Photophysics and applications in biosensor for 4′-N,N-dimethylamino-2-trans-styryl-(6-chloroquinoline)

Article abstract of DOI:10.1016/j.cplett.2007.06.122

A push-pull compound with intramolecular charge transfer (ICT) characteristic, 6-Cl-2-StQ-NMe₂, was synthesized in this work. The emission maxima of 6-Cl-2-StQ-NMe₂ correlates excellently with the static polarity of the environment. In an aqueous solution, 6-Cl-2-StQ-NMe₂ was observed to form an 'H type' aggregate in the ground state, but deaggregation occurred in the excited state. We find that 6-Cl-2-StQ-NMe₂ can be used as a biosensor. Quantitative analysis showed that a linear relationship exists when bovine serum albumin (BSA) concentration is between 40 μg/ml and 5000 μg/ml. While monitoring the conformational change of BSA, the denaturation reaction was observed when the temperature exceeded 60 °C.

Full text of DOI:10.1016/j.cplett.2007.06.122

Chemical Physics Letters 444 (2007) 71–75
www.elsevier.com/locate/cplett
Photophysics and applications in biosensor for
40-N,N-dimethylamino-2-trans-styryl-(6-chloroquinoline)
*
Shun-Li Wang , Ju-Mei Lin
Department of Applied Chemistry, National ChiaYi University, ChiaYi 600, Taiwan, ROC
Received 22 May 2007; in final form 26 June 2007
Available online 30 June 2007
Abstract
A push–pull compound with intramolecular charge transfer (ICT) characteristic, 6-Cl-2-StQ-NMe2, was synthesized in this work. The
emission maxima of 6-Cl-2-StQ-NMe2 correlates excellently with the static polarity of the environment. In an aqueous solution, 6-Cl-2-
StQ-NMe2 was observed to form an ‘H type’ aggregate in the ground state, but deaggregation occurred in the excited state. We find that
6-Cl-2-StQ-NMe2 can be used as a biosensor. Quantitative analysis showed that a linear relationship exists when bovine serum albumin
(BSA) concentration is between 40 lg/ml and 5000 lg/ml. While monitoring the conformational change of BSA, the denaturation reac-
tion was observed when the temperature exceeded 60 ꢀC.
ꢁ 2007 Elsevier B.V. All rights reserved.
1. Introduction
high sensitivity to the environment and to intermolecular
interactions. Therefore, it is worth to test the ICT com-
pounds as a biosensor. There are two benefits using ICT
compounds as a biosensor in proteins [6,7]. First, they
are neutral and hydrophobic, so they incorporate easily
with protein macromolecules. Second, their emission max-
ima correlate excellently with the static polarity of the envi-
ronment. However, the applications of ICT compounds are
often complicated due to the aggregation of these mole-
cules in aqueous media [8].
It is common for hydrophobic organic molecules to
form various kinds of molecular aggregates in water [9].
In these aggregates, the relative mutual orientation of the
molecules creates a microenvironment that is dramatically
different from those in bulk phase. Thus one can observe an
obvious change in the excited photophysics upon forma-
tion of an aggregate [10–13].
In this Letter, a push–pull compound, 40-N,N-dimeth-
ylamino-2-styryl-(6-chloroquinoline) (6-Cl-2-StQ-NMe2)
possessing ICT characteristic was synthesized, as shown
in Scheme 1. We then studied the photophysics of 6-Cl-2-
StQ-NMe2 in aqueous and mixed aqueous-organic solu-
tions and explored the application of this compound as a
biosensor.
It is important for biochemists to search for various
ways in detecting proteins with high sensitivity in qualita-
tive and quantitative analysis [1]. Fluorescence is the most
sensitive and easily available method to study intermolecu-
lar interactions. Incorporation of a fluorescent molecule
into proteins will change its emission properties during
the recognition event and produce a dramatic fluorescence
spectral change [2]. A good fluorescent biosensor should
possess high fluorescence quantum yield and display obvi-
ous spectral change for reflecting their microenvironment.
For compounds with intramolecular charge transfer
(ICT) characteristic, the dipole moment of the excited state
are much higher than that of the ground state, so the for-
mation and emission of the ICT state will be heavily
affected by the environment. In previous works, we have
studied the photophysics of push–pull styryl systems [3–
5]. With ICT characteristic, these compounds displayed
*
Corresponding author. Fax: +886 5 2717901.
E-mail address: wangshunli@mail.ncyu.edu.tw (S.-L. Wang).
0009-2614/$ - see front matter ꢁ 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.cplett.2007.06.122

72
S.-L. Wang, J.-M. Lin / Chemical Physics Letters 444 (2007) 71–75
Cl
1
N
2
3
6
N
4
7
8
5
1011
6-Cl-2-StQ-NMe2
9
1213
Scheme 1.
2. Experimental
ε
ε
Fig. 2. The relationship between the emission maxima of 6-Cl-2-StQ-
NMe2 and solvent polarity function (the Kirkwood function, fe = (e ꢀ 1)/
(2e+1)) (1, cyclohexane; 2, dibutyl ether; 3, ether; 4, EA; 5, THF; 6,
CH2Cl2; 7, 1-butanol; 8, acetone; 9, ethanol; 10, DMF; 11, CH3CN; 12,
methanol; 13, DMSO) (EXC = 400 nm).
Compound 6-Cl-2-StQ-NMe2 was prepared by follow-
ing the literature procedure [3]. 6-Chloroquinaldine and
4-N,N-dimethylaminobenzaldehyde in acetic anhydride
were refluxed for 1.5 h in the presence of the catalyst
ZnCl2. The solid product was purified by column chroma-
tography and re-crystallized from methanol. Bovine serum
albumin (type A-7906) was purchased from Sigma. All the
solvents were Uvasol grade from Merck or spectrophoto-
metric grade from Aldrich and were used as received.
UV–vis absorption spectra were recorded on a Hitachi
U-3010 spectrometer and fluorescence spectra were
obtained using a Hitachi F-4500 fluorescence spectrometer.
wood function, fe = (e ꢀ 1)/(2e + 1)) is shown in Fig. 2. A
good linear correlation exists between the emission maxima
(cmꢀ1) and the solvent polarity functions. It is important to
note that the solvents used in Fig. 2 include protic solvents
and aprotic solvents. Therefore, the emission maxima of 6-
Cl-2-StQ-NMe2 only respond to the media polarity and is
not influenced by hydrogen bonding between solvent mol-
ecules and 6-Cl-2-StQ-NMe2 [14].
3. Results and discussion
To use 6-Cl-2-StQ-NMe2 as a biosensor, it is necessary
to know its photophysics in aqueous solution. For apolar
molecules in aqueous solution, aggregation is easily
observed because of the hydrophobic interactions. Since
the aggregate of organic compounds often display different
photophysics, it is important to study the fluorescence of 6-
Cl-2-StQ-NMe2 in an aqueous solution.
The absorption and emission spectra of 6-Cl-2-StQ-
NMe2 in various ratios of methanol–aqueous solutions
are shown in Fig. 3. The absorption maxima displayed a
blue-shift when the ratio of aqueous in the mixed solution
was increased, but the emission maxima exhibited opposite
behavior except in a pure aqueous solution. It is obvious to
The fluorescence spectra of 6-Cl-2-StQ-NMe2 in a series
of solvents are shown in Fig. 1. Absorption maxima are not
sensitive to the solvent polarity; however, the fluorescence
spectra display a bathochromic shift in polar solvents. This
is a typical phenomenon in an intramolecular charge trans-
fer (ICT) state. The ground state and excited-state dipole
moments of 6-Cl-2-StQ-NMe2 are 2 D and 15 D, respec-
tively, which were calculated from Stokes shifts and solvent
polarity function [3].
To become an excellent biosensor, the emission maxima
need to be highly affected by the polarity of the environ-
ment. The relationship between the emission maxima of
6-Cl-2-StQ-NMe2 to different solvent polarity (the Kirk-
a
c
e
f g
e
a
b
d
a* c*
d*
e*
e*
250
350
450
Wave length (nm)
550
650
410
460
510
560
610
660
Fig. 3. The absorption and emission spectra of compound 6-Cl-2-StQ-
NMe2 (1.0 · 10ꢀ5 M) in different mixed solvents (v/v0) between methanol
and water. (a) Methanol 100%, (b) methanol 80%, (c) methanol 60%, (d)
methanol 40%, (e) methanol 20% (a, b, c, d, e are absorption spectra; a*,
b*, c*, d*, e* are emission spectra; EXC = 400 nm).
Wave length (nm)
Fig. 1. The emission spectra of compound 6-Cl-2-StQ-NMe2 in different
solvents (EXC = 400 nm): (a) cyclohexane; (b) di-butyl ether; (c) ether; (d)
CH2Cl2; (e) acetone; (f) CH3CN; (g) DMSO.

S.-L. Wang, J.-M. Lin / Chemical Physics Letters 444 (2007) 71–75
73
find a ‘jump’ in the blue-shift of the absorption maxima
when the ratios of water is higher than 80%, and the emis-
sion maxima displays a sudden blue-shift instead of red-
shift. Since the polarity of water is higher than methanol,
it is reasonable to observe a red-shift in the emission max-
ima when the content of water is increased (from 540 nm to
550 nm). The discontinuity of the emission maxima in a
high-water content solution was due to the formation of
aggregates because the poor solvation of water to the
hydrophobic organic molecules. The behavior of the
blue-shift for compound 6-Cl-2-StQ-NMe2 in the absorp-
tion maxima after aggregation corresponds to the forma-
tion of an ‘H’ aggregate [15].
It is important to note two interesting things from
Fig. 3. First, the absorption and the emission maxima of
the aggregates were located at 342 nm and 499 nm, respec-
tively. The huge Stokes shift (9200 cmꢀ1) cannot be
explained simply by a pure ICT emission. Second, the fluo-
rescence of the aggregates displayed a fine structure, but
the monomer exhibited a structureless emission in the polar
solvent (Fig. 1).
There are two types of aggregates: H-aggregates where
molecules are arranged in a head-to-head direction and
are characterized by blue-shifted absorption bands with
respect to those of isolated molecule, and J-aggregates
where the molecules are arranged in a head-to-tail direction
and display a red-shift of the UV absorption maximum
compared to individual molecules. In the ground state, 6-
Cl-2-StQ-NMe2 possesses a slightly dipole moment; there-
fore, the p-interaction between the two quinoline rings
favor a H-type aggregation in an aqueous solution [16].
However, a larger charge separation occurs in the excited
state, which disfavors the H-aggregate. This leads to the
breakup of the aggregate in the excited state, and yields
an excited-state monomer pair. A rigid environment exists
in the monomer pair restraining the solvent relaxation
around the fluorophore and produces a structured emission
spectrum. The mechanism of the excited-state deaggrega-
tion reaction was proposed in Scheme 2.
can be used for monitoring the concentration and confor-
mation change of BSA. However, the low fluorescence
yield of BSA leads to the application intrinsic fluorescence
of BSA become inefficient. Here, we try to explore the pos-
sibility of 6-Cl-2-StQ-NMe2 as a nonvalent protein probe
for BSA [19,20].
The fluorescence spectral changes of 6-Cl-2-StQ-NMe2
before and after the addition of BSA are shown in Fig. 4.
6-Cl-2-StQ-NMe2 displayed a very weak structured fluores-
cence (from aggregate) with the maxima located at 499 nm
in an aqueous solution, but the fluorescence intensity
increased and maxima shifted to 468 nm after the addition
of BSA. In using any of the fluorescence signals to monitor
the content of the protein, it is important to realize that
there may be changes in the absorption spectra as well.
The difference in absorption probably contributes some
errors in the fluorescence intensity change. Therefore, we
used the ratios of the emission intensities to monitor the
content of BSA. The relationship between the ratios of
the emission intensities (468 nm/499 nm) and the BSA con-
centrations was inserted into Fig. 4. This plot shows a lin-
ear relationship when the BSA concentration was between
40 lg/ml and 5000 lg/ml.
Beside the quantitative analysis of BSA concentrations,
we also explored the possibility of using 6-Cl-2-StQ-NMe2
for monitoring the conformational change of BSA. BSA is
a well-known globular protein that aggregates into macro-
molecular assemblies. At room temperature, the tertiary
structure of BSA is well defined and stabilized. As temper-
ature increases, some molecular regions become accessible
to new intermolecular interactions, producing new tertiary
structures through disulfide and noncovalent bonds [21–
24]. The disruption of the native conformation with con-
comitant loss of biological activity is known as denatur-
ation [25].
The normalized fluorescence spectra of 6-Cl-2-StQ-
NMe2 incorporated into BSA in different temperature are
From the point of physiology, serum albumin is one of
the most important blood proteins [17]. The amino acid
sequence of bovine serum albumin (BSA) consists of 582
residues with 17 disulfide bridges, 1 free thiol group at res-
idue 34 (in the polypeptide chain portion that is not
restrained by disulfide bonds), and 2 tryptophan residues
[18]. The tryptophan residues are a fluorescent group and
ICT state
hν
hν
Fig. 4. The emission spectra of compound 6-Cl-2-StQ-NMe2
(2.0 · 10ꢀ5 M) as a function of BSA concentration in aqueous solution
(EXC = 350 nm): curve a–h correspond to 0.004%, 0.007%, 0.01%, 0.04%,
0.07%, 0.1%, 0.3% , 0.5% BSA (W/W0), respectively. Insert figure: the
relation between BSA concentration and intensity ratio of I468 nm/I499nm
(EXC = 350 nm).
hν
H-aggregate
monomer
Scheme 2.

74
S.-L. Wang, J.-M. Lin / Chemical Physics Letters 444 (2007) 71–75
low T.
high T.
the excited-state deaggregation, as suggested in Scheme 2,
and the polarity inside denatured BSA is similar with the
monomer located inside the aggregate. Second, the emis-
sion spectrum of 6-Cl-2-StQ-NMe2 after incorporation into
denatured BSA also displayed a fine structure. Noncova-
lent protein–fluorophore interactions can occur by different
physical mechanisms, including hydrophobic interactions,
electrostatic interactions and hydrogen bonding [20].
Although the exact nature of these interactions is difficult
to determine in our system, the existence of an interaction
restricted the motion of 6-Cl-2-StQ-NMe2, and the rigid
environment around 6-Cl-2-StQ-NMe2 produced a struc-
tured emission spectrum.
420
460
500
540
580
620
Wave length (nm)
Fig. 5. The normalized emission spectra of compound 6-Cl-2-StQ-NMe2
(2.0 · 10ꢀ5 M) with 0.04% BSA as a function of temperature in aqueous
solution (EXC = 350 nm). Insert figure: the relation between temperature
and emission intensity k468 nm (EXC = 350 nm).
4. Conclusion
The emission maxima of compound 6-Cl-2-StQ-NMe2,
possessing ICT characters, correlate excellently with the
static polarity of the environment. In an aqueous solution,
6-Cl-2-StQ-NMe2 formed an ‘H type’ aggregate. Upon
photoexcitation, a deaggregation reaction in the excited
state was observed because of a larger charge separation
occurring in the excited-state disfavored the H-aggregate.
6-Cl-2-StQ-NMe2 is an excellent biosensor in monitoring
the content of protein and the conformational change in
thermal denaturation process. The detection limit of the
BSA concentration was found to be 40 lg/ml, and a con-
formational change was observed beyond 60 ꢀC for BSA
in the thermal denaturation reaction.
shown in Fig. 5. At room temperature, 6-Cl-2-StQ-NMe2
displays a structured fluorescence with the maximum
located at 468 nm. As temperature increases, a new struc-
tured fluorescence with the maximum located at 499 nm
appears. The inserted figure in Fig. 5 is the plot of the emis-
sion intensities (after normalization) at 468 nm vs. the solu-
tion temperature. This clearly indicates a dramatic change
in the emission intensity occurring above 60 ꢀC.
In a previous work, the denaturation temperature of
BSA was recorded using Fourier transform infrared
(FTIR) spectroscopy. The secondary structure of BSA
exhibited an obvious change beyond 60 ꢀC in the heating
process [26]. Therefore, we concluded that the emission
intensity change that occurred beyond 60 ꢀC was from
the denaturation of BSA. Since the emission maxima dis-
play a red-shift after denaturation, we conclude the polar-
ity inside BSA increase after denaturation.
Acknowledgement
Financial support from the National Science Council is
acknowledged.
References
Globular proteins with quaternary structures dissociate
before being denatured [27]. In addition to the disruption
of noncovalent bonds, there is more exposure of the thiol
and hydrophobic groups, which were previously buried,
to the aqueous medium. This consequential exposure of
the hydrophobic core of the molecule into an aqueous envi-
ronment results in a polarity increase around the probe (6-
Cl-2-StQ-NMe2) molecules.
Two important results need to be mentioned in Fig. 5.
First, the absorption maximum of 6-Cl-2-StQ-NMe2 after
incorporation into denatured BSA is located at 400 nm, a
typical absorption of monomer molecule. Therefore, it
was considered that the addition of a protein broke the
assembled structure of the aggregate of 6-Cl-2-StQ-
NMe2. The emission spectrum of 6-Cl-2-StQ-NMe2 after
incorporation into denatured BSA was similar with the
emission spectrum of the aggregate in an aqueous solution.
Since only a monomer molecules exists inside denatured
BSA, this concludes that the emission spectrum of an
aggregate in an aqueous solution is from the emission of
monomer species. This result confirms the occurrence of
[1] C. Sun, J. Yang, L. Li, X. Wu, Y. Liu, S. Liu, J. Chromatogr. B 803
(2004) 173.
[2] A.P. Demchenko, Anal. Biochem. 343 (2005) 1.
[3] S.L. Wang, T.I. Ho, J. Photochem. Photobiol., A 135 (2000) 119.
[4] S.L. Wang, G.-Y. Gao, T.-I. Ho, L.-Y. Yang, Chem. Phys. Lett. 415
(2005) 217.
[5] S.L. Wang, T.-W. Yeh, T.-I. Ho, Chem. Phys. Lett. 418 (2005) 418,
393.
[6] R. Das, D. Guha, S. Mitra, S. Kar, S. Lahiri, S. Mukherjee, J. Phys.
Chem. A 101 (1997) 4042.
[7] Y. Suzuki, K. Yokoyama, J. Am. Chem. Soc. 127 (2005) 17799.
[8] A. Sarkar, S. Chakravorti, Chem. Phys. Lett. 235 (1995) 195.
[9] X.-K. Jiang, Acc. Chem. Res. 21 (1988) 362.
[10] F.H. Quina, E.A. Lissi, Acc. Chem. Res. 37 (2004) 703.
[11] D. Horn, J. Rieger, Angew. Chem., Int. Ed. 40 (2001) 4330.
[12] Y.-B. Jiang, M.-G. Jin, L.-R. Lin, Spectrochim. Acta, Part A 54
(1998) 1927.
[13] B.-K. An, S.-K. Kwon, S.-D. Jung, S.Y. Park, J. Am. Chem. Soc. 124
(2002) 14410.
[14] S.L. Wang, T.C. Lee, T.I. Ho, J. Photochem. Photobiol., A 151
(2002) 21.
[15] M. Kasha, H.R. Rawls, M.A. El-Bayoumi, Pure Appl. Chem. 11
(1965) 371.
[16] C.A. Hunter, J.K.M. Sanders, J. Am. Chem. Soc. 112 (1990) 5525.

S.-L. Wang, J.-M. Lin / Chemical Physics Letters 444 (2007) 71–75
75
[17] P. Relkin, Crit. Rev. Food Sci. 36 (1996) 565.
[18] D.C. Carter, J.X. Ho, Adv. Protein Chem. 45 (1994) 153.
[19] A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem. B 109
(2005) 14683.
[20] F. Welder, B. Paul, H. Nakazumi, S. Yagi, C.L. Colyer, J.
Chromatogar. B 793 (2003) 93.
[23] A. Michnik, K. Michalik, Z. Drzazga, J. Therm. Anal. Calorim. 80
(2005) 399.
[24] V. Militello, V. Vetri, M. Leone, Biophys. Chem. 105 (2003) 133.
[25] C. Scharnagl, M. Reif, J. Friedrich, Biochim. Biophys. Acta 1749
(2005) 187.
[26] S.-L. Wang, S.-Y. Lin, M.-J. Li, Y.-S. Wei, T.-F. Hsieh, Biophys.
Chem. 114 (2005) 205.
[27] O.B. Ptitsyn, Adv. Protein Chem. 47 (1995) 83.
[21] K. Murayama, M. Tomida, Biochemistry 43 (2004) 11526.
[22] T. Kosa, T. Maruyama, M. Otagiri, Pharm. Res. 15 (1998) 449.