J Solution Chem

DOI 10.1007/s10953-012-9912-6

Study of the Interaction between a Water-soluble

Cationic Fluorescent Conjugated Polymer and Bovine
Serum Albumin

Wei Li • Tiantian Liu • Zhilong Li • Yun Wang

Received: 19 January 2011 / Accepted: 29 January 2012

Ó Springer Science+Business Media New York 2012

Abstract The interaction between a water-soluble cationic fluorescent conjugated

polymer (WCFP) and bovine serum albumin (BSA) was studied using UV–Vis absorption,
fluorescence and circular dichroism spectroscopies. The results show that the fluorescence
of BSA is strongly quenched by the WCFP under physiological conditions (pH = 7.4).
The quenching mechanism was found to be static, which was confirmed by the quenching
rate constant (Kq) and UV–Vis absorption spectra. The thermodynamic parameters (DHh,
DSh and DGh) calculated from the complexation constant, determined according to
Lineweaver–Burk equations are 38.6 kJmol-1, 228 Jmol-1K-1 and -29.4 kJmol-1 at
298 K. The principal interaction was proposed to be electrostatic.

Keywords Water-soluble cationic fluorescent conjugated polymer (WCFP) 

Bovine serum albumin (BSA)  Fluorescence spectrum  Interaction

1 Introduction

Water-soluble cationic fluorescent conjugated polymers (WCFP) are used as a novel

fluorescent probe due to their high molar extinction coefficient and fluorescence quantum
yield. As one polythiophene derivative, whose thiophene molecules possess a five-mem-
bered heterocyclic ring and comply with Hückel’s rule, WCFP shows high performance as
a p-conjugated optical electrical material with a moderate energy gap, wide spectral
response, and high environmental and thermal stability [1, 2]. Due to its good water
solubility, WCFP has attracted widespread interest in biology and medicine and has
already been used in micro molecule determination, pH sensing, bio-macromolecule
determination, cell imaging, etc. [3–5]. Ho et al. [6] investigated the highly sensitive and
distinctive detection of iodine compounds with WCFP and Shang et al. [7] established a

W. Li  T. Liu  Y. Wang (&)

College of Science, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
e-mail: yunwang123@126.com

Z. Li
College of Life Science, Tarim University, Ala’er 843300, People’s Republic of China

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new method, involving rapid detection of cysteine with a fluorescent conjugated polymer
modified gold nanoprobe, with a detection limit of 25 nmolL-1, for cysteine.
Bovine serum albumin (BSA), a single polypeptide chain protein, consists of 582 amino
acid residues, among which are 19 tyrosine residues and 2 tryptophan residues [8]. In this
study, we selected BSA as the model protein due to its low cost, stability, unusual ligand-
binding properties and structural homology with human serum albumin (HSA) [9–11].
Many studies have been conducted on the interactions between proteins and drugs.
However, few reports are found about the interaction between protein and WCFP, and up
to now, the effect of water-soluble WCFP on proteins remains unclear.
In this paper, the interaction between WCFP and BSA was investigated by UV–Vis
absorption and fluorescence spectroscopy. The mechanism of the reaction was further
verified by CD spectroscopy. After that, the modified Stern–volmer equation and the
Lineweaver–Burk equation were used to analyze the data of fluorescence spectra. Finally,
the thermodynamic parameters of the binding reaction were calculated and the interaction
mechanism is discussed. The findings will contribute considerably to establishing a WCFP-
based bio-chemistry sensor.

2 Experimental

2.1 Apparatus

The fluorescence spectra were recorded using a LS-55 luminescence spectrometer (Perkin–
Elmer, USA) and the UV–Vis absorption spectra were acquired on a Nicolet Evolution 300
UV–Visible spectrophotometer (THERMO, USA). The temperature was adjusted using a
CS501 Super constant temperature water bath (Shanghai Sunshine Experimental Instru-
ment Co., Ltd.). CD spectra were obtained using a J-810 Circular Dichroism Chiroptical
Spectrometer (Jasco, Japan), with a wavelength range from 190 to 250 nm, and a cell path-
length of 0.1 cm.

2.2 Reagents

The WCFP used in this experiment was a polythiophene derivative and was prepared
according to the literature [6]. Shown in Fig. 1 is the molecular structure of WCFP with a
monomer molecular weight of 285.57 gmol-1and the concentration of WCFP was cal-
culated according to the monomer molecular weight. BSA (M = 68,000 gmol-1) was
purchased from Shanghai Bio Life Science & Technology Co., Ltd. All other chemicals
used were of analytical-grade. Ultrapure water (resistivity[18 MXcm) was prepared from
an Aquapro Ultrapure Water Systems (Ever Young Enterprises Development Co., Ltd.,
Chongqing, PR China).

Fig. 1 Molecule structure of

WFCP

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2.3 Procedure

2.00 mL of Tris–HCl buffer solution (pH = 7.4), 1.00 mL of the BSA solution and an
appropriate volume of WCFP solution were transferred into a 10.00 mL colorimetric tube.
The mixtures were diluted to the mark with ultrapure water and then were shaken. The
solution was kept static for at least 15 min before measuring the spectrum. The fluores-
cence spectra were obtained at an excitation and emission wavelength of 280 and
342.5 nm, respectively, and with an excitation and emission slit width of 10 nm. The
UV–Vis absorption spectra were obtained in the range of 200–600 nm, with a 1.0 9
1.0 cm-quartz cuvette on a Thermo Nicolet Corporation Model Evolution 300 (America).

3 Results and Discussion

3.1 Fluorescent Quenching Spectra of BSA with WCFP

BSA possesses a strong intrinsic fluorescence due to its tryptophan residues [12, 13],
tyrosine residues, phenylalanine residue, etc. As shown in Fig. 2, the fluorescent intensity
is reduced in the presence of WCFP. Moreover, with increasing WCFP concentration the
fluorescent intensity of BSA decreases, while the wavelength of the maximum emission of
BSA did not show an obvious change, nor did the fluorescence peak shape when the
concentration of WCFP is less than 5 9 10-6 molL-1.
Fluorescent quenching mechanisms can be divided into static and dynamic quenching
[14]. Dynamic quenching is caused by the collision between the quencher molecules and the
excited state of fluorescence molecules, while static quenching results from non-luminous
ground-state complexes formed between the fluorescent and the quencher molecules [15].
Dynamic and static quenching can be distinguished by the quenching rate constant.
To confirm the quenching mechanism, it was assumed to be dynamic and the fluores-
cence data were analyzed by the modified Stern–Volmer equation [16, 17]:


F0 =ðF0 ¼ F Þ ¼ 1=fa þ 1=ðKSV ½QÞ ¼ 1=fa þ 1= Kq s0 ½Q ð1Þ

where F0 and F are the fluorescence intensities of BSA in the absence and presence of
WCFP, respectively, [Q] is the quencher concentration, KSV is the dynamic quenching

Fig. 2 Fluorescence spectra of

BSA in the presence of WFCP
with different concentrations
(0.1 molL-1, pH = 7.4 Tris–
HCl buffer). The BSA
concentration was fixed at
2.0 9 10-7 molL-1. From a to
h: WCFP (910-6 molL-1): 0;
1.3; 1.6; 2.0; 2.5; 3.2; 4.0; 5.0

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constant and Kq is the dynamic quenching rate constant. KSV was calculated to be
1.48 9 105 Lmol-1.
s0 is the average lifetime of BSA without WCFP. Provided that the typical fluorescence
lifetime of the bio-macromolecule is 1.0 9 10-8 s [18], the dynamic quenching rate
constant Kq is calculated to be 1.48 9 1013 Lmol-1s-1.
However, according to previous literature [19], the dynamic fluorescence quenching rate
constant between a bio-macromolecule and a fluorescence quencher is usually below
2.0 9 1010 Lmol-1s-1, while the fluorescence quenching rate constant between BSA and
WCFP is calculated to be 1.48 9 1013 Lmol-1s-1 which is far larger than 2.0 9
1010 Lmol-1s-1. This result indicates that the fluorescence quenching was not initiated by
dynamic collision but by the formation of a complex resulting in static quenching [20].
UV–Vis absorption spectroscopy was used to investigate the complex formation by the
method described in previous literature [21, 22]. The absorption spectra of BSA with and
without WCFP were recorded and are presented in Fig. 3, in which the UV absorption peak
can be seen to be much larger than those of WCFP and BSA, and even the sum of both of
them. The results indicated that WCFP and BSA form a complex. The static quenching
mechanism was further confirmed by CD spectroscopy.
CD spectroscopy was used to investigate the conformation and conformational changes
of proteins and polypeptides in aqueous solution as described in previous literature [23].
The CD spectra (Fig. 4) of native BSA are characterized by two main negative bands at
208 and 222 nm [24]. Shown in Table 1 are fractional contents of the secondary structure
of BSA (fa, fb, fturn and frandom) with and without WCFP. Compared with the native BSA,
the a-helical content of BSA–WCFP decreased from 28.1 to 17.8 %, while the content of
random coil decreased from 22.3 to 18.8 %. The reduction in a-helix structures confirmed
the partial deformation of the a-helical structure.

3.2 Thermodynamic Parameters for Fluorescent Quenching and the Binding Mode

The experimental data of static quenching were fitted to the Lineweaver–Burk equation [16]:
1=ðF0  FÞ ¼ 1=F0 þ 1=ðKLB F0 ½QÞ ð2Þ
where KLB is the static quenching constant, describing the dose–effect relationship in the
static quenching when the bio-macromolecules and fluorescence quencher molecules have
achieved a balanced combination.

Fig. 3 The UV–Vis absorption

spectra of (a) BSA–WCFP,
(b) the sum of WFCP and BSA,
(c) BSA and (d) WCFP. The
concentrations of WCFP and
BSA are 1.0 9 10-5 and
1.0 9 10-6 molL-1,
respectively

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Fig. 4 CD spectra of BSA in the

absence (a) and presence (b) of
WCFP. The concentrations of
BSA and WCFP are 2.0 9 10-6
and 2.5 9 10-5 molL-1,
respectively

Table 1 Fractional contents of the secondary structure of BSA (fa, fb, fturn and frandom) with and without
WCFP
Systems Fractions of secondary structure (%)

fa fb fturn frandom

BSA 28.1 46.5 3.1 22.3

BSA–WCFP 17.8 55.9 7.6 18.8

As shown in Fig. 5, a good linear relationship is found between 1/(F0 - F) and 1/[Q],
and the slopes of curves indicate the static fluorescent quenching binding constant KLB at
different temperatures. The standard state enthalpy change (DHh) of the binding reaction
can be held as a constant if the temperature does not vary significantly and DHh, DGh and
DSh can then be calculated according to the following equations:
InK2h =K1h ¼½1=T1  1=T2 DH h =R ð3Þ

DGh ¼ RT lnK ð4Þ

h h h
DG ¼ DH  TDS ð5Þ
where R is the gas constant, T is the temperature (K) and K is the equilibrium constant.
Table 2 lists the thermodynamic parameters for the interaction between WCFP and BSA,
where DHh [ 0 indicates that the interaction between WCFP and BSA is endothermic, and
DGh \ 0 indicates that the standard state reaction is spontaneous. DSh [ 0 indicates the
increase of entropy caused by the WCFP and BSA binding. The results are in agreement
with the analysis conducted above, suggesting that the fluorescent quenching procedure is
static.

3.3 Discussion of the Interaction Mechanism

The interaction forces between quencher and bio-macromolecule can be hydrophobic,

electrostatic interactions, van der Waals interactions, hydrogen bond and so on [25, 26].
The isoelectric point of BSA is 4.6 [27], and thus in the pH = 7.4 Tris–HCl buffer, BSA

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Fig. 5 Curves from

Lineweaver–Burk equation at
different temperatures

Table 2 Binding constants (KLB) and thermodynamic parameters for the interaction between BSA and
WCFP
T (K) KLB (9105 Lmol-1) DHh (kJmol-1) DSh (Jmol-1K-1) DGh (kJmol-1)

283 0.62 38.6 228 -26.0

298 1.52 -29.4
308 2.31 -31.7

and WCFP are oppositely charged, indicating that the electrostatic force may play a key
role in the interactions.
According to a previous study [28], WCFP can be used in a rapidly and highly sensitive
detection of DNA due to its large numbers of positive charges, and the interaction is
mainly electrostatic between DNA and WCFP. To investigate the interaction between
WCFP and BSA, the effect of ionic concentration in the reaction system was studied. As
shown in Fig. 6, fixing the concentration of WCFP and BSA and increasing the concen-
tration of NaCl in the reaction system causes the relative fluorescence intensity ratio
(F0/F) to decrease steadily.
The results showed that the ionic strength has a strong influence on the interaction
between WCFP and BSA, and the electrostatic force plays a major role in this interaction,
which is in line with a previous finding [28] that the interaction between WCFP and BSA is
mainly electrostatic.

Fig. 6 Effect of NaCl

concentration on the relative
fluorescence intensity (F0/F).
The concentrations of WCFP and
BSA are 2.0 9 10-6 and
2.0 9 10-7 molL-1,
respectively

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4 Conclusions

The interaction between WCFP and BSA was investigated with UV–Vis absorption
spectra, fluorescence spectra and CD spectra. WCFP was used as a novel fluorescence
probe in the bio-macromolecule determination. The quenching rate constant indicates that
the probable quenching mechanism is a static quenching procedure. Therefore, electro-
static interactions can be assumed to be the main driving force for the interaction between
WCFP and BSA, which provides a foundation for the application of WCFP as a probe in
the bio-system research.

Acknowledgments We gratefully acknowledge the financial support for the project from the Fundamental
Research Funds for the Central Universities (No.2009JC007) and Hubei Province Natural Science Foun-
dation (No. 2008CDB040).

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