A large stokes-shifted fluorescent dye synthesized as a new probe for the determination of protein

Article abstract of DOI:10.1007/s10895-016-1851-y

A novel fluorescent dye, 1-(2-hydroxyethyl)-4-((E)-2-(3-benzothiazol-2yl-9-ethyl-carbazole-3yl)vinyl) pyridinium bromide, was synthesized for determination of protein and its structure was characterized by ¹H NMR. Photophysics of the new probe in different solvents has been delineated in this paper, the new fluorescent molecular dye exhibited a large stokes-shifted and fluorescence quantum yields in organic solvent. The photostability and thermostability of the new dye were also studied and the results suggested the stable was excellent. The interactions of the dye with bovine serum albumin (BSA) , Human serumal bumin (HSA) and calf thymus deoxyribonucleic acid (ctDNA) were studied by fluorescence and absorption spectroscopy. The binding constant for BSA, HSA and DNA were calculated to be 8.91?×?10⁷, 1.86?×?10⁶ and 2.9?×?10⁴, respectively. The experimental results indicated a potential value of the new dye for biomarker.

Full text of DOI:10.1007/s10895-016-1851-y

J Fluoresc
DOI 10.1007/s10895-016-1851-y
ORIGINAL ARTICLE
A large stokes-shifted fluorescent dye synthesized as a new probe
for the determination of protein
Dayong Lin1 & Xuening Fei1,2 & Ran Li2 & Yingchun Gu2,3 & Yalin Tang3
Jianguo Zhou2 & Baolian Zhang2
&
Received: 1 March 2016 /Accepted: 30 May 2016
#
Springer Science+Business Media New York 2016
.
.
.
Abstract A novel fluorescent dye, 1-(2-hydroxyethyl)-
4-((E)-2-(3-benzothiazol-2yl-9-ethyl-carbazole-3yl)vinyl)
pyridinium bromide, was synthesized for determination of
Keywords Spectra properties Fluorescent probe Protein
Specific
1
protein and its structure was characterized by H NMR.
Photophysics of the new probe in different solvents has been
delineated in this paper, the new fluorescent molecular dye
exhibited a large stokes-shifted and fluorescence quantum
yields in organic solvent. The photostability and thermostabil-
ity of the new dye were also studied and the results suggested
the stable was excellent. The interactions of the dye with bo-
vine serum albumin (BSA) , Human serumal bumin (HSA)
and calf thymus deoxyribonucleic acid (ctDNA) were studied
by fluorescence and absorption spectroscopy. The binding
constant for BSA, HSA and DNA were calculated to be
8.91 × 107, 1.86 × 106 and 2.9 × 104, respectively. The exper-
imental results indicated a potential value of the new dye for
biomarker.
Introduction
Fluorescent molecular probe is widely used for labeling DNA
and protein [1, 2]. In recent years, lots of fluorescent molecu-
lar probes, such as coumarin [3], fluorescein [4], BODIPY [5],
naphthalimide [6], rhodamine [7] and Cyanines [8], were
studied by researchers. Cyanine dyes, which were previously
used as spectral sensitizer in photographic [9], are now of
interest as fluorescent molecular probe in labeling DNA and
protein [10, 11]. It is mainly attributed to the outstanding ad-
vantages of cyanine dyes, such as their relative stability, high
molar extinction coefficients, narrow spectrum width, high
sensitivity, and low aggregation [12, 13]. Cyanine dyes are
molecules containing polymethine bridge between two nitro-
gen atoms with a delocalized charge. With the increase of
methine chain, the electronic excitation energy of the dye will
be reduced. It is beneficial to the increase of absorption wave-
length, which can cause the reduction of the background in-
terference of the fluorescent probe. However, the stability of
the probe would become weaker with the lengthening of the
conjugated system. To overcome this problem and obtain a
kind of probe with not only longer emission wavelength but
also better stability is a popular research topic. Nowadays,
researchers introduced a rigid ring in the methine chain, such
as crown-ether ring [14], cyclohexene [15, 16] and squaric
acid [17].
* Xuening Fei
xueningfei@126.com
* Yingchun Gu
jvgugugu@126.com
* Yalin Tang
tangyl@iccas.ac.cn
1
School of Envirnmental Science and Engineering, Tianjin University,
92 Weijin Road, Nankai District, Tianjin 300072, China
Based on these ideas, our group synthesized a series of
novel styryl cyanine dyes with a carbazole bridged chain in
the methine chain based on thiazole orange [18–20]. The spec-
tral characteristic of these dyes were excellent. In this article,
we report our recent research in fluorescent molecular probe, a
2
School of Science, Tianjin Chengjian University, No.26 Jinjing
Road, Xiqing District, Tianjin 300384, China
3
Institute of Chemistry, Chinese Academy of Sciences (ICCAS),
Zhongguancun North First Street 2, Beijing 100190, China

J Fluoresc
novel styryl cyanine dye with a carbazole bridged. With the
introduction of carbazole, the stokes shift of the molecular
probe was larger than the traditional probe, the photostability
and thermostability of the molecular probe was brilliant. It can
be used as an excellent fluorescent probe in biological
labeling.
for the sample and reference respectively, and Ax and AS are
the absorbance for the sample and reference at the excitation
wavelength respectively. The standard used in this study is
Fluorescein in NaOH (0.1 mol/L) aqueous solution with a
fluorescence quantum yield of 0.95 [24]. The synthesis of
the title compound is shown in Scheme 1.
Synthesis
Experimental
General
Synthesis of 3-benzothiazol-6- formyl −9-ethylcarbazole (3)
The synthesis procedure of compound 3 was based on the Ref
1
[18]. Yield: 35 %. H NMR (400 MHz, CDCl3) δ (ppm):
In this article, all materials and solvents were purchased and
used without further purification. The UV/Vis absorption
spectra were scanned on a Perkin Elmer LAMBDA 35 spec-
trometer. Fluorescence spectra were recorded on a Hitachi
F-7000 spectrometer. The excitation and emission slits were
5 nm and the PTM voltage of the spectrometer were 700 V. 1H
NMR spectra were obtained on a Bruker (400 MHz) spec-
trometer. Chemical shifts were reported in parts per million
(ppm) down field from TMS (tetramethylsilane) using
DMSO-d6 or CDCl3 as a solvent.
1.43–1.46 (t, J = 7.40 Hz, 3H), 4.35–4.40 (m, 2H), 7.30–
7.34 (t, J = 7.20 Hz, 1H), 7.42–7.48 (m, 3H), 7.86 (d,
J = 8.00 Hz, 1H), 7.99–8.04(t, J = 9.20 Hz, 2H), 8.22(d,
J = 8.40 Hz, 1H), 8.64 (s, 1H), 8.82 (s, 1H), 10.06 (s, 1H).
Synthesis of 1-(2-hydroxyethyl)-4-methylpyridinium bromide
(4)
4-Methylpyridine (2 g, 21 mmol) and 2-bromoethanol (2.6 g,
21 mmol) were dissolved in toluene. The mixture was stirred
overnight under a refluxing condition. After cooling down to
room temperature, the solvent were removed to get the desired
compound 4. Yield: 80 %. 1H NMR (400 MHz, DMSO-d6) δ
(ppm):8.94 (d, J = 6.4 Hz, 2H), 7.98 (d, J = 6.4 Hz, 2H), 5.55
(br, 1H), 4.64 (t, J = 4.8 Hz, 2H), 3.81 (t, J = 4.8 Hz, 2H), 2.60
(s, 3H).
Preparation of stock solutions
For absorption or fluorescence measurements, DMSO were
used for the solvent to obtain stock solutions (10 mL), then
the stock solutions were diluted to the desired concentration
(5 × 10−6 mol/L).
BSAwere dissolved directly into PBS buffer (pH = 7.4) to get
the stock solutions (5 × 10−4 mol/L) and then various amounts of
stock solutions were used for dye–protein complexes. Finally, the
dye was diluted to the desired concentration.
Calf thymus DNA were purchased from Sigma Chemical
Co. and dissolved in 10 mM Tris–HCl buffer (pH = 7.4). The
purity of DNA was checked by scanning the absorption spec-
Synthesis of the novel probe (5)
A solution mixture of 3 (0.1 g, 0.28 mmol), 4 (0.05 g,
0.23 mmol) and piperidine (2 drops) in ethanol (30 mL) was
heated to reflux overnight. After cooling down to room tem-
perature, the solvent was removed by rotary evaporation. The
crude product was purified by silica gel column chromatogra-
phy. Yield: 35 %.1H NMR (400 MHz, DMSO-d6) δ (ppm):
9.00 (s, 1H), 8.91 (s, 1H), 8.85 (d, J = 3.4 Hz, 2H), 8.21–8.28
(m, J = 14 Hz, 4H), 8.17 (d, J = 3.8 Hz, 1H), 8.07 (s,
J = 4.2 Hz, 1H), 7.93 (d, J = 4.0 Hz, 1H), 7.85 (q,
J = 12 Hz, 2H), 7.67 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 8.0 Hz,
1H), 7.45–7.49 (m, J = 8.0 Hz, 1H), 5.28 (t, J = 5.2 Hz, 1H),
4.53–4.60 (m, J = 14 Hz, 4H), 3.88 (q, J = 7.2 Hz, 2H), 1.38(t,
J = 7.2 Hz, 3H).
tra and the ratio of the absorbance at 260 and 280 nm, A260
/
A280 = 1.83. The result could meet the purity condition of
DNA according to Ref [21]. The DNA concentration in
Tris–HCl buffer were determined by the molar extinction co-
efficient ε = 6600 M−1 cm−1 at 260 nm [2, 22].
Fluorescence quantum yields
The relative fluorescence quantum yields of the compound
were determined by comparison with a standard solution with
the following eq. [23]:
Results and discussion
ꢀ
ꢁ
2
nX
nS
AS FX
⋅ ⋅
ϕX ¼ ϕS⋅
The spectral properties of compound 5 in different solvent
AX FS
In which nX and nS are the refractive indexes of the sample
and reference, FX and FS are the area of fluorescence spectra
Solvent effects on the absorption and fluorescence spectra of
compound 5 were measured in different solvents. The spectra

J Fluoresc
Scheme 1 Synthesis of the new
probe
were shown in Fig. 1 the spectra data and parameters of sol-
vents were summarized in Tables 1 and 2.
Therefore, we propose that these photophysical properties varia-
tions are due to an intramolecular chargetransfer (ICT) process
[26]. The largest λem and the strongest intensity were all obtained
in DMSO. However, the intensity was the lowest in water solu-
tion. The possible reason was that the bond between the carba-
zole and pyridinium was rotated and water molecules made the
twist angle increase. It destroys the coplanarity of the molecule,
thereby resulting in the increase of the nonradiative decay pro-
cess and finally the obviously quenching occurred. We further
explored the relationship between dipole moment of the solvents
and λem, the result was shown in Fig. 3
From Fig. 3 we can see that the λem increased with the
increasing of the dipole moment. The possible reason is that
the solvent with larger dipole moment could result in the re-
duction of energy difference between excited state and ground
state, which as a result lead to a red-shift of compound 5 [27].
Table 2 indicated that Stokes shift were large in all cases, even
up to 140 nm in CH3CN., and changed in different solvents. The
stokes shift were mainly influenced by two factors, refractive
Figure 1 (a) indicated that the maximum absorption wave-
length (λmax) of compound 5 in different solvents was around
430 nm. In CH2Cl2, λmax was the biggest and located at
455 nm. In aprotic polar solvents, the λmax of compound 5
was red-shifted with the increasing of solvent polarity. In ad-
dition, we can see that λmax of the dye has the same trend with
the refractive index of the solvent in aprotic polar solvents.
The relationship between f (n) and ν was further shown in
Fig. 2. We can see that ν was decreased with the increase of
f (n) in different solvents, which is in accordance with the
Bayliss equation [25]. From the fig. we can also find that the
absorbing intensity was stronger for the solvent with higher
polarity, which is possible due to the energy of the anti-
bonding orbital and bonding orbital of the molecular are both
reduced for the solvent with higher polarity
Figure 1 (b) showed that compound 5 has different maximum
emission wavelength (λem) and intensity in different solvents.
a
b
DMSO
CH3OH
C2H5OH
DMF
0.16
Ethyl acetate
DMSO
5000
DCM
C2H5OH
DMF
CH3CN
4000
0.12
CH3OH
DCM
CH3CN
3000
2000
1000
0
Ethyl acetate
0.08
H2O
H2O
0.04
0.00
450
500
550
600
650
700
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)
Fig. 1 Absorption (a) and Emission (b) spectra of compound 5

J Fluoresc
Ethyl acetate
2.38
2.36
2.34
2.32
2.30
2.28
Table 1 The solvent properties and UV–vis characters of compound 5
Solvent
nD20 a ε d
f(n) b
λ
max/nm ν c/10−3 cm−1
CH3OH
H2O
1.329
1.340
1.344
1.361
32.7
0.169 434
0.173 440
0.175 430
0.181 438
2.304
2.273
2.326
2.283
2.375
2.208
2.320
2.315
80.1
37.5
24.3
DMSO
CH3CN
C2H5OH
DMF
Ethyl acetate 1.372
6.02 0.185 421
CH2Cl2
DMF
1.424
1.430
1.479
8.9
38.3
47.2
0.203 453
0.205 431
0.221 432
0.18
0.19
0.20
f (n)
0.21
0.22
DMSO
Fig. 2 Relationship between f (n) and ν
a Refractive index
b f(n ) = ( n 2 –1)/(2n 2 + 1)
c ν = 1/ λmax
d Dielectric constant
Photostability of compound 5
The good photostability is an important requirement for the
application as biomedical fluorescent labels. In this study, we
used iodine-tungsten lamp (500 W) as the illuminant. The
NaNO2 aqueous solution (50 g/L) was used as the cold trap,
which can decrease the effect on the stability caused by the
strong heat from illumination. On the other hand, the solution
could also filtrate most of the UV-lights, whose wavelength is
shorter than 400 nm. The photostability experiment was per-
formed in DMSO at room temperature without natural light
[29]. The absorption intensity of the compound was measured
after exposing to light with different time. The curve between
the intensity at λmax and the exposure time was shown in Fig. 5.
Figure 5a showed that the change is no prominent after
different exposure time. After irradiation of 7 h, the dye
showed only 2.6 % photofading. The result indicated that
the dye can be exposed to the strong light for a long time
and still keep its intensity, which can make it as an excellent
probe for application in many fields.
index and dielectric constant. The effect of solvents on fluores-
cent spectra could be stated by Lippert equation [28].
ꢀ
ꢁ
2
ε‐1
n2‐1
μ*‐μ
a3
Δν ¼ νem‐νabs
ε‐1
¼
‐
þ constant
hc 2ε‐1 2n2 þ 1
n2‐1
2ε‐1 2n2 þ 1
Δf ¼
‐
Where νabs and νem are wavelengths of the excitation and
emission respectively, so νem -νabs is stokes shift. The h is
Planck constant and c is light speed. The a is the cavity radium
that encapsulate the molecule. a is a constant when there is no
hydrogen bond between the molecule and solvent. μ and μ*
are the dipole moment of ground state and excited state. The
Δf is called orientation polarizability.
The relationship between stokes shift and Δf of compound 5
in three different solvents were shown in Fig. 4. We can see that
the stokes shift is approximately proportional to Δf, which is
consistent with the Lippert equation. But for other solvents, there
is a deviation between the relationship of Δν and Δf.
In addition, the rate constant of photoreaction could be
calculated based on the photooxidation mechanism and
counld be expressed as [30]:
−d½DyeŠ=dt ¼ k1 ½Dye Š ½O2 Š
ð1Þ
Table 2 The solvent properties and fluorescent characters of
compound 5
580
575
570
Solvent
μ a
Δ f
λ
em (nm)
Stokes shift
Φ b
CH3OH
H2O
1.70
1.82
3.92
1.69
1.88
1.20
3.82
3.96
0.323
0.324
0.318
0.308
0.270
0.267
0.288
0.274
563
575
571
562
540
553
566
568
129
135
141
124
119
100
135
136
0.24
0.02
0.25
0.44
0.55
0.75
0.39
0.47
DMSO
DMF
CH3OH
565
CH3CN
C2H5OH
Ethyl acetate
CH2Cl2
DMF
560
555
550
545
540
535
C2H5OH
CH2Cl2
DMSO
1.0
1.5
2.0
2.5
3.0
3.5
4.0
a Dipole moment
Dipole moment
b Fluorescence quantum yield
Fig. 3 The influence of dipole moment on the λem of compound 5

J Fluoresc
Thermostability of compound 5
140
120
100
H2O
We also measured the thermostability of the probe at 80 °C in
water bath. The concentration of the dye was 5 × 10−6 mol/L
in DMSO. The absorption intensities of the compound after
different time were measured to explore the thermostability.
The results were shown in Fig. 6.
The results indicated that the absorption intensity reduced
little after heating 6 h, so we can get that the thermostability of
the dye solution is excellent.
CH3OH
C2H5OH
CH2Cl2
0.27 0.28
0.26
0.29
0.30
0.31
0.32
0.33
f
Fig. 4 The relationship between stokes shift and orientation
polarizability (Δ f) of compound 5 expressed by the Lippert equation
Spectral properties of compound 5 in complex with ctDNA
We studied the interaction between the probe (5 × 10−6 mol/L)
and different concentrate ctDNA (0 μM, 1 μM , 4 μM, 8 μM,
12 μM,16 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM,
40 μM) in 10 mM Tris–HCl buffer (pH = 7.4) and the results
were shown in Fig. 7.
Figure 7 shows that the intensity of the compound was
weak without the addition of ctDNA. Interestingly, the inten-
sity increased noticeably with the addition of ctDNA. One
reason for this may be due to the strong binding formed be-
tween the cationic N-pyridinium unit and the anionic phos-
phate of DNA. The binding can hinder the rotations around
various bonds and thereby decreases the possible nonradiative
process. When the concentration of ctDNA is stable, the
fluoresence intensity of ctDNA is increased with the com-
pound 5 increase and the emission wavelength was changed
little.
in which [O2] is a constant of about 3 × 10–4 mol/ L, so the
following equation can be obtained:
−d½DyeŠ0=dt ¼ k ½DyeŠ
ð2Þ
ð3Þ
ln½DyeŠ0=½DyeŠ ¼ kt
t
In the equation, [Dye]0 and [Dye]t were the original con-
centration of dye and the concentration after being illuminated
for t mins, respectively. Based on Lambert–Beer’s law, there
was a linear relation between concentration and the maximum
absorption under low concentration. So eq. (3) could be
predigested as below:
lnð A0=AtÞ ¼ kt
ð4Þ
In the equation, A0 is the maximum absorbance of dye before
irradiation, At is the maximum absorbance after being illuminat-
ed and k is the constant of the photodegradation reaction. The
result was shown in Fig. 5b. The photoreduction rate constant
could then be obtained from the slope, the result is
5.64 × 10−5 mol/Min, which is much little than those of cyanines
[31].
In addition, we defined the degree of signal enhancement
as (F-F0)/F0, where F0 and F are the fluorescent intensities at
570 nm in the absence and presence of DNA respectively, and
the there is a linear ship between the fluorescence intensity
were shown in Fig. 8 and also a linear ship between (F-F0)/ F0
and the concentration of ctDNA (not shown). The fluorescent
intensity increased remarkably with the addition of DNA, so
a
b
0.40
0.030
0.025
0.020
0.015
0.010
0.005
0.000
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0
200
400
-1
0
1
2
3
4
5
6
7
8
Time (min)
Time (h)
Fig. 5 The curve of (a) photostability and (b) lightfastness of the dye

J Fluoresc
0.4
0.2
0.0
Spectral properties of compound 5 labeled with protein
Spectral properties of compound 5 labeled with BSA
To study the spectral properties of compound 5 labeled with
BSA, we measured the absorption and fluorescence spectra of
the compound 5 (5 × 10−6 mol/L) with the addition of different
concentration of BSA (0 μM, 1 μM, 4 μM, 8 μM, 12 μM,
16 μM, 20 μM, 24 μM, 32 μM, 36 μM, 40 μM) in phosphate
buffer (pH = 7.4) at room temperature and the result was
shown in Fig. 9.
0
1
2
3
4
5
6
7
Time (h)
From Fig. 9, we can see that the absorbance and fluorescent
intensity of compound 5 increased with the addition of BSA,
accompanied by a blue-shift of the maximum absorbance and
emission wavelength. There are two possible factors effect on
increasing of intensity. Firstly, with the addition of BSA, the
compound was included into a hydrophobic cavity with lower
polarity inside BSA molecule. The other reason is that the
binding between BSA and the compound inhibited the rota-
tional freedom of compound 5 and caused higher intensity. All
these effects together caused the enhancement of the com-
pound’s absorbance and fluorescent intensities. The blue shift
of the maximum wavelength can be explained by the reduc-
tion of ground state energy of the compound when there is
interaction between the dye and BSA. These results jointly
confirmed that the compound can interact with BSA protein.
In order to further study the binding interaction of dye with
BSA,The quenching experiment was also carried out
preliminarily.
Fig. 6 The curve of thermostability
the bonding association constant could be agree with the
Benesi-Hildebrand equation [32]:
1
1
1
1
¼
þ
Â
Fx‐F0
F
max‐F0
F
max‐F0 K½DNAŠ
Where Fx and F0 represent the fluorescent intensity of the
probe in the presence and absence of DNA, respectively. Fmax
is the maximum fluorescent intensity of the probe in the pres-
ence of DNA. [DNA] is the concentration of DNA added. The
equation could be simplified as:
F
max‐F0
1
¼ 1 þ
Fx‐F0
K½DNAŠ
From the Fig. 10, we can see that there is a strong endog-
enous. With the addition of the dye, the fluorescent intensity
of BSA decreased sharply, indicating an interaction between
the dye and BSA [33].
The fluorescent quenching effect is due to the molecular
interactions. Both the main of static and dynamic quenching
Plots of (Fmax − F0)/ (Fx − F0) against [DNA] for the probe
showed remarkable linear dependence (Fig.10), justifying the
validity of the used equation and hence confirming the one-to-
one association between DNA and the probe. The binding
constant (Ka) were determined from the slope of the corre-
sponding plots, which came as 2.9*104 M−1.
a
b
[ctDNA]/µM
2000
1500
1000
500
0
40
300
0
1
4
8
12
16
20
24
28
32
36
40
[dye]*106
12
M
0
200
100
0
2
4
6
8
10
12
0
0
300
320
340
360
380
400
420
500
600
700
Wavelength (nm)
Wavelength (nm)
Fig. 7 The fluorescence spectra of (a) compound 5 with different concentration of ctDNA (0 μM, 1 μM , 4 μM, 8 μM, 12 μM,16 μM, 20 μM, 24 μM, 28 μM,
32 μM, 36 μM, 40 μM) and (b) ctDNA with different concentration of the compound 5 (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 12 μM)

J Fluoresc
a
b
50
40
30
20
10
0
2000
1500
1000
500
0
0
10
20
30
40
0.0
0.2
0.4
0.6
0.8
1.0
[DNA]*106 M
1/[DNA] 10-6 M-1
Fig. 8 a Linear range between fluorescence intensity and the concentration of ctDNA (0 μM, 1 μM , 4 μM, 8 μM, 12 μM,16 μM, 20 μM, 24 μM,
28 μM, 32 μM, 36 μM, 40 μM) , b The linearity of (Fmax − F0)/ (Fx − F0) against [DNA]
require molecular contact between the fluorophore and
quencher. If the quenching effect between dye and BSA was
known to be dynamic quenching, it could be described by the
Stern-Volmer equation: [34]
BSA. Therefore, quenching data are presented as plot of [lg
(F0-F)/ F] versus lg [Q]. The liner fitting was carried out using
origin 8.0. The Ka was obtained to be 8.91 × 107 from the
intercept and n was obtained to be about 2 from the slope of
1.68(Fig. 10b). So the complex of the dye and BSA is stable
and one mole of BSA is binded to 2 mol of the dye, which
showed that the BSA can be labeled with the dye in the paper.
F0=F ¼ 1 þ kd ½QŠ
In this equation F0 and F are the fluorescent intensities in
the absence and presence of quencher, respectively, Kd is the
quenching constant and [Q] is the concentration of the
quencher. Therefore, quenching data are presented as plot of
F0/F versus [Q]. But, in our experiment, there was not a linear
relationship between F0/F and [Q] (inserted in Fig.10a). So, it
is not a dynamic quenching.
Spectral properties of compound 5 labeled with HSA
In order to determine its labeling of protein, its spectral prop-
erties of the compound with HAS was also studied. The ab-
sorption and fluorescence spectra were shown in Fig.11.
From the Fig.11, it can be seen that the Absorption and
fluorescence intensities increased with the HAS addition.
And the wavelength shifted blue a little, which is the same
to that of BSA. The reasons are the same to those of BSA.
These results confirmed that the compound can interact with
HSA protein.
If it is static quenching, it could be described by the
Scatchard equation: [35]
lgðF0−F Þ=F ¼ lgKa þ nlg½QŠ
In this equation, Ka is the association constant between the
dye and BSA, n is binding stoichiometry between dye and
a
b
[BSA]/µM
4000
3000
2000
1000
0
0
1
4
8
12
16
20
24
32
36
40
[BSA]/µM
40
0.10
40
0
0.05
0
0.00
500
600
700
400
500
Wavelength (nm)
600
Wavelength (a.u.)
Fig. 9 Spectral properties of compound 5 labeled with BSA (0 μM, 1 μM, 4 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, 32 μM, 36 μM, 40 μM)

J Fluoresc
a
b
[dye]*106 M
0
1400
1200
1000
800
600
400
200
0
-0.4
-0.8
-1.2
-1.6
1.5
1.4
1.3
1.2
1.1
1.0
0
2
4
6
8
10
12
y=1.68x+7.95
[dye]*106
M
12
-5.6
-5.4
-5.2
-5.0
-4.8
300
350
400
450
Wavelength (nm)
log[Q]
Fig. 10 a Fluorescence spectra of BSA (5 × 10−6 mol/L) with the addition of the dye ( 0, 2, 4, 6, 8, 10 and 12 × 10−6 mol/L). b The plot of [lg (F0-F)/ F]
versus lg [Q]
In order to further study the binding interaction of dye with
HSA,The quenching experiment was also carried out prelim-
inarily to study the labeling reaction between the dye and HAS
(Fig. 12).
the quenching data are presented as plot of F0/F versus [Q].
But, in our experiment, there was not a linear relationship
between F0/F and [Q](inserted in Fig.12a). So, it is also not
a dynamic quenching.
From the Fig. 12, we can see that there is a strong
endogenous. With the addition of the dye, the fluores-
cent intensity of HSA decreased sharply with the fluo-
rescent wavelength shifted red a little, which indicated
that there is an interaction occurred between the dye
and HSA.
The fluorescent quenching effect is due to the molecular
interactions. If the quenching effect between dye and HSA
was known to be dynamic quenching, it could also be de-
scribed by the Stern-Volmer equation mentioned above and
If it is static quenching, it could be described by the
Scatchard equation mention above. Therefore, quenching data
are presented as plot of [lg (F0-F)/ F] versus lg [Q]. The Ka
was obtained to be 1.86 × 106 from the intercept and n was
obtained to be about 1 from the slope of 1.32(Fig. 12b). So the
complex of the dye and HSA is stable and one mole of HSA is
bound to 1 mol of the dye, which showed that the HSA can be
labeled with the dye in the paper.
It is indicated that the stability constant between the com-
pound and BSA or HSAwas much larger than that between it
a
b
[HSA]/µM
0.15
40
6000
4000
2000
0
[HSA]*106 M
0.10
40
0.05
0
0
0.00
500
600
700
400
500
600
Wavelength (nm)
Wavelength (nm)
Fig. 11 a Absorption and b Fluorescence spectra of the dye with different concentration of HSA (0 μM, 1 μM, 4 μM, 8 μM, 12 μM, 16 μM, 20 μM,
24 μM, 32 μM, 36 μM, 40 μM)

J Fluoresc
a
b
0.0
-0.3
-0.6
-0.9
-1.2
1500
[dye]*106 M
0
1.4
1.2
1.0
1200
900
600
300
y=1.32x+6.27
0
2
4
6
8
10
12
[Q]
12
0
300
350
400
450
500
-5.6
-5.4
-5.2
-5.0
-4.8
Wavelength (nm)
log[Q]
Fig. 12 a Fluorescence spectra of HSA (5 × 10−6 mol/L) with the addition of the dye (form 0 to 7: 0, 2, 4, 6, 8, 10 and 12 × 10−6 mol/L). b The plot of [lg
(F0-F)/ F] versus lg [Q]
51578360) and Technology Development Foundation Plan Project of
Tianjin Colleges (20130505).
and ctDNA. In addition, the labeling experiment showed that
the fluorescence intensity of the dye labelled with HSA and
BSA apparently higher than that labeled with ctDNA at the
same concentration. So the novel fluorescent dye could inter-
act with protein more stable than ctDNA. Above all, the title
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