2-(2′-Hydroxyphenyl)-benzothiazole (HBT)-terpyridine conjugate: A highly specific ICT based fluorescent probe for Zn2+ ions and its application in confocal cell imaging

Article abstract of DOI:10.1016/j.jphotochem.2017.10.009

A new fluorescent sensor based on benzothiazole-terpyridine conjugate is reported. It functions as a fluorescent sensor that is highly selective for the Zn²⁺ ions via internal charge transfer (ICT) mechanism in DMSO: H₂O medium. The

Full text of DOI:10.1016/j.jphotochem.2017.10.009

Accepted Manuscript
Title: 2-(2’-Hydroxyphenyl)-benzothiazole (HBT)-terpyridine
conjugate: A highly specific ICT based fluorescent probe for
Zn2+ ions and its application in confocal cell imaging
Authors: Sunanda Sahana, Gargi Mishra, Sri Sivakumar,
Parimal K. Bharadwaj
PII:
S1010-6030(17)31148-6
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2017.10.009
JPC 10933
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
4-8-2017
6-10-2017
7-10-2017
Please cite this article as: Sunanda Sahana, Gargi Mishra, Sri Sivakumar, Parimal
K.Bharadwaj, 2-(2’-Hydroxyphenyl)-benzothiazole (HBT)-terpyridine conjugate: A
highly specific ICT based fluorescent probe for Zn2+ ions and its application
in confocal cell imaging, Journal of Photochemistry and Photobiology A:
Chemistry https://doi.org/10.1016/j.jphotochem.2017.10.009
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2-(2'-Hydroxyphenyl)-benzothiazole (HBT)-terpyridine conjugate: A highly
specific ICT based fluorescent probe for Zn2+ ions and its application in
confocal cell imaging
Sunanda Sahanaa, Gargi Mishrab, Sri Sivakumarb and Parimal K. Bharadwaja*
aDepartment of Chemistry, Indian Institute of Technology Kanpur, Kanpur-208016, India
bDepartment of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur-208016,
India
* E-mail: pkb@iitk.ac.in
Graphical abstract
A benzothiazole-terpyridine conjugate is designed for Zn2+ ions sensor via internal charge transfer
(ICT) mechanism with 42 nm blue-shift in emission spectra with a very low detection limit 35 nM
within a biological pH range of 6-8.
Zn2+
Highlights




One benzthiazole based chemosensor for Zn2+probe has been synthesized.
1:1 stoichiometry of probe to Zn2+ was confirmed by ESI-MS.
Exhibited a good binding constant and lowest detection limit towards Zn2+
The probe is also able to function within a broad biological pH range of 6-8.
ABSTRACT
1

A new fluorescent sensor based on benzothiazole-terpyridine conjugate is reported. It functions as
a fluorescent sensor that is highly selective for the Zn2+ ions via internal charge transfer (ICT)
mechanism in DMSO: H2O medium. The 42 nm blue-shifted emission upon addition of Zn2+ to
the sensor is attributed to the capture of Zn2+ by the terpyridine moiety that inhibits ICT from the
1
terpyridine to the benzthiazole moiety. The sensing is supported by the Job’s plot, ESI-MS, H
NMR titration experiments. Importantly, this probe shows high selectivity over other biologically
relevant metal ions. Also, anions do not show any effect. In addition, the detection limit is found
to be up to 35 nM which is acceptable within the EPA (US) limit. The probe is also able to function
within a broad biological pH range of 6-8.
Keywords: Fluorescence; Internal Charge Transfer; Terpyridine; Zn2 + ions detection
1. Introduction
The zinc ions, as the second most abundant and essential trace element in the human body
after iron, play a significant role in various fundamental biological processes, such as
neurotransmission, cellular metabolism, enzyme regulation, gene expression and so on [1-233].
The disorder of Zn2+ metabolism leads to many neurological diseases, including Alzheimer’s
disease (AD), cerebral ischemia, and epilepsy [4-56]. There have been continuous efforts [7] to
have better fluorescence chemosensors for the Zn2+ ions as direct and accurate spatiotemporal
concentration of this metal ion can be made via fluorescence signaling in purposely built systems.
[8-9101112].
Intramolecular charge-transfer (ICT) mechanism has been widely used as the basis for the
design of ratiometric fluorescent sensors because metal binding to sensors induces a large shift in
the absorption and fluorescence maxima [13-141516]. Ratiometric analysis reduces the
environment-dependence, photo bleaching, variations in excitation intensity, emission collection
2

efficiency, and the physical or chemical fluctuations in the sample. Because of the intermolecular
charge transfer (ICT) effect, ICT fluorophores with conjugated electron donor (D) and receptor
(A) are able to display visible ICT absorption band and large Stokes shift in emission maxima.
When a fluorophore contains an electron-donating group (donor) conjugated to an electron-
withdrawing group (acceptor), it undergoes ICT from the donor to the acceptor upon excitation by
light. The consequent change of the dipole moment leads to a larger Stokes shift, which is
influenced by the microenvironment of the fluorophore. If the electron-rich terminal of the
fluorophore interacts with a cation, a partial positive charge is photo-generated adjacent to the
cation, and that affects the absorption or emission spectral wavelength of the fluorophore with an
ICT excited state. So when a cation interacts with the donor group, the ground state is more
stabilized than excited state, and this leads to blue shift of the absorption and emission spectra.
Alternatively when a cation interacts with the electron withdrawing group (acceptor group), the
excited state is more stabilized by the cation than in the ground state, and this leads to a red- shift
of the absorption and emission spectra [17]. This mechanism is quite general, and therefore, the
development of novel fluorescent sensors based on typical ICT fluorophore is an attractive and
promising strategy [18-1920].
A series of novel heterocycle-based fluorescent dyes with donor-π-bridge-acceptor- (D-π-A)
structural motif is reported, where benzothiazole serves as a strong electron-acceptor unit[21-
2223].The strong electron withdrawing capability arises due to the unsymmetrical nature of the
benzothiazole unit with nonzero dipolar moment and partially push-pull character. 2, 2’: 6’, 2’’ -
Terpyridine unit has gained much interest in recent years as typical metal-binding domain in the
fields of supramolecular and coordination chemistry as well as materials science [24,25]. The
3

terpyridine unit possessing three near-coplanar nitrogen atoms, which has been widely used as
building block for transition metal cations in supramolecular chemistry [26-27].
In this work, the ICT fluorophore, 2-(2’-Hydroxy-5’-methylphenyl) benzothiazole has been
synthesized. To meet the demand of high selectivity toward Zn2+, a strong chelator, i.e., 4-[2,2':6',
2''-terpyridin]-4'-yl-benzenamine moiety has been incorporated into benzothiazole moiety to
design D-π-A motif. When the terpyridine moiety coordinates to a Zn2+, the electron density of
that moiety will be decreased, lowering the electron-donating ability of the amine in the sensor
leading to its detection.
2. Experimental
2.1. Materials and methods
Reagent grade, 2-hydroxy-5 methyl benzaldehyde and all metal perchlorate salts were acquired
from Aldrich Chemicals (USA) and were used as received. 2-acetylpyridine, 4-nitrobenzaldehyde,
2-aminobenzenethiol, KOH, NH4OH, SnCl2, hexamine and the solvents were procured from S. D.
Fine Chemicals (India). All the solvents were purified prior to use following standard procedures.
The perchlorate metal salts are used in titration experiments. Chromatographic separations were
done by column chromatography using silica gel (100-120 mesh) and basic alumina from S. D.
Fine Chemicals (India). The variation of pH was achieved with dilute hydrochloric acid and
sodium hydroxide. The reagents required for confocal cell imaging and MTT assay studies like
Phosphate buffer saline, gelatin, Dulbecco’s-modified eagle’s medium (DMEM), penicillin-
streptomycin antibiotic, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)
were also procured from Gibco.
4

All the synthesized compounds were characterized by various spectroscopic methods. Both
1H NMR (500 MHz) and 13C NMR spectra (125 MHz) of the compounds were recorded on a JEOL
DELTA2 spectrometer in CDCl3 with TMS as the internal standard. The ESI-MS data were
obtained from a WATERS Q-Tof Premier Mass Spectrometer. UV-vis spectra were recorded on a
Shimadzu 2450 UV-vis spectrophotometer at 21 °C. Fluorescence emission spectra were obtained
on a Perkin-Elmer LS 50B Luminescence Spectrometer at 21 °C. The pH of different solutions
were measured by using a pH meter model Eco testr pH I by Thermo Scientific (USA).
2.2. UV-vis and fluorescence spectroscopic studies
Luminescence properties of L2 were evaluated in DMSO: H2O (3:2, v/v, 10 mM HEPES
buffer, pH = 7)). Stock solution of L2 was prepared at a concentration of 10−3 M in 25 mL of
DMSO and then diluted to the desired concentration. Stock solutions of various ions were prepared
at the concentration of ~10−4 M in 25 mL distilled water and then diluted to the desired
concentrations. Absorbance and fluorescence spectral data were recorded 1 min after the addition
of the ions. For fluorescence measurements, excitation wavelength was done at 360 nm (slit width
= 10/10 nm) and emissions were acquired from 400 nm to 700 nm.
2.3. Synthesis
The probe L2 was synthesized in several steps as illustrated in Scheme 1.
2.3.1. Synthesis of 4′-(4-nitrophenyl)-2,2′:6′,2’’-terpyridine (A)
The compound A was synthesized from a known procedure [28]. In a 500 mL round bottom flask,
a mixture of 2-acetylpyridine (5 g, 41.5 mmol) and 4-nitrobenzaldehyde (3.17 g, 21 mmol) in
CH3OH (170 mL) was stirred vigorously with 15% aq. KOH (15 mL) and conc. NH4OH (160 mL)
for 3 days at room temperature. The deep brown colored precipitate was filtered and washed with
5

distilled water and then with cold methanol. The precipitate was further dissolved in 100 mL ethyl
acetate. The organic fraction was washed with 1% NaHCO3 solution three times. The organic layer
was dried over anhydrous sodium sulphate. After complete removal of the solvent under vacuum,
the crude brown product was recrystallized from absolute ethanol to give the pure product (Yield:
34%). m.p. 210–211 0C; 1H NMR (500 MHz, CDCl3)  (ppm): 8.74 (m, 4H), 8.69 (d, J = 6.85 Hz,
2H), 8.37 (d, J = 8 Hz, 2H), 8.05 (d, J = 8 Hz, 2H), 7.90 (t, J = 7.4 Hz, 2H), 7.37 (t, J = 7.45, 2H)
(Fig. S1). 13C NMR (125 MHz, CDCl3, 25 °C, TMS) (ppm): 119.04, 121.51, 124.26, 128.39,
137.11, 145.58, 149.15, 155.78 (Fig. S2). ESI-MS: m/z (%): 355.1198 (100%) [M+H]+(Fig. S3).
Elemental analysis: calculated (%) for C21H14N4O2: C 71.16, H 3.98, N 15.82; found: C 71.55, H
3.76, N 15.75.
2.3.2. Synthesis of 4′-(4-aminophenyl)-2,2′:6′,2’’-terpyridine (B)
In a 100 mL round bottom flask a mixture of compound A (0.46 g, 1.3 mmol) and SnCl2 (1.536 g,
6.8 mmol) were taken in concentrated hydrochloric acid (12 mL) and then heated at 80° C for 8 h
under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was poured
into cool water and adjusted to pH 8–9 by addition of NaHCO3 solution. The reaction mixture was
then extracted from 100 mL dichloromethane (three times). The organic layer was dried over
anhydrous sodium sulphate. After complete removal of solvent a brown solid was obtained which
was purified by column chromatography (basic alumina, eluting with hexane /ethyl acetate = 1:1)
o
to give the pure product (Yield: 62%). m.p. 253–254 C; 1H NMR (500 MHz, CDCl3)  (ppm):
8.72 (d, J = 4 Hz, 2H), 8.67 (s, 2H), 8.66 (d, J = 8 Hz, 2H), 7.86 (t, J = 8 Hz, 2H), 7.78(d, J = 8.55
Hz, 2H), 7.33 (t, J = 6.85 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H) (Fig. S4). 13C NMR (125 MHz, CDCl3,
25 °C, TMS) (ppm): 115.32, 117.88, 121.44, 123.75, 128.47, 136.90, 149.15, 155.72 (Fig. S5).
6

ESI-MS: m/z (%): 325.1440 (100%) [M+H]+(Fig. S6). Elemental analysis: calculated (%) for
C21H16N4: C 77.81, H 4.97, N 17.28; found: C 77.88, H 4.93, N 17.32.
2.3.3. Synthesis of 2-(2’-benzothiazolyl)-4-methyl- phenol (C)
To a 30 mL ethanolic solution of 2-aminobenzenethiol (109 mg, 1.00 mmol), 2-hydroxy-5
methylbenzaldehyde (136 mg, 1.00 mmol) was added at room temperature under nitrogen
atmosphere. The mixture was allowed to stir for 30 min at RT and then treated with hydrogen
peroxide (30%) (2.31 mL, 74.8 mmol) and hydrochloric acid (37.5%) (1.14 mL, 37.3 mmol). The
reaction mixture was stirred for 3h to obtain a yellow-green precipitate. The reaction mixture was
then poured into crushed ice and then extracted three times with ethyl acetate (EtOAc). The organic
layer was dried over anhydrous sodium sulphate and the crude product was purified by column
chromatography (silica gel, 100 -200 mesh, EtOAc: hexane = 4:96, v/v) as a pale-yellow
1
crystalline solid. (yield :51 %). Mp 127-128 ºC. H NMR (500 MHz, CDCl3) (ppm): 10.60 (s,
1H), 8.07 (d, J = 7.85 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 8.62 Hz, 1H), 7.52 (t, J = 5.65
Hz, 1H), 7.39 (s, 1H), 7.06 (d, J = 9.0 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 2.36 (s, 3H) (Fig. S7). 13C
NMR (125 MHz, CDCl3, 25 °C, TMS) (ppm): 20.53, 117.80, 121.59, 122.13, 125.58, 126.80,
128.40, 133.94, 150.40, 158.40, 165.58 (Fig. S8). ESI-MS: m/z (%): 242.0647 (100%)
[M+H]+(Fig. S9). Elemental analysis: calculated (%) for C14H11NOS: C 69.75, H 4.59, N 5.81;
found: C 69.78, H 4.53, N 5.72
2.3.4. Synthesis of 3-(2-benzothiazolyl)-2-hydroxy-5-methyl- benzaldehyde (D)
In a 250 mL round bottom flask, compound C (0.5 g, 2.19 mmol) was dissolved in 50 mL
trifluoroacetic acid and then hexamine (1.8 g, 12.85 mmol) was added in portion wise. The
resulting solution was refluxed for 36 h at 145 °C under N2 atmosphere till it turned dark brown.
The reaction mixture was then allowed to cool to RT and poured into100 ml 4 (N) HCl solutions.
7

The reaction mixture was extracted with ethyl acetate (EtOAc). The organic layer was dried over
anhydrous sodium sulphate and the crude product was purified by column chromatography
(hexane:ethyl acetate 5:1, v/v) to afford the pure product as a yellow solid (yield: 67%). m.p 176-
178 ºC. 1H NMR (500 MHz, CDCl3) (ppm): 12.95 (s, 1H), 10.69 (s, 1H), 8.80 (s, 1H), 7.98 (d, J
= 10.5 Hz, 1H), 7.91 (d, J = 9.55 Hz, 1H), 7.63 (s, 1H), 7.52 (t, J = 9.05 Hz, 1H), 7.43 (t, J= 9.52
Hz, 1H), 2.61 (s, 3H) (Fig. S10). 13C NMR (125 MHz, CDCl3, 25 °C, TMS)  (ppm): 20.33,
117.80, 121.59, 122.13, 125.58, 126.80, 133.13, 150.59, 158.40, 166.80 (Fig. S11). ESI-MS: m/z
(%): 270.0588 (100%) [M+H]+(Fig. S12). Elemental analysis: calculated (%) for C15H11NO2S: C
66.89, H 4.12, N 5.03; found: C 66.83, H 4.13, N 5.02.
2.3.5. Synthesis of L2
In 100 mL round bottom flask compound D (0.5 g, 1.85 mmol) was dissolved in a mixed solvent
containing 25mL absolute ethanol and 5 mL dry chloroform. Then compound B (0.66 g, 2.03
mmol) was added and the reaction mixture was refluxed for 48 h under nitrogen atmosphere. A
deep orange colored precipitate was obtained. The precipitate was filtered off and washed with
ice-cold absolute ethanol. The product was recrystallized from ethanol (Yield: 74%). m.p. 230–
231 0C.
1H NMR (500 MHz, CDCl3) (ppm):10.78 (s, 1H), 8.82 (s, 2H), 8.78(s, 1H), 8.75 (d, J= 6.5 Hz,
2H), 8.70 (d, J = 8.0 Hz, 2H), 8.11 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 8.0
Hz, 2H), 7.91 (d, J=6.6 Hz, 2H), 7.89 (t, J= 6.82 Hz, 2H), 7.52 (t, J= 7.11 Hz, 1H), 7.45 (s, 1H)
7.41(t, J= 6.45 Hz, 1H), 7.37 (t, J= 6.70 Hz, 2H), 7.29 (s, 1H) 2.34 (s, 3H) (Fig.S13).
CRzCDCl3CS(ppm):29.77,105.11,110.08,118.69,121.49,121.54,1
21.85,123.99,128.61,134.94,137,141.54,145.69,149.24,153.99,157.72, 168.61 (Fig. S14). ESI-
8

MS: m/z (%): 576.1874 (100%) [M+H]+(Fig. S15). Elemental analysis: calculated (%) for
C36H25N5OS: C 75.11, H 4.38, N 12.17; found: C 75.20, H 4.33, N 12.13.
2.4 Cell imaging
For potential application of the probe L2 in biological samples, the intracellular confocal imaging
studies of L2 have been carried out in the presence of Zn2+. For this purpose, HeLa cells were
cultured in DMEM medium containing penicillin/streptomycin (1% v/v) and FBS (10% v/v) in
5% CO2 at 37 °C. About 104 cells were seeded in each well of the 24 welled tissue culture plates
and incubated for 6 h in a CO2 incubator. After 6 h, HeLa cells were incubated with zinc perchlorate
salt (10 µM) dispersed in HEPES buffer (10 mM) which was added to the cell culture media. After
18 h, the media was removed from the plate and fresh media with L2 (10 µM) was added and
incubated for 1 h. After 1 h, cells were washed with PBS, and fixed with 4 % formaldehyde
solution for 20 min. The cells were imaged with confocal microscope Zeiss LSM 710. The images
of the cell were recorded upon excitation at 360 nm. For green emission 488 nm argon laser was
used and for red emission 543 helium neon laser was used.
2.5 MTT assay for cell viability assessment
For cytotoxicity assessment of biosensor L2, the HeLa cells (cervical cancer cell line) are chosen
which have been used for testing toxicity of biosensors under vitro conditions [29,30]. Different
concentrations of the sensor, ranging from (0-100 µM) of stock solutions were added to the
cultured HeLa cells. A set of untreated control cells were also kept which were devoid of any
exposure to any concentration of the sensors. 104 cells were seeded in each well of 96 well plates
and exposed to different concentrations of sensors (0- 100 µM) for 24 h. After 24 h, MTT (0.5
µg/ml) dispersed in plain DMEM media was added into each well and plate was incubated at 37
oC for 4 h. After 4 h, media containing dye was removed and 100 µl of DMSO was added to each
9

well and incubated for 20 min to dissolve the formazon crystals. The purple complex formed was
read spectrophotometrically by taking absorbance of each at 376 nm using ELISA reader (Thermo
scientific, Multiskan ELISA, USA). The percentage of cell viability was calculated as: (mean
experimental absorbance/mean control absorbance) × 100%. No significant difference was
observed between the percentages of viable cells in the sensor-treated and control samples
indicating thereby that the chemosensor L2 is non-cytotoxic and suitable for biological use.
3. Result and discussion
3.1. Photophysical properties
The photophysical properties of compound L2 were measured using UV-vis and fluorescence
spectroscopy in DMSO: H2O (3:2, v/v, 10 mM HEPES buffer, pH = 7) medium. The binding,
recognition, and selectivity of L2 toward metal ions, including Cd2+, Zn2+, Na+, K+, Ca2+, Mg2+,
Mn2+, Fe2+, Hg2+, Pb2+, Ag+, Al3+, Fe3+, Ni2+, Cu2+, Co2+, and Cr3+ were investigated. The
absorption spectra of benzthiazole-terpyridine dye in presence of different metal ions are shown
in Fig. 1a. In general the dye L2 displays an intense absorption spectrum at 467 nm corresponds
to intramolecular-charge transfer (ICT) from the terpyridine moiety to the electron-withdrawing
benzothiazole group. Upon addition of Zn2+ the color of the solution changes from colourless to
yellow and discernible 91 nm blue shift of absorption peak is observed. This hypsochromic shift
can be primarily ascribed due to the attachment of terpyridine moiety of dye molecule with Zn2+
via three near-coplanar nitrogen atoms. A similar but less pronounced blue shift (34 nm) is also
observed in presence of Cd2+ ions. The other metal ions do not show significant shift.
The emission spectrum of free L2 exhibits a band with maxima positioned around 542 nm upon
excitation at 360 nm. The examined alkali, alkaline earth and most transition metal ions show no
10

changes relative to L2, whereas the emission intensity of L2 is completely quenched by Cu2+, Cr3+,
and Fe3+ ions. Interestingly, Zn2+ shows a 42 nm blue-shift of the band due to intramolecular charge
transfer (ICT) from the Zn2+ coordinated terpyridine core to electron-withdrawing benzthiazole
moiety at higher energy region. Therefore, L2 behaves as a ratiometric fluorescent sensor for the
Zn2+ ions
.
To utilize L2 as an effective ion-selective fluorescence chemosensor for Zn2+, the effect of
competing metal ions was investigated. For this purpose, L2 was treated with Zn2+ in the presence
of other various biologically and environmentally relevant metal ions in excess concentration in a
mixed DMSO/water solution. Relatively low interference is observed for the detection of Zn2+ in
the presence of other metal ions (Fig. 2a). Thus L2 can be used as a selective fluorescent sensor
for Zn2+ in the presence of most competing metal ions. The possible interference of anions was
also probed by the competitive experiments which clearly demonstrate that the emission profile of
-
-
2-
L2-Zn2+ solution remains unchanged by the presence of common anions such as NO3 , NO2 , SO3
2-
2-
-
3-
, SO4 , CO3 , HCO3 , F-, Cl-, Br-, I-, OAc-, PO4 , and CrO42- (Fig.2b).
The titration of Zn2+ with L2 (2 µM) shows a strong fluorescence enhancement with the increase
of the Zn2+ concentration range (0-50 µM). The fluorescence titration curve reveals that the
fluorescence intensity at 542 nm is totally diminished and at 500 nm, increased linearly with
increasing concentration of Zn2+ ions (Fig.3b). In the case of absorption titration, where the
absorption peak at 376 nm is gradually intensified with increasing concentration range (5-50 µM)
(Fig.3a).
11

The method of continuous variation (Job’s plot) was also done to prove the 1:1 stiochiometry (Fig.
4). The binding mode of Zn2+ions with receptor L2 is also proved by mass spectroscopy (Fig.S16).
-
The mass spectrum shows an intense peak at m/z 739.33 corresponding to the [L2 +Zn2++ OClO3
]+ complex which not only confirms the binding of Zn2+ ions with receptor L2 but also proved the
1:1 stoichiometry of host and guest species.
The association constant for Zn2+ was estimated to be 9.95 × 105 M-1 on the basis of linear fitting
of the fluorescence titration (Fig. 5a) curve using Benesi−Hildebrand plot and demonstrated that
1:1 stoichiometry (host/guest) is the most stable species in the solution [31]. To evaluate the
sensing properties of L2 for Zn2+, the limit of detection (LOD) of L2 for Zn2+ was also measured.
The detection limit of the probe for Zn2+ was evaluated from the fluorescence titration and
calculated to be 35 nM (Fig.5b) using the equation DL = K×Sb1/S ,where K = 3, and Sb1 is the
standard deviation of 10 replicate blank solution and S is the slope of the calibration curve in the
lower region.
The direct evidence to determine the mode of complexation, 1H NMR titrations of L2 with various
concentrations of Zn2+ was also studied in CDCl3. As shown in Fig. 6, the characteristic peaks of
terpyridine protons at 8.76, 8.72, 8.66, 7.47 ppm shifted to a lower field after the coordination of
L2 with Zn2+ ions. Similar shifts upon metal binding are observed in other cases [32]. However
the proton signal at 10.6954 ppm due –OH group in benzthiazole moiety was almost unaltered
suggesting phenolate O atom not bound to Zn2+ ions. These observed downfield shifts of various
protons suggest 1:1 binding stoichiometry for L2: Zn2+ and tri-coordination of Zn2+ to L2 through
three near-coplanar nitrogens of terpyridine unit. The tetra-coordination of Zn2+ is satisfied by
perchlorate counter anion.
3.3. Reversibility
12

In order to obtain a better understanding of the interactions between L2 and Zn2+, we have studied
the chemical reversibility of the binding of L2 to Zn2+ in DMSO: H2O (3:2, v/v, 10 mM HEPES
buffer, pH = 7) (Fig. 7). As seen by the naked eye, the yellow color of a solution of L2-Zn2+
gradually disappeared followed by red shift of emission spectra upon the addition of excess
disodium EDTA (10 equivalents).
3.4. Density functional theory
In parallel to the experimental study, to further understand the electronic structures of L2 and L2-
Zn2+ complex DFT calculation were performed using the Gaussian 09 programme [33]. The L2
was optimized with the B3LYP [34-3536373839] functional and 6-311+G (d,p) basis set. For Zn2+
bound species with L2, LANL2DZ for Zn2+ and 6-31g*+ for the rest of the atoms were used for
optimization. The optimized structure of L2 with Zn2+ is shown in Fig. 8.The bond lengths of Zn-
N bonds are similar to the reported complex [40].
3.5. Effect of pH
In addition to metal ion selectivity, the pH-insensitivity of fluorescence in near neutral and weak
acidic media is of importance for practical applications both in environmental and biological
analysis. Thus we have measured the fluorescence intensity of L2 at various pH values in the
presence and absence of Zn2+. The fluorescence intensity of the L2 at 542 nm decreases under
acidic condition due to protonation of nitrogen atoms of terpyridine moiety. In the neutral to
alkaline medium it shows moderate fluorescence intensity at 542 nm (Fig. 9a). Interestingly, L2
shows good fluorescence sensing ability to Zn2+ at 500 nm over a wide range of pH values from 6
to 9. As shown in Fig. 9b, L2 shows little sensing ability to Zn2+ at a pH below 6.0, which may be
13

due to protonation, but exhibits satisfactory Zn2+ sensing abilities when the pH is increased to the
range 6 -8.
3.6. Confocal Imaging Studies
The Zn2+ detection capability of probe L2 in living cells was evaluated by confocal microscopy.
HeLa cells incubated with L2 (10 µM) exhibits bright fluorescence in red channel upon excitation
at 360 nm but very weak fluorescence in green channel. However a bright fluorescence signal is
observed in green channel when the cells strained with L2 and Zn2+ (10 µM) , which suggests that
probe is cell membrane permeable and can be efficiently applied in imaging of Zn2+ in living cells
(Fig. 10). The cells incubated with both L2 and Zn2+ show considerable weak fluorescence in red
channel. Hence, this approach of bio sensing using L2 of Zn2+ ions can potentially be used for in
vitro and in vivo diagnostics of accumulation of Zn2+ ions.
Additionally, MTT assessment for finding the toxicity of the biosensor was carried out to check if
sensor itself induces any cytotoxicity in cells. It was observed that the proliferation of HeLa cells
is not affected by the addition of this sensor in the in vitro cell culture conditions in the relevant
working concentrations (5-20 µM). Thus the sensor induces negligible toxicity in HeLa cells and
the cell proliferation is almost ~ 90 % of the negative control (samples which did not contain any
biosensors) (Fig.11). From this result it can be concluded that sensor is not cytotoxic and also able
to perform fluorescence based detection of Zn2+ ions in vitro conditions.
4. Conclusion
In summary, we have presented a fluorescent sensor with terpyridine substituent groups as a
fluorescent ratiometric sensor for detection of Zn2+based on an internal charge transfer (ICT)
mechanism in DMSO: H2O medium. The 42 nm blue-shift emission upon addition of Zn2+ to L2
is attributed to the capture of Zn2+ by a terpyridine moiety leading to diminished electron donating
14

1
ability. The Zn2+ ion sensing mechanism is also supported by the Job’ plot, ESI-MS, H NMR
titration experiments. Importantly, this probe shows high selectivity over other anions; moreover,
this selectivity is retained even in the presence of other biologically relevant metal ions. A
comparison study for recent ICT based chemosensors for Zn2+ is also shown (Table 1 in SI). In
addition the detection limit of L2 was found to be upto 35 nM which is acceptable within the
USEPA limit. The probe is also able to work in a broad biological pH range of 6-8.
Acknowledgements
This work was supported by Department of Science and Technology, New Delhi, India (No.
SERB/CHM/20130239 to P. K. B). S.S. thanks Council of Scientific and Industrial Research
(CSIR) India for senior research fellowship. For Gaussian calculation HPC 2010 facility of IIT
Kanpur is gratefully acknowledged.
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Scheme 1. Synthetic scheme of the probe L2
Fig. 1. (a) Absorption spectra of L2 (10 µM) in DMSO: H2O (3:2, v/v, 10 mM HEPES buffer, pH
= 7) in the presence of 3 equivalents of various metal ions. (b) Fluorescence spectra of L2 (10 µM)
in DMSO: H2O (3:2, v/v, 10 mM HEPES buffer, pH = 7) in the presence of 3 equivalents of various
metal ions. λex =360 nm; slit = 10 /10 nm
Fig. 2. (a) Selectivity of the dye L2 (10 μM) for the Zn2+ in DMSO: H2O (3:2, v/v, 10 mM HEPES
buffer, pH = 7) in the presence of 10 equivalents of various metal ions. Red bars indicate the
emission responses of the dye with 10 equiv. of the metal ions of interest, and blue bars represent
the final integrated fluorescence response after the addition of 3 equivalents of Zn2+ to each
solution containing other metal ions (b) Selectivity of the dye L2 (10 μM) for the Zn2+ in DMSO:
H2O (3:2, v/v, 10 mM HEPES buffer, pH = 7) in presence 10 equivalents of different anions. λex=
360 nm; slit = 10/10 nm
19

Fig.3. (a) Absorption titration (b) fluorescence titration of L2 with increasing Zn2+ ions
concentration in DMSO: H2O (3:2, v/v, 10 mM HEPES buffer, pH = 7) Arrow indicates the
increasing trend in Zn2+ ion concentration (0-20 µM). λex = 360 nm ; slit = 10 /10 nm
Fig. 4. Job’s plot for determination of binding stoichiometry between L2 and Zn2+
20

Fig.5. (a) Binding constant plot of L2 for Zn2+ (b) Linear response curve of L2 at 500 nm depending
on the Zn2+ ion concentration for determination of lowest detection limit
3.2. 1H NMR titration
21

Fig. 6. Proposed binding mode of L2 with Zn2+.(a) L2 only, (b) L2 and 0.5 equivalent of Zn2+, (c)
L2 and 1 equivalent of Zn2+
Fig. 7. Reversible nature of L2 in Zn2+ sensing process in presence of excess of EDTA
Fig.8 DFT Optimized structure of L2-Zn2+ complex
22

Fig. 9. The fluorescence intensity changes (a) of L2 at 542 nm and (b) of L2-Zn2+ solution at 500
nm under various pH conditions. λex = 360 nm; slit = 10 /10 nm.
20 µm
20 µm
20 µm
20 µm
20 µm
20 µm
23

<InlineShape2>
Fig.10. Biosensing of Zn2+ in HeLa cells. Fluorescence images of HeLa cells first incubated with
L2 (10 μM) for 30 min and then zinc perchlorate (10 μM) for 1 h, (a) fluorescence image of L2 in
green channel (b) fluorescence image of L2 in red channel (c)The overlay images of (a) &(b), (d)
fluorescence image of L2 after incubated with zinc perchlorate in green channel (e) fluorescence
image of L2 after incubated with zinc perchlorate in red channel (f) The overlay images of (d) &
(e). Scale bar is 20 µm
Fig.11. Cellular cytotoxicity assessment of different concentrations of sensor L2 in HeLa cell lines
24