mM phosphate buffer
solution, pH 7.0) and ct DNA (10 μg/mL) is very strong; however,
if ct DNA is pretreated with Fenton’s reagent, which generates
HO to cleave DNA, then the FL of ensemble of 5 and ct DNA
3
fragments becomes very weak. In the meantime, when the
cleavage process is performed after ct DNA detection by 5 is
completed, the FL of the ensemble of 5 and ct DNA is also
decreased distinctly. Clearly, the probe is selective: it works for
native DNA but not its fragments. More importantly, apart from
HO , nuclease can also cleave DNA into fragments.28b Therefore,
3
it is possible to employ 5 to follow the DNA cleavage process by
nucleases, leading to the development of a label-free fluorescence
nuclease assay.
Previous studies of DSA derivatives indicated that the res-
tricted intramolecular rotations between the 9,10-anthrylene
core and the vinylene moiety are the main origin of the AIE
properties.21 How, then, is the AIE effect of 5 associated with the
(27) (a) Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M. Langmuir 2000, 16,
922. (b) Gull, N.; Chodankar, S.; Aswal, V. K.; Sen, P.; Khan, R. H.; Kabir-ud-Dina
Colloids Surf., B 2009, 69, 122.
(28) (a) Shigenaga, M. K.; Park, J.-W.; Cundy, K. C.; Gimeno, C. J.; Ames, B.
N. Methods Enzymol. 1990, 186, 521. (b) Tang, Y.; Feng, F.; He, F.; Wang, S.; Li, Y.;
Zhu, D. J. Am. Chem. Soc. 2006, 128, 14972.
Langmuir 2010, 26(9), 6838–6844
DOI: 10.1021/la904727t 6843

Article
Lu et al.
Conclusions
In this work, we designed DSA-based new compounds with
different AIE aggregation properties through the tuning of
chemical structures. Dye 2 is a phenol-moiety-containing com-
pound that shows aggregation at pH values smaller than 10 to
result in a high FL intensity with a pKa of 9.94. 4 is an amine-
containing compound that starts to aggregate at slightly basic
conditions, resulting in a pKa of 6.90. Thus, 4 might be suitable for
pH sensing in a biological environment. Dye 5 is ammonium-salt-
containing compound. Because it is very soluble in water, this
compound exhibits no AIE phenomenon, but 5 can interact
strongly with protein or DNA to amplify its emission. Therefore,
5 is an excellent fluorescent turn-on biological probe for protein
and DNA detection. The detection limits for BSA and ct DNA
can reach 500 and 100 ng/mL, respectively. Probe 5 is also
selective, which works for native BSA and ct DNA but not their
denatured forms. Therefore, we not only developed a few new
compounds showing the AIE phenomena but also controlled AIE
through environmental stimulation and demonstrated that the
new AIE molecules are suitable for pH and biomacromolecular
sensing. Moreover, studies on their further applications in bio-
logical science and technology are currently in progress.
Figure 8. FL spectrumof(a) 5(2.5 μMin10mMphosphate buffer
solution, pH 7.0) and those in the presence of (b) ct DNA (10.0 μg/
mL) and (c) ct DNA (10.0 μg/mL), which was pretreated with HO
(generated from 14 μM FeSO4 and 3.6 mM H2O2) at 37 °C for
30 min.
3
bioprobing process? In the buffer solutions, the cationic am-
phiphilic dyes bind to the biomacromolecules (protein and
DNA) via supramolecular interactions such as electrostatic
interaction and the hydrophobic effect.11 For the native BSA, it
contains hydrophobic binding sites such as hydrophobic pock-
ets. When the dye molecules bind to the hydrophobic regions of
BSA chains and enter into the hydrophobic pockets of their
folding structures, the intramolecular rotations of the dye
molecules are frozen, inducing them to emit as aggregation
does. For ct DNA, the formation of a 5-ct DNA complex is
driven by the electrostatic interaction between the ammonium
group in 5 and the negative phosphate backbones in ct DNA
and the possible hydrophobic interaction between nucleosides
and aryl rings in 5. These intermolecular forces suppress
intramolecular rotations of the dye molecules, accordingly
activating their AIE processes. Moreover, these conclusions
can be proven by the phenomenon in which the emissions of 5
are turned off, when the native structures of biomacromole-
cules are destroyed (including BSA unfolding by CTAB and
Acknowledgment. We are grateful to Dr. Yanqing Tian at the
Biodesign Institute of Arizona State University for helpful
discussions. This work was supported by the 973 Program
(2009CB623605), the NSFC (grant no. 50673035), the State
Key Development Program for Basic Research of China (grant
no. 2009CB623605), the Program for New Century Excellent
Talents in Universities of the China Ministry of Education, and
the 111 Project (grant no. B06009).
Supporting Information Available:
Absorption and
emission spectra of 4 in acetonitrile and an acetonitrile/water
mixture. SEM image of nanoaggregates of 4 in acetonitrile/
water mixtures. FL spectra of 2 at different pH values in an
acetonitrile/water mixture. pH dependence of 4 in aceto-
nitrile/water mixtures at differing pH. Change in the FL
spectrum of 5 with the addition of ct DNA to an aqueous
phosphate buffer. This material is available free of charge via
the Internet at http://pubs.acs.org.
DNA cleavage by HO , see Figures 6 and 8).
3
6844 DOI: 10.1021/la904727t
Langmuir 2010, 26(9), 6838–6844