1-(Carbamoylmethyl)-3H-indolium squaraine dyes: Synthesis, spectra, photo-stability and association with BSA

Article abstract of DOI:10.1016/j.dyepig.2009.10.002

Novel, squarylium dyes containing 1-(alkylcarbamoylmethyl)-2,3,3-trimethyl- 3H-indolium groups were synthesized and their UV/Vis and fluorescence spectra, aggregation, photo-stability and association with bovine serum albumin were studied. The absorption and emission max wavelengths of the dyes in different solvents were in the range 619-653?nm. Compared to a typical ethyl squarylium dye, the introduction of alkylcarbamoylmethyl groups reduced aggregation and improved molar extinction coefficient, fluorescence quantum yield and photo-stability in water. Moreover, the fluorescence intensity of the dyes increased upon the addition of BSA in pH 7.0 phosphate buffer solution. An excellent linear relationship (r²?=?0.9982) was obtained between fluorescence intensity and bovine serum albumin concentration.

Full text of DOI:10.1016/j.dyepig.2009.10.002

Dyes and Pigments 85 (2010) 43–50
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
1-(Carbamoylmethyl)-3H-indolium squaraine dyes: Synthesis, spectra,
photo-stability and association with BSA
*
Bingshuai Wang, Jiangli Fan, Shiguo Sun, Li Wang, Bo Song, Xiaojun Peng
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:
Novel, squarylium dyes containing 1-(alkylcarbamoylmethyl)-2,3,3-trimethyl- 3H-indolium groups were
synthesized and their UV/Vis and fluorescence spectra, aggregation, photo-stability and association with
bovine serum albumin were studied. The absorption and emission max wavelengths of the dyes in
different solvents were in the range 619–653 nm. Compared to a typical ethyl squarylium dye, the
introduction of alkylcarbamoylmethyl groups reduced aggregation and improved molar extinction
coefficient, fluorescence quantum yield and photo-stability in water. Moreover, the fluorescence intensity
of the dyes increased upon the addition of BSA in pH 7.0 phosphate buffer solution. An excellent linear
relationship (r2 ¼ 0.9982) was obtained between fluorescence intensity and bovine serum albumin
concentration.
Received 6 July 2009
Received in revised form
1 October 2009
Accepted 2 October 2009
Available online 28 October 2009
Keywords:
Squarylium dye
Aggregation
Ó 2009 Elsevier Ltd. All rights reserved.
Photo-stability
Fluorescence
Bovine serum albumin
Linear relationship
1. Introduction
Fig. 1) improved photo-stability markedly [13]. In contrast, the
water-solubility of fluorescent probes is a significant issue in bio-
Squaraine dyes possess good photoconductivity [1], high
extinction coefficient [2], and intense fluorescence [3]. These
features make them useful in a variety of applications such as
photoconductive materials [4], organic solar cells [5] and optical
discs [6]. The dyes also enjoy use as long-wavelength fluorescent
probes and labels in biological assay, since their absorption and
fluorescence spectra lie in the visible red and near-infrared regions
(NIR), which are outside the self-absorption and self-luminescence
regions of biological objects [7,8].
Squarylium dyes derived from 2,3,3-trimethyl-3H-indolium and
its derivatives are important as some of these dyes (such asIandIIin
Fig. 1) have been used as non-covalent protein probes of high
fluorescence intensity [7,9,10]; however, the dyes display poor
photo-stability and low water-solubility. To improve the photo-
stability of squarylium dyes, much effort has been devoted to
molecular structure adjustment of the benzene ring of the indoli-
nium salt. The introduction of an electron-withdrawing groups such
as chloro and nitro affords considerable protection against fading
[11,12]. Recently, the present authors found that electron-with-
drawing groups on the N-benzyl group of III (Y ¼ CO2H or NO2 in
logical applications; whilst the introduction of charged groups such
as sulfonate (III, IV in Fig. 1) into the dyes can increase water-solu-
bility, that accompanying ionic charge can lower the dye's binding
potency [14]. Although some progresses had been achieved through
the introduction of nonionic groups into polymethine cyanine dyes
[15,16], the improvement in squaraine dyes has been limited.
This paper concerns novel 2,3,3-trimethyl-3H-indolium squar-
aine dyes that contain alkylcarbamoylmethyl groups (Fig. 2) and
the effects which the moderate nonionic hydrophilicity and high
electron-withdrawing ability of such groups have, upon the water-
solubility and photo-stability of squarylium dyes.
2. Experiments
2.1. Materials and general methods
Deionized water was redistilled before use. Other chemicals
used for the experiments were of analytical grade. Mass spectral
determinations were made on HP1100 API-ES mass spectrometry.
1H NMR spectra were recorded on a Varian 400 MHz NMR spec-
trometer. Fluorescence measurements were performed on a PTI-C-
700 Felix and Time-Master system. Visible spectra were measured
on an HP-8453 spectrophotometer.
* Corresponding author. Tel.: þ86 0411 39893899; fax: þ86 0411 39893800.
E-mail address: pengxj@dlut.edu.cn (X. Peng).
0143-7208/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2009.10.002

44
B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
Fig. 1. Squarylium dyes from 2,3,3-trimethyl-3H-indolium.Ă
2.2. Determination of quantum yield
concentrations relative to 1
aggregated molecules.
mM can indicate the relative amount of
The relative fluorescence quantum yields were determined by
using Rhodamine B (
¼ 0.56 in ethanol) as standard [17] and were
calculated through the following equation [18]:
f
2.4. Photo-stability
The photo-stability tests were carried out in quartz cells (10 mm
in width) where sample solutions were irradiated with a 500 W
Iodine-tungsten (I/W) lamp at room temperature. The distance
between the cells and the lamp was 350 mm. The 5 mM solutions of
the dyes in CH3CN were radiated and the irreversible bleaching of
the dyes at the absorption peak was monitored as a function of time.
Fx
¼
FsðFx=FsÞðAx=AsÞð
l
exs=
l
exxÞðnx=nsÞ2
where
F
¼ quantum yield; F ¼ integrated area under the corrected
emission spectrum (in Ep units); A ¼ absorbance at the excitation
wavelength;
lex ¼ the excitation wavelength; n ¼ the refractive
index of the solution (because of the low concentrations of the
solutions (10ꢀ7–10ꢀ8 mol/L), the refractive indices of the solutions
are replaced with those of the solvents.); and the subscripts x and s
refer to the unknown and the standard, respectively.
2.5. Redox potential
The redox potential was measured on BAS 100B elec-
torchemical analyser. A three- electrode cell was composed of
a glass carbon as working electrode, a platinum wire as counter
electrode, and Ag/Agþas reference electrode (0.01 M AgNO3). The
supporting electrolyte was TBAPF6 (0.1 M tetra n-butylammonium
hexafluorophosphate) and the concentrations of the dyes were
10ꢀ3 M in CH3CN.
2.3. Aggregation
The absorption spectra display no obvious change when the
concentrations of the dye solutions are lower than 1
when the concentration is higher than 40 M, the dyes tend to
deposit from the solutions. So 1–40 M were chosen as the testing
mM. Otherwise,
m
m
concentration ranges for all dyes except dye 3b. The absorption
density of dye 3b solution would exceed the maximum limit (<3) of
2.6. Dye–BSA association
the absorption instrument in high concentrations (>20
mM), so 3b
The association of 2 mM dye (3a, 3b, III) and 100 mM BSA was
was kept in 1–20 M. The formation of aggregation leads to
m
conducted in order to ascertain the association time. The fluores-
a change in the wavelength maxima and shape of the absorption
band which can be used to recognize the occurrence of aggregation.
The absorption intensity is proportional to the concentrations of
the dyes in low concentration, but the aggregation will make the
absorption density of the monomer decrease, so the spectra are
normalized (each spectrum data divided by its maximum absorp-
tion value) in order to facilitate direct comparisons on the shapes of
the spectra. The loss of monomer absorption density in higher
cence-time curves were presented in Fig. 3 and which shows the
Fig. 3. The time-dependence curves of dye 3a (black line), 3b (red line) and III (blue line)
Fig. 2. Structures of 2,3,3-trimethyl-3H-indolium squaraine dyes.Ă
associated with 100 mM BSA.

B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
45
fluorescence intensities of the dyes reach the balance after 20 min,
so the association time of 20 min was chosen.
quaternized salt (chloride) 2c. Reaction time: 48 h; Yield: 41%; Red
powder.
The concentrations of the dyes were kept constant at 2
m
M. After
1-(methyl acetate-2-yl)-2, 3, 3-trimethyl-3H-indolium quater-
nizedsalt(bromide)2d. Reactiontime:24h;Yield:56%;Fawnpowder.
BSAwas added for 20 min, the fluorescence spectra of the dyes were
recorded with increasing the BSA concentration until the fluores-
cence intensity of dye–BSA become saturated in pH ¼ 7.0 phosphate
buffer solution (PBS). Dye–BSA associations in different pH buffer
solutions were also studied in order to compare the effect of pH.
2.7.3. Synthesis of dyes 3a–3e
2.7.3.1. Dye 3b. Squaric acid (114 mg, 1 mmol) was heated under
reflux in a mixture of toluene (10 mL) and n-butanol (10 mL). Upon
dissolution of the squaric acid, a pyridine (5 mL) solution of 2b
(680 mg, 2.2 mmol) was added and, after refluxing for 3 h, the
reaction mixture was cooled to room temperature and the solvents
were removed by rotary evaporation. The residue was treated with
diethyl ether (100 mL) and the precipitate was filtered. Further
purification was achieved using column chromatography emplying
ethyl acetate-acetone mixture as eluent. When the solvents were
removed, a blue solid powder was obtained which was recrystal-
lized from acetone, providing bright green crystals (248 mg)
2.7. Synthesis
The synthetic routes of the dyes are shown in Fig. 4. Indolium
quaternized salts 2a and 2e were synthesized according to the
literature procedure [19]. To serve as a reference, the well-known
squarylium dye 3a (R ¼ CH2CH3) was also synthesized. Yield: 75%.
Dye 3a: 1H NMR(400 MHz, Acetone-d6, ppm):
d
1.36 (t, 6H, J ¼ 7.2 Hz,
CH3CH2),1.74 (s,12H, C(CH3)2), 4.18 (q, 4H, CH3CH2), 5.88 (s, 2H, CH),
7.15 (t, 2H, J ¼ 7.6 Hz, Ar-H), 7.25 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.34 (t, 2H,
J ¼ 8.0 Hz, Ar-H), 7.47 (d, 2H, J ¼ 7.6 Hz, Ar-H). ESI-MS, m/z: 453.3
[M þ H]þ.
collected in 40% yield. 1H NMR (400 MHz, CDCl3, ppm):
d1.14 (t, 6H,
J ¼ 6.8 Hz, CH3CH2), 1.38 (t, 6H, J ¼ 6.8 Hz, CH3CH2), 1.739 (s, 12H, C
(CH3)2), 3.42 (q, 4H, CH3CH2), 3.51 (q, 4H, CH3CH2), 5.09 (s, 4H, CH2–
N), 5.75 (s, 2H, C–CH]C), 6.79 (d, 2H, J ¼ 7.6 Hz, Ar-H), 7.13 (t, 2H,
J ¼ 7.6 Hz, Ar-H), 7.26 (t, 2H, J ¼ 7.6 Hz, Ar-H), 7.33 (d, 2H, J ¼ 7.6 Hz,
Ar-H). ESI-MS, m/z: 623.3 [M þ H]þ.
2.7.1. Synthesis of intermediates 1b and 1c
A chloroform solution (10 mL) of 2-chloroacetyl chloride (4.52 g,
40 mmol) was added dropwise to a chloroform (10 mL) solution of
appropriate alkyl amine (40 mmol) with stirring at 0–10 ꢁC, then the
solution was heated to reflux for 2 h, the solvent was removed by
a rotary evaporator to get the chloromethylamides as yellow liquids.
2.7.3.2. Dye 3b1. Squaric acid (114 mg, 1 mmol) was heated under
reflux in a mixture of toluene (10 mL) and n-butanol (10 mL). Upon
dissolution of the squaric acid, a pyridine (5 mL) solution of inter-
mediate 2e (301 mg, 1 mmol) was added. After refluxing for 0.5 h,
a further pyridine (5 mL) solution of intermediate 2b (371 mg,
1.2 mmol) was added. After refluxing for 3 h, the reaction mixture
was cooled to room temperature and the solvent removed by rotary
evaporation. Theresiduewas treated withdiethyl ether (100 mL)and
the resulting precipitate was filtered. Further purification was ach-
ievedusingcolumnchromatographyemplyingethylacetate-acetone
mixture as eluent. Thesolventswere removed anda luesolid powder
(225 mg) was obtained in 43% yield. 1H NMR (400 MHz, Acetone-d6,
2.7.2. General procedure for the synthesis of indolium
quaternized salts 2b, 2c and 2d
2,3,3-Trimethyl-3H-indolenine (3.20 g, 20 mmol) and appro-
priate halide (40 mmol) were mixed in toluene (15 mL) under
nitrogen in 50 mL flask and refluxed for 24–48 h until the solid
appeared around the flask. The ensuing mixture was cooled to
room temperature and added to diethyl ether (100 mL); the
precipitate was filtered and washed with ethyl acetate (20 mL ꢂ 3).
The corresponding compound was used in the next reaction
without further purification. The following indolium quaternized
salts were prepared:
ppm):
d
1.07 (t, 6H, J ¼ 6.8 Hz, CH3CH2),1.39 (t, 6H, J ¼ 6.8 Hz, CH3CH2),
1.72 (s, 6H, C(CH3)2), 1.75 (s, 6H, C(CH3)2), 3.38 (q, 2H, CH3CH2), 3.63
(s, 3H, CH3–N), 3.64 (q, 2H, CH3CH2), 5.12 (s, 2H, CH2–N), 5.70 (s, 1H,
C–CH]C), 5.84 (s,1H, C–CH]C), 7.06 (d,1H, J ¼ 7.6 Hz, Ar-H), 7.13 (m,
2H, Ar-H), 7.25 (m, 2H, Ar-H), 7.33 (t,1H, J ¼ 7.6 Hz, Ar-H), 7.44 (d, 2H,
J ¼ 7.2 Hz, Ar-H). ESI-MS, m/z: 524.3 [M þ H]þ.
1-(N, N-diethylacetamide-2-yl) ꢀ2, 3, 3-trimethyl-3H-indolium
quaternized salt (chloride) 2b. Reaction time: 48 h; Yield: 52%; Red
powder.1-(N-propylacetamide-2-yl)-2, 3, 3-trimethyl-3H-indolium
Fig. 4. Synthesis of the squarylium dyes.Ă

46
B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
Table 1
2.7.3.3. Dye 3c. The syntheses and purification of 3c were the same
as that of 3b. However, analyzing the MS and 1H NMR of the
compound, we found the crystal was not our target compound 3c
but 3c1. The reason was discussed in the part 3.
Spectral data of the dyes in different solvents.
dye solvent Absorption/ Stokes
3
( ꢂ 105 molꢀ1 cmꢀ1 L) fa
Emission (nm) Shift (nm)
Yield: 25 %. 1H NMR (400 MHz, CDCl3, ppm):
d0.84 ((t, 3H,
3a Chloroform 633/643
10
15
10
9
2.17
2.42
2.28
2.43
0.5
0.19
0.12
0.23
0.072
0.005
Acetone
DMSO
Methanol
Water
632/647
642/652
627/636
621/632
J ¼ 7.6 Hz, CH3CH2CH2), 1.47 (m, 2H, CH3CH2CH2), 1.69 (s, 6H, C
(CH3)2), 1.77 (s, 6H, C(CH3)2), 3.21 (q, 2H, CH3CH2CH2), 3.49 (s, 2H,
CH2–N), 3.64 (s, 3H, CH3–N), 5.94 (s, 2H, C–CH]C), 7.06 (d, 2H,
J ¼ 6.8 Hz, Ar-H), 7.15 (t, 1H, J ¼ 7.6 Hz, Ar-H), 7.20 (t, 1H, J ¼ 7.6 Hz,
Ar-H), 7.27–7.39 (m, 4H, Ar-H). ESI-MS, m/z: 510.3 [M þ H]þ.
11
3b Chloroform 633/643
10
12
10
9
1.79
2.06
1.99
2.27
1.57
0.16
0.16
0.28
0.16
0.022
Acetone
DMSO
Methanol
Water
633/645
643/653
627/636
622/633
2.7.3.4. Dye 3d. Syntheses and purification of 3d were the same as
that of 3b and blue-purple crystal (254 mg) was collected. However,
analyzing the MS and 1H NMR of the compound, we found the
crystal was not our target compound 3d but 3e. The reason was
discussed in the part 3. The yield of dye 3e was 60%. 1H NMR
11
3b1 Chloroform 633/642
9
9
11
9
2.25
2.53
2.34
2.42
1.20
0.11
0.12
0.17
0.076
0.013
Acetone
DMSO
Methanol
Water
632/641
642/653
626/635
620/631
(400 MHz, CDCl3, ppm): d1.78 (s, 12H, C(CH3)2), 3.59 (s, 6H, CH3–N),
5.93 (s, 2H, C–CH]C), 7.03 (d, 2H, J ¼ 7.6 Hz, Ar-H), 7.15 (t, 2H,
11
J ¼ 7.6 Hz, Ar-H), 7.35 (m, 4H, Ar-H). ESI-MS, m/z: 425.2 [M þ H]þ.
3c1 Chloroform 637/647
10
10
15
10
12
2.53
2.85
2.70
2.78
0.96
0.20
0.13
0.25
0.078
0.011
Acetone
DMSO
Methanol
Water
632/642
638/653
626/636
619/631
3. Results and discussion
3.1. Synthesis
a
:
lex: 600 nm, error ca. ꢃ10%.
The novel dyes were synthesized by condensation of squaric
acid and two equivalents of 3H-indolium salts in n-butanol-
toluene-pyridine mixed solvent. In the cases of 2a (N-ethyl-2,3,3-
trimethyl-3H-indolium) and 2b (N-(N'N'-diethylcarbamoyl)methyl-
2,3,3-trimethyl-3H-indolium), the reactions provided moderate
yields of the respective dyes (3a, 3b). However, for the reactions of
2c (N-(N'-propylcarbamoyl) methyl-2,3,3-trimethyl-3H-indolium)
and 2d (N-ethoxycarbonyl)methyl-2,3,3-trimethyl-3H-indolium),
the expected products (3c and 3d) were not obtained. The sepa-
rated products were unsymmetrical 3c1 and symmetrical 3e,
respectively, where N-carbamoylmethyl or N-ethoxycabonylmethyl
groups were changed to N-methyl substituent.
The reason might be that the methylene linking nitrogen cation
(Nþ) and carbonyl group are so reactive, just like the cases in
literatures [20,21], that in basic and heating conditions, N-carba-
moylmethyl (2c) or N-ethoxycabonylmethyl (2d) groups on 2,3,3-
trimethyl-3H-indolium are easy to be converted into N-methyl
indolium(Fig. 5).
however, the spectra of the dyes are much different. In order to
facilitate comparisons of the spectra in water and acetone, the
spectra in acetone are normalized relative to corresponding spectra
in water. As shown in Fig. 6, the absorption spectrum of 3a becomes
broad with two round peaks at 550 nm and 675 nm and the spectra
have several nanometers blue-shift comparing to those obtained in
organic solvents. The peaks are characteristic of assembly of
cyanine dyes into non-covalent dimers and higher aggregations
[22,23]. In the case of dye 3b, the absorption spectrum in water has
no obvious round peak except 10–20 nm blue-shift. The shapes of
the spectra are similar with those obtained in organic solvents. The
aggregation of 3a leads to the fluorescence intensity of only one
eighth of that of 3b (Fig. 6) and
e value 30% of that of 3b in water
(Table 1).
3.3. Aggregation
The electron-withdrawing abilities of the carbonyl groups of 2b,
2c and 2d are in an order of 2d > 2c > 2b, which correlates with the
yields of dyes: 3d < 3c1 < 3b. In order to confirm the mechanism,
2d (2 mmol) was treated 2 h in the same conditions as those of
synthesizing dyes, where a colorless transparent crystal with green
fluorescence was obtained. The crystal was confirmed to be the
N-methyl-2,3,3-trimethyl-3H-indolium (2e) by mass spectrometry
and 1H NMR spectroscopy.
The aggregation of cyanine dyes is extremely favored by strong
attractive dispersion forces derived from the high polarizability of
3.2. Spectral properties
The spectra data of the dyes in different solvents are listed in
Table 1. In same organic solvents, the spectral shapes of the dyes are
almost the same. Typical absorption and emission spectra of the
dyes in organic solvents are illustrated in Fig. 6. In aqueous solution,
Fig. 6. Comparisons on the spectra of 5 mM dye 3a and 3b in water and acetone. 3a in
water (black lines), 3b in water (red lines), 3a in acetone (blue lines, normalized), 3b in
acetone (green lines, normalized), inset: the spectra of 3a and 3b in acetone before
being normalized.
Fig. 5. Decomposition of N-carbonylmethyl-2,3,3-trimethyl-3H-indolium.Ă

B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
47
Fig. 7. Normalized absorption spectra of dye 3a, 3b, 3b1 and 3c1 in different concentrations in water. a–f: 1 (black line), 5 (red line), 10 (blue line), 20 (dark cyan line), 30 (magenta
line) and 40 M (dark yellow line), inset: absorption spectra before being normalized.
m
the chromophoric chain in aqueous solution. Moreover, the high
dielectric constant of water facilitates the aggregation process [24–
30]. In this paper, the aggregates were detected through recording
the absorption spectra of increasing concentration of the dyes in
water in order to facilitate the formation of aggregates. Normalized
absorption spectra of dye 3a, 3b, 3b1 and 3c1 are shown in Fig. 7.
When the concentration of 3a in water is increased, the absorption
band becomes broad at the expense of a rise of relative intensity of
the short-wavelength shoulder at 580 nm and of the long-wave-
length part of the spectrum (675 nm), which reveals the formation
of aggregates [23]. In the same manner, dye 3b1 and 3c1 form lower
aggregations from their normalized absorption spectra. Neverthe-
less, the normalized absorption spectra of dye 3b overlap
completely which implies that few aggregations have formed.
Fig. 9. The redox spectra of dye 3a (black line), 3b (red line), 3b1 (blue line) and 3c1
(dark cyan line) with scan rate of 100 mV/s and the concentrations of the dyes were
10ꢀ3 M in CH3CN.
Fig. 8. Comparisons on the photo-stability of the dyes in CH3CN.Ă

48
B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
In the other hand, the loss of monomer absorption intensity for
M dyes resulted from the aggregations relative to 1 M dyes was
aggregations of the dyes more remarkably than the mono-
alkylcarbamoylmethyl one.
20
m
m
51.9%, 12.5%, 33.5% and 45.5% for 3a, 3b, 3b1 and 3c1, respectively.
All the differences suggest that the introduction of the carba-
moylmethyl group can inhibit the aggregation of the squarylium
dyes in water which is beneficial to their application in aqueous
media, and the dialkylcarbamoylmethyl group can decrease the
3.4. Photostability
The results of photo-stability of the dyes are shown in Fig. 8.
After irradiation by I/W lamp for 7 h, the absorption intensities of
Fig. 10. Fluorescence spectra of 2
mM dye 3a, 3b, 3b1, 3c1 and III associated with BSA in pH ¼ 7.0 PBS at room temperature.Ă

B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
49
Fig. 11. left: Sigmoidal fit of the fluorescence intensity of 2
right: The best linear relationship range for each dye.
mM dye–BSA at 645 nm (dye III at 650 nm) and BSA concentrations in pH ¼ 7.0 PBS, inset text: the errors of linear fit;
3a, 3b, 3b1 and 3c1 decrease to 86%, 95%, 92% and 94% from 100%
respectively. The photo-stabilities of the novel dyes with N-carba-
moylmethyl group are better than that of the well-known dye 3a.
The decomposition pathway of cyanine dyes had been depicted
in the literature. Singlet oxygen was an important oxidation species
which could be produced in situ by irradiation of dyes [31–33] and
bleach cyanine dyes through destroying the conjugation of the
dyes. On the other hand, it was reported that quenching of singlet
oxygen by cyanine dyes decreased as the oxidation potential of the
dye increased [34].
The fluorescence of the dyes at 645 nm (dye III at 650 nm) is
plotted as a function of BSA concentration (Fig. 11 left). Better linear
relationship (r2 ¼ 0.9976) between the fluorescence intensity of dye
3b and the BSA concentration (0–2 mM) can be obtained than those
of the other dyes. The aggregation of dyes and nonlinear and
sigmoidal calibration curves are two of the disadvantages for the
reagents used for the fluorescence spectrometry and for the other
analytical methods during the protein measurement [35]. The
introduction of N-carbamoylmethyl group into squarylium dyes
can provide a way to resolve these problems.
As seen in Fig. 9, the redox spectra of dye 3a, 3b, 3b1 and 3c1
were examined. The first oxidation potentials of 3b, 3b1 and 3c1 are
more positive than that of 3a which is likely due to the electron-
withdrawing N-carbamoylmethyl substituent. This suggests that
these new dyes should be less susceptible to reaction with singlet
oxygen, i.e. the oxidation of the new dyes by singlet oxygen will not
be easy to carry out. The other reason for this might be that the
carbamoylmethyl groups are larger and sterically hinder attack by
both singlet oxygen and other oxidation species which contribute
to the photo-fading of the dyes.
The better linear range for each dye was studied and the data are
presented as following: 0.072–0.2
mM for 3a, 0.2–3
mM for 3b,
0.024–0.4 M for 3b1, 0.096–1
m
mM for 3c1 and 0.2–1 for III,
respectively (Fig. 11 right). The sensitivity of dye 3b is lower than
other dyes because of the lower fluorescence enhancement.
However, the linear range is the largest (0–3
is very sensitive in low concentrations of BSA, while the linear
range is very small (0.072–0.2 M). The fluorescence backgrounds
mM). Contrarily, dye 3a
m
of these dyes are very low except dye III which is the most soluble in
water. Fig. 11 shows the relationships on larger scale. It illustrates
that less water soluble dye (3a) has high fluorescent response with
small linear range in lower BSA concentrations, while higher water-
solubility dyes (3b and III) have lower fluorescent response with
wider linear range in higher concentration of BSA.
3.5. Dye–BSA association
It was suggested that these squarylium dyes occupy a common
hydrophobic binding site on protein to form dye–protein complex
[10], which may enhance the fluorescence of the dyes and be used
for the determination of proteins. The fluorescence spectra of dye
3a, 3b, 3b1, 3c1 and III (Y ¼ COOH) associated with different
concentration of BSA are shown in Fig. 10. The fluorescence inten-
sities of the dyes increased by adding BSA in pH 7.0 PBS as expected.
Three different pH buffer solution (pH ¼ 5 Na2HPO4/citric acid,
pH ¼ 7 PBS and pH ¼ 9 borax) were used to study the effect of pH
on the dye–BSA association. Fig. 12 indicates the pH have different
effects. In the cases of dye 3a and 3b, the pH affects mostly the state
of BSA. At pH 5, BSA may be protonated so that the dye–BSA
associations of dye 3a and 3b are weakened and the fluorescence
intensity become lower. At pH 7.0 and pH 9.0, the interactions are
Fig. 12. Plot of the fluorescence of 2
mM dye–BSA at 645 nm (dye III at 650 nm) in pH ¼ 5, 7, 9 buffer solutions as a function of BSA concentrations (0–2
mM).Ă

50
B. Wang et al. / Dyes and Pigments 85 (2010) 43–50
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much similar and show high fluorescent enhancement. In the case
of dye III (Y ¼ CO2H), the 3 pH have the different effects on dye–BSA
interaction. At pH 9, the deprotonation of the dye carboxyl group
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In summary, wehavesynthesized newsquaraine dyes containing
N- alkylcarbamoylmethyl-2,3,3-trimethyl-3H-indolium and tested
their properties such as fluorescence, aggregation, photo-stability
and fluorescent response to BSA. The results showed that the
introduction of alkylcarbamoylmethyl group decrease the aggre-
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yields of the dyes in water. Among the dyes, 3b with two 1-(N',
N'-diethylcarbamoyl)methyl- 2,3,3-trimethyl-3H-indolium groups
has good fluorescence quantum yield, high photo-stability and
excellent linear relationship between fluorescent intensity and BSA
concentration. The pH of thebuffer solution fortestinghas dissimilar
effect to the different dye–BSA associations. These positive results
arising from the introduction of alkylcarbamoylmethyl group would
be worthwhile to design new squaraine dyes with powerful func-
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This work was supported by NSF of China (20706008, 20725621
and 20872012), National Basic Research Program of China
(2009CB724706), Ministry of Education of China (Program for
Changjiang Scholars and Innovative Research Team in University,
IRT0711 and Cultivation Fund of the Key Scientific and Technical
Innovation Project, 707016).
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