A chemosensor for micro- to nano-molar detection of Ag+ and Hg2+ ions in pure aqueous media and its applications in cell imaging

Article abstract of DOI:10.1039/c7dt02524f

The pyridine substituted thiourea derivative PTB-1 was synthesized and characterized by spectroscopic techniques as well as by single crystal X-ray crystallography. The metal ion sensing ability of PTB-1 was explored by various experimental (naked-eye, UV

Full text of DOI:10.1039/c7dt02524f

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Patil, S. K. Sahoo, C. P. Pradeep, A. Churakov, F. Yu, L. Chen, C. Redshaw, A. A. Patil and U. D. Patil, Dalton
Trans., 2017, DOI: 10.1039/C7DT02524F.
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1
7
January 2016 Pages 1–398
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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
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a Zr-metal–organic
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ARTICLE
+
2+
†
Chemosensor for micro to nano-molar detection of Ag and Hg
ions in pure aqueous media and its applications in cell imaging
a
a
c
d
Jitendra P. Nandre , Samadhan R. Patil , Suban K. Sahoo , Chullikkattil P. Pradeep , Andrei
e
f
f
g
a
b
Churakov , Fabiao Yu , Lingxin Chen* , Carl Redshaw , Ashok A. Patil *, Umesh D. Patil*
The pyridine substituted thiourea derivative PTB-1 was synthesized and characterized by spectroscopic techniques as well
as by single crystal X-ray crystallography. The metal ion sensing ability of PTB-1 was explored by various experimental
1
(naked-eye, UV-Vis, fluorescence, mass spectrometry and
H NMR spectroscopy) and theoretical (B3LYP/6-
3
1G**/LANL2DZ) methods. PTB-1 exhibited a highly selective naked-eye detectable color change from colorless to dark
+
brown and UV-Vis spectral changes for the detection of Ag with a detection limit of 3.67 µM in aqueous medium. The
Received 00th July 2017,
Accepted 00th July 2017
+
detection of Ag ions was achieved by test paper strip and supported silica methods. In contrast, PTB-1 exhibited a 23-fold
2
+
enhanced emission at 420 nm in the presence of Hg ions with a nano-molar detection limit of 0.69 nM. Finally, the sensor
2
+
DOI: 10.1039/x0xx00000x
PTB-1 was applied successfully for the intracellular detection of Hg ions in a HepG2 liver cell line, which was monitored
by use of confocal imaging techniques.
www.rsc.org/
+
useful for the recovery of Ag from waste water, the latter
+
Introduction
mostly results from photographic industries. However, the Ag
ion has only moderate coordination ability which makes it
The synthesis and development of colorimetric and
fluorescence chemosensors for heavy transition metal ions is
gaining significant impetus due to their biological and
quite difficult to be separated from other chemically similar
metal ions.
1
-5
Additionally, mercury has been considered as the most
toxic metal ion amongst the heavy transition metal ions to
environmental importance. Among the transition metal ions,
+
2+
Ag and Hg ions have attracted considerable attention. The
excessive intake and long-term accumulation of silver ions can
1
3-15
humans,
and causes severe neurotoxic, immunotoxic and
6
genotoxic effects. Mercury contamination of ecosystems
-9 occurs through a number of sources, which include volcanic
and oceanic discharges, burning of fossil fuels and solid waste
lead to insoluble precipitates in the eyes and skin and also can
7
deactivate the normal functions of sulfhydryl enzymes.
+
Regular feasting of Ag can produce anemia, growth
1
6-17
incineration.
Mercury can be accumulated in human body
retardation, cardiac enlargement and degenerative changes in
1
animals. In addition, very strong Ag ion recognition is
0
+
through the food chain. When it is engrossed in the human
body, it can cause serious and lasting damage to the kidneys,
endocrine system, brain, nervous system and immune system
1
11
11-12
essential for
Ag-based radio immunotherapy
and is
1
8-19
when present at a very low concentration.
At present, a number of methods have been developed to
+
2+
detect Ag and Hg ions such as inductively coupled plasma
detectors, inductively coupled plasma mass spectrometry (ICP-
MS), fluorescence anisotropy assays, quantum dot based
assays, atomic absorption spectrometry (AAS) and inductively
2
0-22
coupled plasma atomic emission spectrometry (ICP-AES).
+
2+
Although these technologies can detect Ag and Hg ions
selectively with high sensitivity, they require exclusive and
sophisticated instrumentation, highly trained hands and
complex sample-preparation steps, which are major blockages
in their everyday use. On the contrary, the naked-eye
detection
method
allows
detection
up
to
micro/submicromolar levels and that too without involving any
expensive/sophisticated instruments. Silver and mercury are
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+
2+
normally found as ions (Ag and Hg ) in water. Therefore,
there is a high demand for highly selective and sensitive
+
2+
chemosensors for detecting Ag and Hg ions from aqueous
solution. However, most of the previously reported
chemosensors for this purpose have a number of downsides
viz. poor detection limit, tedious synthetic procedures,
intervention from other transition metal ions, long response
times, use of organic solvents, and most the reported sensors
Scheme 1 Synthesis of chemosensor PTB-1
+
2+
are selective either only for Ag or Hg ions only (Table S1).
Furthermore, the fluorescence sensing of cations, such as
2
+
3+
2+
2+
Hg , Fe , Pb and Cu is a particularly challenging task since
these ions mostly act as quenchers via electron transfers and
facilitated intersystem crossing processes. Therefore, it
remains a challenge to develop a “turn-on” fluorescence
2
+
sensor for Hg that can be applied in aqueous medium and
applied to living systems. Although there are several sensors
+ 2+
capable of recognizing Ag and Hg ions individually, to the
Fig. 1 Molecular structure of PTB-1 Displacement ellipsoids are drawn at the 50 %
probability level. Selected bond lengths (Å) and angles (o): S(1)-C(1) 1.6686(18); O(1)-
C(11) 1.224(2); N(1)-C(11) 1.385(2); N(1)-C(1) 1.390(2); N(1)-H(1) 0.83(2); N(2)-C(1)
best of our knowledge, only around 5-10 % of them are 1.329(2); N(2)-C(2) 1.407(2); N(2)-H(2) 0.82(2); N(3)-C(6) 1.330(3); N(3)-C(2) 1.334(2);
C(11)-N(1)-C(1) 128.03(17); C(11)-N(1)-H(1) 115.4(13); C(1)-N(1)-H(1) 115.5(13); C(1)-
selective and sensitive for both. Additionally, among the
2
+
N(2)-C(2) 133.00(17); C(1)-N(2)-H(2) 113.6(16); C(2)-N(2)-H(2) 113.3(16); O(1)-C(11)-
N(1) 121.54(18); O(1)-C(11)-C(12) 121.22(16); N(1)-C(11)-C(12) 117.25(16).
Intramolecular hydrogen bond is shown as dashed line: N(2)...O(1) 2.592(2); N(2)-
reported sensors most are chemodosimeters for Hg ions.
Thus, the development of sensitive and selective methods for
+
2+
the determination of trace amounts of Ag and Hg ions in H(2)...O(1) 144(2).
aqueous media is of considerable importance for both
environmental protection and human health. From literature,
we also note that the presence of sulfur (as a soft Lewis base)
+ 2+
is highly desirable in the ligands to be utilized for Ag and Hg
Naked-eye selectivity study of PTB-1
The recognition properties of PTB-1 were studied
experimentally toward different metal ions by naked-eye, UV-
Visible, and fluorescence methods. During the naked-eye
experiments (Figure 2), the colourless solution of PTB-1 [2 mL,
ion recognition.
Considering the above facts and as a part of our on-going
23-27
research into the design and synthesis of chemosensors,
-
4
we report herein a new pyridine substituted thiourea based
5
х 10 M, in CH OH:H O (20:80, v/v)] turned to dark brown
3 2
+
selectively only in the presence of Ag among the other tested
sensor PTB-1 (Scheme 1) for the selective and sensitive
+
2+
detection of Ag and Hg ions from 100 % aqueous solution.
+
2+
2+
+
+
2+
2+
3+
2+
3+
metal ions (Cs , Ba , Hg , K , Li , Cu , Cd , Al , Co , Cr ,
2
+
+
2+
2+
3+
2+
Mg , Na , Ni , Fe , Fe , Mn , Ca , Pb and Zn (200 µL, 1 х
2+
2+
2+
-2
0
1
M, in H
2
O). No significant color change of PTB-1 was
Results and discussion
observed in the presence of the other examined cations even
2
when present in excess, except for Hg which exhibited a
+
Synthesis of PTB-1
The synthesis of PTB-1 was achieved by using a simple slight yellow coloration. These results clearly indicated the
+
nucleophilic attack by the primary amine group of pyridine to selective colorimetric response of PTB-1 towards Ag .
the isothiocyanato group of phenyl isothiocyanate under reflux Furthermore, the concentration dependent naked-eye sensing
3
0
+
conditions in acetone (Scheme 1). The structure of PTB-1 was of Ag ion was performed (Figure S5), and we observed visible
1
characterized by IR, H-NMR, C-NMR spectroscopy, and color changes from colourless to brown color, even at low
13
-4
+
HRMS spectrometry (Figure S1, S2, S3, and S4). Finally, a concentration i.e. up to 5 х 10 M of Ag ions. Encouraged by
‡
+
suitable crystal of PTB-1 for single crystal X-ray diffraction was these Ag responses shown by PTB-1, the qualitative and
obtained from acetone, and the molecular structure is shown quantitative metal ion sensing ability of PTB-1 was determined
further by use of spectrophotometric methods.
in Figure 1.
2
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-
5
-2
Fig. 3 UV-Vis absorption spectra of PTB-1 (2 mL, 4 х 10 M) in CH
3
OH:H
2
O (20:80, v/v) (A) upon addition of 5 equivalents of various metal cations (40 µL, 1 х 10 M, in H
2
O);
+
-3
(
2
B) with the successive addition of 0-10 equivalents of Ag ions (1 х 10 M, in H O).
UV-Visible absorption study of PTB-1
new peak appeared at m/z = 491.2671 corresponding to the
+
The changes in the absorption spectrum of PTB-1 in complex [{(PTB-1)-2H + 2Ag} + Na] .
-5
+
CH
3
OH:H
2
O (20:80, v/v) (2 mL, 4 х 10 M) in the absence and
Based on the 1:2 stoichiometry for PTB-1.Ag
+
presence of 5 equivalents of different metal cations (40 µL, 1 х complexation, the binding constant (Ka) of PTB-1 with Ag was
- 31
0 M, in water) are shown in Figure 3A. The absorption bands determined using the Benesi-Hilderbrand plot analysis, by
2
1
of PTB-1 were significantly and selectively affected by the using the following equation (1).
+
addition of Ag ions. The receptor PTB-1 has two distinct
+
absorption bands at 210 nm and 265 nm. Upon addition of Ag
ions, the absorption bands of PTB-1 at 210 nm and 265 nm
were merged together and appeared as a broad band having
absorption maxima at 243 nm along with the advent of a
broad band between 350-600 nm. This observed wide-ranging
0
Where, A and A are the absorbance of the PTB-1 solution in
band between 350-600 nm is responsible for the generation of
+
the presence and absence of Ag ions, Amax is the saturated
the dark brown color. A slight spectral change of PTB-1 was
+
absorbance of PTB-1 in the presence of excess amounts of Ag ,
2
+
observed with Hg with the appearance of a new broad
absorption band having maxima at 214 nm. However, no
noticeable spectral changes were observed with the other
tested cations.
+
+
-1
Ag ] is the concentration of Ag ions added (mol L ). Finally,
[
+
the plotting of 1 / ΔA vs 1 / [Ag ] showed a linear relationship
Figure S9), and the binding constant (Ka) was determined
(
-1
from the slope and was estimated to be 15,963 M for the
+
PTB-1.Ag complexation.
To gain more insight into the cation chemosensing
properties and mechanism of PTB-1, absorption titration was
+
performed with Ag ions. As shown in Figure 3B, upon
+
sequential addition of Ag ions (0-10 equivalent, 1 х 10 M, in
-3
-
5
H
2
O) to the PTB-1 solution [2 mL, 4 х 10 M, in CH
20:80, v/v)], the intensity of the absorption band centered at
43 nm was uninterruptedly amplified with the diminishing of
3 2
OH:H O
(
2
the band at 265 nm. Also, a new broad band appeared
between 350 to 600 nm. From the absorption titration data, a
linear dependence of absorption at 243 nm was observed as a
+
function of the Ag concentration (Figure S6), and thereby the
+
stoichiometry of PTB-1 with Ag could be estimated to be 1:2.
Moreover, the mole ratio plot (Figure S7) and the Job’s plot
(
Figure 4) support the formation of a complex between PTB-1
+
and Ag in a 1:2 binding stoichiometry. Furthermore, direct
evidence for the formation of a 1:2 complex was obtained
from the ESI-MS spectra of PTB-1 in presence of 2.0
+
equivalents Ag in methanol/water (20:80, v/v) (Figure S8). The
-5
Fig. 4 Jobs plot for determining 1:2 stoichiometry of PTB-1 [4 х 10 M, in
+
O (20:80, v/v)] and Ag ions (4 х 10 M, in H
-5
main characteristic MS peak was observed at m/z = 258.0714 CH
+
PTB-1+H] ) for pure PTB-1. However, on addition of 2.0
+
equivalents of Ag , the peak at 258.0714 disappeared and a
3
OH:H
2
2
O).
([
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Further, the limit of detection (LOD) and limit of The cation binding affinity of PTB-1 was found to be ≈ 1,00,000
-1
2+
quantification (LOQ) of the receptor PTB-1 was calculated. M for Hg . Based on the fluorescence titrations, the LOD and
2
+
According to the IUPAC definition, the LOD and LOQ was LOQ of PTB-1 for Hg were found to be 0.69 nM and 0.02 nM,
calculated using the relationship LOD = (3.3 x standard respectively, and these values are an improvement on
deviation)/slop and LOQ = (10 x standard deviation)/slop. To reported sensors (Table S1). Moreover, the linear response
calculate the relative standard deviation, the absorption range of the chemosensor PTB-1 was calculated by plotting
measurements of ten blank samples were recorded. The ratio of fluorescence intensity vs. the increasing concentration
+
2+
+
calibration curves (absorbance vs [Ag ]) were plotted (Figure of Hg and Ag . The chemosensor exhibited a dynamic
2
S6), and then the obtained slope was used to calculate the LOD response range for Hg (Figure S14a) from 1.0 x 10 to 1.8 x
+
LOQ. The obtained LOD and LOQ value of PTB-1 for Ag were 10 M and 1.2 x 10 to 2.4 x 10 M for Ag (Figure S14b).
+
-7
-5
-6
-5
+
&
found to be 3.67 µM and 0.11 µM respectively
.
To examine the selectivity and reversibility measurement
+
2+
of PTB-1 to Ag and Hg , we carried out emission reversible
titrations by using an EDTA (Fig. S15). Upon addition of
Emission spectroscopic study of PTB-1
The cation binding behavior of the PTB-1 was also equimolar amount of EDTA solution [DMSO:H O (90:10, v/v)]
2
2
investigated by fluorescence spectroscopy. We observed a to the solution of PTB-1:Hg [4 х 10 M, in CH OH:H O (20:80,
+
-5
3
2
-5
remarkable fluorescence enhancement of PTB-1 [2 mL, 4 х 10
M, in CH OH:H O (20:80, v/v)] at 420 nm (λ = 290 nm) upon significant decrease in the fluorescence signal at 420 nm was
v/v)], the color changed from yellow to colorless and
3
2
ex
2
addition of Hg ions (40 µL, 1 х 10 M, in H O), while no observed (Fig. S15a). These results indicating that PTB-1
+
-2
2
2
significant changes were observed in the presence of other detects Hg ions reversibly. Sequential alternate addition of
+
2
tested metal ions (Figure 5A). The emission data of PTB-1 with Hg and EDTA solutions were repeated for several times,
+
2
+
Hg exhibited an ≈ 23-fold fluorescence enhancement at 420 indicates only a slight decrease in the emission intensity,
nm. In the fluorescence titration experiment (Figure 5B), up to suggesting the reusability of the PTB-1 (Fig. S15b). In contrast,
2
the addition of increasing amounts of Hg ions from 0-40 µL the alternate addition of equimolar solution of Ag and EDTA
+
+
-3
+
(
0-0.5 equivalent, 1 х 10 M, in H O), the emission bands of solutions [DMSO:H O (90:10, v/v)] to the solution of PTB-1:Ag
2 2
-5
PTB-1 at 420 nm increased dramatically, whereas less changes [4 х 10 M, in CH OH:H O (20:80, v/v)] showed no change in
2
3
in emission intensity were observed up to the addition of 80 µL color and fluorescence intensity (Fig. S15c,d). These results
+
0-1 equivalent) and a decrease was observed after the indicating that PTB-1:Ag complex was irreversible with EDTA.
(
2
addition of 1 equivalent (80 µL) of Hg ions to the 10
+
equivalents (800 µL). This suggested the formation of a host- Possible recognition mechanism
guest complex of 2:1 stoichiometry. To confirm this The molecular structure of PTB-1 and its complexes with Ag
+
2
stoichiometry, the Job’s plot analysis (Figure S10) and mole and Hg were computed by applying the DFT method (Figure
+
+
ratio plot (Figure S11) were performed. More direct evidence S16). The different possible binding modes of PTB-1 with Ag
2
+
for the formation of this 2:1 complex was evaluated from the and Hg were tested by considering the binding stoichiometry
ESI-MS spectra of PTB-1 in the absence and presence of 1.0 obtained from the Job’s plots and other analyses. As shown in
2
equivalent Hg in methanol/water (20:80, v/v) (Figure S12). Figure S16, the N, S and O atoms of PTB-1 are used on
+
+
The peaks at m/z = 715.0420 [{2(PTB-1)-2H+Hg}+H] and m/z = complexation with Ag , whereas a tetrahedral environment
+
+
58.0714 ([PTB-1+H] ) corresponded to the complex and the was provided by the pyridine-N and S atoms of two PTB-1
2
2
receptor alone, respectively. Then, the binding constant (Ka) of molecules to complex with Hg . On complexation of PTB-1
+
2
+
+
2+
PTB-1 with Hg was determined by a Benesi-Hildebrand plot with Ag and Hg , the interaction energy (E = Ecomplex-Ereceptor
-
int
2
analysis by using the fluorescence titration data (Figure S13). EHg /Ag ) was lowered by -390.86 kcal/mol and -263.86
+
+
4
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kcal/mol respectively, which indicates the formation of a possibility of fluorescence quenching through PET in the (PTB-
2
+
stable complex. The plots of the frontier molecular orbitals of 1)
2
.
Hg complex.
Further, we carried out H NMR spectroscopic titration
analyzed (Figure 6). The chelation of PTB-1 with metal ions experiments on PTB-1 solutions with various equivalents of
2
+
+
1
the receptor PTB-1 and its complexes with Hg and Ag were
2
+
+
resulted in the lowering of the band gap between the highest Hg and Ag ions separately (Figure S17-S18). On addition of
2
+
occupied molecular orbital (HOMO) and the lowest 0.5 equiv. of Hg ions, it was observed that the peaks due to
unoccupied molecular orbital (LUMO) due to the the two NH protons of PTB-1 at 13.12 and 9.07 ppm
intramolecular charge transfer (ICT), which may be responsible disappeared, whilst a broad peak appeared at 16.13 ppm
for the naked-eye detectable color change and appearance of probably due to the OH group (Figure S17). In addition, some
a new absorption spectral band in the visible region. Also, the of the aromatic protons of PTB-1 exhibited an up-field shift
+
calculated band gap for the PTB-1.(Ag )
2
complex was found to (from 8.83, 8.46 & 7.78 ppm to 8.46, 8.05 & 7.67 ppm,
2
+
be lower than the (PTB-1)
2
.Hg complex. These calculated respectively) while some other aromatic protons exhibited a
results corroborated well with the metal ion selectivity down-field shift (from 7.92, 7.54 & 7.18 to 8.06, 7.58 & 7.32
2
observed by UV-Vis absorption spectroscopy. Further, to ppm, respectively) in the presence of Hg ions accompanied
+
2
understand the fluorescence enhancement of PTB-1 with Hg , by a broadening of the peaks. Similar NMR studies conducted
+
+
the electron density distribution of the HOMO and LUMO in on PTB-1 by adding various equivalents of Ag also exhibited
2
+
2
PTB-1 and the (PTB-1) .Hg complex was analyzed. In the free the disappearance of both the NH protons of PTB-1 at 13.12
+
receptor, the HOMO was localized mainly over the S atom and and 9.07 ppm (Figure S18); 2.0 equiv. of Ag ions are required
the LUMO was distributed uniformly over the receptor which for the complete disappearance of these peaks. However, in
2
allowed for the photoinduced electron transfer (PET) process contrast to the case of Hg addition, no broad peak at 16.12
+
+
from the chelating moiety to the fluorophore moiety. Upon ppm appeared on addition of Ag ions. In addition, some of the
2
complexation with Hg , the electron density over the S atom aromatic protons of PTB-1 revealed an up-field shift (from 8.83
+
2
of PTB-1 was transferred to the Hg ions which inhibited the & 8.46 ppm to 8.37 & 8.06 ppm, respectively) whilst others
+
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+ 2+
exhibited a downfield shift (from 7.92, 7.54 & 7.18 ppm to pH range for the use of PTB-1 for detecting Ag and Hg ions
+
.99, 7.59 & 7.51 ppm, respectively) on addition of Ag ions, was 5 - 9.
7
which was accompanied by broadening of peaks. It is
noteworthy that, some aromatic protons of PTB-1 experience Test strips and supported silica based applications of PTB-1
slight net increase in shielding may be attributed to anisotropic In order to ensure that PTB-1 is potentially of practical use,
2
+
+
PTB-1 loaded test strips were prepared to detect Ag ions from
effect and conformation changes after coordination with Hg
+
or Ag .
33
an aqueous environment. The desired test strips were
Direct evidence for the formation of complexes of the PTB- prepared by soaking small strips of cellulose paper (Whatman
2
+
+
-2
with Hg and Ag was obtained from HR-MS analysis. HR-MS No. 42) in the solution of PTB-1 (1х10 M) in methanol and
1
spectra of pure PTB-1 exhibited a characteristic MS peak at were dried in air. When these strips were treated with 10 mL
+
m/z = 258.0714 corresponding to [(PTB-1) + H] (Figure S4). aqueous solution of Ag (1х10 M), the colorless strips sharply
+
−4
2
+
However, on addition of 0.5 equivalents of Hg ion, a new turned to a dark brown color (Figure 8A and supporting video).
peak appeared at m/z = 715.0420, corresponding to the The rapid color change of the test strip in solution clearly
+
species [{2(PTB-1) − 2H + Hg} + H] , which confirmed the inferred the practical application of PTB-1. To evaluate the
2
formation of a 2:1 complex of PTB-1 with the Hg ion (Figure applicability of the PTB-1 loaded test strips for the quantitative
+
+
+
S12). Similarly, on addition of 2.0 equivalents of Ag , the determination of Ag in aqueous solution, the test strips were
+
parent ion peak of PTB-1 at m/z = 258.0714 disappeared and a immersed in different concentrations of Ag shows a clear
new peak appeared at m/z = 491.2671 corresponding to the color change (Figure S20). The detection limit of the test strips
+
species [{(PTB-1) − 2H + 2Ag} + Na] , suggesting the formation was found to be 1 x 10 M, allowing for sensitive detection of
-6
+
of a 1:2 complex (Figure S8). Additionally, FT-IR comparative Ag ions.
2
studies of PTB-1 and PTB-1.Hg complex were performed
+
+
The sensing of Ag by PTB-1 also worked on a solid support.
(
Figure S19). The shifting of the –NH, C=S and C=O bands also The silica gel (60-120 mesh, 5.0 g, colorless) was soaked in
−2
supports the proposed binding mechanism.
PTB-1 (in methanol, 5 mL, 1х10 M) and then the solvent was
removed. A very faint off-white color for the silica gel
appeared, which indicated that the adsorption of the receptor
Interference and pH effects
The UV-Vis and emission responses of receptor PTB-1 to on the surface. When it was treated with 10 mL aqueous
+
various possible interfering metal ions and its selectivity for solution of Ag (1х10 M), the colorless solution promptly
−4
+
2+
Ag and Hg ions were tested. To study this, two equivalents turned to a dark brown color (Figure 8B & supporting video).
+
of Ag and Hg ions (16 µL, 1 х 10 M, in H O) were added to The instantaneous color change of the solid silica gel in
2+
-2
2
-5
the solution containing PTB-1 [4 х 10 M, in CH OH:H O solution clearly inferred the practical application of PTB-1 for
3
2
+
20:80, v/v)] and two equivalents of the other competitive the qualitative detection of Ag in aqueous media.
(
-2
metal ions (16 µL, 1 х 10 M, in H O) of interest; their
2
absorption and emission spectra were recorded. From the Live cells imaging study of PTB-1
+
absorption study, the Ag selectivity of PTB-1 was not
interfered by the presence of the other tested ion, including
2
+
2+
Hg (Figure 7A). Similarly, the fluorescent sensing of Hg by
chemosensor PTB-1 was found to be barely affected by a
number of commonly co-existing miscellaneous competitive
+
cations, including Ag (Figure 7B). Additionally, the UV-Vis and
fluorescence responses of the sensor PTB-1 in acidic and basic
pH regions were examined. It was observed that the optimum
Fig. 9 Fluorescence confocal microscopic images of living HepG2 cells incubated with
2
+
Hg : (A) cells loaded with 10 μM of PTB-1 for 20 min as control, (C) cells in ‘A’ loaded
2+
with Hg 100 μM for 0.5h, (D) overlay of fluorescence channels in ‘C’, (B and E) bright
field images of ‘A’ and ‘C’, respectively. The scale bar is 20 μm.
The fluorescent behavior of PTB-1 was applied to the
2
+
intracellular detection and monitoring of Hg in a HepG2 liver
cell line i.e. the human hepatocellular liver carcinoma cell line.
After being incubated with PTB-1 (10 μM) in RPMI 1640 for 20
min, the cells in Figure 9A were imaged by a confocal
6
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fluorescence microscope. As expected, no fluorescent image spectra were recorded on a Perkin-Elmer Spectrum One FT-IR
was observed. Then, the HepG2 cells loaded with PTB-1 were spectrometer as potassium bromide pellets and nujol mulls,
2
incubated with 100 μM of Hg for 0.5 h in RPMI 1640 medium unless otherwise mentioned. IR bands are expressed in
+
o
-1
1
at 37 C, and then washed with RPMI 1640 to remove excess frequency (cm ). The H and C NMR spectra were recorded
13
2
+
Hg ions and were imaged (Figure 9C). As shown in Figure 9C on a Jeol JNM-ECX 500 MHz multinuclear probe NMR
and 9D, there was a significant increase in the intracellular spectrometer at ambient temperature in DMSO with TMS as
fluorescence emission intensity compared with the control internal standard and chemical shifts are reported in ppm. The
cells in Figure 9A and 9B, which indicated the ability of PTB-1 abbreviations s, d and t stand for singlet, doublet and triplets,
2
+
to detect intracellular Hg ions. Moreover, the fluorescence respectively. Mass spectra were recorded on a Bruker
-5
stability of PTB-1 and PTB-1 + 0.5 eq. Hg (II) [(2 mL, 4 х 10 M) Compact HD mass spectrometer. UV-Vis spectra were
in CH OH:H O (20:80, v/v)] was also evaluated (Figure S21), the recorded on a U-3900 spectrophotometer (Perkin Elmer Co.,
3
2
fluorescence intensity hardly effected within the range from 0 USA) with a quartz cuvette (path length=1 cm). Fluorescence
to 3 h. On this basis, we suggested that PTB-1 owned good spectra were recorded on a Fluoromax-4 spectrofluorometer
fluorescence stability.
To access the potential toxicity of PTB-1, MTT assays
(HORIBA JobinYvon Co., France).
3
4
6
were carried out. HepG2 cells (10 cells/mL) were planted into Synthesis of PTB-1: In a 100 mL three necked flask fitted with a
6-well microtiter plates in DMEM with 10% fetal bovine reflux condenser and dropping funnel, ammonium thiocyanate
serum (FBS). Plates were maintained at 37 °C in a 5% CO /95% (5.0 gm, 0.0656 mol) in 25 mL of dry acetone was placed. To
air incubator for 12 h. Then the cells were incubated for 24 h this solution, benzoyl chloride (9.23 gm, 0.0656 mol) in 10 mL
at 37 °C in a 5% CO /95% air upon different concentrations of dry acetone was added drop wise using a dropping funnel.
9
2
2
probe of 0 μM to 100 μM respectively. MTT solution (5.0 After completion of the addition, the reaction mixture was
mg/mL, PBS) was then added to each well. After 4 h, the refluxed for 15 to 20 min. To this refluxed solution, 2-amino
remaining MTT solution was removed, and 200 μL of DMSO pyridine (6.18 gm, 0.0656 mol) dissolved in 15 mL of dry
was added to each well, shaking 10 min to dissolve the acetone was added drop wise. Refluxing was continued for 2 h,
formazan crystals at room temperature. Absorbance was and the reaction progress was monitored by TLC. After
measured at 490 nm in a TECAN infinite M200pro microplate completion of the reaction, the reaction mixture was cooled to
reader. The high cells viability of PTB-1 indicated that the room temperature and was poured into ice cold water with
probe displayed low cytotoxicity to living cells (Figure S22).
vigorous stirring. The yellow colored solid was filtered, washed
with cold water (3 х 20 mL) to give the crude product. The pure
PTB-1 was afforded after recrystallization from acetone.
Crystal structure of PTB-1
5 4
The pyridine substituted thiourea fragment (C H
11 3
N)-NH-C(=S)- Yield: 91 %; Mol. Formula: C13H N OS; Mol. Weight: 257.06 g;
0
-1
NH-C(=O)- is planar within 0.103(1) Å (Figure 1). This Physical Nature: Yellow solid; m.p.: 108-110 C; IR (cm ) [KBr]:
conformation is stabilized by a short intramolecular hydrogen 3277, 3224, 3005, 1678, 1597, 1550, 1438, 1284, 1251, 1157,
.
..
…
1
3
bond N(2)-H(2) O(1) with an N O separation of 2.592(2) Å. 1109, 885; H NMR (500 MHz, CDCl , δ ppm): 7.18 (t, J = 7.5
The phenyl ring C(12)-C(17) is slightly skewed around the Hz, 1H, Ar-H ), 7.54 (t, J = 7.5 Hz, 2H, Ar-H), 7.65 (t, J = 7.5 Hz,
C(11)-C(12) bond; the torsion angle O(1)-C(11)-C(12)-C(13) is 1H, Ar-H), 7.78 (t, J = 7.5 Hz, 1H, Ar-H), 7.92 (d, J = 10.0 Hz, 2H,
equal to 25.6(3)°. According to the Cambridge Structural Ar-H), 8.46 (d, J = 5.0 Hz, 1H, Ar-H), 8.83 (d, J = 10 Hz, 1H Ar-H),
3
2
13
3
Database, all the bond lengths and angles in this structure 9.07 (s, 1H, NH), 13.12 (s, 1H, NH); C NMR (125 MHz, CDCl , δ
exhibit ordinary values. In the structure, adjacent molecules ppm): 116.0, 121.4, 127.5, 129.2, 131.6, 133.7, 137.69, 148.6,
…
are connected as centrosymmetric dimers via N(1)-H(1) S(1) 151.2, 166.4, and 176.9; HRMS (ESI): m/z: Calculated for
+
OS: [M+H] 258.06, Found: 258.0714.
hydrogen bonds (Figure S23).
C
13
H
11
N
3
Experimental
X-ray diffraction studies: Crystal data for PTB-1
:
C
13
H
11
N
3
O
1
S
1
,
M
=
257.31, monoclinic, a=5.2573(8),
Materials and measurements
3
b=20.338(3), c=11.7359(17) Å,
space group P2 /n, Z=4, D =1.362 g/cm , F(000)=536, μ(MoK
.249 mm , colourless plate with dimensions ca.
.32 0.09 0.01. Total of 8081 reflections (2343 unique, Rint
.0418) were measured on a Bruker SMART APEX II
radiation,
-scan mode at 150 K. The structure was
β=90.393(2)°, V=1254.8(3) Å ,
All the starting reagents and metal perchlorates were
purchased either from S.D. Fine chemicals or Sigma Aldrich
depending on their availability, and were used as received. All
the solvents were of spectroscopic grade and were used
without further treatment. The purity of the compounds and
the progress of reactions were determined and monitored by
means of analytical thin layer chromatography (TLC). Pre-
coated silica gel 60 F254 (Merck) on alumina plates (7х3 cm)
were used and visualized by using either an iodine chamber or
a short UV-Visible lamp. Melting points were recorded on the
Celsius scale by open capillary method and are uncorrected. IR
3
1
c
ꢀ
)=
-
1
0
0
0
×
×
=
diffractometer (graphite monochromatized MoK
ꢀ
λ=0.71073 Å) using
ω
solved by direct methods and refined by full matrix least-
2
squares on F with anisotropic thermal parameters for all non-
2
8
hydrogen atoms. All H atoms were found from difference
Fourier synthesis and refined isotropically. The final residuals
were: R
1
= 0.0373, wR
2
= 0.0797 for 1720 reflections with I>2σ(I)
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ARTICLE
Journal Name
and 0.0628, 0.0886 for all data and 207 parameters. Goof= micromolar (3.67 µM) and nanomolar (0.69 nM)
+
-
3
1
.006, maximum ∆ρ= 0.229 e
×
Å .
concentrations, respectively. The sensor PTB-1 recognized Ag
2
+
and Hg
with
a
1:2 and 2:1 binding stoichiometry,
Computational methods: The Gaussian 09W computer respectively. Confocal microscopy images indicate that PTB-1
2
program was used for all theoretical calculations.
9
2+
can be used for detecting changes in Hg levels within living
+
Optimization of PTB-1 and its complexes with Ag and Hg
2+
cells. Also, PTB-1 can be applied for colorimetric detection of
+
were carried out without symmetry constraints by applying the Ag in the aqueous media by naked-eye using test strip and
B3LYP/6-31G(d,p) method in the gas phase. The basis set silica support methods.
LANL2DZ was used for Ag and Hg atoms.
Acknowledgements
Spectroscopic Study: The aqueous stock solutions of cations of
-
2
-1
concentration 1 х 10 mol L were prepared from their
The author Dr. U. D. Patil is grateful for the financial support
from the Department of Science & Technology, New Delhi,
India (Reg. No. SB/FT/CS-039/2013). X-ray diffraction studies
were performed at the Centre of Shared Equipment of IGIC
RAS. The author Dr. F. Yu, and L. Chen are grateful to ‘The
National Natural Science Foundation of China’ (NO. 21275158)
and ‘The program of Youth Innovation Promotion Association’,
Chinese Academy of Sciences.
corresponding salts i.e. AgClO
Ca(NO .4H O, Cd(ClO .H O, Co(ClO
CsNO , KNO , NaNO , Fe(ClO .xH O, HgCl
Mn(ClO .H O, Ni(ClO .6H O, Fe(ClO .H O, Pb(ClO
Zn(ClO .6H O and Cu(ClO .6H
for all spectroscopic studies after appropriate dilution. The
4
, Al(ClO
4
)
3
.9H
O, Cr(ClO
, LiBr, Mg(ClO
.3H
2
O, Ba(ClO
4
2
4
2
2
) ,
3
)
2
2
4
)
2
2
4
)
2
.6H
2
4
)
3
.6H
O,
3
3
3
4
)
2
2
2
2
) ,
4
)
2
2
4
)
2
2
4
)
3
2
4
)
2
O,
4
)
2
2
4
)
2
2
O. These solutions were used
-2
-1
stock solution of PTB-1 (1.0 х 10 molL ) was prepared in
-5
-1
methanol and then diluted to 4 х 10 molL with CH
20:80, v/v) mixed solvents. For the spectroscopic (UV-Vis and
fluorescence) titrations, the required amount of the diluted Notes
3 2
OH:H O
(
-5
-1
receptor PTB-1 [2 mL, 4 x 10 molL , in CH
v/v)] was taken directly into the cuvette and the spectra were
recorded after each successive addition of cations (0-240 µL, 1 the link below) by e-mailing data_request@ccdc.cam.ac.uk, or
3
OH:H
2
O (20:80,
‡
CCDC-986043 contains the supplementary X-ray data for this
paper. These data can be obtained free of charge via (please use
-3
-1
by contacting: The Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge. CB21EZ, UK. Fax: +44(0)1223-336033.
www.ccdc.cam.ac.uk/data_request/cif.
2
x 10 molL , in H O) by using a micropipette. The interaction
+
2+
of PTB-1 with Ag and Hg ions were completed in less than 2
minutes, and therefore all readings were taken 2 minutes after
the addition.
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0
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1
2
3
4
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| Dalton Trans., 2017, 00, 1-8
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