New pyrazolo-quinoline scaffold as a reversible colorimetric fluorescent probe for selective detection of Zn2+ ions and its imaging in live cells

Article abstract of DOI:10.1016/j.jphotochem.2017.03.035

A simple colorimetric fluorescent chemosensor 1-[5-(2-chloro-6-methylquinolin-3-yl)-3-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethan-1-one (L) based on pyrazoline has been designed, synthesized and characterized. The sensing properties of L were studied using UV–visible and fluorescence spectrophotometric analyses. L showed excellent selectivity towards Zn²⁺ ions over other metal ions in aqueous media through “OFF-ON” type process and a possible photoinduced electron transfer (PET). The viability of L to Zn²⁺ has been demonstrated by live cell imaging.

Full text of DOI:10.1016/j.jphotochem.2017.03.035

Accepted Manuscript
Title: New pyrazolo-quinoline scaffold as a reversible
colorimetric fluorescent probe for selective detection of Zn2
ions and its imaging in live cells
+
Authors: Kasirajan Gayathri, Krishnaswamy Velmurugan,
Raju Nandhakumar, Mahalingam Malathi, Sadasivam
Mathusalini, Palathurai Subramaniam Mohan, Ramasamy
Shankar
PII:
S1010-6030(17)30126-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.03.035
JPC 10586
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
28-1-2017
25-3-2017
26-3-2017
Please cite this article as: Gayathri Kasirajan, Velmurugan Krishnaswamy, Raju
Nandhakumar, Malathi Mahalingam, Mathusalini Sadasivam, Mohan Palathurai
Subramaniam, Shankar Ramasamy, New pyrazolo-quinoline scaffold as a reversible
colorimetric fluorescent probe for selective detection of Zn2+ ions and
its imaging in live cells, Journal of Photochemistry and Photobiology A:
Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.03.035
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New pyrazolo-quinoline scaffold as a reversible colorimetric fluorescent probe
2
+
for selective detection of Zn ions and its imaging in live cells
a
b
b*
Kasirajan Gayathri , Krishnaswamy Velmurugan , Raju Nandhakumar , Mahalingam
c
a
a*
Malathi , Sadasivam Mathusalini , Palathurai Subramaniam Mohan , Ramasamy
d
Shankar
a
Department of Chemistry, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.
Department of Chemistry, Karunya University, Coimbatore 641 114, Tamil Nadu, India.
Department of Chemistry, Bannari Amman Institute of Technology, Sathyamangalam 638 401,
b
c
Tamil Nadu, India
d
Department of Physics, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.
*Corresponding author. Tel.: +919363211210
E-mail address: ps_mohan_in@yahoo.com, psmohan59@gmail.com (P.S. Mohan).
nandhakumar@karunya.edu (R. Nandhakumar).
Graphical abstract
O
CH3
Zn2+
CH3
Zn2+
OH
N N
O
CH3
CH3
N
N
O
N
EDTA
Cl
N
Cl
1

Highlights


Pyrazoline based chemosensor was synthesized that selectively detects zinc ion.
Naked eye color change from colorless to yellow is due to OFF-ON process,
photoinduced electron transfer (PET) and CHEF.
2
+



The limit of detection (LOD) of probe with Zn ion is 2.9 nM.
Density functional theory calculations further supports the fluorescence change.
2
+
The sensitivity of probe is demonstrated successfully for selective imaging of Zn in
living cells.
Abstract
A simple colorimetric fluorescent chemosensor 1-[5-(2-chloro-6-methylquinolin-3-yl)-3-
2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl]ethan-1-one (L) based on pyrazoline has been
(
designed, synthesized and characterized. The sensing properties of L were studied using
UV–Visible and fluorescence spectrophotometric analyses. L showed excellent selectivity
2
+
towards Zn ions over other metal ions in aqueous media through “OFF-ON” type process and a
2
+
possible photoinduced electron transfer (PET). The viability of L to Zn has been demonstrated
by live cell imaging.
Keywords
Quinoline; Chemosensor; Zinc; Colorimetric; Fluorimetric; Bioimaging
1
. Introduction
In recent years, development of chemosensors witnessed remarkable progress in the
detection of transition and heavy metal ions and it has widespread applications in the field of
analytical chemistry, biology and environmental processes[1-10]. Chemosensors based on cation
induced fluorescence changes have advantage of possessing high sensitivity, selectivity,
2

simplicity and instantaneous response [8, 11-13]. Neurobiological processes relevant to human
2
+
health and diseases are primarily controlled by Zinc ions. Zn is the second most abundant and
2
+
3+
essential element after Fe and Fe ions in human and performs a variety of functions. The
2
+
decrease in concentration of Zn in human body causes diseases such as Alzheimer’s disease,
β-thalassemia, Parkinson’s disease, ischemic stroke and epilepsy[14-19]. Zinc decreases copper
burden and has been used effectively to treat Wilson’s disease and also in the treatment of
hepatic encephalopathy, sickle cell disease, diabetes and malignancy. The cell culture studies
showed that zinc activates nuclear factor kappa B in T helper cells and deficiency of it reduces
the binding of nuclear factor kappa B to deoxyribonucleic acid, leading to decreased gene
expression of interleukin 2 and its production[20, 21]. Hence, there is a need for the development
of artificial chemosensors for recognition of Zn2+ ions in environmental as well as biological
samples. However, selective recognition of zinc in the presence of other cations is a tedious task
1
0
as closed shell d electronic configuration totally affects the redox and spectral features.
Recently, fluorescent sensors for zinc ions have made their mark in the field of molecular
recognition. Several zinc sensors have been developed based on different fluorophores such as
quinoline, fluorescein, coumarin, peptide, pyrene and proteins with apparent dissociation
constants in the micro to nano molar ranges[22-32]. Particularly, more attention has been paid to
quinoline fluorophore due to its strong binding ability with metal ions, altering the molecular
size based on coordinating systems and high quantum yield which plays a vital role to detect
metal ions in biological and living systems[33, 34]. Nevertheless, only a few researchers have
reported on detection of Zn2+ ion through dual pathways such as colorimetric and fluorescent
turn ‘off-on’ signaling system[35-39]. In this connection, there are number of issues to be
addressed while devising suitable zinc sensors such as selectivity, binding ratio, suitability in live
2
+
cells. Therefore, designing a suitable sensor capable of detecting Zn ions via colorimetric and
fluorescent sensing methods is desirable in the field of environmental and biological sciences.
Hence our efforts were directed to the preparation of suitable sensor by following a simple route
and easily accessible chemicals.
Herein, we report a quinoline-pyrazoline chemosensor (L), that selectively detect Zn2+
ion, using colorimetric and fluorescent techniques. This sensor L is designed in such a way that
quinoline acts as a fluorophore, which is covalently attached to a pyrazoline and hydroxy phenyl
3

rings through a C-C spacer unit. The hetero atoms of pyrazoline ring containing ‘N’, acetyl ‘O’
2
+
and hydroxy phenyl ‘O’ act as binding sites to recognize Zn ion. This is the first example of
quinoline-pyrazoline based sensor L for selective detection of Zn2+ ion by colorimetric and
fluorimetric methods.
2
. Experimental
2
.1. Reagents and instruments
All reagents were of analytical grade and doubly distilled water was used throughout the
analysis. 2'-hydroxyacetophenone was obtained from Sigma Aldrich. Other reagents were
1
purchased from commercial suppliers and used without further purification. H NMR and
13C NMR spectra were recorded on a Bruker 400 MHz and 100MHz spectrometer and DMSO
solution with tetramethyl silane (TMS) as internal standard. LC–MS was recorded on a
LC–MSD-Trap-XCT Plus based on infusion methods. A Nicolet Avatar Model FT-IR
−
1
spectrophotometer was used to record IR spectra (4000–400 cm ). Microanalyses were
examined on a Vario EL III model CHNS/O analyzer (Vario, Germany) and Scanning Electron
microscope (SEM) studies were performed on JEOL model JSM- 6390. UV–visible absorption
spectra were measured using JASCO-V- 630 spectrophotometer. Fluorescence measurements
were performed on JASCO FP-6600 spectrofluorometer equipped with quartz cuvettes of 1 cm
path length. The excitation and emission slit width were 5.0 nm. All emission spectra were
-
6
recorded at 24 ± 1 °C. Stock solutions for analysis were prepared (2 ×10 M for compound L
EtOH/H O, 1:1 (v/v), HEPES = 50 mM, pH = 7.2) immediately before the experiments. The
solutions of metal ions were prepared from nitrate salts of Na , K , Al , Cu , Cd , Hg , Pb ,
(
2
+
+
3+
2+
2+
2+
2+
2
+
2+
2+
2+
2+
3+
2+
2+
2+
3+
2+
3+
+
Zn , Co , Ni , Ca , Mn , Cr , Ba , Mg , Fe , Fe , Zr , Ce and Ag .
2
.2. Synthesis of 1-[5-(2-chloro-6-methylquinolin-3-yl)-3-(2-hydroxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl]ethan-1-one (L)
Chalcone 3 was easily prepared from compound 1 and 2 in 79 % yield, according to the
literature[40]. An equimolar mixture of compound 3 (3.2 g, 0.01 mol) and hydrazine hydrate (0.5
4

g, 0.01 mol) were taken in a 100 ml reaction flask in the presence of glacial acetic acid (15 ml)
and refluxed at 120° C for 6 h. After completion of reaction, it was cooled and poured into
crushed ice. The resulting precipitate was filtered and recrystallized from ethyl acetate to yield
probe L. Pale yellow solid; Mp. 232 ºC; Yield: 74.33 %; FT-IR (KBr): 3430 (-OH), 1664 (C=O)
-1
1
and 1101 cm (C-N). H NMR (DMSO-d6, 400 MHz) δ ppm: δ 10.18 (s, 1H, OH), 8.03 (s, 1H,
Ar-H), 7.81-7.85(m, 2H, Ar-H), 7.63(d, 1H, J = 8.4 Hz, Ar-H), 7.56(d, 1H, J = 8 Hz, Ar-H),
7
.32(t, 1H, J = 6.8 Hz, Ar-H), 6.88-6.97(m, 2H, Ar-H), 5.79 (dd, 1H, J = 12 Hz, J = 5.2 Hz, CH
in pyrazoline moiety), 4.07-4.14 (m, 2H, CH in pyrazoline moiety), 2.48(s, 3H, COCH ),
); 13C NMR (DMSO-d6, 100 MHz) δ ppm: 178.07, 160.93, 152.10, 143.68,
2
3
2
1
1
.38(s, 3H, CH
3
37.14, 135.69, 135.02, 128.33, 127.83, 126.10, 125.52, 125.34, 124.77, 124.12, 123.67, 122.09,
+
09.90, 34.45, 31.15, 25.35, 17.83; LCMS (ESI ) m/z Calculated: 379.11, Found: 378.2
+
[
(M-1) ]; Calculated For C21
H
18
N
3
O Cl (379.11): C, 66.40; H, 4.78; N, 11.06, Found: C, 67.54; H,
2
4.90; N, 11.49 %.
2
.3. Theoretical studies
Both probe L and L-Zn2+ were optimized using density functional theory (DFT) by
employing B3LYP[41] functional and LANL2DZ[42-44] basis set. The optical absorption and
2
+
emission spectra for L and L-Zn were calculated using time dependent density functional
theory (TDDFT). All structures were optimized in ethanol medium using PCM model[45]. To
calculate emission spectra, the structures were optimized in excited state using
2
+
TD-B3LYP/LANL2DZ basis set. The frequency calculations were done for L and L-Zn in
order to ensure optimized ground state and the computation was carried out using G09 software
program[46].
2
.4. Cytotoxicity study by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay and fluorescence imaging
A cytotoxicity of probe L was carried out on mouse neuroblastoma (N2A) cancer cell
lines which were obtained from National Center for Cell Science (NCCS), Pune, India. Cell
5

viability was carried out using the MTT assay method[47]. Cells were maintained in minimum
essential Eagle’s medium (Sigma Aldrich, India) supplemented with 10 % fetal bovine serum
(FBS), streptomycin (100 µg/ml), penicillin (100 U/ ml) and essential amino acids (Himedia,
3
India). The cells were plated at a density of 7×10 in 96-well cell cultured plate and incubated in
a humidified chamber with 95 % air, 5 % CO and 100 % relative humidity for 24 h prior to the
2
addition of compound. The probe L was dissolved in dimethyl sulphoxide (DMSO) and diluted
in the respective medium with 1% FBS containing the probe at various concentrations and
incubated at 37 ºC with 95 % air, 5 % CO and 100 % relative humidity for 48 h. After 48 h, 10
2
µL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated
for 4 h at 37 ºC. The medium with MTT was then flicked off and as a result formazan crystals
were dissolved in 100 µL of DMSO. The absorbance was then measured at 570 nm using a
microquant plate reader (Bio-Tek Instruments).
4
For bioimaging, cells were seeded at a density of 2×10 in 24-well cell culture plate and
allowed to settle for 12 h. When cells attained 80 % confluence, the media was changed. Cells
2
+
were treated with L (10 µM) alone for 3 h and then treated with Zn (10 µM) and the plate was
incubated for 3 h in a humidified incubator at 37 ºC with 95 % air and 5 % CO . The
2
fluorescence of probe L localized was captured under inverted fluorescence microscope (EVOS,
Life Technologies, USA).
2
.5. Scanning Electron Microscope (SEM) studies
The probe L and L+Zn2+ were dissolved in small amount of acetone. By using drop-
casting method, the samples were coated over silicon bare wafer and placed in the hot air oven at
0 °C for 5 hours to get thin film samples. Then before taking SEM images Pt was coated by
6
sputtering to avoid charging effect. The film uniformity is evenly agglomerated which can be
seen in lower magnification of SEM images.
3
. Results and Discussion
The fluorescent probe L was conveniently synthesized from 2-chloro-6-methylquinoline-
3
-carbaldehyde[48] by two steps, as shown in Scheme 1. The reaction between quinoline
6

carbaldehyde (1) and 2'-hydroxyacetophenone (2) in ethanol afforded compound 3 by Claisen-
Schmidt type of condensation process. Subsequently, 3 and hydrazine hydrate were reacted in
the presence of glacial acetic acid under reflux condition to obtain L in reasonable yield through
addition and cyclization process. The structure of L was confirmed by standard spectroscopic
1
13
and analytical techniques such as NMR ( H and C) and mass spectrometry (Fig. S1-S3).
Scheme 1.
The selective detection of probe L was done by using UV- visible absorption spectrum in
EtOH-H O (1:1 v/v, HEPES= 50 mM, pH=7.2) solution. The spectrum showed two intense
2
2
+
absorption bands centered at 280 and 323 nm (Fig. 1). Upon addition of Zn ion (50 equiv) to L,
considerable changes in the two absorption bands occurred followed by a new shoulder band
appearing at 380 nm. Also, a significant color change from colorless to pale yellow was observed
by naked eye which is due to d-d transition between metal to ligand charge transfer (MLCT)
+
+
2+
2+
2+
2+
2+
2+
2+
complex. Addition of other metal ions such as Na , K , Cu , Cd , Ni , Hg , Pb , Co , Ca ,
2
+
3+
2+
2+
2+
3+
3+
2+
3+
+
Mn , Cr , Ba , Mg , Fe , Fe , Al , Zr , Ce and Ag to L did not show any spectral and
2
+
color changes. The results reveal that probe L coordinate well with Zn ion in EtOH-H
v/v, HEPES= 50 mM, pH=7.2) solution, with an apparent color change. Furthermore, the
sensing behavior of L was examined with Zn2+ based different counter ions such as ZnSO
Zn(NO , ZnCl , Zn(CH COO) as shown in supporting information (Fig. S11). L bind with
2
O (1:1
4
,
3
)
2
2
3
2
2
+
different salts of Zn and the emission intensity were found to be almost similar in all the cases.
These observations show that the counter ions do not play an important role in the selectivity of
2
+
2+
Zn using L. This indicates that probe L shows selective emission enhancement towards Zn .
Fig. 1.
In continuation of the above findings, UV-Vis titration experiments were carried out by
2
+
consecutive addition of Zn ions (0 to 25 equiv.) to L. The absorbance bands at 280 and 323 nm
2
+
of L steadily decreases with an increase in the concentration of Zn ions. Simultaneously, an
7

isosbestic point and a shoulder peak were observed at 358 and 380 nm respectively (Fig. 2),
2
+
which indicates the formation of a coordination complex between L–Zn . Thus, probe L acts as
2
+
a ratiometric and naked eye sensor for Zn ions via colorimetric method.
Fig. 2.
Metal ion binding ability of L was carried out with various transition metal ions by using
fluorescence spectrometric technique (Fig. 3). The emission spectra of L showed a weak band at
2
+
4
08 nm with quantum yield. Upon addition of Zn ions to solution L, a small red shift with
fluorescence enhancement was observed. The change in spectral wavelength from 408 to 418 nm
is due to an inhibition of photo-induced electron transfer (PET) process and restricted C=N
isomerization mechanism[49, 50]. This 10 nm red shift is clearly shown in the normalized
+
+
2+
2+
spectrum (Fig. S4). Under identical conditions, addition of other metal ions (Na , K , Cu , Cd ,
2
+
2+
2+
2+
2+
2+
3+
2+
2+
2+
3+
3+
2+
3+
+
Ni , Hg , Pb , Co , Ca , Mn , Cr , Ba , Mg , Fe , Fe , Al , Zr , Ce and Ag ) to L,
did not show any significant changes in the emission profiles. Thus, probe L can selectively
2
+
senses Zn ions by fluorescence “turn off-on” process.
Fig. 3.
The effect of pH on probe L and L-Zn2+ with different pH values were recorded by
fluorescence spectra. The experiments revealed that L showed a weak fluorescence intensity
2
+
between pH 0 and 14, whereas in the presence of Zn , fluorescence intensity of L increased
dramatically between pH 5 and 8 and reached the maximum at pH 7 (Fig S5). Therefore, we
have chosen physiological pH for all biological studies like live cell imaging.
The time-dependent fluorescence intensity changes of L with Zn2+ was studied and
2
+
results are shown in Fig S6. It showed that L completely coordinates with Zn in almost 5 min
and after that no noticeable changes was observed. Therefore, we have selected the incubation
time of 15 minutes for all spectral studies.
To find the practical applicability of L, interference studies were carried out in the
2
+
presence of 50 equiv. of Zn , treated with same equiv. of other potential metal ions is shown in
8

Fig. 4. No obvious interference in emission spectrum was found on comparison with other
competitive metal ions besides Zn . In general, most of the Zn sensors found Cd2+ ion
2
+
2+
2
+
2+
interferences due to same periodic behavior with Zn . Surprisingly, L-Zn complex eliminated
2
+
the influence of Cd ion by blocking PET, CHEF and restricted C=N isomerization mechanism.
These results establishes that L strongly coordinates with Zn2+ ion which could be used to
2
+
2+
distinguish Zn from Cd ion in real applications.
Fig. 4.
To evaluate the chemosensing behavior of L, fluorescence titration experiments were
2
+
2+
carried with various concentrations of Zn ions. Upon gradual addition of Zn from 0 to 25
equiv., the fluorescence intensity of L at 408 nm steadily increased with a small red shift. The
2
+
fluorescence changes of L upon addition of Zn is shown in Fig. 5.
Fig. 5.
The quantum yield φ[51] was calculated by using the following equation,
φx = φs(F /F )(A /A
x s s x
)(η2
x
/η2
).
s
where, x and s indicate the unknown and standard samples, φ-quantum yield, F is the integrated
fluorescence intensity, A-absorbance, and η- refractive index of the solvent.(Quinine sulphate
[
52] as standard in 0.1 M H
2
4
SO [φ=0.54]). The quantum yield of L is 0.019, and on
2
+
2+
complexation with Zn , the value significantly increased to 0.214. The binding ratio of L-Zn
(Host-Guest) complex was investigated by Job’s plot continuous variation methods [53] is shown
2
+
in Fig. 6. The maximum mole fraction of L was appeared at 0.5, which supported a 1:1 (L: Zn )
binding stoichiometry. It was further advocated by Benesi–Hildebrand nonlinear curve fitting
method [54, 55](Fig. 7). The binding constant of L-Zn2+ complex was found to be
4
-1
Ka = 1.88 x 10 M and the limit of detection (LOD) of probe L is 2.9 nM, calculated using 3δ/S
[
56]. The high sensitivity and low detection limit of L could be used as a trace level
2
+
2+
identification of Zn in real level environmental samples. Moreover, Zn ion concentration in
human body ranges from 3 nM to 10 μM and can be monitored by this probe L [57, 58]. The
9

advantage of preparing the above compound and its detection limit is realized when compared
with other zinc sensors reported in the literature with their corresponding detection limit (Table
1
).
Fig. 6.
Fig. 7.
Table 1.
2
+
1
The interaction of the probe L with Zn was investigated through H NMR spectroscopy.
2
+
This experiment was performed to further support the coordination structure for Zn complex
1
2+
with L in DMSO-d
6
. Fig. 8 shows the H NMR spectra of L with and without Zn . All the
1
2+
chemical shifts were identified by the H NMR titration analysis. During the addition of Zn to
2
+
probe L and upon increasing the concentration of Zn ions gradually ( 0.5, 1.0, 1.5 and 2.0
equiv.) the hydroxyl peak at 10.15 ppm decreases and almost disappeared on addition of 2.0
2
+
equiv. of Zn . In addition, small upfield shifts were noticed in the aromatic region and for the
methyl and the methoxy protons. These shifts clearly advocates the complex formation between
2
+
the probe L and Zn wherein the hydroxyl and C=N groups are involved in the coordination.
Fig. 8.
−
1
As shown in Fig. 9, FT-IR spectrum of L, showed characteristic bands at 3430 cm ,
-
1
-1
1
6
664 cm and 1101 cm assigned to -OH, pyrazoline C=O and C-N stretching respectively [59-
2
+
-1
3]. On addition of Zn to L, two bands shifted downward from 3430 and 1664 cm to 3314
-1
-1
-1
and 1632 cm respectively and C-N stretching band at 1101 cm shifted to 1112 cm , which is
due to decrease in electron density via the coordination of –OH, C=O and C=N groups with Zn2+
(see Scheme 2).
Fig. 9.
1
0

Furthermore, in order to understand the morphological differences, the Scanning Electron
Microscope (SEM) images of probe L and L-Zn2+ was captured as shown in Fig. 10. The
chemosensor L shows a random fibre network structure with microholes. However, upon probe
2
+
L on complexation with Zn ions, the fibre network structure with microholes were completely
2
+
covered and agglomerated. The chemical composition of the L- Zn complex is measured by the
EDX analysis (Fig. 10 and Table S1) which clearly showed the presence of carbon (C), oxygen
2
+
(
O), chlorine (Cl) and Zn elements in the prepared L- Zn Complex.
Fig. 10.
The recognition reversibility of L was further verified with EDTA by fluorescence
2
+
experiments. The addition of Zn (50 equiv.) to probe L showed remarkable enhancement in its
2
+
fluorescence intensity. Upon addition of EDTA (50 equiv.) to L-Zn solution, emission intensity
2
+
get quenched and almost reached the intensity of original receptor L due to EDTA-Zn complex
2
+
formation. This indicates, Zn recognition process is reversible (Fig. 11).
Fig. 11.
2
+
In order to evaluate absorption and emission behaviors of probe L and L-Zn complex,
we have performed quantum chemical TDDFT calculations using Gaussian 09 program. The
optimized geometries and TD spectra of L and L-Zn2+ complex are shown in supporting
information (Fig. S7 and S8). For probe L, absorption peak at 311 nm with oscillator strength
0
.523 a.u was generated from HOMO to LUMO+1. While in the case of L-Zn2+ complex,
transitions from HOMO-2 to LUMO and HOMO-1 to LUMO had contributed absorption values
at 344 nm and 370 nm respectively. In computed spectrum, L showed emission at 342 nm and
for L-Zn2+ complex, it was around 363 nm with oscillator strength 0.280 a.u. The difference
from experimental wavelength is due to the basis set[64] used. The calculated absorption and
emission spectra, orbital transition, wave length and its oscillator strength, are shown in
2
+
supporting information (Table S2). Binding of Zn leads to the lowering of energy gap of L
from 3.982 eV (HOMO-LUMO+1) to 1.908 eV (HOMO-LUMO+4), 3.346 eV (HOMO-1 -
1
1

LUMO) and 3.595 eV (HOMO-2-LUMO). This might be the cause for formation of absorption
bands at 344 nm and 370 nm[65].
Fig. 12.
For probe L, bond distances of N
1
-N
2
, N
2
-C10, C10-C11, C10-O
2
and C
4
-O were calculated
1
2+
as 1.420 Å, 1.386 Å, 1.522 Å, 1.262 Å and 1.392 Å respectively. After complexation with Zn ,
the donor sites were strained and changes in bond lengths were observed as 1.411 Å, 1.362 Å,
1
.500 Å, 1.292 Å and 1.422 Å respectively. Further the coordination bond lengths of nitrogen
atom of pyrazolyl and oxygen atoms of pyrazolyl and phenyl rings were found to be 2.085 Å,
.076 Å and 2.106 Å respectively (Fig. S7). Hence it is clearly evident that, in probe L, HOMO
2
is highly localized in the phenyl ring and a considerable amount of electron distribution is
present in pyrazoline ring while LUMO is distributed around the quinoline ring (Fig. 12). After
2
+
binding of Zn , HOMO is distributed around quinoline ring and LUMO is resided around
2
+
phenyl and pyrazoline rings. In LUMO+2, the electrons are centered on Zn and in the case of
HOMO-2, it spread over the entire molecule and this could be the cause for fluorescence
2
+
enhancement of L upon addition of Zn . The plots of frontier molecular orbitals of L and L-
2
+
Zn are shown in Figs S9 and S10.
Based on the results obtained, a plausible sensing mode for detection of Zn2+ by L is
2
+
shown in Scheme 2. Probe L exhibited weak fluorescence intensity and addition of Zn to L,
leads to fluorescence enhancement and turn-on response with a red shift. This enhancement
might be due to the combination of photo-induced electron transfer (PET) process and chelation-
2
+
enhanced fluorescence (CHEF)[66] and the chelation of L with Zn made the complex more
rigid, which totally restrict C=N isomerisation[67, 68]. The 1:1 binding stoichiometry of L-Zn2+
complex was confirmed by titration studies, Job’s plot and Benesi-Hildebrand nonlinear square
curve fitting methods. Above results reveals that Zn2+ ion coordinates with C=N, C=O, -OH
groups in 1:1 binding mode.
Scheme. 2.
1
2

The potential of receptor L was studied as 'ready to use indicator strips'. The experiments
2
+
were done by using filter paper test strips dipped in solution of receptor L and L-Zn . Both the
2
+
strips were kept under exposure to UV light, the color of L-Zn paper is changed to bright blue
2
+
(
Fig.13). Thus, L could be used as 'ready to use indicator strips' for detecting Zn ions in EtOH-
H
2
O mixture (1:1 v/v, HEPES= 50 mM, pH=7.2).
Fig. 13.
To determine the practical applicability in biological systems, probe L was initially tested
for bioimaging studies in N2A cell lines (Neuroblastoma cell lines from mouse). For this the cell
viability was assessed by MTT assay in vitro. Three independent triplicates were done to
determine the sensitivity of the probe L and the medium not containing the probe was served as
the control. The percentage of cell inhibition was determined using the formula
%
of inhibition = [mean OD of untreated cells (control) / mean OD of treated cells] × 100
and a graph was plotted as the percentage of cell inhibition versus concentration and from this
the corresponding IC50 (the concentration that causes a 50% reduction of the cell growth) value
was calculated as 37.10 ± 2.86 µM (Fig. 14). The concentration of probe L required for live cell
imaging was determined by MTT assay and it was decided as 10 µM, which was less than IC50
value (37.10 ± 2.86 µM).
Fig. 14.
The fluorescence images recorded in green and red channels are shown in Fig. 15. The
N2A cells incubated with probe L alone for 3 h displayed a weak intracellular fluorescence.
Surprisingly, an enhanced intracellular fluorescence was observed in cells containing L
2
+
incubated with Zn for 3 h. From fluorescence and bright field measurements, it is confirmed
that cells were sustainable throughout the imaging experiments. This reveals that probe L shows
2
+
good cell permeability and also used to detect Zn ions in living cells.
1
3

Fig. 15.
Conclusion
We have designed and synthesized a new reversible colorimetric fluorescent probe L,
pyrazolo-quinoline conjugate, which showed enhanced fluorescence intensity in the presence of
Zn2+ ions over other potentially competing metal ions in aqueous media. Thus L exhibits its
advantage of using as a colorimetric, ratiometric and naked eye zinc sensor based on PET and
2
+
4
-1
CHEF mechanism. The binding constant of L-Zn complex was found to be K
a
= 1.88× 10 M
2
+
and the limit of detection (LOD) of probe L with Zn ions is 2.9 nM. The binding mode of probe
2
+
L with Zn was supported by DFT and TDDFT studies. Further, L has been used for live cell
2
+
imaging of Zn ions in N2A cells under physiological conditions.
Acknowledgement
P.S.M thanks, DRDO-BU-CLS (DLS/86/50071/DRDO-BU center /Phase-II) for
providing financial support and instrument facility.
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0

-6
Fig. 1. UV-Vis absorption changes of L (2×10 M) in the presence of different metal ions (50 equiv.) in
EtOH-H O (1:1 v/v, HEPES= 50 mM, pH= 7.2) solution.
2
2
1

-6
Fig. 2. Changes in absorption intensity of L (2×10 M) solution (EtOH-H O, 1:1 v/v, HEPES= 50 mM,
2
2
+
pH= 7.2) upon addition of different amount of Zn (0-25 equiv.).
2
2

-6
Fig. 3. Fluoresence changes of L (2×10 M) solution (EtOH/H O, 1:1 v/v, HEPES= 50 mM, pH= 7.2) in
2
the presence of various metal ions (50 equiv.).
2
3

-6
Fig. 4. Metal ions competition analysis of L (2X10 M) in EtOH/H O, 1: 1 v/v, HEPES = 50 mM,
2
pH=7.2. The blue bars represent fluorescence emission of L and 50 equiv. of other metal ions. The pink
bars represent fluorescence changes that occur upon addition of 50 equiv. of other metal ions to solution
2+
containing L and Zn (50 equiv.).
2
4

2
+
Fig. 5. Fluorescence spectra of L obtained upon addition of Zn (0-25 equiv.)in EtOH/H O mixture (1:1,
2
v/v) using HEPES buffer at pH=7.2.
2
5

2+
Fig. 6. Job's plot for L with Zn ions in EtOH/H O (1:1 (v/v), HEPES = 50 mM, pH= 7.2) solution.
2
2
6

2+
Fig. 7. Benesi- Hildebrand plot of L-Zn complex (1:1) stoichiometry.
2
7

O
CH3
CH3
N
OH N
N
Cl
1
2+
Fig. 8. Partial H- NMR spectra of L and L- Zn .
2
8

2
+
Fig. 9. FT-IR spectra of L and L-Zn .
2
9