Chromogenic sensing of biological thiols using squarylium dye

Article abstract of DOI:10.1016/j.saa.2013.10.066

A new highly selective probe for biothiols, squarylium dye (SQ), was designed and synthesized. The probe displayed a color change from blue to colorless upon reaction with biothilos such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The competition experiments revealed that no obvious interference was observed by performing the titration with the mixtures of Cys and other amino acids. The results indicated that SQ was highly selective for Cys detection. Moreover, SQ could also serve as a naked-eye probe for Cys with a minimum detectable concentration of approximately 4 μM.

Full text of DOI:10.1016/j.saa.2013.10.066

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 642–645
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Short Communication
Chromogenic sensing of biological thiols using squarylium dye
In-Sub Shin a, Seon-Yeong Gwon a, Sung-Hoon Kim a,b,
⇑
a Department of Advanced Organic Materials Science and Engineering, Kyungpook National University, Daegu 702 701, Republic of Korea
b School of Chemistry Science & Technology, Zhanjiang Normal University, Zhanjiang 524048, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
A new biothiol probe SQ was
synthesized.
The probe displayed a color change
from Cys, Hcy and GSH.
SQ was highly selective for Cys
detection.
O
HO
HO
SR
O
HO
HO
OH
OH
N
RSH
2+
OH
N
N
N
OH
O
SQ
HO
SQ could serve as a ‘‘naked-eye’’
probe for Cys with a minimum
concentration of approximately 4 lM.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2013
Received in revised form 7 October 2013
Accepted 17 October 2013
Available online 26 October 2013
A new highly selective probe for biothiols, squarylium dye (SQ), was designed and synthesized. The probe
displayed a color change from blue to colorless upon reaction with biothilos such as cysteine (Cys), homo-
cysteine (Hcy) and glutathione (GSH). The competition experiments revealed that no obvious interfer-
ence was observed by performing the titration with the mixtures of Cys and other amino acids. The
results indicated that SQ was highly selective for Cys detection. Moreover, SQ could also serve as a
‘‘naked-eye’’ probe for Cys with a minimum detectable concentration of approximately 4 lM.
Keywords:
Chemosensor
Cysteine
Ó 2013 Elsevier B.V. All rights reserved.
Homocysteine
Glutathione
Biothiols
Squarylium dye
Introduction
important role in the detoxification of xenobiotic compounds and
in the antioxidation of reactive oxygen species and free radicals
[5]. The determination of biothiols in body fluids is very important
from the biological and pharmacological stand point. Some Cys and
Hcy analyses have been developed in conjunction with HPLC [6],
capillary electrophoresis [6], immunoassay [6], colorimetric and
fluorescence detection [7–11], etc.
Recently, colorimetric sensors are popular due to their capabil-
ity to detect analyte by the naked eye without resorting to any
expensive instruments. Therefore, the design of ratiometric and
colorimetric biothiol probes has been the focus of numerous re-
search efforts because of their remarkable importance in the qual-
itative and quantitative detection. Squarylium dyes and derivatives
are 1,3-disubstituted compounds synthesized from squaric acid
and two equivalents of various types of electron donating carbocy-
cles or heterocycles such as azulene [12], pyrroles [13], or hetero-
cyclic methylene bases [14]. Squarylium dyes are expected to have
Naturally occurring thiols exhibit a variety of structures and
important physiological properties. In which, Cys, Hcy, GSH have
the similar structures (Fig. 1).
Biological thiols such as Cys, Hcy and GSH play crucial roles in
the cellular antioxidant defense system [1]. Their abnormal levels
have been directly linked to some diseases and cancers. Deficiency
of Cys would lead to many diseases, such as hematopoiesis de-
crease, leucocyte loss [2], and psoriasis, while Hcy is a risk factor
for cardiovascular [3] and Alzheimer’s disease [4]. GSH is a major
component of the cellular antioxidant system, and it plays an
⇑
Corresponding author at: Department of Advanced Organic Materials Science
and Engineering, Kyungpook National University, Daegu 702 701, Republic of Korea.
Tel.: +82 53 950 5641; fax: +82 53 950 6617.
E-mail address: shokim@knu.ac.kr (S.-H. Kim).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.10.066

I.-S. Shin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 642–645
643
HS
1.0
O
O
0.8
Cys
Hcy
GSH
SQ
SH
NH
HN
HS
0.6
COOH
H
H
H
0.4
0.2
0.0
H2N
COOH
H2N
H2N
COOH
COOH
Cysteine(Cys)
Homocysteine(Hcy)
Glutathione(GSH)
Fig. 1. Structure of biothiol.
0
10
20
30
40
significant technological applications in many areas such as elec-
trophotography [15–18], solar energy conversion [19], optical
recording [20], nonlinear optics [21], and electroluminescence
[22]. We now present a squarylium dye (SQ), having chromogenic
responses for fast, selective and sensitive detection of biothiols in
aqueous media.
Time (min)
Fig. 2. Time dependant absorption intensity change of SQ (4 lM) in the absence
and presence of 1 equiv. Cys, Hcy, GSH at 654 nm in a mixture of DMSO and HEPES.
(9:1,v/v, pH7.4).
Experimental
1.2
1.0
0.8
0.6
0.4
Synthesis of SQ
0.9
0.2
N-phenyldiethanolamine 1 (3.62 g, 20 mmol) and squaric acid 2
(1.14 g,10 mmol) was heated under reflux for 24 h in a mixture of
60 ml of n-butanol/benzene (5:5/v:v). Water was removed azeo-
tropically using a Dean–Stark trap. The reaction mixture was
cooled to room temperature. The product is collected by filtration
and washed with n-hexane to give 1.05 g (24%) of pure product. 1H
NMR (400 MHz, DMSO-d6): d 3.64–3.67 (m, 20H), 7.00 (d, J = 9.32,
4H), 8.10 (d, J = 9.2, 4H). EA: Anal.Calcd. for C24H32N2O6: C, 64.85;
H, 7.26; N, 6.30. Found: C, 64.99; H, 6.32; N, 6.02%. IR (Kbr):
0.0
0.0
0.2
0.4
0.6
0.8
1.0
[Cys]/[SQ]
Cys
0.6
0.3
0.0
m
= 3358 cmꢁ1 (OH), 1756 cmꢁ1 (C@O), 1374, 1353 cmꢁ1 (ArAN).
FAB Mass: 440.1.
500
600
Wavelength (nm)
700
Results and discussion
Fig. 3. Absorption spectra change of SQ(4 lM) upon addition of Cys (0–1 equiv.) in
DMSO/HEPES (9:1v/v, pH7.4). Each spectrum is recorded 10 min after Cys addition.
Inset:change in absorption intensity profile of SQ at 654 nm.
Symmetrical squarylium dyes are normally synthesized by con-
densation of squaric acid 2 and electron rich aromatic or heteroar-
omatic compounds. SQ dye used in this work was synthesized from
N-phenyldiethanolamine 1 and squaric acid 2, according to previ-
ous described procedures [15–18] (Scheme 1).
In order to demonstrate the potential application of SQ as a
highly colorimetric probe for biothiols, absorption spectral studies
of SQ in the presence of biothiols were performed.
At first, the time course of the reaction between probe SQ and
biothiols was studied by monitoring the absorption intensities of
the reaction mixture at 654 nm (Fig. 2).
SQ presents a sharp and intense absorption band at 654 nm.
Upon treatment with 1 equiv. of Cys in pH 7.4 DMSO/HEPES(4-
(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid) (9:1, v/v) at
room temperature, the absorption band at 654 nm decreased within
10 min. After 30 min the decrease of the absorption intensity of SQ
induced by Hcy/GSH leveled off. Therefore, all samples were equil-
ibrated for 30 min before measurements. Changes in the UV–visi-
ble absorption spectra upon additions of Cys to the solution of
SQ are shown in the Fig. 3.
We examined the kinetic profiles of the reaction under first-
order reaction with 1 equiv. of Cys, Hcy and GSH over probe SQ.
The first-order rate constant k was calculated according to the
following equation:
lnðAt ꢁ A Þ=ðAi ꢁ A Þ ¼ kt
ð1Þ
1
1
OH
HO
OH
OH
O
O
O
2+
N
N
N
HO
OH
O
HO
OH
SQ
Scheme 1. Synthesis of SQ.

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I.-S. Shin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 642–645
0
-1
-2
-3
-4
-5
-6
-7
-8
1.2
(a)
Cys
Hcy
GSH
Leu, Pro, Val, Met, Thr, Gln,
Ala, Asp, Nor, IIe, Trp, Phe,
Glu, Gly, Cystine, Homocystine
0.8
His, Arg, Lys
0.4
Hcy, GSH
Cys
0.0
0
5
10
15
20
25
30
400
600
Time (min)
Wavelength (nm)
(b)
Fig. 4. First-order decoloration kinetic plot upon addition of 1 equiv. of Cys, Hcy
and GSH.
where Ai is the absorbance at 654 nm, and At is the absorbance
at 654 nm at time t after addition of biothiols. A and k refer to
absorbance at 654 nm after 1 h and first-order rate constant,
1
Fig. 6. (a) Absorption spectra of SQ (4
amino acids. (b) Photos of SQ in the presence of different amino acids.
l
M) upon addition of 1 equiv. of various
respectively. In the color changing process, the kinetic analysis pre-
dicts the logarithm of the difference between A and At at time t to
1
be linear with time, the slope giving the decoloration rate constant,
k. First-order plots according to Eq (1) are shown in Fig. 4. The first-
order decoloration rate constants for Cys, Hcy ans GSH were deter-
mined as k = ꢁ77.9 ꢂ 10ꢁ2 minꢁ1, ꢁ16.8 ꢂ 10ꢁ2 minꢁ1, ꢁ16.6 ꢂ
10ꢁ2 minꢁ1, respectively, which indicates that the reaction occurs
more readily in the following order: Cys >> Hcy ’ GSH.
SQ dyes is highly susceptible to nucleophilic attack by thiols. This
abrupt decrease in the absorption intensity signified a high initial
reaction rate of nucleophilic attack of the Cys on the four-atom ring
of the SQ. The 1H NMR experiments were carried out to confirm the
Cys-containing SQ (Fig. 5).
When 1 equiv. of Cys was added into SQ, aromatic protons (8.06
and 6.97 ppm) disappeared whereas new peaks centered at 7.41,
SQ dyes are electrophiles because of their formally electron-
deficient central four-membered ring. The four-membered ring of
Fig. 5. 1H NMR spectral change of SQ upon addition of Cys. (a) SQ only, (b) SQ and 1 equiv. Cys in a mixture of DMSO-d6 and D2O (9:1,v/v).
O
HO
HO
SR
O
HO
HO
OH
OH
N
RSH
2+
OH
N
N
N
OH
O
SQ
HO
Scheme 2. Proposed sensing mechanism of biothiols.

I.-S. Shin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 642–645
645
recognition of biothiols gave obvious decoloration from blue,
which was clearly visible the naked eye. Moreover, the competition
experiments showed that no obvious interference in the absorp-
tion from other amino acids was observed.
60
50
40
30
20
10
0
Acknowledgements
This work was supported by the NationalResearch Foundation
(NRF) grant funded by the Korea Government (MSIP) (2008-
0062617). This research was supported by a grant from the Funda-
mental R&D Program for Core Technology of Materials (10037193)
funded by the Ministry of Trade, Industry & Energy, Republic of
Korea. This research was supported by Kyungpook National Uni-
versity Research Fund, 2013.
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Fig. 7. A0/A654 change profile of SQ (4
bars represent the addition of 1 equiv. of various amino acids to a 4
SQ. Black bars represent the addition of 1 equiv. of Cys to the above solution,
respectively. 1, Cys; 2, HCy; 3, GSH; 4, Val; 5, Pro; 6, Lev; 7, Thr; 8, Met; 9, Gln; 10,
Nor; 11, His; 12, Gly; 13, Ala; 14, Arg; 15, Ile; 16, Trp; 17, Phe; 18, Asp; 19, Lys; 20,
Cystine; 21, Glu; 22, Homocystine.
l
M) to various amino acids (1 equiv.). Gray
lM solution of
7.29, 6.54 ppm appeared which could be assigned to the protons of
non-equivalent aromatic rings. A possible mechanism was pro-
posed as shown in Scheme 2.
Chemosensors that can detect analyte of interest by the naked
eyes are of particular interest because of the convenience in its
use. To evaluate the selectivity of SQ for Cys, Hcy and GSH, we
measured the absorption intensity changes of SQ by adding differ-
ent amino acids to the SQ. The absorption intensity of SQ decreased
only by the addition of Cys, Hcy and GSH.
The other amino acids such as Ala, Met, Val, Pro, Leu, Thr, Gln,
Asp, Nor, Ile, Trp, Phe, Glu, Gly, Cystine, Homocystine, His, Arg,
Lys did not cause any significant changes in the absorption inten-
sity (Fig. 6).
To examine the selectivity of SQ toward biothiols, changes in
absorption spectra of SQ by addition of various amino acid were
measured. As shown Fig. 7, no obvious decoloration were observed
upon addition of other amino acids compared with the decolor-
ation of SQ with Cys, Hcy and GSH.
Conclusions
In conclusion, we developed
a sensitive chemosensor for
biothiols using the highly chromogenic squarylium (SQ) dye. The