A novel pyrazole based single molecular probe for multi-analyte (Zn2+ and Mg2+) detection in human gastric adenocarcinoma cells

Article abstract of DOI:10.1039/c6ra22869k

A new fluorescent sensor, 5-methyl-1H-pyrazole-3-carboxylic acid (6-methoxy-naphthalen-2-ylmethylene)-hydrazide (PYN), composed of a naphthalene group as the fluorogenic unit and a pyrazole carbohydrazide as the binding unit for metal ions has been designed and synthesized. The sensor shows excellent selectivity and sensitivity with a fluorescence enhancement towards Zn²⁺ and Mg²⁺ over other cations in aqueous acetonitrile solution. Turn-on fluorescent enhancements (FE) as high as ～49 fold and ～41 fold in mixed media for Zn²⁺ and, Mg²⁺ respectively were noticed. The signal enhancement of the sensor is based on chelation-enhanced fluorescence (CHEF) effect of PYN-Zn²⁺/Mg²⁺ with the inhibition of the photoinduced electron transfer (PET) effect. Moreover, the Job's plot established 1?:?1 stoichiometry of the complex formation between PYN and Zn²⁺ or Mg²⁺ ions. The limit of detection for Zn²⁺ and Mg²⁺ is as low as 2.2 × 10^?7 M and 3.9 × 10^?7 M respectively. PYN exhibited a second mode of selectivity for Zn²⁺ as it displaces Mg²⁺ from the PYN-Mg²⁺ complex. Density Functional Theory (DFT) calculations have been performed in order to show the structure and electronic properties of PYN and its complexes [PYN-Zn²⁺/Mg²⁺]. Cell imaging experiments confirmed that PYN can be used for monitoring intracellular Zn²⁺ and Mg²⁺ levels in living cells in vitro.

Full text of DOI:10.1039/c6ra22869k

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Page 1 of 29
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DOI: 10.1039/C6RA22869K
2
+
2+
A novel pyrazole based single molecular probe for multiꢀanalyte (Zn and Mg ) detection
in Human gastric adenocarcinoma cells
a
b
c
c
Anamika Dhara, Nikhil Guchhait *, Indrani Mukherjee , Abhishek Mukherjee , Subhash
a
Chandra Bhattacharya *
a
Department of Chemistry, Jadavpur University, Raja S. C. Mallick Road, Kolkata 700 032,
India
b
Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700 009, India
c
Drug Development Diagnostic and Biotechnology Division, CSIRꢀIndian Institute of Chemical
Biology, Kolkata 700032, India
Abstract
A new fluorescent sensor, 5ꢀMethylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylic acid (6ꢀmethoxyꢀ
naphthalenꢀ2ꢀylmethylene)ꢀhydrazide (PYN), composed of a naphthalene group as the
fluorogenic unit and a pyrazole carbohydrazide as the binding unit for metal ions has been
designed and synthesized. The sensor shows excellent selectivity and sensitivity with a
2
+
2+
fluorescence enhancement towards Zn and Mg over other cations in aqueous acetonitrile
solution. Turnꢀon fluorescent enhancements (FE) as high as ~49 fold and ~41 fold in mixed
2
+
2+
media for Zn and, Mg respectively were noticed. The signal enhancement of the sensor is
2
+
2+
based on chelationꢀenhanced fluorescence (CHEF) effect of PYNꢀZn /Mg with the inhibition
of the photoinduced electron transfer (PET) effect. Moreover, the Job’s plot established 1:1
2
+
2+
stoichiometry of the complex formation between PYN and Zn or Mg ions. The limit of
2
+
2+
ꢀ7
ꢀ7
detection for Zn and Mg is as low as 2.2 X 10 M and 3.9 X 10 M respectively. PYN
2
+
2+
2+
exhibited a second mode of selectivity for Zn as it displaces Mg from the PYNꢀMg

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complex. Density Functional Theory (DFT) calculations have been performed in order to show
2
+
2+
the structure and electronic properties of PYN and its complexes [PYNꢀZn /Mg ]. Cell
2
+
imaging experiments confirmed that PYN can be used for monitoring intracellular Zn and
2
+
Mg levels in living cells in vitro.
Introduction
Metal ion detection, both in the environment and in biological organisms, continues to be
1
a thrust area of research in various fields of science. In particular, sensing multiꢀion responsive
2
molecular sensors with multiple emission modes are challenging task. Chemosensor with
multiple analytes are costꢀeffective and highly advantageous for realꢀtime applications.
Fluorescent probe with selectivity for multimetal ions have received a great deal of attention in
recent years. For example, multimetal ion sensors include those that show a different response to
3
+
3+
3
3+
3+
4
2+
+
5
2+/ 3+
6
3+
2+
7
2+
2+
8
2+
2+
9
Cr /Al , Cr /Fe , Mn /Ag , Fe Fe , Fe /Cu , Cu /Zn , Cu /Hg , and
2
+
3+ 10
Zn /Al
Mobile zinc ions, the second most abundant transition metal ion, takes part in a critical
role in biological processes such as neurotransmission, signal transduction, mammalian
.
1
1
reproduction and gene expression.
Zinc imbalance is closely associated with many health
problems, such as prostate cancer, delayed sexual maturation and impotence, type 2 diabetes
mellitus (T2DM), Wilson’s disease, amyotropic lateral sclerosis (ALS) and ageꢀrelated macular
1
2
degeneration (AMD). In addition, free zinc ion is also involved in regulation of programmed
1
3
cell death (i.e., apoptosis). To gain an insight into the crucial role of zinc ion in biological
processes, a variety of fluorescent zinc sensors have been accepted and many of them have been
14
effectively applied in vivo.
It is seen in the literature that various fluorophores such as
quinoline, naphthalimide, bipyridyl, bodipy, rhodamine, pyrene, phenanthroline, coumarin, etc.

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2
+
14
have been used as fluorescent Zn sensors. Despite their interesting features, fluorescent zinc
sensors have some intrinsic drawbacks, especially their short emission lifetimes, resulting in
signal contamination by autofluorescence and scattered light, and hence greatly reducing the
1
0
signal credibility. Since the zinc ion itself is spectroscopically silent due to its 3d electron
1
5
configuration, the development of highly sensitive and selective fluorescent sensors for zinc
2
+
16
ions is therefore of vital importance. Some available Zn sensors
have difficulty in
2
+
2+
distinguishing Zn from Cd , since cadmium is in the same group of the periodic table and has
similar properties. Therefore, the design of a highly selective and sensitive fluorescence sensor
2
+
2+
for Zn detection without interference from other metal ions, especially Cd , is one of the most
vital objectives.
Similarly, magnesium is the second most plentiful cation in intracellular fluids and fourth
1
7
most abundant element in the human body.
It is the heart of chlorophyll molecule, which
plants use to convert carbon dioxide to glucose. Most enzyme activities, cell functions, and
1
8
electrolytes levels are directly or indirectly dependent on magnesium.
It controls neuronal
activity, neuromuscular transmission, cardiac excitability, and blood pressure. From the health
point of view, the daily intake of magnesium for an adult human should reach ca. 300 mg, which
1
9
2+
may come from green vegetables, dairy products, grains and seafood. A low Mg activity has
2
0
been observed in the patients suffering from migraine, and cardiac diseases. On the other hand,
its oversupply in severe cases can cause coma and death. Mineral drinking water may also supply
21
2+
magnesium in fair quantities. The standard method for the determination of Mg in drinking
2
2
2+
water is EDTA complexometric titration. Usually, earlier designs for Mg fluorescent sensors
2
+
were based on several metal chelating structures that have high affinity toward Mg ; these
2
3
24
25
26
sensors include βꢀdiketone, crown ether, polyether, calix[4]arene, etc. However, these

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synthetic fluorescent sensors require timeꢀconsuming and synthetic difficulties with limited
sensitivity or selectivity.
Naphthalene is extensively employed as molecular probes for the study of complex
biological systems because of its short fluorescence lifeꢀtime, low fluorescence quantum yield,
2
7
and ability to act as a donor as well as an acceptor. On the other hand, pyrazole derivatives are
well known five membered heterocycles with a broad spectrum of capable biological activity,
28
namely, as anticancer agents. Furthermore, pyrazoles are recognized as important motifs to be
used as ligands in coordination chemistry, as building blocks in heterocyclic synthesis, as optical
brighteners, and UV stabilizers, and also as units to build supramolecular and photoinduced
2
9
electron transfer systems. So far, much effort has been devoted for the designing of various
2
+
2+
30
chemosensors specific for both Zn and Mg detection. A huge number of fluorescent
chemosensors for zinc or magnesium ions that utilize metalꢀligand coordination have been
3
1
developed because of their high sensitivity and simple operation. However, there are small
number of fluorescent chemosensors reported so far that can selectively sense both of them.
Herein, we present the design, synthesis, spectral properties, and cell imaging studies of
2
+
2+
PYN, a new “turnꢀon” single fluorescent probe that allows Zn and Mg species in a semiꢀ
2
+
2+
32
aqueous system. However, in the presence of these metal ions (Zn and Mg ) the PET
process is restricted and fluorescence intensity is greatly enhanced due to the formation of a rigid
3
3
chelated complex, causing the chelationꢀenhanced fluorescence effect (CHEF). According to
2
+
2+
this significant fluorescence signal change, PYN could detect Zn and Mg sensitively,
ꢀ7
selectively and quantitatively with a wide detection range, a low detection limit (2.2 X 10 M
ꢀ7
2+
2+
and 3.9 X 10 M for Zn and Mg respectively). Furthermore, the obtained probe PYN can be

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2
+
2+
used to detect the Zn and Mg ions in cells by bioimaging. To the best of our knowledge, such
type of unique chemosensor is rare in literature.
Experimental section
General
Chemicals and solvents were purchased from SigmaꢀAldrich Chemicals Private Limited
and were used without further purification. The metal salts used in the present investigation are
as follows: Zn(ClO ) .(H O) , CrCl .(H O) , Cu(ClO ) .(H O) , Fe(ClO ) .(H O) ,
4
2
2
6
3
2
6
4 2
2
6
4 2
2
6
Ni(ClO ) .(H O) , Co(ClO ) .(H O) . NaClO ·H O, KClO .H O, Ca(ClO ) .4H O , Pb(ClO ) .
4
2
2
6
4 2
2
6
4
2
4
2
4 2
2
4 2
3
H O, HgCl , Cd(ClO ) ·6H O, Mg(ClO ) .6H O, Al (SO ) ,.16H O Mn(ClO ) .6H O, GaCl ,
2 2 4 2 2 4 2 2 2 4 3 2 4 2 2 3
InCl etc. The absorption and emission spectra were recorded on Hitachi UV–Vis Uꢀ3501
3
spectrophotometer and PerkinꢀElmer LS55 fluorimeter, respectively. In all measurements the
–
5
sample concentration was maintained at 2×10 M in order to avoid aggregation and reabsorption
effects. All NMR spectra were recorded with TMS as internal standard on Bruker, AV 300/500
Supercon Digital NMR system. Mass spectroscopic analysis of the ligand was performed in a
QTOF Micro YA263 ESIꢀTOF mass spectrometer using methanol as solvent. Fluorescence
lifetimes were measured by the method of Time Correlated Singleꢀ Photon Counting (TCSPC)
using a HORIBA Jobin Yvon Fluorocubeꢀ01ꢀNL fluorescence lifetime spectrometer. The sample
was excited using a laser diode at 375 nm and the signals were collected at the magic angle of
3
4
54.7° to eliminate any considerable contribution from fluorescence anisotropy decay.
The
typical time resolution of our experimental setup is ~ 100 ps. The decays were deconvoluted
2
using DASꢀ6 decay analysis software. The acceptability of the fits was judged by χ criteria and
visual inspection of the residuals of the fitted function to the data. Mean (average) fluorescence
35
lifetimes were calculated using the following equation :

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2
i
α iτ
∑
=
τ av
α iτ
i
∑
(1)
th
in which α is the preꢀexponential factor corresponding to the i decay time constant, τ .
i
i
Caution: Although no problems were encountered during the preparation of perchlorate salts,
suitable care should be taken when handling such potentially hazardous compounds.
Synthesis
5
ꢀMethylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylic acid hydrazide (C) was prepared following an
3
6
established method. 5ꢀMethylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylic acid hydrazide, C (1 g, 7.13 mmol) in
0 mL methanol was added dropwise to the methanolic solution (5 mL) of 6ꢀMethoxyꢀ
2
naphthaleneꢀ2ꢀcarbaldehyde (1.32 g, 7.13 mmol) with constant stirring. The resulting solution
was refluxed for 5 hours and cooled to room temperature. Excess methanol was removed by
rotary evaporator to have a colourless high viscous liquid. It was then kept in ice bath. After a
while, the liquid froze to a microcrystalline white solid. The solid was filtered off, washed
thoroughly with cold methanol and dried in vacuo over fused CaCl . Yield (1.45 g, 65.9%). Mp
2
0
1
>
250 C (decomp.). HꢀNMR (500 MHz, DMSOꢀd ): δ (ppm), 2.31 (s, 3H, ꢀCH ), 3.91 (s, 3H, ꢀ
6 3
OCH ), 6.53 (s, 1H, ArꢀH), 7.23 (t, 1H, ArꢀH, J = 5 Hz), 7.38 (d, 1H, ArꢀH, J = 2.2 Hz), 7.92ꢀ
3
7
1
1
.87 (m, 3H, ArꢀH), 8.00 (s, 1H, CH=N), 8.61 (s, 1H, py), 11.61 (s, 1H, ring py NH), 13.09 (s,
1
3
H, ꢀCONH); CꢀNMR (300 MHz, DMSOꢀd ): δ (ppm), 10.76, 30.97, 40.83, 55.57, 79.63,
6
05.18, 105.57, 119.38, 123.82, 127.57, 128.57, 130.45, 135.61, 147.88, 158.57, 206.36; ESIꢀ
+
MS: m/z calculated for C H N O [M+H] 309.13, found 309.16; Anal. Calc. for C H N O :
1
7
16
4
2
17 16
4
2
C, 66.34; H, 5.28; N, 18.18; Found: C, 66.22; H, 5.23; N, 18.17.

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Scheme 1. Synthesis of the probe PYN.
2
+
Synthesis of the PYN-Zn complex
Zn(ClO ) .6H O (0.24 g, 0.6486 mmol) was dissolved in 5 mL of ACN and to this
4
2
2
solution PYN (0.2 g, 0.6486 mmol) was added. After three hours of stirring at room temperature,
2
+
the solution was dried using rotary evaporator which yielded a white PYNꢀZn complex. The
2
+
1
13
resulting complex (PYNꢀZn ) is purified by recrystallization and characterized by H NMR, C
NMR, FTꢀ IR, TOFꢀMS (Figs. S19, S21, S23, S8, ESI).
2
+
Synthesis of the PYN-Mg complex
Mg(ClO ) .6H O (0.21 g, 0.6486 mmol) was dissolved in 5 mL of ACN and to this
4
2
2
solution, PYN (0.2 g, 0.6486 mmol) was added. After three hours of stirring at room
2
+
temperature, the solution was dried using rotary evaporator which yielded a white PYNꢀMg
2
+
complex. The resulting complex (PYNꢀMg ) is purified by recrystallization and characterized
1
13
by H NMR, C NMR, FTꢀ IR, TOFꢀMS (Figs. S20, S22, S24, S9, ESI).
Determination of the fluorescence quantum yield
The fluorescence quantum yields were determined using anthracene as a reference with a
37
known Φ value of 0.27 in EtOH. The area of the emission spectrum is integrated using the
R
software available in the instrument and the quantum yield is calculated according to the
3
8
following equation :

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2
S
2
Φ /Φ = [A /A ] × [(Abs) /(Abs) ] ×[η /η ]
(2)
S
R
S
R
R
S
R
where Φ and Φ are the fluorescence quantum yield of the sample and reference, respectively;
S
R
A and A are the area under the fluorescence spectra of the sample and the reference,
S
R
respectively; (Abs) and (Abs) are the corresponding optical densities of the sample and the
S
R
reference solution at the wavelength of excitation; η and η are the refractive index of the
S
R
sample and the reference, respectively.
Cyclic voltammetry
The cyclic voltammetric measurements were performed with a Sycopel model
AEW21820F/L instrument. The experiments were carried out in a standard threeꢀcomponent cell
equipped with a glassy carbon working electrode, a Pt wire as an auxiliary electrode and
Ag/AgCl reference electrode. The cyclic voltammograms were acquired in a N ꢀbubbled
2
acetonitrile solution containing ~ 0.2 M tetraethylammonium perchlorate (TEAP) as supporting
electrolyte. The scan speed was 100 mV/s.
Calculation for detection limit
39
2+
2+
The detection limit (DL) of PYN for Zn /Mg was determined using the following
equation:
DL = 3Sb/S
(3)
Where Sb is the standard deviation of the blank solution; S is the slope of the calibration curve.
Cell culture
Human gastric adenocarcinoma (AGS) cells were purchased from National Center for
Cell Science, Pune, India, and used throughout the experiments. Cells were cultured in DMEM
(Gibco BRL) supplemented with 10% FBS (Himedia), and antibiotic mixture (1%) containing
PSN (Himedia) at 37 ºC in a humidified incubator with 5% CO2.

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Cell imaging
For imaging experiments, cells were grown in 6ꢀwell plates at 70ꢀ80% confluence. Cells
were then incubated in DMEM containing different concentrations (500 nM, 1 µM, 10 µM) of
PYN for 30 min at 37 ºC. Cells were then washed with PBS followed by the addition of 10 µM
2
+
2+
Zn and Mg ions and incubation for 10 min. Bright field and fluorescence images were
captured at 20X magnification by a fluorescence microscope (Olympus, 1x70) using Camedia
software (Chicago, MI, USA) (E20P 5.0 Megapixel) and processed using Adobe Photoshop
version 10.0. Also fluorescent intensity was monitored after the addition of TPEN (50 µM in the
incubation media).
Cell cytotoxicity assay
To assess the cytotoxicity of the PYN, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,Sꢀ
diphenyltetrazolium bromide (MTT) assay was performed with AGS cells as per the procedure
4
0
3
described earlier. After treatment of overnight culture of AGS cells (10 cells in each well of
96ꢀwell plate) with PYN (0.5, 1, 10 and 100 ꢁM) for 12 h, 10 ꢁl of a MTT solution (1mg/ml in
PBS) was added in each well and incubated at 37 ºC for 3 h. Culture media were then removed
and 100ꢁl of acidic isopropyl alcohol was added into each well. The intracellular formazan
crystals (blueꢀviolet) formed were solubilized with 0.04 N acidic isopropyl alcohol and
absorbance of the solution was measured at 595 nm wavelength with a microplate reader
(Model: THERMO MULTI SCAN EX). The cell viability was expressed as the optical density
ratio of the treatment to control.
Results and discussions
3
6
The synthesis and characterization of the compound (C) has been published earlier.
The target compound PYN was easily synthesized by simple Schiff base condensation of 5ꢀ

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Methylꢀ1Hꢀpyrazoleꢀ3ꢀcarboxylic acid hydrazide (C) with 6ꢀMethoxyꢀnaphthaleneꢀ2ꢀ
1
13
carbaldehyde (Scheme 1). The structure of compound PYN was characterized by H NMR, C
NMR, FTꢀ IR, TOFꢀMS and CHN elemental analyses (Figs. S1ꢀS4, ESI).
With an objective to evaluate the potential use of the probe PYN as a chemosensor, we
investigated its interaction with various metal ions using perchlorate as a counter anion under
+
+
UVꢀVis and fluorescence measurements. Among the competitive metal ions, such as Na , K ,
2
+
2+
2+
3+
3+
2+
2+
3+
2+
2+
2+
3+
3+
2+
2+
2+
Ca , Mg , Pb , Al , Cr , Mn , Fe , Fe , Co , Ni , Cu , Ga , In , Zn , Cd and Hg ,
o
2+
tested in 7 : 3 (v/v) aqueous acetonitrile solution (10 mM TrisꢀHCl, pH 7.2 at 25 C), only Zn
2
+
and Mg respond to the probe PYN in the ground and excited states. The probe PYN (5 µM)
itself exhibited three absorption bands at 325 nm, 274 nm and 282 nm. The absorbance of PYN
(5 µM) at 325 nm, 274 nm and 282 nm gradually decreases and a new absorption band appears at
2
+
3
75 nm with addition of increasing concentrations of Zn (Fig. S5, ESI). In contrast, titration of
2
+
PYN (5 µM) with concomitant addition of Mg resulted in increase in the absorption bands at
25 nm, 274 nm and 282 nm whereas simultaneously a new band appeared at 253 nm with
3
increased in intensity (Fig. S6, ESI). However, no such significant change in PYN spectrum was
observed with above mentioned metal cations (Fig. S7, ESI).
2
+
2+
The hostꢀguest recognition abilities of PYN with Zn and Mg ions further investigated
via a fluorescence spectroscopic method. The excitation wavelength was selected at 370 nm. No
change in fluorescence was observed with various Groups 1, 2 and transition metals, while
2
+
2+
exposure to Zn or Mg produced a large increase in fluorescence for the probe PYN (Fig. 1).
Under physiological conditions, the probe PYN (5 µM) exhibited maximum emission around
2
+
2+
4
36 nm with very low quantum yields of 0.003. However, upon gradual addition of Zn or Mg
2
+
resulted in a remarkably enhanced fluorescence at 456 nm (sky blue emission for Zn ) or 472

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2
+
nm (blue emission for Mg ) with increasing concentration (0ꢀ1.6 equiv), of the metal ions (Figs.
2
+
2+
2
and 3). The plots of emission intensity of PYN as a function of added Zn and Mg are
presented in inset Figs. 2 and 3 respectively. Therefore, the probe PYN’s fluorogenic sensing
2
+
2+
ability could be observed by the naked eye in the presence of Zn or Mg with the enhancement
of a clearly observed sky blue and blue emission respectively when illuminated with 254 nm
UVꢀlight (Inset Fig. 4). The red shift of the emission band is accompanied by 49ꢀfold
2
+
fluorescence enhancement for Zn (Φ, 0.15). Similarly 41ꢀfold fluorescence enhancement (Φ,
2
+
0
.12,) is observed for Mg . Relative fluorescence enhancement of PYN in the absence and
presence of various other metal ions was shown in Fig. 4.
Fig. 1. Fluorescence spectra (excitation at 370 nm) of the probe PYN (5 ꢁM) in 7:3 (v/v) MeCNꢀ
o
water solution (10 mM TrisꢀHCl, pH 7.2 at 25 C), by addition of 1.6 equiv. different metal ions
+
+
2+
2+
2+
3+
3+
2+
2+
3+
2+
2+
2+
3+
3+
2+
2+
(
Na , K , Ca , Mg , Pb , Al , Cr , Mn , Fe , Fe , Co , Ni , Cu , Ga , In , Zn , Cd ,
2
+
Hg ).

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Fig. 2. Emission spectra of the probe PYN (5 µM) in the presence of increasing concentrations
2
+
o
of 1.6 equiv. of Zn in 7:3 (v/v) MeCNꢀwater solution (10 mM TrisꢀHCl, pH 7.2 at 25 C).
Excitation was performed at 370 nm. Inset: fluorescence intensity at 456 nm as a function of
2
+
Zn concentration.
Fig. 3. Emission spectra of the probe PYN (5 µM) in the presence of increasing concentrations
2
+
o
of Mg in 7:3 (v/v) MeCNꢀwater solution (10 mM TrisꢀHCl, pH 7.2 at 25 C). Excitation was
2
+
performed at 370 nm. Inset: fluorescence intensity at 472 nm as a function of Mg
concentration.
Fig. 4. Bar graph shows the relative emission intensity of the probe PYN (5 ꢁM) upon treatment
o
with various metal ions in 7:3 (v/v) MeCNꢀwater solution (10 mM TrisꢀHCl, pH 7.2 at 25 C).
2
+
2+
Inset: fluorescence change after the addition of Zn and Mg metal ions under UV lamp.
2
+
2+
The reason for the addition of Zn or Mg to the solution PYN resulting in fluorescence
enhancement is due to suppression of photoinduced electron transfer effect (PET) from the lone
2
+
2+
pair electron of the pyrazole used for metal (Zn /Mg ) binding (Scheme 2).

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n+
Scheme 2. Proposed binding mode of PYN with M (n= Zn, Mg).
Additional support for the proposed binding process and PET mechanism between the
pyrazole carbohydrazide and naphthalene moiety is thermodynamically feasible which is evident
from the electrochemical data. PET is the most widely employed mechanism for the design of
fluorescence probes. Classical PET probes for metal cations contain three parts: fluorophore,
spacer and ionophore. Ionophores are normally electron donors (e.g. aminoꢀcontaining group),
while fluorophores are electron acceptors. In this paper pyrazole carbohydrazide is ionophores
and naphthalene moiety is fluorophore. The reduction potential of the fluorophore, Ered
(fluorophore) is ꢀ 0.71V (reversible), while the oxidation potential of the fluorophore is observed
at around 1.08 V (reversible). With the pyrazole moiety, one irreversible oxidation wave was
3
4, 41
observed at 0.46V (vs. Ag/AgCl). Therefore, the free energy change for the PET process
(ꢂGPET) is ꢀ46.06 kcal/mol in acetonitrile according to ꢂGPET = 23.06 [E ox (recep) ꢀ E red
(fluoro)] ꢀ E0,0 ,where, Eox (recep) and Ered (fluoro) represent the oxidation potential of the
receptor moiety and the reduction potential of the fluorophore, respectively, whereas E0,0
represents the energy of the fluorescent state. The value of E0, 0 used in the calculation has been
estimated from location of the first peak position in the fluorescence spectrum. Negative ꢂG
value dictates the thermodynamic feasibility of the PET process in the studied system.
4
2
To determine the stoichiometry, a Job’s plot obtained from the emission data showed a
2
+
2+
1
:1 association of PYN with both Zn and Mg (Figs. 5 and 6 ). Furthermore, the result is
2
+
2+
consistent with the ESIꢀMS experiment of PYN with Zn and Mg (Figs. S8 and S9, ESI). A

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2
+
ꢀ
+ +
peak at m/z 488.17 assigned to [PYN + Zn + ClO + H O ꢀ H ] is observed (calcd. 488.10).
4
2
2
+
Also, the positiveꢀion mass spectrum of PYN upon addition of 1 equiv of Mg showed the
2
+
ꢀ
+ +
formation of the [PYN + Mg + ClO ꢀ H ] complex [m/z: 430.97; calcd, 430.05].
4
2
+
Fig.5 Job’s plot for the binding of probe PYN and Zn , where the intensity at 456 nm was
2
+
plotted against the mole fraction of Zn .
2
+
Fig.6 Job’s plot for the binding of probe PYN and Mg , where the intensity at 472 nm was
2
+
plotted against the mole fraction of Mg .
4
3
2+
2+
The binding constant (K ) for PYNꢀZn /Mg complexs were calculated from the fluorescence
a
titration by mathematical models such as BenesiꢀHildebrand. We thus start with the equation:

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n+
1
/ (I ꢀ I ) = 1/{K (I ꢀ I ) [M ]} + 1/ (I ꢀ I )
(4)
0
a
∞
0
∞
0
in which I , I and I are the emission intensities, respectively, in the absence of, at an
0
∞
n+
intermediate and infinite concentration of the metal ion (M ).
n+
2+
2+
A linear regression for the plot of 1/[IꢀI ] vs. 1/[M ] for Zn and Mg ions dictates a 1:1
0
association between PYN and each of the two metal ions. Assuming a binding interaction
between the two partners, the strength of binding can be quantitatively evaluated in terms of
6
ꢀ1
binding constant as K = intercept to slope ratio of the BenesiꢀHildebrand plot = 7.01 ×10 M
a
2
+
4
ꢀ1
2+
for Zn and 3.23 × 10 M for Mg (Figs. S10 and S11, ESI). Consequently the free energy
change for the process of complexation reaction is calculated to be ꢂG = ꢀRT ln K = ꢀ 39.02 kJ
a
ꢀ1
2+
ꢀ1
2+
mol for Zn and ꢀ 25.52 kJ/mol for Mg , i.e. a negative free energy change which dictates
the thermodynamic feasibility for the occurrence of the binding interaction. Also a relatively
2
+
stronger binding of PYN with Zn ion is manifested through a higher binding constant value as
2
+
2+
compared with Mg ion. Stronger binding implies higher rigidity of PYNꢀZn complex than
2
+
PYNꢀMg complex which causes the inhibition of different nonꢀradiative decay processes
(PET) resulting in enhancement of fluorescence.
For a probe to be extensively used in the detection of specific targets, reversibility is an
important issue. To examine whether the sensing process of PYN is reversible, 1 equiv of a
sodium salt of ethylendiaminetetraacetic acid (EDTA) solution was added into the solution of
2
+
2+
PYN, which was preincubated with 1 equiv of a Zn /Mg solution. After the addition of an
EDTA solution, the initial emission intensity of PYN was almost recovered immediately from
2
+
2+
PYNꢀZn and PYNꢀMg complexes (Figs. S12 and S13, ESI). It indicates the decomplexation
2
+
2+
2+
2+
of PYNꢀZn and PYNꢀMg as EDTA strips away Zn and Mg from the binding zone.

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2
+
For practical purposes, the detection limit of probe PYN for the analysis of Zn and
2
+
Mg ions were also an important parameter. We have determined the standard deviation (Sb) of
the blank solution (Fig. S14, ESI). In addition, the fluorescence titration profile also
ꢀ7
2+
ꢀ7
2+
demonstrates that PYN has a detection limit of 2.2 X 10 M for Zn and 3.9 X 10 M for Mg
2
+
2+
ions (Figs. S15 and S16, ESI), which are comparable to the other reported Zn and Mg
4
4
chemosensors.
The interference of other competing metal ions was investigated by the competition
2
+
2+
experiment which was conducted with Zn and Mg in the presence of other competing ions
Fig. S17 and S18, ESI). The fluorescence intensity of the complex was hardly affected by other
coexistent metal ions. These results indicated that PYN could be used as an efficient and
(
2
+
2+
selective fluorescence probe for Zn and Mg in the presence of a wide range of other
competing cations.
2
+
Based on the fluorimetric experiments, the affinity of PYN with Zn is much stronger
2
+
2+
2+
2+
than that with Mg , when Zn was added to the solution of PYNꢀMg ensemble, Mg was
2
+
displaced by Zn , resulting in a significant decrease emission intensity at 472 nm and an
increase of a blueꢀshifted emission band centered at 456 nm (attributed to the formation of a
2
+
2+
PYNꢀZn complex) (Fig. 7). This suggests that [PYNꢀMg ] can be used as a second optical
2
+
2+
response for sensing Zn . However, the addition of increasing concentrations of Mg to PYNꢀ
2
+
Zn , no response was observed (Fig. 8). These results revealed that PYN can be employed as a
2
+
dualꢀmode Zn sensor via the competitive metal ion displacement approach.

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2
+
2+
Fig. 7 Fluorescence spectra of PYNꢀMg upon addition of Zn in 7:3 (v/v) MeCNꢀwater
o
solution (10 mM TrisꢀHCl, pH 7.2 at 25 C.
2
+
2+
Fig. 8 Fluorescence spectra of PYNꢀZn upon addition of Mg in 7:3 (v/v) MeCNꢀwater
o
solution (10 mM TrisꢀHCl, pH 7.2 at 25 C.
1
To evaluate the binding of metal ion to the probe PYN, HꢀNMR titration experiments in
1
DMSOꢀd are carried out (Fig. 9). H NMR titration experiment of PYN shows that the proton
6
at δ 12.94 ppm corresponding to ꢀCONH peak of the probe PYN disappears on adding
2
+
2+
Zn /Mg . The signal at δ 8.42 ppm corresponding to azomethine proton (C) is slightly shifted
to upfield. If metals bind to the immine nitrogen then a downfield shift is expected. Now slight
upfield shift of other protons (b, d and aromatic hydrogens) may be explained in terms of

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deprotonation of the amide proton which leads to increase in electron density over the probe and
as a consequence upfield shift is observed.
1
2+
2+
Fig. 9 The HꢀNMR spectra of PYN upon the addition of 1 equiv. of (1) Zn and (2) Mg
in DMSOꢀd6.
2
+/
2+
Further, IR spectra of probe PYN with Zn Mg ion also confirms the proposed mechanism
Figs. S23 and S24, ESI) as in both cases the immine peaks remains almost unaffected.
Therefore, these findings clearly support that the complexation occurs between the amide
(
2
+
2+
carbonyl oxygen with Zn /Mg and pyrazole N atoms to the metal center
Timeꢀresolved fluorescence decay studies for PYN were carried out in the absence and
2
+
2+
presence of Zn and Mg ions using TCSPC technique (Fig. 10). A laser excitation source of
375 nm was used and decay luminescence kinetics was monitored at 465 nm. The fluorescence
decay behavior of PYN further confirms PETꢀinduced fluorescence quenching. The decay
behaviour of the bare fluorophore (PYN) or its metal complex is found to be complicated and is
best fitted to triexponential functions. The fluorescence decay curve of PYN consisted of a shortꢀ
lived major component (~0.06 ns, ~93%) which clearly represents the lifetime of the PETꢀ
3
4, 35
2+
2+
quenched fluorophore
. In the presence of Zn and Mg , rigidification of the framework
upon binding of these two metal ions results in lowering the nonradiative decays. Therefore,

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average lifetime (τ ) enhances significantly upon the addition of these two cations (Table 1,
av
ESI).
2
+
2+
Fig. 10 Timeꢀresolved fluorescence decay of PYN (red), PYNꢀZn (olive green) and PYNꢀMg
(blue).
2
+
2+
To better realize the nature of the coordination of Zn /Mg with PYN, energyꢀoptimized
2
+
2+
structures of PYN and PYNꢀZn /Mg were obtained on Density Functional Theory (DFT)
calculations at the B3LYP level using 6ꢀ311G** basis set for probe (PYN) and LANL2DZ basis
4
5
set for metal complexes using the Gaussian 09 program. The DFT computed molecular
2
+
2+
structure of PYN and its PYNꢀZn /Mg complexs were shown in Fig. 11a. The energy gaps
between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular
2
+
2+
orbital (LUMO) of the probe PYN and corresponding Zn and Mg complexes were found to
be 3.95 eV, 3.23 eV and 3.38 eV respectively (Fig.11b). These results exhibited that both the
2
+
HOMO and LUMO of the PYNꢀZn complex was better stabilized than those of PYN and
2
+
PYNꢀMg complex (Table 2, ESI).

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2
+
Fig. 11a Stereoscopic view of the energy optimized structures of (a) PYN (b) PYNꢀZn and
2
+
(
c) PYNꢀMg .
Fig. 11b HOMOꢀLUMO energy gaps for respective compounds and interfacial plots of the
2
+
2+
orbitals of (a) PYN (b) PYNꢀZn (c) PYNꢀMg .
To investigate the potential biological application of the probe PYN in living cells, we
2
+
2+
performed intracellular Zn and Mg dual imaging of AGS cells by fluorescence microscopy.
After incubation with DMSOꢀcontaining PYN (500 nM, DMSO:water = 1:99) for 30 min at 37
0
2+
C, cell could not show any recognizable fluorescence. However, addition of exogenous Zn
2
+
and Mg ions (10 µM each) separately to the cells showed blue (Fig. 12a) and green
fluorescence (Fig. 12b) respectively. These experiments indicate PYN can discriminate

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2
+
2+
intracellularly between Zn and Mg with blue and green fluorescence, respectively. Moreover,
with increasing concentration of probe PYN (500 nM, 1µM & 10 µM) fluorescence intensity
2
+
was enhanced concentrationꢀdependently. The fluorescence signal of PYN in presence of Zn
2
+
and Mg ions may be utilized as a signature of selective sensor response. Hence, these results
indicate that the Probe PYN is an efficient candidate for monitoring changes in the intracellular
2
+
2+
Zn and Mg ions concentration under biological conditions.
Fig. 12 a (AꢀI), (AꢀII) & (AꢀIII) are the bright field images of the Probe PYN (500 nM, 1 µM &
10 µM) treated cells respectively. (BꢀI), (BꢀII) & (BꢀIII) represent fluorescence images treated
2
+
with different concentrations of the Probe PYN, in absence of Zn ions. (CꢀI), (CꢀII) & (CꢀIII)
indicate fluorescence images treated with different concentrations of Probe PYN in addition of
2
+
Zn (10 µM) ions. The samples were excited at 375 nm.

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Fig. 12 b (DꢀI), (DꢀII) & (DꢀIII) are the bright field images of 500 nM, 1µM & 10 µM Probe
PYN treated cells respectively. (EꢀI), (EꢀII) & (EꢀIII) represent fluorescence images treated with
2
+
different concentrations of Probe PYN alone in absence of Mg ions. (FꢀI), (FꢀII) & (FꢀIII)
indicate fluorescence images treated with different concentrations of Probe PYN in addition of
2
+
Mg (10 µM) ions. For all imaging, the samples were excited at 375 nm.
The MTT assay results confirm that PYN has no significant cytotoxic effects on
adenocarcinoma cells (AGS) up to 12 h of treatment (Fig.13). Thus the PYN has promising
2
+
2+
potential in dual sensing of Zn and Mg in vitro.
105
100
95
90
85
80
0
0.5
1
10
100
Sensor concentration (ꢀM)
Fig. 13 % cell viability of AGS cells treated with different concentrations (0.5ꢀ100 µM) of PYN
for 12 hrs determined by MTT assay. Results are expressed as mean ± S.D of three independent
experiments.

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Conclusions
In conclusion, we have successfully developed a low cost and easily synthesize Schiffꢀ
2
+
2+
base probe PYN for the selective detection of Zn and Mg showing ‘turnꢀon’ fluorescent
response without any interference from other tested metal ions. The probe followed a turnꢀon
2
+
mechanism with the inhibition of PET and the activation of CHEF. Moreover, Zn displaces
2
+
2+
Mg from the PYNꢀMg complex, demonstrating that a dualꢀmode PYN sensor can be
2
+
employed to detect Zn through two distinct processes based on the CHEF and competitive
metal ion displacement approaches. The proposed fluorescence probe has a low sensing limit as
2
+
ꢀ7
2+
ꢀ7
well as a high selectivity towards Zn (2.2 X 10 M) and Mg (3.9 X 10 M). Furthermore, the
2
+
2+
probe PYN is found to be efficient in reporting the presence of both Zn and Mg ions inside
the cell without showing any cytotoxicity.
Acknowledgements
A. D. acknowledges the financial support provided by University Grants Commission,
India (Award letter No. F.4ꢀ2/2006(BSR)/CH/14ꢀ15/0163, dated May, 2015) through the Dr. D.
S. Kothari Post Doctoral Fellowship (DSKPDF).
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