A fluorescent "turn-on" sensor for the biologically active Zn2+ ion

Article abstract of DOI:10.1016/j.ica.2014.05.014

A linear Schiff-based receptor (L) containing two imino phenol units was developed for the selective fluorescent sensing of Zn²⁺ with a discriminating enhancement of 12-folds over the other surveyed cations. Within the huge plethora of Zn²

Full text of DOI:10.1016/j.ica.2014.05.014

Accepted Manuscript
A novel fluorescent “turn-on” sensor for the biologically active Zn2+ ion
Kundan Tayade, Suban K. Sahoo, Shweta Chopra, Narinder Singh, Banashree
Bondhopadhyay, Anupam Basu, Nilima Patil, Sanjay Attarde, Anil Kuwar
PII:
S0020-1693(14)00292-8
http://dx.doi.org/10.1016/j.ica.2014.05.014
ICA 15996
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 April 2014
16 May 2014
17 May 2014
Please cite this article as: K. Tayade, S.K. Sahoo, S. Chopra, N. Singh, B. Bondhopadhyay, A. Basu, N. Patil, S.
Attarde, A. Kuwar, A novel fluorescent “turn-on” sensor for the biologically active Zn2+ ion, Inorganica Chimica
Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.2014.05.014
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A novel fluorescent “turn-on” sensor for the biologically active Zn2+ ion
KundanTayadea, Suban K. Sahooc, Shweta Chopraf, Narinder Singhd, Banashree
Bondhopadhyaye, Anupam Basue, Nilima Patila, Sanjay Attardeb, Anil Kuwar*a
aSchool of Chemical Sciences, North Maharashtra University, Jalgaon- 425001 (MS) India.
bSchool of Environmental and Earth Sciences,North Maharashtra University, Jalgaon-425001
(MS) India.
cDepartment of Applied Chemistry, SV National Institute Technology, Surat-395007
(Gujarat) India.
dDepartment of Chemistry, Indian Institute Technology Ropar-140 001 (Panjab) India.
eMolecular Biology and Human Genetics Laboratory Department of Zoology The University
of Burdwan, Burdwan, West Bengal, India.
fCentre for Nanoscience & Nanotechnology, UIEAST, Panjab University Chandigarh, India.
Abstract
A novel linear Schiff-basedreceptor (L) containing two imino phenol units was
developed for the selective fluorescent sensing of Zn2+ with a discriminating enhancement of
12 folds over the other surveyed cations. Within the huge plethora of Zn2+receptors, this
receptor is quite worth to mention because of its simple synthesis, low cost, no interference of
Cd2+ and the judicious response availed with discernment. The spectroscopic (UV-Vis,
fluorescence and mass) results, few mathematical models for determining analytical
parameters such as binding constant (Ka), binding stoichiometry, detection limit etc and the
frontier molecular orbitalswere analyzedto explore the sensing ability of L and the
mechanism.
Keywords: Fluorescent ‘turn-on’ sensor, Zn2+, Schiff base, DFT.
* Corresponding authors (A.Kuwar): E-mail: kuwaras@gmail.com.

1. Introduction
Molecular detection is the foundation for understanding many biological functions [1-
5]. In the recent years, the growth of artificial receptors for recognizing cationic, neutral, and
anionic species are growing because of their important roles in a wide range of biological,
environmental, and chemical course of actions [6-10].These receptors are suitably derived
using different light-emitting groups to develop chemosensors which can be applied for the
qualitative and quantitative detection of the target species.Chemosensors based on
colorimetric and fluorescent responses with possible applications in biological corridor have
received immense interest because of many advantageous features like simplicity, high
sensitivity and selectivity, easy of detection and tunability [11-14].
The transition metal ions play an important role inmany fundamental physiological
processes in organisms ranging frombacteria to mammals [15]. It is therefore significant to
understand their environmental advantages and risks to the living systems. Zinc is a
ubiquitous and indispensable element having second highestabundance after iron in the
humanbody,which playsmultiple roles in both extra- and intra-cellular functions [16]. The
total concentration of Zn2+ in different cells varies from nano to millimolar range.The
excessiveconcentration of Zn2+ above cellular needs causestoxicity and also displaces other
essential metalions that act as co-factors in enzyme-catalyzed reactions [17].Therefore, the
development of fluorescent chemosensors to selectively detect and monitor intracellular
Zn2+ ions is certainly an important issuein the recent years.In this context, numerous efforts
have been devoted for the development of such chemosensors displaying phenomena like
quenching, enhancement or ratiometric responseswith the help of different signalling
mechanisms such as photoinduced electron transfer (PET), C=N bond isomerisation, excited-
state intramolecular proton transfer (ESIPT) [18].

2-Hydroxyacetophenone is a popular substrate for nucleophilic addition reactions by
virtue of its carbonyl group which can be activated by the neighbouring phenolic hydroxyl
group of the same molecule through intramolecular hydrogen-bonding [19]. Based on this
concept, chemosensors with 2-hydroxyacetophenone functionality as the binding site have
been developed for the selective detection of different cationsand anions [20]. In this paper,
we have designed and developed a Schiff base receptor using 2-hydroxyacetophenone
scaffold for the fluorometric determination of Zn2+ by fluorescence ‘turn-on’ response
induced by the inhibition of PET process and/or C=N isomerization.
2.Results and Discussion
The receptor L was synthesised by adopting the reported method [21] by Schiff base
condensation reaction of the corresponding 1-(2-hydroxyphenyl)ethanonewith 1,2-
diaminoethanein ethanolic medium (Scheme 1). The 1H NMR spectra of receptor L displaysa
significant downfield signal at 15.94 ppm for the phenolic protons, indicating the presence of
intramolecular hydrogen bonding between the phenolic-OH and the neighbouring imine-N
atom (Figure S1-3).
ꢀ
Scheme 1. Synthesis of receptor L.
The ability of L to complex differentcations was explored with the absorption and
fluorescence spectrophotometer in mixed MeCN/H2O (99:1, v/v) solution, use of this solvent
mixture has solved the solubility problem of ligand and allowed it to be used as a
chemosensor. Among all the tested cations (Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+,
Cd2+, Bi2+) as their nitrate salts, only Zn2+ was found to cause obvious spectral changes in the
absorption spectra of L (Fig. S4) and no particular changes were observed in the presence of

other cations. The free receptor L exhibited two absorption peaks with maxima at254 nm and
→
320 nm which were attributed to π→π* and n π* transitions associated with the azomethine
chromophore and the imine nitrogen atom in conjugation with the phenol group
respectively.Also, the existence of receptor L predominantly in the enolimine form can be
depicted from the absence of absorption band above 350 nm. On selective interaction of Zn2+
with the imine-N and phenolate ions of L, the absorption bands of L were appreciably red-
shifted due to the intramolecular charge transfer (ICT) process which brings the lowest
excited state closer to the highest ground state. The absorption spectral titration of L with the
successive addition of Zn2+ was shown in Fig. 1. With the addition of Zn2+, the receptor
bands at 254nm and 320nm were attenuated while new bands appeared at longer wavelength
274nm and 358 nm (∆λ = 20 nm and 38 nm respectively). The spectral titrations exhibited
four isosbestic points at 241 nm, 260 nm, 296 nm and 333 nm which clearly indicate the
formation of a well- defined L.Zn2+ complex and result was further supported by the addition
of 0 to 300 µL of zinc nitrate solution that produces a noticeable colour change.
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
200
220
240
260
280
300
320
340
360
380
400
Wavelength (nm)

Fig. 1.Spectrophotometric titration of L (0.1mM, 2000 µL) upon successive additionof Zn2+
(1mM, 0-300 µL) in MeCN/H2O (99:1, v/v).
The DFT computed molecular structure of L and its L-Zn2+ complex was shown in
Fig. 2. The optimized structure of Lindicates the presence of intramolecular hydrogen bonds
of length 1.628 Å. Also, the enolimine (OH) form of L was found to be energetically more
stable than the ketoenamine (NH) formby -8.62 kcal/mol.The optimized structure of L-Zn2+
complex (Fig. 2) produce a distorted tetrahedral geometry with the average Zn-N and Zn-O
bond lengths of 2.096 Å and 1.936 Å respectively. The band gap between the highest
occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of L
was lowered on complexation with Zn2+ which caused the red-shift in the absorption band of
L (Fig. 3). Furtheranalysis of frontier molecular orbitals (FMOs) plots of L and its L-Zn2+
complex indicate the intramolecular charge transfer occurred between the two iminophenol
units on complexation with Zn2+.
Fig. 2. Optimized structure of receptor (a) L and its (b) L.Zn2+ complex.

Fig. 3. The DFT computed HOMO and LUMO diagram of (a) L and (b) its L-Zn2+ complex.
The fluorescence properties of L was studied upon addition of 0.5 equiv. of Cr3+,
Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+, Bi2+ in MeCN/H2O (99:1, v/v) solution
(Fig.4). The receptor L showed weak fluorescence emission at ~500 nm upon excitation at
320 nm presumably due to the PET from the imine-N to the fluorophore and/or the C=N
bonds isomerization in the excited states. Upon addition of Zn2+, prominent fluorescence
enhancement was observed at 450 nm with the ratiometric fluorescence responses (∆λ = 50
nm, anti-strokes or blue shift) (Figure S5). In contrast, no appreciable change in the emission
behaviour of L was observedwith other tested metal ions including Cd2+. The suitable
coordination geometry conformation of the chelating Schiff-based receptor Land the selective
binding of Zn2+ close to fluorophore may have influenced the imine and hydroxyl moieties.
Consequently, a large chelation enhanced fluorescence (CHEF) effect was observed upon
complexation with Zn2+ which inhibited the PET process from the electron-donating group to
the fluorophore and/or the C=N isomerisation [22]. Further, the fluorescence response (Fig.5)

of L indicates the unique selectivity for the Zn2+with 12 folds enhancement among the other
tested metal ions.
5.00E+06
Zn(II)Ions
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
Other metal
350
400
450
500
550
600
Wavelength (nm)
Fig. 4.Fluorescence spectra of L (0.1 mM) upon addition of 100 µL of respective metal ions
(1.0mM).
5.00E+06
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
Various Metal Ions
Fig. 5. Fluorescence response of L (0.1mM) in MeCN/H2O (99:1, v/v) uponaddition of
different metal ions (100µM, 0.5 equiv.).
The practical applicability of Las selective fluorescence sensor for Zn2+ was
investigated by performing a competitive experiment for the estimation of Zn2+ (1 equiv.) in
the presence of other cations such as Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+,
Bi2+(3 equiv.). No significant difference in the fluorescence responses of L for Zn2+ was

observed by comparing the intensity with the co-existence of other metal ions (Fig. 6). The
unique binding for Zn2+ suggests an interesting view to develop receptor Las a fluorosensor
for Zn2+even in the presence of other metal ions including Cd2+.
6.00E+06
4.00E+06
2.00E+06
0.00E+00
Metal ions
Competative ions
Competative ions + Zn(II) ion
Fig. 6.Interference of respective metals in Zn2+ ion detection with receptorL.
Fluorometric titration for L was next carried out by successive addition of Zn2+ to
obtain the analytical parameters such as binding constant and detection limit (Fig. 7). The
fluorescent intensity increases continuously at 450 nm accompanied by a blue shift of 50 nm
in the emission profile of L. According to the IUPAC definition, the limit of detection (DL =
3Sb/m, Sb: standard deviation of blank, m: slope of the regression line) was 16 µM [23],
which can be obtained from the linear range of the regression curve covering concentration of
Zn2+ from 5-100 µM (Fig. 8). The lower detection limit is of importance for the practical use
of the targeted receptor L as selective and significant sensor for Zn2+ ions.

6.00E+06
5.00E+06
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
350
400
450
500
550
600
Wavelength (nm)
Fig. 7.Fluorescence spectra of L (0.1 mM) upon continuous addition of increasing amount of
Zn2+ (0–200 µL) in MeCN/H2O (99:1, v/v) solution (λex = 320 nm).
5.50E+06
y = 6E+09x + 5E+06
R² = 0.9467
5.40E+06
5.30E+06
5.20E+06
5.10E+06
5.00E+06
4.90E+06
0
0.00002
0.00004
0.00006
0.00008
0.0001
0.00012
[G]
Fig. 8.Normalized fitting for determining the limit of detection.
The binding stoichiometry between receptorL and Zn2+ was determined by the Job’s
continuous variation method (Fig. 9) [24]. The results indicate the formation of a 1:1 complex
which was also confirmed by the normalized plot (Figure S6). The binding constant (Ka) for
the L.Zn2+ complex was calculated from the fluorescence titration experiments (Fig. 7) by
mathematical models such as Benesi–Hildebrand, Scatchard and Connor’s linear fitting
methodologies [25] and was found to be about (1.72 0.28) × 105 M-1 (Fig. 10-12).

0.00140
0.00120
0.00100
0.00080
0.00060
0.00040
0.00020
0.00000
0
0.2
0.4
0.6
0.8
1
XH
Fig. 9. Job’s plot between L and Zn2+, wherethe concentration of [HG] was calculated as
[HG] = ∆F/Fo×[H]
0.0000005
y = 1E-12x + 2E-07
R² = 0.942
0.0000004
0.0000003
0.0000002
0.0000001
0
0
50000
100000
1/[G]
150000
200000
250000
Fig. 10.Benesi-Hildebrand Plot of 1/∆F versus 1/[G] , Ka = Intercept/slope = 2.00 × 105 M-1.
6E+11
y = -158169x + 9E+11
R² = 0.9689
5E+11
4E+11
3E+11
2E+11
1E+11
0
0.00E+00 1.00E+06 2.00E+06 3.00E+06 4.00E+06 5.00E+06 6.00E+06
∆F
Fig. 11.Scatchard Plot of 1/∆F versus 1/[G] , Ka = slope = 1. 58 × 105 M-1.

0
-200000
-400000
-600000
-800000
-1000000
-1200000
-1400000
y = 158169x - 2E+06
R² = 0.9689
0.00E+00 2.00E+00 4.00E+00 6.00E+00 8.00E+00 1.00E+01 1.20E+01 1.40E+01
F/F0
Fig. 12.Connor’s fitting Plot of 1/∆F versus 1/[G] , Ka = slope = 1. 58 × 105 M-1.
The recognition behaviour of Lwith Zn2+was further confirmed by FT-IR
spectroscopy, which gives important sign of differentiation between the spectra of receptor L
and its complexwith Zn2+ in the region of 4000–450 cm−1 (Figure S7). In the IR spectra, the
stretching frequency of νC=Nfor the free receptor L appeared at 1606 cm−1 but when the
receptor Lcoordinated with Zn2+, alone pair transfer occurred from C=N to Zn2+ which
induces extreme shift in the IR peak of νC=N to 1609 cm−1[26]. It also confirmed the
involvement of nitrogen atom of L in the complex formation. Next, the IR of free receptor L
showed a broad band at 3396 cm-1 which can be assigned for νOH group. On
complexationwith Zn2+, deprotonation of OH group took place which resulted in the
disappearance of νOH band. Further, the MALDI/TOF–MS data confirmed the formation of
1:1 complex between the receptorL and Zn2+ ion [(MS (ESI): m/z (Calculated)
C18H18ZnN2O2.SO4: 456.70, found 456.78](Figure S8).
To justify the specific and sensitive response of L for Zn2+, L was utilized for in vitro
cell imaging assay. A very faint fluorescence was observed from the cells under UV filter
when the cells were incubated with L (0.011µM) only for 2 hours where as no fluorescence
was observed in the cell when incubated with Zn2+ (25µM) only. However, an enhancement

in the intensity was observed when the cells were incubated using both receptor L (0.011µM)
and Zn2+ (25µM) simultaneously under same experimental condition (Figure13). Therefore,
these results demonstrate that L can be used for the detection of Zn2+ in biological samples
like cells.
Fig. 13: The images were taken in an inverted fluorescence microscope (Leica DMI6000B)
under 20X objective. A) Phase contrast image of the control cells; B) Phase contrast image of
the cells treated with L (0.011µM) only; C) Phase contrast image of the cells treated with
both L (0.011µM) and Zn2+ (25µM). D) Phase contrast image of the cells treated with Zn2+
(25µM) only;E) Fluorescence image of the control cells under UV filter; F) Fluorescence
image of the cells treated with L (0.011µM) for 2 hours. G) Fluorescence image of the cells
treated with L (0.011µM) for 2hours and Zn2+ (25µM) for 1 hour. H) Fluorescence image of
the cells treated with Zn2+ only.
In conclusion, we have successfully developed a low cost and easy to synthesize
Schiff-base receptor L for the selective detection of Zn2+ showing ‘turn-on’ fluorescent
response with the detection limit of 16 µM without any interference from other tested metal
ions. The receptor L can also be used as a chromogenic sensor owing to the noticeable colour
change in the presence of Zn2+ ions.
3.Experimental
3.1 Materials and methods

Chemicals were purchased from Sigma Aldrich and were used without further
purification.The fluorescence and UV-Visible spectra were recorded on a Fluoromax-4
spectrofluorometer with a 5 nm slit width and a Shimadzu UV-24500 spectrophotometer.
Thenitrate salts of the metal ions were used in this study. Ultrapure water with a Millipore
Purification System (Milli-Q water) was used throughout the analytical experiments. The1H-
NMR spectra were recorded on a Varian NMR mercury System 300 spectrometer operating
at 300 MHz in DMSO-d6 and IR spectra were recorded on a Perkin Elmer 27 spectrometer in
the solid state by preparing KBr discs.
All stock and working solutions were prepared in ultrapure water and with
spectroscopic grade MeCN. A stock solution of receptor L (1.0 mM) in MeCN was prepared
and the corresponding working solution (0.1mM) was prepared simply by diluting with
MeCN/H2O (99:1, v/v). Stock solutions of cations (10 mM) in H2O were prepared and the
corresponding working solutions (1 mM) were prepared by diluting with MeCN/H2O (99:1,
v/v).
3.2 Synthesis of receptor L
Receptor Lwas synthesized by reacting one mole of 1,2-diaminoethane with (0.6 g,
10 mM) with two moles of 1-(2-hydroxyphenyl)ethanone(2.72 g, 20 mM) in ethanol. The
mixture was stirred and refluxed for 3 hrs. Receptor L was obtained in good yield and having
1
appearance of yellow bright coloured crystals. 86 %, m.p. 130-1310C. H-NMR (400 MHz,
CDCl3, ppm) δ=2.40 (s, 6H, 2-CH3), 4.00 (s, 4H, 2-CH2), 6.81 (t, 2H, -Ar-H), 6.92-
6.94(d2H, Ar-H), 7.28(t, 2H, Ar-H), 7.53-7.55(d, 2H, Ar-H), 15.94(s, 2H, Ar-OH).13C NMR
(100 MHz, CDCl3, ppm) δ =12.7, 44.9, 70.5, 76.6, 97.4, 105.2, 108.7, 121.5, 122.0, 124.3,
126.3, 133.8, 135.9, 151.8, 153.6, 164.5, 164.6; LC-MS: [(M+H+), C18H19N3OS]= 297.10
found 297.20.
3.3Spectroscopic analysis

The fluorescence titration experiments were carried out with a Fluoromax-4
spectrofluorometer in MeCN/H2O solvent system at room temperature (298 K) with the aim
of determining the binding constant (Ka) for receptor L.Zn(II) ion in this solvent system. The
fluorescence intensity was recorded at λex= 320 nm alongside a reagent blank. The excitation
and emission slits were both set to 5.0 nm.These titration experiments were accomplished
through a stepwise addition of metal salt solutions (0.02 ml, 1mM, guest) to a solution of
receptor L (2 ml, 0.1mM, host) in MeCN/H2O (99:1, v/v) in the cell. Simultaneously, the UV-
visible titration experiments were carried out in MeCN/H2O (99:1, v/v) solvent system at
room temperatures to determine the cations selectivity of Lin the ground state.
3.4. Computational method
All computations were performed using the Gaussian09W program [27]. Full
geometry optimization of L and its Zn2+ complex were carried out using the DFT method at
the B3LYP level of theory in the gas phase. The 6-31G(d,p) basis set was assigned for C, H,
N and O atoms. The LANL2DZ basis set with effective core potential was employed for the
Zn atom. The vibrational frequency calculations were performed to ensure that the optimized
geometries represent the local minima of potential energy surface.
3.5. Cell Culture:
HeLa cells were procured from NCCS, Pune (India) and grown in Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1%
L-glutamine-penicillin-Streptamycin at 37 °C and 5% CO2. Cells were placed on glass
coverslips in 12-well plate and allowed to adhere for overnight.
3.6. Cellular Imaging:
At the time of experiment, complete media was replaced with serum free medium.
The cells were incubated with L (0.011µM) for 2 hours. After 2 hours of incubation with L,
the cells were then incubated with Zn2+ (25µM) for further 1 hour. The cells were washed

twice with Phosphate Buffer Saline (1X PBS) and then fixed with 100% methanol for 5
minutes and again washed with 1X PBS for 10 minutes. The cover slip was then mounted on
a glass slide using glycerol and observed under fluorescence microscope (Leica DMI 6000B)
using 20X objective under UV filter. The fluorescence images of cells were captured through
an attached CCD camera using LAS software.

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Highlights
• Synthesis of 2-hydroxyacetophenone functionalized
Schiff base receptor.
• DFT calculations performed.
• Recognition and sensing of metal ions.
• Fluorescent “turn-on” sensor for Zn2+.
• In vitro cell imaging assay.

Graphical Abstract -Synopsis
A novel linear Schiff-based receptor (L) containing two imino phenol units was
developed for the selective fluorescent sensing of Zn2+ with a discriminating enhancement of
12 folds over the other surveyed cations. Within the huge plethora of Zn2+receptors, this
receptor is quite worth to mention because of its simple synthesis, low cost, no interference of
Cd2+ and the judicious response availed with discernment. The spectroscopic (UV-Vis,
fluorescence and mass) results, few mathematical models for determining analytical
parameters such as binding constant (Ka), binding stoichiometry, detection limit etc and the
frontier molecular orbitals were analyzed to explore the sensing ability of L and the
mechanism.

GRAPHICAL ABSTRACT
A novel fluorescent “turn-on” sensor for the biologically
active Zn2+ ion
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