Green synthesis of a benzothiazole based 'turn-on' type fluorimetric probe and its use for the selective detection of thiophenols in environmental samples and living cells

Article abstract of DOI:10.1039/c6ra07046a

We have developed an efficient turn-on type fluorescent chemodosimeter for the detection of aromatic thiols in aqueous media. The probe was successfully synthesized by condensation of 2-aminothiophenol with salicylaldehyde followed by S_NAr with

Full text of DOI:10.1039/c6ra07046a

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PAPER
Green synthesis of a benzothiazole based ‘turn-on’
type fluorimetric probe and its use for the selective
detection of thiophenols in environmental samples
and living cells†
Cite this: RSC Adv., 2016, 6, 52790
a
a
a
b
Dipratn G. Khandare, Mainak Banerjee,* Rishabh Gupta, Nupur Kumar,
b
a
a
Anasuya Ganguly, Deepak Singh and Amrita Chatterjee*
We have developed an efficient turn-on type fluorescent chemodosimeter for the detection of aromatic
thiols in aqueous media. The probe was successfully synthesized by condensation of 2-aminothiophenol
N
with salicylaldehyde followed by S Ar with 2,4-dinitrochlorobenzene in one-pot in a micellar medium.
The function of the probe is attributed to the thiol driven cleavage of the dinitrophenyl ether linkage of
probe 1 to release 2-(2-hydroxyphenyl)benzothiazole (2) which shows strong greenish fluorescence by
excited-state intramolecular proton transfer (ESIPT). The probe is highly selective for aromatic thiols in
the presence of several aliphatic thiols, including biologically important thiol-containing amino acids.
The utility of the probe was successfully demonstrated in the detection of thiophenols in real samples
and in living cells. A hazardless synthetic procedure, cost effective single-step synthesis, high selectivity
and sensitivity, fast signal transduction and low limit of detection (3.3 ppb) are some of the key merits of
this analytical tool.
Received 17th March 2016
Accepted 24th May 2016
DOI: 10.1039/c6ra07046a
www.rsc.org/advances
particular, the uorimetric techniques have gained immense
attention because of their operational simplicity, cost-
1
. Introduction
Thiophenols, although having a broad synthetic utility as effectiveness, high sensitivity and selectivity, fast signal trans-
1
5
insecticides, pesticides, in pharmaceuticals and in amber dyes,
duction etc. Amongst available techniques for uorimetric
2
6–9
belong to a class of highly toxic and pollutant materials. In fact, detection of thiols, a large number of probes are designed for
thiophenols are much more toxic than their aliphatic selective detection of biological thiols. However, uorimetric
analogues, e.g., for sh, thiophenols have a median lethal dose probes for selective detection of aromatic thiols are not many
LC50) of 0.01 to 0.4 mM. Due to their high toxicity and ease of as compared to biothiols because of their inefficiency to
7,8
8,9
3
(
8a–e
entrance into the human body by inhalation and skin absorp- discriminate between aromatic and aliphatic thiols.
This
tion, thiophenols exacerbate grave systemic and central nervous imposes restriction to monitor only aromatic thiols in envi-
injuries like burning sensation in the throat, eyes and nose, ronmental and biological samples. In addition, some of these
headache, dizziness, coughing, wheezing, laryngitis, shortness methods are not passably sensitive or selective, or involve
4
of breath, muscular weakness, paralysis, coma and even death.
relatively long synthetic route and thus, not cost effective. Some
Thus, development of efficient techniques for the selective and other available methods for the detection of aromatic thiols
1
0
11
sensitive detection of these toxic compounds in the environ- include HPLC technique, UV-detection and GC-MS methods,
12
ment is highly desirable.
optical spectroscopy using gold nano-particles as the probe,
The eld of sensing and imaging agents is growing fast, chemically modied barium titanate beads using surface-
largely driven by the development of optical techniques. In enhanced Raman scattering (SERM), etc. However, these
techniques suffer from expensive instrumentation and
13
requirement of highly trained technicians.
Due to present environmental concerns use of greener
solvents, nontoxic reagents and environment friendly condi-
a
Department of Chemistry, BITS, Pilani – K. K. Birla Goa Campus, NH 17B Bypass
Road, Zuarinagar, Goa 403726, India. E-mail: amrita@goa.bits-pilani.ac.in;
mainak@goa.bits-pilani.ac.in; Fax: +91-832-2557-033; Tel: +91-832-2580-320; +91-
14
tions for any synthetic process has become imperative.
8
32-2580-347
b
Surprisingly, these issues are mostly ignored while the synthesis
Department of Biological Sciences, BITS, Pilani – K. K. Birla Goa Campus, NH 17B
Bypass Road, Zuarinagar, Goa 403726, India
of molecular sensors. Although signicant efforts have been
Electronic supplementary information (ESI) available: Synthetic procedure of made towards preparation of water soluble probes in order to
15
†
compound 2; real sample analysis data; scanned copies of NMR and HR-MS
reduce or eliminate the use of toxic organic solvents, there are
spectra of 1 and 2. See DOI: 10.1039/c6ra07046a
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hardly any attempts to maintain a “green” protocol during the 2.2 Instruments
synthesis of the probe molecule. In fact, the amount of organic
NMR spectra were recorded on Bruker AV400 NMR spectrom-
eter and mass spectra were obtained from Bruker micro TOF-Q
II 10330 HRLCMS (ESI ). Fluorescence spectra were taken on
solvents used during synthesis is oen signicantly higher than
the amount required for analytical studies. In this work, we
intended to introduce the concept of green chemical
approaches in synthesizing the molecular probes. Thus, as
a part of our continued efforts on the development of uores-
cent chemosensors for environmentally and/or biologically
+
a JASCO FP-6300 spectrouorimeter with the slit width of 5 nm
for both excitation and emission. Absorption spectra were
recorded on a JASCO V570 UV/Vis/NIR spectrophotometer at
room temperature. IR spectra were recorded on IR Affinity-1
FTIR spectrophotometer, Shimadzu. The reactions were moni-
tored by thin layer chromatography (TLC) carried out on 0.25
mm silica gel on aluminium plates (60F-254) using UV light (254
or 365 nm) and could be tracked by naked eye.
16
toxic analytes, we report, herein, an environment friendly
synthesis of a molecular probe which is highly selective and
sensitive to aromatic thiols.
A large number of uorescent probes have been con-
structed by exploiting the high nucleophilicity of the thiol
6
–9
group. In many of these cases strongly electron-withdrawing
,4-dinitrobenzenesulfonyl (DNBS) group has been used as the
uorescence quencher by getting attached with the uo-
2.3 Synthetic procedure
2

2.3.1 Synthesis of probe 1. In a 25 mL of round-bottomed
8
rophore unit as sulphonate or sulphonamide. This strategy is ask was taken o-aminothiophenol (76 mL, 0.71 mmol) and
effective in building chemosensors for selective detection of salicylaldehyde (76 mL, 0.71 mmol) in 1 mL of water followed by
thiophenols, however, aliphatic thiols are oen capable of addition of CTAB (26 mg, 0.071 mmol). The reaction mixture
8
e
8a–d
detaching this moiety partially or fully,
from the uo- was stirred at room temperature for 1 h to give 2-(2-hydrox-
rophore at the prevalent condition because of the high lability yphenyl)benzothiazole (2). The completion of the reaction was
of DNBS in the presence of –SH group making discrimination monitored by TLC. To the same reaction mixture 4 mL of water
of thiophenols from other thiols difficult. We envisioned, use and K CO (490 mg, 3.55 mmol) were added followed by addi-
2 3
of 2,4-dinitrophenyl (DNP) group with reduced reactivity will tion of 2,4-dinitrochlorobenzene (142 mg, 0.71 mmol). The
be more effective in obtaining higher selectivity towards thi- reaction mixture was sonicated for 5 min and stirred at room
ophenols and the corresponding probe molecule will be temperature for another 6 h. With the progress of the reaction
unperturbed by the presence of aliphatic thiols. The probe was solid product started separating out. Aer completion of the
constituted by protection of the free hydroxyl group of 2-(2- reaction, excess water (10 mL) was added and the residue was
hydroxyphenyl)benzothiazole with DNP. We assumed, the ltered, washed with water, and air dried. The crude product
aromatic thiols will remain partly in thiolate form at physio- thus obtained was recrystallized from rectied spirit to afford
1
logical pH and behave as much stronger nucleophile than probe 1 (180 mg, 64%). H NMR (400 MHz, CDCl
3
): d (ppm) 6.93
aliphatic thiols. It would selectively cleave the ether linkage of (1H, d, J ¼ 7.0 Hz), 7.23–7.27 (1H, m, merged with solvent peak),
the probe with ease releasing uorescent 2-(2-hydroxyphenyl) 7.36–7.39 (1H, m), 7.45–7.53 (2H, m), 7.56–7.59 (1H, m), 7.83
benzothiazole, which will start emitting light by excited-state (1H, d, J ¼ 6.2 Hz), 8.00 (1H, d, J ¼ 6.2 Hz), 8.24 (1H, dd, J ¼ 6.0,
1
3
1
7
intramolecular proton transfer mechanism (ESIPT). The 1.2 Hz), 8.48 (1H, dd, J ¼ 6.0, 1.2 Hz), 8.90 (1H, d, J ¼ 2.0 Hz);
C
availability of the phenolic proton is essential for ESIPT to NMR (100 MHz): d (ppm) 117.82, 121.49, 122.09, 122.21, 123.45,
operate, which is unavailable in probe 1 due to formation of 125.70, 126.53, 126.58, 127.41, 128.94, 131.29, 132.48, 135.49,
dinitrophenyl ether and the probe becomes nonuorescent. 139.43, 141.79, 150.70, 152.67, 155.63, 160.86; IR: 3098, 2914,
ꢀ
1
When the probe comes in contact with thiophenols under the 2855, 1610, 1536, 1342, 1251, 1145, 1099, 1067, 962 cm ; ESI-
+
appropriate conditions, spontaneous trans-etherication MS: m/z 394 [M + H] ; HRMS (ESI): m/z calcd for C19
occurs resulting in release of uorescent 2-(2-hydroxyphenyl) [M + H] 394.0453, found 394.0452.
H
11
N
3
O
5
S
+
benzothiazole (2). Probe 1 was obtained by mixing 2-amino-
thiophenol, benzaldehyde and 2,4-dinitrochlorobenzene in
a single-pot in micellar medium and its sensing property was
determined.
2
.4 General procedure for the sensing studies
A stock solution of probe 1 (1 mM) was made in DMF. The
uorescence and absorbance studies were done by adding thi-

ophenol to probe 1 in 45% DMF–PBS buffer solution (pH ¼ 7.2)
and incubating at room temperature for 15 min. To check the
selectivity of the probe, the same procedure was followed with
different thiol containing amino acids, 2-mercaptoethanol and
other thiophenol derivatives. For equivalence study, the
concentration of thiophenol was changed from 0–3.0 equiv. and
2
. Experimental section
2.1 Chemicals and materials
All the reagents were purchased from commercial suppliers and
used without further purication. AR grade solvents were used
throughout the experiments. Doubly distilled water was used
for the uorimetric studies. Thiophenol, 2-aminothiophenol, 4-
aminothiophenol were purchased from Spectrochem Pvt. Ltd.

uorescence spectra were recorded.
2.5 Cytotoxicity
(India), SD Fine chemicals (India), Sigma Aldrich (India), Cytotoxicity test of probe 1 was performed using MTT assay.
respectively.
HeLa cell lines were grown in Dulbecco's Modied Eagle's
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Medium (DMEM) supplemented with 10% (v/v) fetal bovine
2.7.3 Detection of thiophenol in soil samples. To detect
ꢁ
serum at 37 C under humid condition with 5% CO . Cells were aromatic thiols in soil samples it was contaminated with
2
4
seeded in a 24 well plate at a density of 5 ꢂ 10 cells per well. measured amount of thiophenol from 1 mM stock solution in
Aer 24 h of incubation, the cells were supplemented with ethanol. In 3 mL DMF–PBS (pH ¼ 7.2) containing probe 1 (1
probe 1 at different concentrations (10 mM, 50 mM and 100 mM; mM) was added soil sample (90 mg) pre-spiked with thiophenol
these solutions were prepared by adding 2 mL, 10 mL and 20 mL (1.2 mM) in ethanol. This sample was incubated at room
from 10 mM stock solutions of probe in 5% DMF–water). The temperature for 15 min, aer which the solution was ltered for
control cells were treated with 0.1% of DMF. Aer incubation uorescence measurement.
ꢀ1
for 24 h, 60 mL of MTT (5 mg mL in phosphate buffer saline)
was added per well and incubated for 4 h. Aer incubation,
media was removed from the wells and 0.5 mL of DMSO was
3
. Results and discussions
added to each well. Absorbance was measured at 570 nm and 3.1 Synthesis and spectral characterization of probe 1
cell viability was expressed as relative absorbance (%) of the
sample vs. control cells.
Probe 1 was synthesized by condensation of 2-aminothiophenol
with salicylaldehyde followed by aromatic nucleophilic substi-
tution (S Ar) with 2,4-dinitrochlorobenzene in one-pot in
N
2
.6 Fluorescence imaging of living cells
a micellar medium with 64% overall yield (Scheme 1). The
1
13
HeLa cells were seeded in a 6 well plate in Dulbecco's modied formation of probe 1 was well-supported by H NMR, C NMR,
Eagle's medium (DMEM) supplemented with 10% fetal bovine and HR-MS. As expected, the characteristic peak of aromatic
serum for 24 h. HeLa cells were then incubated with thiophenol proton anked by two nitro groups appeared at the most
1
ꢁ
(
1 mM) in the culture medium for 30 min at 37 C. Aer washing downeld position (at d 8.90 ppm) in H NMR as a doublet with
coupling constant (J) 2.0 Hz, which is attributed to meta-
coupling. In C NMR, separate peaks appeared for each of
0 min at 37 C. Aer incubation, the cells were again washed the 19 carbons in the expected region indicating attachment of
with PBS buffer for three times to remove the remaining thio-
phenol, the cells were further incubated with probe 1 (1 mM) for
3
1
3
ꢁ
with PBS buffer for three times and the uorescence images 2,4-dinitrophenyl unit to 2-(2-hydroxyphenyl)benzothiazole
+
were obtained using Nikon eclipse TS100 microscope tted with part. The molecular ion peak ([M + H] ) appeared as the base

uorescence attachment and using a FITC lter.
peak at m/z 394 rmly indicating formation of probe 1. In
addition, HR-MS data for [M + H] of probe 1 closely matched
+
with the expected value. In a separate reaction, 2-(2-hydrox-
2
.7 Real sample analysis
yphenyl)benzothiazole (2) was isolated from the micellar
2.7.1 Detection of thiophenol in water samples. The pres-
1
13
medium in high yield (88%) and characterized by H NMR,
C
ence of thiophenol was detected by spiking known amount of
thiophenol in tap water. The water sample was ltered through
microltration membrane. The pH of water sample was
adjusted to 7.2 using PBS. Next, 1.5 mL of water sample was
spiked with two different concentrations of thiophenol (1.5 mM
NMR and HR-MS. Probe 1 was used for the detection of
aromatic thiols.
3.2 Effect of solvent and pH
and 3.0 mM) in DMF. These samples were further treated with To establish proper condition for thiolysis of dinitrophenyl
probe 1 (30 mL, 1 mM) and diluted to 3 mL using DMF and water moiety a thorough screening of solvent system was done as well
as per requirement. The solutions were incubated at room as the effect of pH was studied. In this direction, experiments
temperature for 15 min and the uorescence spectra were were performed varying different solvent systems to choose the
measured.
.7.2 Thiophenol vapor detection by paper strips and TLC tion reaction. The reactions were carried out by adding thio-
best reaction medium for this aromatic nucleophilic substitu-
2
plates. A paper strip of probe 1 was prepared by dipping a rect- phenol in a solution of probe 1 of a given solvent system in 1 : 1
angular shape Whatmann lter paper in the probe solution (1 molar ratio under neutral condition at room temperature. We
mM) in DMF for few seconds and then it was placed in chemical used different proportions of organic solvents in water. Among
storage cabinet where the uncapped bottle of thiophenol was DMF, THF and ethanol it was found that 45% DMF–water is
stored and allowed to get exposed to its vapor for 15 min. As most suitable for the system to smoothly undergo S Ar reaction.
N
a control experiment another paper strip was dipped in the Therefore, all further experiments were carried out in this
same solution and kept in the storage cabinet without exposure solvent system. To check the effect of pH on probe 1, the
of thiophenol for 15 min. The strip in contact with thiophenol
showed strong uorescence aer 15 min of exposure. In
a similar manner, probe 1 modied TLC plate (0.25 mm silica
gel on aluminium plate) was prepared by spotting one drop of
probe solution (1 mM) in DMF, allowed to soak properly, and
then placed in the chemical storage cabinet for 15 min where an
uncapped bottle of thiophenol was preplaced. The spotted area
in TLC plate showed strong uorescence. Like paper strips
similar control experiment was conducted for TLC plates.
Scheme 1 Synthesis of probe 1.
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reaction was done at various pH (pH 1–9). In a separate study,
several solutions of probe 1 at different pH were prepared in
45% DMF–water and thiophenol was added to it, and uores-
cence output of the resulting solution was measured aer 15
min. Fig. 1 shows the uorescence responses of probe 1 without
and with thiophenol as a function of pH. The experiment
revealed that the probe is ineffective in the detection of thio-
phenol at acidic pH (pH ¼ 1–6) as expressed by very low uo-
rescence response from the solution. However, the same
reaction when carried out under neutral condition using 45%
DMF–PBS buffer (pH 7.2), to our delight, signicant increase in

uorescence intensity was noticed. Under the prevailing
condition the cleavage of the ether bond resulted in formation
of uorescent compound 2. On the other hand, under basic
condition (pH $ 8), the probe 1 showed appreciable uores-
cence response which is presumably because of the initiation of
the aromatic nucleophilic substitution reaction in the presence
Fig. 2 Fluorescence response of a probe 1 (30 mM) upon addition of
thiophenol (0–90 mM) in 45% DMF : PBS (pH 7.2) [excitation wave-
length is 395 nm]. Inset: plot of the increment in emission against no.
of excess hydroxide ions to produce back phenol 2 responsible of equiv. of thiophenol.
for ESIPT. However, probe 1 could work very efficiently for
aromatic thiols at neutral pH and ambient temperature, which
would enable the use of the probe in the detection of these mostly used up by addition of just 1 equiv. of thiophenol.
pollutants in biological and environmental samples directly.
However, 3 equiv. of thiophenol was required to reach the actual
saturation point. This indicates that the reaction slows down
towards the end and excess thiophenol is required for complete
conversion, which is a common phenomenon for most of the
nucleophilic substitution reaction.
3.3 Thiophenol sensing by probe 1: spectrophotometric
studies and mechanistic aspect
To understand the sensing characteristic of probe 1 an equiv-
alence study was conducted. In a typical experiment, the uo-
rescence response of probe 1 upon addition of 0–3 equiv. of
thiophenol in 45% DMF–PBS buffer was measured. The solu-
tion was incubated for 15 min aer addition of each portion of
thiophenol before measuring the uorescence response of the
corresponding solution. An intense greenish blue uorescence
was observed at lmax 462 nm which gradually intensied with
increasing concentration of thiophenol (Fig. 2). The uores-
cence enhancement at 462 nm was up to 16 fold. The probe
responded linearly till addition of 1 equiv. of thiophenol. Upon
addition of more amount of thiophenol nominal increase in
To validate the sensing mechanism of probe 1, a relatively
large scale reaction was carried out by adding thiophenol to
probe 1 under the prevailing condition and the product was
isolated and characterized by H NMR and mass spectroscopy.
It was observed that the spectra of the product obtained from
1

uorimetric study exactly match with the spectra of compound
2; thus, conrming that the non-uorescent probe 1 was con-
verted back to strongly uorescent compound 2 by thiophenol
driven nucleophilic substitution reaction. It is well-established
that 2-(2-hydroxyphenyl)benzothiazole (2) shows uorescence
by excited state intramolecular proton transfer (ESIPT). Since
the sensing condition use neutral pH the newly liberated ben-
zothiazole derivative (2) remains in the protonated form. The
internal H-transfer occurs from phenolic –OH to –N– of ben-
zothiazole moiety of the molecule in the excited state which
causes large electron delocalization from one aromatic ring to
other and the molecule becomes uorescence active. The above
study clearly indicated that probe 1 is highly useful for the
detection of aromatic thiols by spontaneous “ESIPT”.

uorescence response was seen indicating that the probe is
3.4 Selectivity of aromatic thiols over other thiols
To conrm the selectivity of probe 1 towards thiophenol, it was
treated with a broad variety of species under the standard
condition and corresponding uorescence response was recor-
ded. The probe was separately treated with several L-amino
acids such as glycine, alanine, leucine, argenine, histidine,
proline, and thiol containing cysteine and glutathione. This
study also included an aliphatic thiol, 2-mercaptoethanol, and
aromatic competing nucleophiles phenol and aniline. It
revealed that each of the above mentioned species educed very
Fig. 1 Fluorescence response of probe 1 (30 mM) with (1 equiv.) and
without thiophenol at different pH (pH 1–9.0) (lex ¼ 365 nm).
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nominal uorescence response indicating that the probe is thiophenol) and the linearity was obtained from 9 ꢂ 10ꢀ M of
8
inactive to aliphatic thiols and nucleophiles under the prevail- thiophenol. Thus, the detection limit (LOD) and limit of
ꢀ
8
ing reaction condition (Fig. 3 and S1 of ESI†). The same study quantication (LOQ) of probe 1 was considered as 3 ꢂ 10
M
ꢀ
8
was extended to other aromatic thiols such as 2-amino- (3.3 ppb) and 9 ꢂ 10 M (10 ppb), respectively (Fig. 4).
thiophenol, 4-aminothiophenol, pyridine-2-thiol, 4-bromothio-
phenol and 4-nitrothiophenol. It was observed that 1 could
3
.6 Real sample analysis
efficiently sense the presence of most of the aromatic thiols
showing a profound uorescence response. However, probe 1
was found to be inactive to 4-nitrothiophenol indicating its
inefficacy for the detection of aromatic thiols having strong
electron withdrawing groups in the ring (Fig. 3 and S1 of ESI†).
This is an expected outcome because the nucleophilicity of
aromatic-SH group is signicantly reduced for 4-nitro-
Probe 1 could express its potential applicability as an efficient
analytical tool in the detection of thiophenols especially in the
environment. Therefore, probe 1 was applied for qualitative/
quantitative detection of thiophenol levels in several real
samples.
3.6.1 Detection of thiophenol in water samples. To
demonstrate the applicability of this probe for monitoring the
contamination of thiophenol in industrial effluent, a proof-of-
concept experiment was performed on water samples pre-
spiked with thiophenol. Thus, water samples (1 mL) were
collected from tap and river, ltered through microltration
membrane and then spiked with known quantities of thio-
phenol (1 mM) in DMF and marked as sample A and B. To these
samples, DMF and PBS were added to make the nal concen-
trations of thiophenol as 1.5 mM for sample A and 3.0 mM for
sample B in 45% DMF–PBS solution (pH 7.2). These samples
were further treated with probe 1 (30 mM). Aer 15 min of
incubation at room temperature, the uorescence responses of
the solutions were recorded and the intensity at 462 nm was
plotted onto the standard curve. As shown in Fig. 5, our analytic
tool worked efficiently in the detection of pre-spiked thiophenol
in water samples with high recovery (97–100.2%) [see ESI, Table
S1†].
N
thiophenol and therefore, the probe does not undergo S Ar
reaction at room temperature. In a separate competitive
experiment, the probe was exposed to 1 equiv. of thiophenol in
the presence 5 equiv. each of phenol, aniline and 2-mercap-
toethanol and corresponding uorescence response was
measured. As showed in Fig. 3 the presence of other nucleophile
could cause little effect in spontaneous detection of thiophenol
by probe 1.
3.5 Limit of detection
The limit of detection (LOD) is one of the most important
properties of any probe that needs to be assessed to understand
potential applicability of the probe as an analytical tool for real
sample analysis. It was observed that probe 1 is well verge to
detect trace level of thiophenols in water. Under the standard
condition probe 1 was found to respond to thiophenols below
micro molar concentration range (from 3 ꢂ 10ꢀ M of
8
3.6.2 Detection of thiophenol vapour by paper strips and
TLC plates. As thiophenol is volatile liquid and the resulting
vapor can cause severe health issues like headache and irrita-
tion, it is of immense importance to detect its vapors in the
atmosphere. The paper strip of probe 1 was prepared by soaking
it in a solution of probe 1 in DMF and then kept in a chemical
storage cabinet for 15 min where an uncapped bottle of thio-
phenol was preplaced. As a control experiment one more paper
Fig. 3 Maximum fluorescence response of probe 1 in 45% DMF–PBS
(
pH ¼ 7.2) upon addition of different amino acids, aliphatic thiols, other
nucleophiles (1 ¼ blank; 2 ¼ cysteine, 3 ¼ argenine, 4 ¼ leucine, 5 ¼ L-
proline, 6 ¼ alanine, 7 ¼ glycine, 8 ¼ histidine, 9 ¼ glutathione, 10 ¼
ꢀ
phenol, 11 ¼ aniline, 12 ¼ 2-mercaptoethanol, 13 ¼ HSO
3
, 14 ¼ thi-
ophenol, 15 ¼ pyridine-2-thiol, 16 ¼ 2-aminothiophenol, 17 ¼ 4-
aminothiophenol, 18 ¼ 4-bromothiophenol and 19 ¼ thiophenol in
the presence of 5 equiv. of other nucleophiles) in the presence of Fig. 4 A fluorescence intensity plot of the probe 1 against a low
a fixed concentration of probe 1 (30 mM) [excitation wavelength ¼ concentration range of thiophenol. The straight line was obtained
ꢀ8
3
65 nm].
from 9 ꢂ 10 M of thiophenol (lex ¼ 365 nm).
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probe 1 was used for the estimation of pre-spiked thiophenol in
a soil sample. In a typical experiment, 3 mL of probe 1 (30 mM)
in 45% DMF–PBS (pH 7.2) was added to soil sample (90 mg) pre-
spiked with known quantity thiophenol (1.2 mM) in ethanol.
This sample was mixed thoroughly, incubated at room
temperature for 15 min, aer which the solution was ltered for

uorescence measurement. Again, the concentration of thio-
phenol was cross-examined by plotting the uorescence inten-
sity value on the standard curve. A near equal level of
concentrations of thiophenol indicated that the probe is equally
effective in determination of aromatic thiols in the soil samples
as well.
3.7 Cytotoxicity and uorescent cell imaging
Another potential application of probe 1 could be in the
detection of the thiophenols in living cells. For this purpose, the
cytotoxicity of probe 1 was rst examined and it was used for the
detection of aromatic thiols in thiophenol contaminated HeLa
cells.
Fig. 5 Maximum fluorescence response of probe 1 towards the real
samples spiked with thiophenol (lex ¼ 365 nm).
strip was dipped in the same probe solution and placed in
storage cabinet without thiophenol. The whole strip, which was
placed in the cabinet with uncapped bottle of thiophenol,
started glowing under exposure of long wavelength UV light
3.7.1 Cytotoxicity assay. As a part of the standard protocol
the cytotoxicity of probe 1 was assessed on HeLa cell line using
MTT assay before uorescent cell imaging studies. In the
concentration range 1–20 mM of probe 1 hardly any cell death
was seen aer 24 h of incubation indicating the probe molecule
is non-toxic even at much higher concentration than that
required for the detection studies (see ESI, Fig. S3†).
(365 nm), in contrast, no uorescence was seen from the other
paper strip (Fig. 6). Similarly, probe 1 modied TLC plate
showed intense blue uorescence spot under long wavelength
UV light upon exposure to thiophenol for sometimes. Therefore,
it can be concluded that the vapours of thiophenol could
spontaneously react with probe 1 even on paper strip or TLC
plate, and converts it back to the highly uorescent compound
3.7.2 Fluorescence imaging of living cells. The usefulness
of probe 1 for uorescence imaging of thiophenols in the living
cells was investigated. Therefore, HeLa cells were pre-treated
with thiophenol (1 mM) in a growth medium and then incu-
bated with probe 1 (1 mM) for 30 min. A bright uorescence was
observed when the cell-line was placed under a Nikon eclipse
TS100 microscope (Fig. 7g). By contrast, in a control experi-
ment, incubation of HeLa cells with only probe 1 provided no
signicant uorescence (Fig. 7e). These observations indicate
that probe 1 is cell membrane permeable and able to interact
with the intercellular thiophenol as well to produce uores-
cence signals. In a separate control experiment, the HeLa cells
were pre-treated with thiophenol (1 mM), then incubated with N-
ethylmaleimide (1 mM, as a thiol scavenger), and then treated
with probe 1 (1 mM). As expected, no marked uorescence signal
(2). In a similar manner the TLC plates and paper strips were
also employed for the selective detection of aqueous thio-
phenol. For this purpose the aqueous solution of thiophenols
and other analytes were spotted over TLC plates at the same
area where the probe 1 were spotted. Similarly, the paper strips
soaked with probe 1 was dipped in aqueous solution of
respective analytes. In both the cases only the solution con-
taining thiophenol showed bright uorescence under long
wavelength UV light (354 nm) indicating that the same tech-
nique is equally applicable for the detection of thiophenols in
the solution phase as well (see ESI, Fig. S2†).
3.6.3 Detection of thiophenol in soil samples. To examine
whether the probe can detect thiophenol contamination in soil
Fig. 6 (i) Showing the effect of vapors of aromatic thiols on probe 1 Fig. 7 Detection of thiophenol in HeLa cells with probe 1. Phase
before and after exposure to thiophenol on TLC plate and (ii) showing contrast (a–d) and fluorescence images (e–h) of the HeLa cells after
the effect of vapors of aromatic thiols on the paper strips spiked with incubating with probe 1 (1 mM), thiophenol (1 mM), probe 1 (1 mM) in
probe 1 ((A) spiked with probe 1, (B) spiked with probe 1 and kept in presence of thiophenol (1 mM), probe 1 in presence of thiophenol (1
a cabinet containing open bottle of thiophenol).
mM) and N-ethylmaleimide (1 mM), respectively.
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was observed from cells (Fig. 7h), indicating the selective reac-
tion of probe 1 with thiophenol. These preliminary studies
revealed that probe 1 is useful for in vitro imaging to monitor
thiophenols in biological samples as well.
H. C. Goicoechea and R. Babiano, Anal. Methods, 2013, 5,
30–49; (c) M. E. Jun, B. Roy and K. H. Ahn, Chem.
Commun., 2011, 47, 7583–7601; (d) J. Chan, S. C. Dodanil
and C. J. Chang, Nat. Chem., 2012, 4, 973–984; (e) X. Chen,
G. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2012, 41,
4610–4630.
4
. Conclusion
6
(a) S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev.,
2013, 42, 6019–6031; (b) X. Chen, Y. Zhaou, X. Peng and
J. Yoon, Chem. Soc. Rev., 2010, 39, 2120–2135; (c) H. Peng,
W. Chen, Y. Cheng, L. Hakuna, R. Strongin and B. Wang,
Sensors, 2012, 12, 15907–15946; (d) C. Yin, F. Huo,
J. Zhang, R. Mart ´ı nez-M ´a n˜ ez, Y. Yang, H. Lv and S. Li,
Chem. Soc. Rev., 2013, 42, 6032–6059.
In summary, we have rationally designed a novel, “turn-on” type
uorimetric sensor for the detection of aromatic thiols in

aqueous systems. The probe was synthesized in high yield via
a one-pot method in water maintaining environment friendly
conditions all throughout. The function of the probe is based on
simple thiol driven cleavage of the dinitrophenyl ether linkage
of probe 1 to release uorescent 2-(2-hydroxyphenyl)benzo-
thiazole (2). The probe utilizes strong nucleophilic character of
aromatic thiols for their selective detection among many other
interfering species including thiol containing amino acids.
Probe 1 is effective for thiophenol (PhSH) and other aromatic
thiols with electron donating substituents but inactive to
aromatic thiols with strong electron withdrawing substituents
7 For selected recent references see: (a) B. Tang, Y. Xing, P. Li,
N. Zhang, F. Yu and G. Yang, J. Am. Chem. Soc., 2007, 129,
11666–11667; (b) X. Chen, S.-K. Ko, M. J. Kim, I. Shin and
J. Yoon, Chem. Commun., 2010, 46, 2751–2753; (c) V. Hong,
A. A. Kislukhin and M. G. Finn, J. Am. Chem. Soc., 2009,
131, 9986–9994; (d) M. H. Lee, J. H. Han, P.-S. Kwon,
S. Bhuniya, J. Y. Kim, J. L. Sessler, C. Kang and J. S. Kim, J.
Am. Chem. Soc., 2012, 134, 1316–1322; (e) Y.-Q. Sun,
M. Chen, J. Liu, X. Lv, J.-F. Li and W. Guo, Chem.
Commun., 2011, 47, 11029–11031; (f) M.-Y. Jia, L.-Y. Niu,
Y. Zhang, Q.-Z. Yang, C.-H. Tung, Y.-F. Guan and L. Feng,
ACS Appl. Mater. Interfaces, 2015, 7, 5907–5914; (g) L. Wang,
T. Yao, S. Shi, Y. Cao and W. Sun, Sci. Rep., 2014, 4, 5320,
DOI: 10.1038/srep05320; (h) Z. Chen, D. Lu, Z. Cai, C. Dong
and S. Shuang, Luminescence, 2014, 29, 722–727; (i) L. Yi,
H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem., Int.
Ed., 2009, 48, 4034–4037; (j) Y. Li, Y. Duan, J. Li, J. Zheng,
H. Yu and R. Yang, Anal. Chem., 2012, 84, 4732–4738; (k)
Y. Yue, C. Yin, F. Huo, J. Chao and Y. Zhang, Sens.
Actuators, B, 2016, 223, 496–500; (l) F. J. Huo, Y. Q. Sun,
J. Su, J. B. Chao, H. J. Zhi and C. X. Yin, Org. Lett., 2009,
11, 4918–4921; (m) Y. Yang, F. Huo, C. Yin, A. Zheng,
J. Chao, Y. Li, Z. Nie and R. Mart ´ı nez-M ´a n˜ ez, Biosens.
Bioelectron., 2013, 47, 300–306.
(e.g. 4-nitrothiophenol). The advantage of this probe is that it
shows excellent results under neutral condition at room
temperature facilitating its wide scope of applications at phys-
iological pH. Notably, the probe is found to be capable of
detecting pre-spiked thiophenols in water, soil and living cells.
We presume, the probe with green synthetic prole, cost
effective single-step synthesis, high selectivity and sensitivity,
ꢀ
8
fast signal transduction and low limit of detection (3 ꢂ 10 M)
would certainly be
applications.
a potential candidate for practical
Acknowledgements
A. C. thanks DST (India) (project no. SR/FT/CS-092/2009) for

nancial support. M. B. thanks CSIR (India) (project No.
02(0075)/12/EMR-II) for research grant. D. G. K. is indebted to
DST (India) for fellowship.
8
(a) J. Bouffard, Y. Kim, T. M. Swager, R. Weissleder and
S. A. Hilderbrand, Org. Lett., 2008, 10, 37–40; (b) H. Guo,
Y. Jing, X. Yuan, S. Ji, J. Zhao, X. Li and Y. Kan, Org.
Biomol. Chem., 2011, 9, 3844–3853; (c) X.-D. Jiang, J. Zhang,
X. Shao and W. Zhao, Org. Biomol. Chem., 2012, 10, 1966–
1968; (d) Z. Wang, D.-M. Han, W.-P. Jia, Q.-Z. Zhou and
W.-P. Deng, Anal. Chem., 2012, 84, 4915–4920; (e) W. Sun,
W. Li, J. Li, J. Zhang, L. Du and M. Li, Tetrahedron Lett.,
2012, 53, 2332–2335; (f) X. Shao, R. Kang, Y. Zhang,
Z. Huang, F. Peng, J. Zhang, Y. Wang, F. Pan, W. Zhang
and W. Zhao, Anal. Chem., 2015, 87, 399–405; (g) D. Kand,
P. S. Mandal, T. Saha and P. Talukdar, RSC Adv., 2014, 4,
59579–59586.
Notes and references
1
(a) K.-M. Roy, in Ullmann's Encyclopaedia of Industrial
Chemistry, Wiley, New York, 7th edn, 2007; (b) J. C. Love,
L. A. Estroff, J. K. Kriebel, R. G. Nuzzo and
G. M. Whitesides, Chem. Rev., 2005, 105, 1103–1170.
E. Bingham, B. Cohrssen and C. H. Powell, in Patty's
Industrial Hygiene and Toxicology, John Wiley & Sons, Inc,
New York, 5th edn, 2001, p. 722.
2
3
4
(a) T. P. Heil and R. C. Lindsay, J. Environ. Sci. Health, Part B,
1989, 24, 349–360; (b) E. J. Fairchild and H. E. Stokinger, Am.
Ind. Hyg. Assoc. J., 1958, 19, 171–188.
(a) R. Munday, Free Radical Biol. Med., 1989, 7, 659–673; (b)
T. P. Heil and R. C. Lindsay, J. Environ. Sci. Health, Part B,
9 (a) J. Li, C.-F. Zhang, S.-H. Yang, W.-C. Yang and G.-F. Yang,
Anal. Chem., 2014, 86, 3037–3042; (b) S. K. Malwal, A. Labade,
A. S. Andhalkar, K. Sengupta and H. Chakrapani, Chem.
Commun., 2014, 50, 11533–11535; (c) D. Kand, P. K. Mishra,
T. Saha, M. Lahiri and P. Talukdar, Analyst, 2012, 137,
3921–3924; (d) W. Lin, L. Long and W. Tan, Chem.
Commun., 2010, 46, 1503–1505; (e) W. Jiang, Q. Fu, H. Fan,
1
989, 24, 349–360; (c) P. Amrolia, S. G. Sullivan, A. Stern
and R. Munday, J. Appl. Toxicol., 1989, 9, 113–118.
5
(a) R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger and
T. Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936–3953; (b)
M. J. Culzoni, A. M. de la Pena, A. Machuca,
52796 | RSC Adv., 2016, 6, 52790–52797
This journal is © The Royal Society of Chemistry 2016

View Article Online
Paper
RSC Advances
J. Ho and W. Wang, Angew. Chem., Int. Ed., 2007, 46, 8445–
448; (f) X.-L. Liu, X.-Y. Duan, X.-J. Du and Q.-H. Song,
Alternative Solvents for Green Chemistry, RSC Publishing,
2nd edn, 2013.
8
Chem.–Asian J., 2012, 7, 2696–2702; (g) X. Liu, F. Qi, Y. Su, 15 X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2014, 114, 590–
W. Chen, L. Yang and X. Song, J. Mater. Chem. C, 2016, 4, 659.
320–4326; (h) W. Zhao, W. Liu, J. Ge, J. Wu, W. Zhang, 16 (a) D. G. Khandare, H. Joshi, M. Banerjee, M. Majik and
4
X. Meng and P. Wang, J. Mater. Chem., 2011, 21, 13561–
A. Chatterjee, Anal. Chem., 2015, 87, 10871–10877; (b)
A. Chatterjee, D. G. Khandare, P. Saini, A. Chattopadhyay,
M. S. Majik and M. Banerjee, RSC Adv., 2015, 5, 31479–
31484; (c) D. G. Khandare, H. Joshi, M. Banerjee,
M. S. Majik and A. Chatterjee, RSC Adv., 2014, 4, 47076–
47080; (d) D. G. Khandare, V. Kumar, A. Chattopadhyay,
M. Banerjee and A. Chatterjee, RSC Adv., 2013, 3, 16981–
16985; (e) S. Hazra, S. Balaji, M. Banerjee, A. Ganguly,
N. N. Ghosh and A. Chatterjee, Anal. Methods, 2014, 6,
3784–3790; (f) V. Kumar, M. Banerjee and A. Chatterjee,
Talanta, 2012, 99, 610–615.
13568.
1
1
1
1
1
0 W. Chen, Y. Zhao, T. Seefeldt and X. Guan, J. Pharm. Biomed.
Anal., 2008, 48, 1375–1380.
1 T. Wang, E. Chamberlain, H. Shi, C. D. Adams and Y. Ma, Int.
J. Environ. Anal. Chem., 2010, 90, 948–961.
2 O. Pluchery, C. Humbert, M. Valamanesh, E. Lacaze and
B. Busson, Phys. Chem. Chem. Phys., 2009, 11, 7729–7737.
3 J. Onuegbu, A. Fu, O. Glembocki, S. Pokes, D. Alexson and
C. M. Hosten, Spectrochim. Acta, Part A, 2011, 79, 456–460.
4 (a) J. C. Warner, A. S. Cannon and K. M. Dye, Environ. Impact
Assess. Rev., 2004, 24, 775–799; (b) R. A. Sheldon, Green 17 V. S. Padalkar and S. Seki, Chem. Soc. Rev., 2016, 45, 169–202,
Chem., 2005, 7, 267–278; (c) F. Kerton and R. Marriott, in and references cited there in.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 52790–52797 | 52797