New water-soluble dicyano-stilbene dyes: One and two-photon fluorescence and photo-response to BSA

Article abstract of DOI:10.1016/j.dyepig.2010.09.005

Two novel quadrapolar stilbene fluorescent dyes with CN groups (DCNs) are reported. Photophysical properties such as fluorescence, intermolecular charge transfer, two-photon absorption action cross section (δ) and fluorescence-response to bovine serum alb

Full text of DOI:10.1016/j.dyepig.2010.09.005

Dyes and Pigments 89 (2011) 330e335
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
New water-soluble dicyano-stilbene dyes: One and two-photon
fluorescence and photo-response to BSA
*
Jingqiang Cui, Jiangli Fan, Xiaojun Peng , Shiguo Sun
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 158 Zhongshan Road, Dalian 116012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel quadrapolar stilbene fluorescent dyes with CN groups (DCNs) are reported. Photophysical
properties such as fluorescence, intermolecular charge transfer, two-photon absorption action cross
section (dV) and fluorescence-response to bovine serum albumin (BSA) are investigated and discussed.
The fluorescence emission is red-shifted and quenched with the increase in polarity of solvents. In
aqueous buffer (pH 7.0), compound 1b has a large blue shift (74 nm) and 37-fold fluorescence
enhancement when interacts with BSA. The response is noticeable by naked eyes. Cations, anions and
DNA have no such responses. It is resulted from the change of environmental polarity from aqueous
solution to hydrophobic cage in protein, and sequentially, the change in the effect of excited intermo-
lecular charge transfer.
Received 7 December 2009
Received in revised form
12 September 2010
Accepted 17 September 2010
Available online 26 September 2010
Keywords:
Fluorescence
Dye
Two-photon absorption action cross section
Molecular switch
Ó 2010 Elsevier Ltd. All rights reserved.
Bovine serum albumin
Stainer
1. Introduction
Meantime, various organic TPEF compounds with high quantum
yields, large TPA cross sections
reported [13e18]. Especially, the CN groups in the molecular core
increases the values significantly [18]. Whereas, few of the stil-
bene derivatives containing CN groups are water-soluble [19,20].
The water-solubility is very important for practical applications for
protein determinations.
Herein we report the synthesis, photophysics, and protein-
responsive properties of water-soluble stilbene derivatives with CN
groups (DCNs) (Fig. 1): there is a 37-fold fluorescence enhancement
of DCN 1b after interaction with BSA.
d, and good stability have been
Protein-responsive fluorophores [1e5] and chromophores are of
scientific and practical interest to detect various proteins such as
bovine serum albumin (BSA) [6e8]. There are two ways to label
proteins with an fluorophore: one is covalent labeling, requiring
a reactive functional group on the label molecule to react to the
proteins; and another is noncovalent labeling, whereby the probe
forms a complex with the proteins via noncovalent interactions,
such as ionic, electrostatic, hydrophobic, and hydrogen bonding.
The noncovalent labeling method is typically a simpler and faster
protocol compared to conventional covalent labeling [9,10].
d
Recently, fluorophores with two-photon excited fluorescence
(TPEF), which are based on molecular two-photon absorption
(TPA), have received increasing interest for its various advantages
such as localized excitation, increased penetration depth, lower
tissue autofluorescence and self-absorption, in addition to the
reduced photodamage and photobleaching [11,12]. A useful fluo-
rophore for such applications should have a large TPA cross-section,
2. Experiments
2.1. Materials and general methods
Deionized water was redistilled before use. All the solvents were
of analytical grade. 1H NMR and 13C NMR spectra were recorded on
a VARIAN INOVA-400 spectrometer with chemical shifts reported
as ppm (in CDCl3, TMS as internal standard). Mass spectrometric
data were obtained on a HP1100LC/MSD MS spectrometer and a LC/
Q-Tof MS spectrometer. Fluorescence measurements were per-
formed on a PTI-700 Felix and Time-Master system, and the slit
width was 5 nm for both excitation and emission. Absorption
spectra were measured on Lambda 35 UV/vis spectrophotometer.
d, in NIReIR region, appreciable solubility in water, high photo-
stability, and sensitive fluorescence-enhancement to special bio-
logical substrates.
* Corresponding author. Tel.: þ86 0411 39893899; fax: þ86 0411 39893800.
E-mail address: pengxj@dlut.edu.cn (X. Peng).
0143-7208/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.09.005

J. Cui et al. / Dyes and Pigments 89 (2011) 330e335
331
RO
O
CN
OR
N
O
O
N
O
RO
NC
OR
1a: R = Et; 1b: R = Na
OR
O
OR
O
RO
O
CN
O
N
O
N
O
NC
OR
O
RO
O
RO
2a: R = Et; 2b: R = Na
Fig. 3. The dependence of the up-converted fluorescence intensity for 1b in H2O on
the incident intensity.
Fig. 1. New stilbene derivatives with CN groups (DCNs).
of hexane in dichloromethane (20e0%) and ethyl acetate in
dichloromethane (0e20%). Compound 1a (296 mg, 32%) yielded as
All pH measurements were made with a Model PHS-3C meter.
Melting points were determined by an X-6 micro-melting point
apparatus and are uncorrected. The fluorescence quantum yields of
DCNs were measured using standard methods on air-equilibrated
samples at room temperature. Quinine bisulfate in 0.05 mol Lꢀ1
a yellow powder. M.p. 194.3e195.4 ꢁC. 1H NMR (CDCl3, 400 MHz)
d:
7.96 (s, 2H), 7.47 (d, J ¼ 8.4 Hz, 4H), 7.21(d, J ¼ 16.0 Hz, 2H), 7.15 (d,
J ¼ 16.0 Hz, 2H), 6.63 (d, J ¼ 8.4 Hz, 4H), 4.26 (s, 8H), 4.25e4.17 (m,
8H), 1.30 (t, J ¼ 8.0 Hz, 12H). 13C NMR (CDCl3, 100 MHz)
d: 171.132,
H2SO4 solution (
F
¼ 0.546) was used as a reference [21]. TPEF
168.732, 149.535, 141.166, 138.786, 134.468, 129.598, 122.674,
119.952, 119.335, 116.849, 114.794, 113.329, 77.517, 77.199, 76.881,
66.465, 61.523, 61.029, 53.974, 14.388. TOF MS (ES): m/z Calcd for
[Mþ], C40H42N4O8, 706.2998, Found: 729.3000.
spectra were measured on SD2000 spectrometer (Ocean Optical).
The TPA cross sections (dTPA) of DCNs were determined by the two-
photon-induced excited fluorescence method [22].
2.2.2. Compound 2a
2.2. Synthesis
Aldehyde
4 [23,24] (0.38 g, 1 mmol), and NaH (130 mg,
5.4 mmol) were dissolved in THF (3 mL), and the solution was
cooled to 0 ꢁC under N2. To this solution, phosphonate 5 (1.46 g,
5.0 mmol) in THF (9 mL) was added dropwise. The reaction mixture
was stirred for 1 h at 0 ꢁC, and then for 12 h at room temperature,
followed by the removal of THF under reduced pressure. Water
then was added to the reaction mixture, and the product was
extracted with dichloromethane (4 ꢂ100 mL). The organic layer
was dried with dry Na2SO4 followed by evaporation of the solvent.
The crude product was separated by column chromatography with
a gradient of hexane in dichloromethane (20e0%) and ethyl acetate
in dichloromethane (0e20%). Compound 2a (326 mg, 35%) yielded
The synthetic routes for DCNs are shown in Fig. 2.
2.2.1. Compound 1a
Aldehyde
3 [23,24] (0.29 g, 1 mmol), and NaH (130 mg,
5.4 mmol) were dissolved inTHF (3 mL), and the solutionwas cooled
to 0 ꢁC under N2. To this solution, phosphonate 5 [25] (1.46 g,
5.0 mmol) in THF (9 mL) was added dropwise. The reaction mixture
was stirred for 1 h at 0 ꢁC, and then for 12 h at room temperature,
followed by the removal of THF under reduced pressure. Water then
was added to the reaction mixture, and the product was extracted
with dichloromethane (4 ꢂ100 mL). The organic layer was dried
with dry Na2SO4 followed by evaporation of the solvent. The crude
product was separated by column chromatography with a gradient
Table 1
Photophysical properties of DCNs in various solvents.
b
b
d
e
Dye Solventa
labs
lOP
Stokes
Fc
lTP
max
Fdmax
max
max
OEt
CN
O
/nm
/nm
shift/nm
/nm
O
O
EtO
EtO P
P OEt
1b
1,4-dioxane 392
510
527
542
565
567
583
455
460
461
118
130
150
169
160
206
101
100
98
0.61 820
0.52 820
0.48 820
0.43 820
0.11 820
0.04 820
0.45 830
0.33 850
0.25 730
601
357
291
251
189
57
259
189
157
EtO
OEt
N
NC
O
CHCl3
THF
MeCN
DMSO
H2O
397
392
396
407
377
5
NaOH/MeOH
1a
1b
NaH/THF
3
O
1,4-dioxane 354
CHCl3
THF
360
363
OEt
O
2b
MeCN
DMSO
H2O
378
388
353
462
473
462
84
85
109
0.12 720
0.09 850
0.06 720
112
89
62
CN
O
EtO
O
EtO P
N
P OEt
O
EtO
OEt
OEt
O
O
NC
a
5
NaOH/MeOH
The numbers in the parenthesis are normalized empirical parameter of solvent
2b
2a
polarity.
NaH/THF
b
c
lmax of the absorption and emission spectra in nm.
4
Fluorescence quantum yields, ꢃ15%.
O
d
e
l
max of the two-photon excitation spectra in nm.
Fig. 2. Synthetic procedures of DCNs.
Two-photon action cross section (10ꢀ50 cm4s/photon GM), ꢃ15%.

332
J. Cui et al. / Dyes and Pigments 89 (2011) 330e335
Fig. 4. One-photon absorption (left), emission spectra (right) of 1b (10 m
mol Lꢀ1).
as a yellow powder. M.p. 184.3e185.4 ꢁC. 1H NMR (CDCl3, 400 MHz)
Fu
¼
FsFuAslexs
h
u=FsAulexuhus
d
: 7.97 (s, 2H), 7.20 (d, J ¼ 16.4 Hz, 2H), 7.17 (s, 2H), 7.16 (d, J ¼ 8.0 Hz,
where
F
is quantum yield; F is integrated area under the corrected
2H), 6.88 (d, J ¼ 16.0 Hz, 2H), 6.86 (d, J ¼ 8.4 Hz, 2H), 4.69 (s, 8H),
emission spectra; A is absorbance at the excitation wavelength; lex
is the excitation wavelength; is the refractive index of the solu-
tion; the subscripts u and s refer to the unknown and the standard,
4.31 (s, 4H), 4.29e4.17 (m, 12H), 1.29 (t, J ¼ 8.0 Hz, 18H). 13C NMR
h
(CDCl3, 100 MHz) d: 171.132, 168.732, 149.535, 141.166, 138.786,
134.468, 129.598, 122.674, 119.952, 119.335, 116.849, 114.794,
113.329, 77.517, 77.199, 76.881, 66.465, 61.523, 61.029, 53.974,
14.388. TOF MS (ES): m/z Calcd for [Mþ], C48H54N4O14, 933.3534,
Found: 933.3537.
respectively.
2.4. Spectroscopic measurements
Stock solutions of DCNs were prepared in DMSO and the OP
absorption and emission spectra (OPEF) were obtained in different
solvent. For absorption spectra of the BSA-responsive measure-
ments, sample solutions were obtained by mixing appropriate
amount of stock solution of DCNs (1 mmol Lꢀ1 in DMSO) and
phosphate buffer (20 mmol Lꢀ1) in water and finally diluted with
buffer to make the solution having desired concentrations of DCNs.
OPEF measurements were carried out similarly. TPEF spectra were
recorded on SD2000 spectrometer (Ocean Optical) with excitation
by using a femtosecond laser (Tsunami, Spectra-Physics) with 70-fs
pulse width and 80 MHz repetition rate.
2.2.3. Compound 1b and 2b
Solution of the ester 1a was saponified under basic conditions to
generate free 1b using a standard protocol [26]. In all cases,
a suspension of the ester (60 mg, 0.049 mmol) and NaOH (120 mg,
3 mmol) in methanol was refluxed overnight under nitrogen, at
which time TLC analysis showed complete consumption of starting
material and was kept into the freezer. The precipitate formed was
filtered and washed with cold methanol and hexane. Solids
obtained were completely soluble in water and was directly used to
prepare stock solutions in Millipore water for further spectroscopic
measurements.
1b. Yellow solid. Yield: 95 %. M.p. 235.5e236.6 ꢁC. TOF MS (ES):
m/z Calcd. for C32H26N4O8 594.1832, Found: 594.1834.
2b. Yellow solid. Yield: 92 %. M.p. 215.3e216.4 ꢁC. TOF MS (ES):
m/z Calcd. for C36H30N4O14 742.1759, Found: 742.1762.
2.5. Measurement of TPA cross section
The TPA cross sections of the DCNs were determined by the two-
photon-induced excited fluorescence method. And it is assumed
that the quantum efficiencies after two-photon excitation are the
sameasthose afterone-photon excitation. TheTPAcross sections are
2.3. Determination of quantum yield
obtained by calibration against fluorescein with known
d value in
The relative quantum yields of DCNs were determined by using
aqueous NaOH solution (pH ¼ 11) at concentrations of 1.0 ꢂ 10ꢀ4
mol$Lꢀ1 for the femtosecond measurement. The samples were dis-
solved in solvents at concentrations of 1.0 ꢂ 10ꢀ4 mol Lꢀ1. The error
of TPEF measurement is about 15%. To ensure that the measured
signals were solely due to TPA, the dependence of TPEF on the
incident intensity was verified in each case to be quadratic. The slope
is approximately two (Fig. 3). Therefore, this is a two-photon
absorption process.
Quinine bisulfate in 0.05 mol Lꢀ1 H2SO4 solution (
F
¼ 0.546) as the
standard and were calculated through the following equation [27]:
2.6. BSA-responsive properties of the DCNs in PBS buffer solutions
A series of PBS buffer solutions (20 mmol Lꢀ1, pH 7.0) containing
various amounts of BSA (0w20 m m
mol Lꢀ1) and 10 mol Lꢀ1 of DCNs
were prepared. The emission spectra of the dyes were recorded
with increasing concentration of the BSA.
2.7. Interferences to the fluorescence response of BSA
To explore further the utility of 1b as a fluorescence probe
Fig. 5. OPEF and TPEF spectra of 1b in water. OP Excitation: 380 nm, TP Excitation:
820 nm.
for BSA, the interference experiments were conducted including

J. Cui et al. / Dyes and Pigments 89 (2011) 330e335
333
Fig. 6. Two-photon action spectra of DCNs: a (1b), b (2b). TP Excitation: 720e890 nm.
various metal cations, anions and DNA (calf thymus) in PBS buffer
solutions.
the different organic solvents. For a certain compound, the spectral
profiles and peak positions of OPEF and TPEF are basically the same.
Although TPEF wavelength has a small blue shift relative to the
OPEF spectrum of dye 1b, the vibronic structures in the OPEF and
TPEF spectra are very similar in water (Fig. 5). This implies that
OPEF and TPEF come from the same fluorescent excited state. The
TPEF emission peak at 485 nm is weaker in comparison with the
profile of the OPEF spectrum. This is due to the higher concentra-
tion (1.0 ꢂ 10ꢀ4 mol Lꢀ1) of 1b used for TPEF determination, and the
2.8. Investigation of the pH dependency of 1b-BSA
The fluorescence responses of 1b-BSA were investigated in three
different pH buffer solutions.
3. Results and discussion
fluorescence quantum yield (F) of 1b in aqueous solution is so
small that the excitation efficiency of TPEF in water is much low.
3.1. Spectral properties
3.1.1. One-photon absorption and emission spectra of DCNs
The photo-physical properties of DCNs in different solvents are
summarized in Table 1. The OPEF spectra show large red-shift with
3.2. TPA cross section
The TPA cross sections dTPA were determined by using the two-
photon-induced fluorescence measurement technique. To avoid
possible complications due to the excited-state excitation, we have
used a mode-locked Ti/sapphire femtosecond (fs) laser pulses. The
pulse width and repetition rate of the laser are 70 fs and 80 MHz,
solvent polarity (P*) [28] in the order: 1,4-dioxane < CHCl3 < THF
< MeCN < DMSO < H2O. For example, the OPEF spectrum of 1b in
aqueous solution is red-shifted for 73 nm to the one in dioxane,
although the absorption has small blue-shifts (Fig. 4). It implies that
there are great changes in the molecular geometry of excited states
before fluorescence emission. On the other hand, the quantum yield
respectively. The values
the solvents from 1,4-dioxane toH2O. For example,
d
VTPA of DCNs decrease with the change in
VTPA of 1b
d
(
F
) of 1b in aqueous solution is much quenched (from 0.61 in
reduce gradually from 601 GM (in 1,4-dioxane) to 57 GM (in H2O)
(Fig. 6), which should be attributed to the excited state charge
transfer. More polar solvent H2O is favorable to balance positive and
negative charge arising from excited state, which will then be
dioxane to 0.04 in water). These photo-physical data indicate that
DCNs can take place an ICT phenomenon between the fluorophore
center and the amino group. Their high sensitivity to polarity
change of environments should benefit for DCNs as fluorescent
sensors.
efficiently deactivated, so the values of
dVTPA of the dyes in H2O are
comparatively small. Significant decreases in
dVTPA of other dye
systems with increased solvent polarity have been reported in
literatures [29]. For practical applications, TP cross section per
molecular weight (dmax/MW) is more valuable. The dmax/MW of 1b
is 2.39 in H2O solvent. Generally, dmax/MW > 1.0 have been found to
3.1.2. Two-photon excited fluorescence (TPEF) spectra of DCNs
The TPEF spectra of DCNs were obtained with excitation at
830 nm by using a mode-locked Ti/sapphire femtosecond laser in
Fig. 7. a. Ratio of fluorescence intensity (1b: F/F0 at 501 nm, 2b: F/F0 at 467 nm) of 10
dye 1b binding with BSA in PBS buffer solutions (pH 7.0) at room temperature. Excitation: 380 nm.
m m m
mol Lꢀ1 dye DCNs in the presence of BSA (2 mol Lꢀ1). b. Fluorescence spectra of 10 mol Lꢀ1

334
J. Cui et al. / Dyes and Pigments 89 (2011) 330e335
Fig. 8. a. Photographs of compound 1b solutions in the absence (left) and the presence (right) of BSA. b. plot of the fluorescence intensity at 501 nm as a function of BSA
concentration.
be good for such applications [30,31]. So, the intensity of the TPF of
1b is strong enough for practical applications, although TPA action
cross section is not very high.
under physiological conditions. This kind of interaction might let
some interference of the cations to the response to proteins.
Addition of various metal ions (50 equiv of Mn2þ, Ca2þ, Mg2þ, Zn2þ
,
Hg2þ, Cu2þ and Ni2þ) into 1b (10 mol Lꢀ1) solution, however, the
m
fluorescence intensity of 1b has little change (Fig. 9a). The results
imply that the responses of 1b to cations are not so strong. Anions
(50 equiv of NOꢀ3 , NOꢀ2 , HCOꢀ3 , CO23ꢀ, HPO24ꢀ, PO43ꢀ, SO24ꢀ, HSO3ꢀ,
SO23ꢀ, SCNꢀ, Clꢀ, Brꢀ, Iꢀ, and Fꢀ) and DNA (calf thymus) also show
nearly no changes in fluorescence intensity of 1b in PBS buffer (pH
7.0) solution (Fig. 9b). Generally speaking, the selectivity between
a protein and a DNA is a difficulty for a staining dye based on the
polarity of environment, as hydrophobic properties in both kinds of
bio-macromolecules are similar. The independence of 1b to DNA
might be from the nonspecific electrostatic repulsion between the
anionic carboxylated groups of 1b and the polyanionic phosphate
backbone of DNA. It is this kind of repulsion that would reduce
binding affinity for 1b and DNA.
3.3. Fluorescence off-on response of DCNs to BSA in aqueous
solutions
When BSA is added into the PBS buffer solution of 1b (pH 7.0),
great fluorescence enhancements are observed (Fig. 7b). As 1b has
a very weak emission in water (
effect of BSA is rather acute. Adding 20
enhancement in fluorescence (
¼ 0.36) is obtained. The fluores-
cence intensity is linearly increased with BSA concentration up to
20
mol Lꢀ1 (R2 > 0.995, Fig. 8b). And obvious emission blue-shifts
F
¼ 0.04) by itself, the switching-on
m
mol Lꢀ1 of BSA, c.a. 37-fold
F
m
(from 575 to 501 nm) are companied with the off-on process. This
significant fluorescence turn-on leads to visual observation of
fluorescence by naked eye from dark to bright green (Fig. 8a). The
fluorescence responses of 1b to BSA are very close to effects of the
polarity change of solvents from water to dioxane (Table 1).
Therefore, the 1b-BSA interaction should be resulted from a great
change in the environments of 1b from hydrophilic (polar) aqueous
solution to hydrophobic (apolar) cage of BSA. Under identical
conditions, no such kind of fluorescence response is observed in the
cases of 2b (Fig. 7a). Although 2b is soluble in water, compared with
1b, it has a branching molecular structure which hinders the
molecule get into the hydrophobic cage of BSA.
3.5. Investigation of the pH dependency of 1b-BSA
We also examined the pH dependency of the emissive properties
of 1b binding with BSA by addition of different pH buffer solution
(pH ¼ 4.00 Na2HPO4/citric acid, pH ¼ 7.00 PBS and pH ¼ 9.18 boric
acid). The addition of BSA showed some fluorescence enhancement
in Na2HPO4/citric acid buffer (pH 4.00), although much greater
fluorescence enhancements were achieved upon titrating the 1b
with BSAin PBS (pH 7.00) and boric acid (pH 9.18) buffers. In all cases,
the fluorescence enhancement increased as more BSA was added
(Fig. 10).
3.4. Interferences to the fluorescence response of BSA
Compound 1b, with multi-carboxyl groups, like a chelating
It is known that BSA undergoes reversible conformational isom-
erization with changes of pH [32]. At pH 4.00, BSA undergoes an
agent, might interact with multivalent metal cations (such as Ca2þ
)
Fig. 9. Ratio of fluorescence intensity (F/F0 at 501 nm) of 10
(b), in PBS buffer solutions (pH 7.0) at room temperature. Excitation: 380 nm.
m m m m )
mol Lꢀ1 dye 1b in the presence of different metal ions (50 mol Lꢀ1) (a), anion (50 mol Lꢀ1) and DNA (100 mol Lꢀ1

J. Cui et al. / Dyes and Pigments 89 (2011) 330e335
335
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4. Conclusion
Two water-soluble one and two-photon fluorescent dyes were
synthesized. Photo-physical properties of them were investigated
by the absorption, OPEF and TPEF spectra. These fluorescent dyes
own the larger TPA action cross section dV in organic solution.
When interacts non-covalently with BSA in aqueous solutions, new
dye 1b provides a dramatic linear-increase in the fluorescence
intensity. Cations, anions and DNA have no such responses. It is
resulted from the change of environmental polarity from aqueous
solution to hydrophobic cage in protein, and sequentially, the
change in the effect of excited intermolecular charge transfer.
[23] Wang J, Qian X. A series of polyamide receptor based PET fluorescent sensor
molecules: positively cooperative Hg2þ ion binding with high sensitivity.
Organic Letter 2006;8:3721e4.
Acknowledgements
[24] Wang J, Qian X, Cui J. Detecting Hg2þ ions with an ICT fluorescent sensor
molecule: remarkable emission spectra shift and unique selectivity. The
Journal of Organic Chemistry 2006;71:4308e11.
This work was supported by NSF of China (207025621 and
20706008), National Basic Research Program of China
(2009CB724706), Ministry of Education of China (Program for
Changjiang Scholars and Innovative Reserch Team in University,
IRT0711; and Cultivation Fund of the Key Scientific and Technical
Innovation Project, 707016). We are also grateful to Professor
Xuanming Duan and Professor Weiqiang Chen (Technical Institute
of Physics and Chemistry, Chinese Academy of Science) for their
help in two-photon fluorescence detection.
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et al. Five orders-of-magnitude enhancement of two-photon absorption for
dyes on silver nanoparticle fractal clusters. The Journal of Physical Chemistry
B 2002;106:6853e63.
ꢀ
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