Colorimetric and fluorescent detection of biological thiols in aqueous solution

Article abstract of DOI:10.1016/j.cclet.2013.01.037

A new colorimetric and fluorescent probe, 2-(2,4-dinitrostyryl)-1,3,3- trimethyl-3H-indolium iodide (DTI), for selective and sensitive detection of biological thiols is reported. In aqueous solution at physiological pH 7.4, biological thiols react with DTI via Michael addition to give the brownish red adduct concomitant with fluorescence emission decrease.

Full text of DOI:10.1016/j.cclet.2013.01.037

Chinese Chemical Letters 24 (2013) 96–98
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Colorimetric and fluorescent detection of biological thiols in aqueous solution
Yin-Hui Li a, Jin-Feng Yang a,b, Chang-Hui Liu a, Ji-Shan Li a, Rong-Hua Yang a,
*
a State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
b Anesthesia Department, Affilicated Cancer Hospital, Xiangya School of Medicine, Central South University, Changsha 410013, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A new colorimetric and fluorescent probe, 2-(2,4-dinitrostyryl)-1,3,3-trimethyl-3H-indolium iodide
(DTI), for selective and sensitive detection of biological thiols is reported. In aqueous solution at
physiological pH 7.4, biological thiols react with DTI via Michael addition to give the brownish red adduct
concomitant with fluorescence emission decrease.
Received 10 December 2012
Received in revised form 5 January 2013
Accepted 10 January 2013
Available online 8 February 2013
ß 2013 Rong-Hua Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Keywords:
Colorimetry
Fluorescence
Biological thiols
Michael addition
1. Introduction
Herein, we have designed and synthesized a new hemicyanine
probe 2-(2,4-dinitrostyryl)-1,3,3-trimethyl-3H-indolium (DTI),
Biological thiols such as glutathione (GSH), cysteine (Cys), and
homocysteine (Hcy) play crucial roles in the cellular antioxidant
defense system. It has been suggested that alternations in the level
of cellular thiols are linked to a number of diseases, such as
leukocyte loss, psoriasis, liver damage, cancer, and AIDS. Addition-
ally, several forms of drug resistance have been correlated with
elevated levels of GSH. Thus, it is important to conveniently
monitor the concentration of biological thiols in physiological
media. A wide variety of detection techniques are applied, such as
high-performance liquid chromatography (HPLC) [1], electro-
chemical assays [2], mass spectrometry [3], surface-enhanced
Raman scattering (SERS) [4], localized surface plasmon resonance
[5], interplasmon coupling in Au nanorods [6], combinatorial
library-based sensors [7], enzymatic methods [8], fluorescent
detection based on synthetic receptors [9] and quantum dots [10].
Among them, optical detection methods have proven most
convenient. By far, various colorimetric and fluorescent probes
for thiol-containing amino acids and peptides are desirable. The
majority of the reported methods are based on redox chemistry or
labeling with chromophores or fluorophores and a combination of
separation techniques, and there is still plenty of room for
improvement in terms of selectivity, sensitivity, and performance
with a different interaction mechanism.
where two unites of indole quinoline and dinitrobenzene are
covalently linked by a double bond. By taking advantage of the
strong electron-withdrawing capacity of the 2,4-dinitrobenzene
moiety, the double bond of DTI could be susceptible to the
nucleophilic biological thiols via Michael addition reaction
(Scheme 1).
2. Experimental
All the solvents and chemicals were of analytical reagent grade
and were supplied by Alfa aesar or Sigma–Aldrich. The DTI probe
was synthesized by one step. 2,4-Dinitrobenzaldehyde (0.392 g,
2 mmol) was dissolved in 10 mL of acetic anhydride, then 1,2,3,3-
tetramethyl-3H-indolium iodide (0.903 g, 3 mmol) and sodium
acetate (0.246 g, 3 mmol) were added. The solution was stirred at
room temperature for 12 h followed by the addition of 20 mL of
diethyl ether. Brown precipitate was obtained. The precipitate was
filtered and washed with cold ethyl acetate three times. Drying the
precipitate in vacuum afforded DTI as a reddish-brown product
(0.62 g, 65%).
The stock solution of 1.0 Â 10À3 mol/L DTI was obtained by
dissolving the compounds in CH3CN. Working solutions were
prepared by sequential dilution of the stock solution with Tris–HCl
solution (50 mmol/L, pH 7.4). All stock solutions of amino acids
were diluted in double distilled water. UV–vis spectra were
recorded in 1 cm path length quartz cuvettes on a Hitachi U-4100
UV/vis spectrophotometer (Kyoto, Japan). Fluorescence emission
* Corresponding author.
E-mail address: yangrh@pku.edu.cn (R.-H. Yang).
1001-8417/$ – see front matter ß 2013 Rong-Hua Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.01.037

Y.-H. Li et al. / Chinese Chemical Letters 24 (2013) 96–98
97
O2N
2000
NO2
DTI
NO2
DTI+ Cys
RSH
N
O2N
I
N
I
SR
1500
1000
500
Scheme 1. Reaction of probe DTI with thiols.
spectra were measured on Hitachi F-7000 Fluorescence spectro-
photometer (Kyoto, Japan). pH value was measured by model 868
pH meter (Orion). Data processing was performed with Sigmaplot
software.
0
3. Results and discussion
4
6
8
10
pH
During the addition reaction, the conjugated system of DTI was
broken, resulting in dramatic changes in both the UV–vis and
fluorescence spectra when probe DTI was treated with Cys as the
model compound of biothiols (Fig. 1A). Free DTI displayed two
absorption bands at 276 nm and 370 nm in aqueous solution (Tris–
HCl buffer, 50 mmol/L, pH 7.4), which were responsible for the
yellow color of the solution. In the presence of Cys, both the
absorbances at 276 nm and 370 nm decreased dramatically while
two new bands at 306 nm and 465 nm appeared with increasing
the concentration of Cys. Meanwhile, in the titration traces, four
clear isosbestic points at 266, 297, 320, 432 nm were observed.
Such shift in absorption behavior changes the color of the resultant
solution from yellow into brownish red, allowing ‘‘naked-eye’’
detection. Similar experimental phenomena were also observed
when we added Hcy or GSH into the solution of DTI. The profound
shift indicates that the conjugation between indole quinoline and
dinitrobenzene was broken due to the plausible Michael addition
of Cys to DTI.
We also noticed that the reaction of DTI with Cys produced an
‘‘on–off’’ type of fluorescence emission at 510 nm with maximum
excitation at 370 nm. In the absence of Cys, DTI showed very strong
fluorescence intensity at 510 nm. Upon addition of Cys to the
solution of DTI, a dramatic turn-off fluorescence response was
observed (Fig. 1B). There was a good linearity between the
fluorescence decrease and Cys concentrations in the range of
1.0 Â 10À7 mol/L to 9.0 Â 10À7 mol/L. The regression equation was
Fig. 2. The fluorescence intensity of DTI at 510 nm in the presence and absence of
Cys under different pH system, ex = 370 nm.
l
showed little change in fluorescence intensity at lower pH (Fig. 2).
But the intensity decreased in alkaline conditions. When Cys was
added to DTI in buffers at various pHs, significant fluorescence
change was detected at pH range of 6.0–8.0. The curves indicated
that DTI responded to biothiols under physiological conditions.
The reaction mechanism was confirmed by using 1H NMR
spectroscopy and electrospray ionization mass spectrometry (ESI-
MS) (data not shown). Upon addition of Cys, a vinyl proton of DTI
around
d
8.4 dramatically disappear with the concomitant
7.1 and 6.7. And the aromatic
appearance of a new peak at
d
d
protons produced corresponding chemical shifts. In addition, the
m/z formula [M+H]+ of the free DTI was found to be 352.0 (calcd.
479.0) in its ESI-MS. After addition of Cys to the DTI solution, a new
peak at m/z 494.9 (DTI+Cys+Na+) was clearly observed, which is
assigned to the 1:1 complex between DTI and Cys.
To examine the selectivity of DTI toward thiol-containing
amino acids, changes in the fluorescence spectra of DTI by addition
of various amino acids were measured in the Tris–HCl buffer
solution (Fig. 3). Significant decrease at 510 nm has been found
upon addition of Hcy or Cys to DTI solution, with an evident color
change from yellow to brownish red. On the other hand, treatment
of up to 50 equiv. of other 19 amino acids (Tyr, Gly, Phe, Met, Leu,
Arg, Pro, Lys, Glu, Gln, Asp, Iso, Ile, Val, His, Ser, Ala, Thr, Try) with
DTI did not induce any obvious change in the fluorescence spectra,
F0/F = 1970.866 À 2144.161 [Cys] (
mmol/L) with a linear coeffi-
cient of 0.989. The detection limit that was taken to be 3 times the
standard derivation of a blank solution was estimated to be
2.6 Â 10À8 mol/L.
60
45
30
15
0
To investigate the effect of pH on the fluorescence response of
DTI to Cys, the fluorescence intensity changes of DTI induced by
Cys were measured at various pHs. In the absence of Cys, DTI
4
1
2
3
6
18 19
17
20 21
16
2
2
5
11
13 14 15
10
12
7
8
9
Fig. 1. Absorption (A) and fluorescence spectra (B) of DTI in the presence of
increasing amounts of Cys in Tris–HCl solution (50 mmol/L, pH 7.4). The arrow
indicates the signal changes as increases in Cys concentrations (0, 0.1, 0.2, 0.3, 0.4,
Fig. 3. The selectivity of DTI toward various amino acids. Gray bars: the ratio of F0/F
of DTI in the presence of various amino acids; Black bars: the ratio of F0/F of DTI in
the presence of the mixture of each amino acid and GSH, (1) Free DTI. (2) Cys. (3)
Hcy. (4) Tyr. (5) Gly. (6) Phe. (7) Met. (8) Leu. (9) Arg. (10) Pro. (11) Lys. (12) Glu. (13)
Gln. (14) Asp. (15) Iso. (16) Ile. (17) Val. (18) His. (19) Ser. (20) Ala. (21) Thr. (22) Try.
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 2.0
of DTI as a function of the concentrations of Cys.
mmol/L). Inset: the ratio of F0/F

98
Y.-H. Li et al. / Chinese Chemical Letters 24 (2013) 96–98
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Acknowledgments
This work was supported by the National Key Natural Science
Foundation of China (No. 21135001), ‘‘973’’ National Key Basic
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