Turn-on Fluorescence Chemosensor for Zn2+ Ion Using Salicylate Based Azo Derivatives and their Application in Cell-Bioimaging

Article abstract of DOI:10.1007/s10895-019-02382-4

The synthesis and optical studies of salicylate based azo derivatives (DPSAD and IPSAD) are reported. The receptors act as a versatile fluorogenic chemosensor for Zn²⁺ causing a selective enhancement of fluorescence over other competing cations

Full text of DOI:10.1007/s10895-019-02382-4

Journal of Fluorescence
https://doi.org/10.1007/s10895-019-02382-4
ORIGINAL ARTICLE
Turn-on Fluorescence Chemosensor for Zn2+ Ion Using Salicylate
Based Azo Derivatives and their Application in Cell-Bioimaging
Mathappan Mariyappan1 & Nelson Malini1 & Jayaraman Sivamani1 & Gandhi Sivaraman1
Muniyasamy Harikrishnan1 & Sepperumal Murugesan1 & Ayyanar Siva1
&
Received: 29 January 2019 /Accepted: 17 April 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The synthesis and optical studies of salicylate based azo derivatives (DPSAD and IPSAD) are reported. The receptors act as a
versatile fluorogenic chemosensor for Zn2+ causing a selective enhancement of fluorescence over other competing cations. The
complex formed between receptors and Zn2+ are identified on the basis of absorption and fluorescence titration and further
confirmed by ESI-MS. DFT/TD-DFT calculations support the observed optical changes happens only upon complexation with
Zn2+ ion. Moreover, receptors are further applied to intracellular sensing and imaging studies.
.
.
.
.
Keywords Azo derivatives Salicylate derivative Chemosensor Intracellular sensing Zinc ion
Introduction
zinc (Zn2+) is the second most abundant metal ion (con-
centration ranging from sub-nM to 0.3 mM) [13] and
makes variety of roles in biological system, such as
gene expression, apoptosis, metalloenzymes regulation
and neurotransmission, [14, 15] neurological diseases,
including Alzheimer’s, ischemia and epilepsy [16–18]
are associated with the disorder of the Zn2+ metabolism
and also involved in carboxy peptidase and carbonic
anhydrase enzymes, which are very important to the
regulation of carbon dioxide (CO2) and digestion of
proteins, respectively. In addition, the elevated levels
of Zn2+ ions in water lead to environmental pollution,
viz., decrease the soil microbial activity causing phyto-
toxic effects, making the water smelly and muddy [19,
20]. Therefore, the design and development of efficient
fluorescent chemosensors for selective detection of Zn2+
ions are more considerable interest recently. Many Zn2+
chemosensors have been reported so far by researchers
using fluorophores such as quinoline and its derivatives
[21–24], coumarin [25], triazole [26], cyanine [27, 28],
rhodamine [29], fluorescein [30, 31], benzophenoxazone
[32], anthracene [33, 34], naphthalimide [35–37],
BODIPY [38, 39], NBD derivatives [40–43], thiazole
derivatives [44] and other near-infrared fluorophores
and so on [45]. However, all these probes contain imine
The aim and synthesis of chemosensor exhibit excellent
selective and sensitive detection of heavy metal ions in
the field of supramolecular, medicinal and environmen-
tal chemistry [1–4]. Recently, detailed consciousness has
been focused on objective of important selective sensor
of metal ions and their recognition of living cell envi-
ronment [5–9] The findings of trace metal ions have
garnered great attention in the past two decades, due
to large potential applications in the areas of life sci-
ences, medicinal and biotechnology [10–12]. It is well
identified that metal ions play an vital role in biological
enzymatic systems. Among the metal ions, specifically
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-019-02382-4) contains supplementary
material, which is available to authorized users.
* Ayyanar Siva
drasiva@gmail.com; siva.chem@mkuniversity.org
1
Supramolecular and Organometallic Chemistry Lab, Department of
Inorganic Chemistry, School of Chemistry, Madurai Kamaraj
University, Madurai, Tamil Nadu 625 021, India

J Fluoresc
Scheme 1 Synthetic route of salicylate based azo derivatives 3 (DPSAD) and 6 (IPSAD)
N- and carbonyl O-donor atoms are used to binding the
metal ions very precisely [46]. Nevertheless, most of the
Zn2+ chemosensors reported have some disadvantages
also such as multi-ion detection [47, 48], a requirement
of organic solvent for sensing [49, 50] and interferences
from other similar metal ion having similar properties
[51–54]. Furthermore, some of the receptors often re-
quire relatively tedious synthetic procedure followed by
their complex formation and purification [5, 55].
Recently, many improvement has been made in the
progress of salicylhydrazide derivative systems with
great diversity in this field. Zhefeng et al., and
Shouzhi Pu et al., was reported salicylhydrazide based
chemosensor for selective sensing for Cu2+ and Zn2+
ions [56, 57]. Furthermore, Murugavel et al., reported
dual sensing ability for picric acid and CO2 capture by
using azo-linked fluorescent triphenylbenzene based co-
valent organic polymers [58]. As a consequence, much
efforts has been dedicated to the development of salic-
ylate based azo chemosensors for the recognition of
Zn2+ ions [26–29].
In this work, simple salicylate based azo derivatives such as 3
(DPSAD) and 6 (IPSAD) were synthesized and they acts as
fluorescent chemosensor for selective detection of Zn2+ in aque-
ous media and living HeLa cells. Further, their photophysical
properties and sensing abilities of the receptors have been ex-
plored systematically through UV-vis absorption and emission
spectra. Both receptors DPSAD and IPSAD demonstrated high
selectivity towards Zn2+ via the turn-on fluorescence mechanism
in the presence of other transition and non-transition metal ions in
aqueous media. To the best of our knowledge, no report on
salicylate based azo derivatives Bturn-on^ fluorogenic probe for
selective and effective detection of Zn2+ ions.
Experimental Section
Materials and Methods
All the chemicals and reagents were used in the present
work as an analytical grade. 4-Iodoaniline and 1,4-
diaminobenzene were acquired from Alfa Aesar. NaNO2,
Fig. 1 Normalized absorption (a)
and emission (b) spectra of
DPSAD (3) and IPSAD (6) in
THF (10 μM)
1.0
120000
a
b
IPSAD
DPSAD
DPSAD
IPSAD
100000
0.8
80000
60000
40000
20000
0.6
0.4
0.2
0.0
400
450
500
550
300
350
400
450
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)

J Fluoresc
Fig. 2 Normalized absorption (a)
and emission (b) spectra of
DPSAD in different organic
solvents
1.0
0.8
0.6
0.4
0.2
0.0
THF
b
1.2
1.0
0.8
0.6
0.4
0.2
0.0
a
DMF
THF
Methanol
DCM
Toluene
DMF
Methanol
DCM
Toluene
400
450
500
550
600
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
HCl, NaOH and metal chloride salts were purchased from
Merck and all the common solvents were received from
laboratory and analytical grade.
JASCO V-630 in 1 cm path length quartz cuvette with a vol-
ume of 2 mL at ambient temperature. All fluorescence studies
were recorded on a Fluoromax-4 Spectrofluorometer
(HORIBA JOBIN YVON) with excitation slit set at 5.0 nm
band pass and emission at 5.0 nm band pass in 1 cm × 1 cm
quartz cell. The ground-state geometries were optimized using
density functional theory with B3LYP hybrid functional at the
basis set level of 6-31G. All the calculations were performed
using Gaussian 09 package.
The melting points were calculated in open capillary tubes
1
and are uncorrected. The H and 13C NMR spectra were re-
corded on a Bruker (Avance) 300 MHz NMR instrument
using TMS as an internal reference, DMSO-d6 as a solvent.
Standard Bruker software was used throughout. Chemical
shift value are given in parts per million (δ-scale) and their
coupling constants are specified in Hertz. Silica gel-G plates
(Merck) were used for TLC analysis with a mixture of n-
hexane and ethyl acetate as mobile phase. Electrospray
Ionization Mass Spectrometry (ESI-MS) analyses were re-
corded in LCQ Fleet, Thermo Fisher Instruments Limited,
United States. ESI-MS was measured in positive ion mode.
The collision voltage and ionization voltage were − 70 Vand
− 4.5 kV, respectively, using nitrogen as atomization and
desolvation gas. The desolvation temperature was set at
300 °C. The relative amount of each part was determined from
the LC-MS chromatogram, using the area normalization
method. UV-vis absorption spectra were analysed on
Synthesis and Characterization
General Procedure a for Synthesis of Compounds 3 (DPSAD)
and 6 (IPSAD)
Aromatic amine was taken in 1:1 ratio of concentrated
hydrochloric acid and water. Sodium nitrite solution was
added drop wisely into the reaction mixture with con-
stant stirring. The mixture was stirred for about 30 min
at a temperature of 0 to 5 °C. The diazotization step was
pursued by coupling of the diazotized amines with drop
1.0
1.0
Fig. 3 Normalized absorption (a)
and emission (b) spectra of
IPSAD in different organic
solvents
b
a
DCM
THF
DMF
MeOH
DCM
MeOH
THF
Toluene
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
Toluene
DMF
300
350
400
450
500
550
600
390
420
450
480
510
Wavelength (nm)
Wavelength (nm)

J Fluoresc
Table 1 The photophysical
values of DPSAD (3) and IPSAD
(6) in different organic solvents
Solvents
DPSAD
abs (nm)
IPSAD
abs (nm)
λ
λ
emi (nm) Stoke’s shift (cm−1
)
λ
λ )
emi (nm) Stoke’s shift (cm−1
Toluene
THF
354
364
358
372
404
448
434
462
470
3496
5151
4888
5236
5823
308
304
306
295
302
391
406
410
416
422
6897
8264
8289
9416
9898
DCM
DMF
Methanol 369
wise addition to the solution of sodium salicylate. The
reaction was stirred at 0–5 °C for about 2 h under basic
medium. The resultant azo derivatives (3 and 6) were
obtained as an orange to red powders after changing
the pH range between 6 and 7.
0.045 mmol), NaNO2 (0.31 g, 0.045 mmol), salicylic acid
(0.63 g, 0.045 mmol), NaOH (0.36 g, 0.091 mmol). Orange
to red solid was obtained (65% yield); m.p 232 °C. FT-IR
(cm−1) 3216, 2241, 2180, 1571, 1456, 1385, 1339, 1297,
1
1250, 1172; H NMR (300 MHz, DMSO-d6) δH = 8.28 (s,
1H), 7.90 (d, J = 7.9 Hz, 2H), 7.81 (d, J = 7.8 Hz, 1H), 7.59
(d, J = 7.58 Hz, 2H), 6.80 (d, J = 6.79 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6) δC 172.73, 168.05, 152.50, 143.98,
140.19, 139.01, 127.42, 124.75, 120.06, 118.55, 97.39; ESI-
MS: m/z calculated for C13H9IN2O3 367.9658; found
368.9732 [M + H]+.
Synthesis of Compound 3 (DPSAD)
Compound 3 (DPSAD) was synthesized by using the above
said general procedure A using 1,4-diaminobenzene 1 (1 g,
0.092 mmol), NaNO2 (1.28 g, 0.184 mmol), salicylic acid
(2.55 g, 0.184 mmol), NaOH (1.48 g, 0.37 mmol). Orange
to red solid was obtained (69% yield); m.p 265 °C. FT-IR
(cm−1) 3227, 3026, 2834, 2525, 1656, 1606, 1480, 1440,
Results and Discussion
1
1382, 1324, 1241, 1205; H NMR (300 MHz, DMSO-d6)
δH = 8.28 (s, 1H), 7.89 (s, 4H), 7.81 (d, J = 7.8 Hz, 2H),
6.79 (d, J = 6.79 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
δC 172.73, 162.04, 152.50, 145.94, 128.21, 124.13, 122.67,
119.83, 117.80; ESI-MS: m/z calculated for C20H14N4O6
406.0913; found 407.0091 [M + H]+.
Synthesis of Fluorophores
The synthetic way of two different salicylate based azo com-
pounds 3 (DPSAD) and 6 (IPSAD) are represented in
Scheme 1. The anilines 1 and 4 were first diazotized using
sodium nitrite in the presence of hydrochloric acid and further
it reacts with salicylic acid to afford compounds 3 and 6 with
69% and 65% yields respectively. Both the final compounds
were thoroughly characterized by various spectral techniques
such as 1H, 13C NMR, FT-IR and ESI-mass spectra.
Synthesis of Compound 6 (IPSAD)
Compound 6 (IPSAD) was synthesized by using the above
said general procedure A using 4-iodoaniline 4 (1 g,
0.4
Fig. 4 UV-vis spectra of DPSAD
a
b
0.4
0.3
0.2
0.1
0.0
+
+
2+ 2+
DPSAD, Na , K , Mg , Ca ,
(10 μM) (a) with various cations
(1 × 10−4 M); (b) upon gradual
addition of Zn2+ (0–20 μM) in
aqueous THF (THF/H2O = 8:2) at
pH = 7.4 [HEPES buffer
2+ 2+ 2+ 2+ 2+
Ba , Mn , Fe , Co , Ni
2+ 2+ 2+ 2+
,
0.3
0.2
0.1
0.0
Cu , Cd , Hg , Pb
2+
Zn
(20 mM)]
2+
DPSAD + Zn
300 350 400 450 500 550 600 650
Wavelength (nm)
700
300
350
400
450
500
550
600
650
Wavelength (nm)

J Fluoresc
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.6
0.4
0.2
0.0
Fig. 5 UV-Vis spectra of IPSAD
(10 μM) (a) with various cations
(1 × 10−4 M); (b) upon gradual
addition of Zn2+ (0–10 μM) in
aqueous THF (THF/H2O = 8:2) at
pH = 7.4 [HEPES buffer
a
b
+
+
2+ 2+
2+
2+
IPSAD, Na , K , Mg , Ca , Ba , Mn ,
2+ 2+ 2+ 2+ 2+ 2+
2+
Fe , Co , Ni , Cu Cd Hg , Pb
,
,
2+
Zn
(20 mM)]
2+
IPSAD + Zn
250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
300 350 400 450 500 550 600 650 700
Wavelength (nm)
Photophysical Properties
the emission spectra, this may be due to the presence
of the donor and acceptor groups present in the probe.
The absorption bands of 3 and 6 has negligible shift
because the ground-state electronic structure and tiny
dipole moments coupled with the ICT transitions are
self-governing of solvent polarity [59]. But in the case
of the emission, band was slightly red shifted upon
varying the solvent polarity from nonpolar to polar
(Figs. 2b and Fig. 3b). This may be due to increase in
charge separation at the excited state. In addition to that
the absorption spectra of Figs. 2a and Fig. 3a showed a
distinct shoulder, thia may be due to the solvent relax-
ation. We did not find any shoulder peak in the emis-
sion spectra. After excitation, the internal charge trans-
fer occurred and causes a highly polar charge separated
emission state which is stabilized most effectively by
the polar solvents [59] and bathochromic shift also ob-
served. Further, we calculated Stoke’s shift values of all
the solvents and are listed in (Table 1), we observed
that the values are gradually increases from non-polar
to polar solvents which indicates that stabilization oc-
curs at highly polar emitting exited state, i.e. the elec-
tronic redistribution occurred upon excitation [60].
The absorption and emission spectra of 3 (DPSAD) and
6 (IPSAD) in THF are somewhat different to each other
(Fig. 1) due to the existence of diazo group in DPSAD
(Scheme 1). Both the compounds exhibit two absorption
maxima, 3 (DPSAD) appeared at 283 nm and 363 nm
while 6 (IPSAD) displayed band at 286 nm and
304 nm. DPSAD shows bathochromic shift from
IPSAD this might be due to azo existence. In the emis-
sion spectra, compound 3 (DPSAD) showed λemi at
448 nm (λexi = 363 nm) and compound 6 (IPSAD) re-
vealed λemi at 388, 406 and 433 nm (λexi = 304 nm).
As shown in Figs. 2a and 3a, the nature of solvent
polarity is strongly influenced the optical properties of 3
(DPSAD) and 6 (IPSAD), this may be due to the pres-
ence of the donor and acceptor groups present in the
receptors. The absorption bands of 3 and 6, are almost
negligible shift was noticed, because the ground-state
electronic structure and tiny dipole moments coupled
with the ICT transitions are self-governing of solvent
polarity. As shown in Figs. 2b and 3b, DPSAD and
IPSAD depends on the nature of solvent polarity in
Scheme 2 Probable binding mode of DPSAD with Zn2+

J Fluoresc
Scheme 3 Probable binding site of IPSAD with Zn2+
Selectivity of DPSAD (3) and IPSAD (6) for Zn2+ over Other
Competitive Ions
addition of Zn2+ ions to the solution of receptors caused
a simultaneous decrease in both the absorption bands
which indicates that the formation of co-ordination be-
tween receptors and metal ion (Schemes 2 and 3).
The availability of -COOH and –OH unit of 3 and 6 can
act as binding sites towards various metal ions such as
K+, Mg2+, Ca2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+,
Cd2+, Hg2+, Pb2+ and Zn2+. The metal binding properties
of DPSAD and IPSAD were examined by absorption and
emission spectra, in aqueous THF (THF/H2O = 8:2) at
pH = 7.4 [HEPES buffer (20 mM)] at ambient tempera-
ture. Initially, the absorption spectrum of DPSAD exhib-
ited two bands at 283 and 363 nm whereas IPSAD
displayed bands at 283 and 304 nm in THF/H2O = 8:2)
at pH = 7.4 [HEPES buffer (20 mM)] at ambient temper-
ature. Then, we added the 20 μM and 10 μM of metal
ion concentrations into the solution of DPSAD and
IPSAD respectively. Under these circumstances metal
ions such as Na+, K+, Mg2+, Ca2+, Ba2+, Mn2+, Fe2+,
Co2+, Ni2+, Cu2+, Cd2+, Hg2+ and Pb2+ unsuccessful to
perform like Zn2+, indicating that both the compounds
(DPSAD and IPSAD) acts as competent and discriminat-
ing sensor for Zn2+ over other essential metal ions
(Figs. 4a and Fig. 5a). To determine the coordination
behaviour of DPSAD and IPSAD with Zn2+, the UV-vis
titration test was carried out alone and given in Figs. 4b
and Fig. 5b. From this spectra, when increasing the
The fluorescence titration of the receptors DPSAD
and IPSAD in the presence of various metal cations
viz., Na+, K+, Mg2+, Ca2+, Ba2+, Mn2+, Fe2+, Co2+,
Ni2+, Cu2+, Cd2+, Hg2+, Pb2+ as well as Zn2+ were
studied in aqueous THF (THF/H2O = 8:2) at pH = 7.4
[HEPES buffer (20 mM)]. When the incremental addi-
tion of addition of 20 μM and 10 μM of metal ion
concentrations to the solution of DPSAD and IPSAD,
the fluorescence enhancement gradually increases with
blue shift upon excitation at 363 nm (Figs. 6a and
Fig. 7). Similarly, when the IPSAD is titrated with
Zn2+, fluorescence enhancement is observed with exci-
tation at 304 nm (Fig. 8a). Further, the test solution
shows an apparent fluorescence change from red to blue
for DPSAD (Fig. 8b) while light yellow to light blue
for IPSAD (Fig. 6b) under the irradiation of UV light at
365 nm. To the selectivity test, the addition of other
metal ions such as Na+, K+, Mg2+, Ca2+, Ni2+, Mn2+,
Cu2+, Cd2+, Hg2+, Ba2+, Fe2+, Co2+ and Pb2+ over the
receptors DPSAD and IPSAD were checked and it does
not show any observable fluorescence changes (Figs. 6a
and Fig. 7a). However, the addition of Zn2+ (0–20 μM)
2500000
Fig. 6 Emission spectra of
2+
DPSAD + Zn
a
b
DPSAD (10 μM) (a) with various
cations (1 × 10−4 M); (b) upon
gradual addition of Zn2+ (0–
20 μM) in aqueous THF (THF/
H2O = 8:2) at pH = 7.4 [HEPES
buffer (20 mM)]. Excitation at
363 nm. Slit width is 5 nm
2000000
1500000
1000000
500000
+
+
2+
2+
Zn
2000000
1500000
1000000
500000
DPSAD, Na , K , Mg
2+ 2+ 2+
,
2+
Ca , Ba , Mn , Fe
,
2+
2+
2+
2+
2+
Cd
Co , Ni , Cu
,
,
2+
Hg , Pb
350
400
450
500
550
600
350
400
450
500
550
Wavelength (nm)
Wavelength (nm)

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Fig. 7 Emission spectra of
b
a
300000
250000
200000
150000
100000
50000
300000
250000
200000
150000
100000
50000
IPSAD (10 μM) (a) with various
cations (1 × 10−4 M); (b) upon
gradual addition of Zn2+ (0–
10 μM) in aqueous THF (THF/
H2O = 8:2) at pH = 7.4 [HEPES
buffer (20 mM)]. Excitation at
304 nm. Slit width is 5 nm
2+
IPSAD + Zn
2+
Zn
+
+
2+ 2+
IPSAD, Na , K , Mg , Ca ,
2+ 2+ 2+ 2+
2+
,
Ba , Mn , Fe , Co , Ni
2+ 2+ 2+ 2+
Cu , Cd , Hg , Pb
400
450
500
550
600
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
induced intense fluorescence augmentation with blue
shift from 448 nm to 410 nm for DPSAD (Fig. 6b).
In case of receptor IPSAD, after titration with Zn2+
(0–10 μM), the intensity of emission band at 388, 406
and 433 nm significantly increases without any change
in emission wavelength (Fig. 7b), which indicates that
the fluorescence intensity changes might be attributed to
combine the effect of chelation enhanced fluorescence
(CHEF) and intramolecular charge transfer (ICT) within
the receptor [25]. From the observed results of the ab-
sorption and fluorescence, it is identified that the recep-
tors (DPSAD and IPSAD) are very selective and sensi-
tive for Zn2+ over other mono/divalent metal ions such
as Na+, K+, Ni2+, Cu2+, Ba2+, Mn2+, Cd2+, Hg2+, Mg2+,
Ca2+, Fe2+, Co2+ and Pb2+.
To test the practical applicability of our newly synthesized
receptors (DPSAD and IPSAD), a competitive binding test
was studied in the presence of varying concentration of Zn2+
with competing analytes. No significant difference was detect-
ed in the presence of other competitive ions over the compar-
ison to a solution containing Zn2+ metal ion (Fig. 9). These
findings suggest that Zn2+ recognition by receptors are hardly
affected by other synchronized metal ions.
Further, we calculated linear dependent coefficient (R2) of
DPSAD and IPSAD with Zn2+ are 0.9976 and 0.9986 respec-
tively with the help of varying concentration of Zn2+ ion (Fig.
S8). The detection limits of DPSAD and IPSAD were mea-
sured to be 6.73 × 10−6 M and 5.07 × 10−6 M for Zn2+ which
was quite appreciable when compared to those previously
reported receptor [61] for Zn2+. The stoichiometry test of
Fig. 8 Fluorescence image of
DPSAD and IPSAD with Zn2+
under illumination of UV light at
365 nm

J Fluoresc
2+
2+
IPSAD + Zn + Metal ions
DPSAD + Zn + Metal ions
IPSAD + Metal ions
DPSAD + Metal ions
b
a
300000
250000
200000
150000
100000
50000
0
20000000
15000000
10000000
5000000
0
Zn Na
K
Mg Ca Ba Mn Fe Co Ni Cu Cd Hg Pb
Metal ions ( M)
Zn Na
K
Mg Ca Ba Mn Fe Co Ni Cu Cd Hg Pb
Metal ions ( M)
Fig. 9 a Selectivity studies of DPSAD and IPSAD with Zn2+ (b) competitive experiments of DPSAD and IPSAD with Zn2+. Excitation was performed
at 363 and 404 nm for DPSAD and IPSAD respectively
DPSAD and IPSAD was carried out with Zn2+ and it was
estimated by Job’s plot using fluorometric titrations with vary-
ing concentration of metal ion. From the Job’s plot analysis,
we observed that DPSAD forms a 1:2 stoichiometric complex
and IPSAD forms 1:1 stoichiometric complex with Zn2+ (Fig.
S9). In order to validate the stoichiometric complex, the effi-
cient bindings of (DPSAD and IPSAD) with Zn2+ were stud-
ied by ESI-MS. The ESI-MS spectra, (Fig. S10 and Fig. S11)
shows peaks at m/z = 530.9247 and 430.8865 corresponding
to [DPSAD+H + 2 Zn2+]+ and [IPSAD+H + Zn2+]+ respec-
tively, which also confirmed the formation of 1:2 complex
for DPSAD and 1:1 complex for IPSAD with Zn2+.
In biological applications, a suitable pH condition was
identified for our newly synthesized the receptors DPSAD
and IPSAD and it was evaluated by recording the fluorescence
spectra over different pH values (Fig. 10). In acidic condition,
(pH 1–4) the fluorescence intensity decreased due to the pro-
tonation of receptor but under basic conditions (pH 10–14),
the fluorescence intensity of probe enhanced due to the depro-
tonation of salicylate (-COOH and –OH) group leading to
extend the conjugation [62–66]. Aweak fluorescence intensity
was observed at intermediate pH (HEPES buffer (20 mM,
pH 7.4). Hence, the chemosensors DPSAD and IPSAD
showed a significant response at a biologically relevant pH
(7.4) which is very close to the physiological pH condition.
DFT Studies
DFT studies were performed for investigating to throw light
on receptor-guest interaction mechanism. The optimized na-
ture of the receptors (DPSAD and IPSAD) and the
Zn2+complex formed by co-ordination with receptors was ob-
tained by DFT/B3LYP-6-31G and B3LYP/LanL2DZ basis set
[67] respectively (Fig. 11). The fluorescence enhancement of
receptor was easily understand after binding with Zn2+, and
also TD-DFT calculations were performed using DFT/
B3LYP-6-31G basis set.
We carried out the quantum mechanical calculations in
larger basis set. The representation is shown in Figs. 12
and Fig. 13 and the explanation is as follows, IPSAD is
Fig. 10 Plot of fluorescence
a
b
intensity against pH for DPSAD
(a) and IPSAD (b) in THF/H2O
system

J Fluoresc
a
b
c
d
Fig. 11 Optimized structure of (a) DPSAD (b) DPSAD-Zn2+ (c) IPSAD (d) IPSAD- Zn2+
an unsymmetrical compound. In the ground state, the
electron distribution of IPSAD represents in the donor
group of azo only. But in the excited state, the electron
flows into the withdrawing group of carboxylic acid (ac-
ceptor). This transition clearly shows the charge transfer
behaviour of IPSAD. After the addition of Zn2+ to
IPSAD, the electron distribution is throughout the mole-
cule in the ground state. In the excited state, electrons
transferred into the metal to ligand through a chelation
effect. The electrons are located in the ground state of
a
b
Fig. 12 DFT-computed molecular frontiers orbitals of DPSAD and DPSAD-Zn2+

J Fluoresc
a
b
Fig. 13 DFT-computed molecular frontiers orbitals of IPSAD and IPSAD-Zn2+
the symmetrical compound DPSAD on the azo side.
When the molecules are excited, the electrons are
transfered throughout the molecule due to charge
transfer. Upon the addition of Zn2+ with DPSAD, the
electrons are located throughout the molecule in the
ground state but in the excited state electrons are
a
b
c
Fig. 14 Live-cell imaging of Zn2+ in HeLa cells; (a) fluorescence image
of cells incubation with DPSAD (1 μM) for 30 min at 37 °C; (b) bright-
field image of DPSAD treated HeLa cells; (c) fluorescence image of
HeLa cells incubated with DPSAD (1 μM) and subsequently treated with
ZnCl2 (10 μM) for 15 min λemi = 448 nm

J Fluoresc
a
b
c
Fig. 15 Live-cell imaging of Zn2+ in Hela cells; (a) fluorescence image of
cells incubation with IPSAD (1 μM) for 30 min at 37 °C; (b) bright-field
image of IPSAD treated Hela cells; (c) fluorescence image of Hela cells
incubated with IPSAD (1 μM) and subsequently treated with ZnCl2
(10 μM) for 15 min λemi = 406 nm
transfered into the metal as well as acceptor of DPSAD
molecule. This observation strongly supports the pro-
posed mechanism.
ceptors reveal fluorescence turn-on response to Zn2+ in aque-
ous medium over the other competing metal ions. Density
functional theory calculation shows that the observed fluores-
cence enhancement of receptors in the addition of Zn2+ is due
to internal charge transfer mechanism. Furthermore, these
molecules can be utilized in living cells for monitoring Zn2+
ions very effectively and quickly.
Live Cell Imaging Studies
Due to the sensitivity of DPSAD and IPSAD, they preferably
suitable for intracellular Zn2+ ion imaging in living cells. We
have deliberated the sensitivity of DPSAD and IPSAD for
Zn2+ in living HeLa cells by fluorescence microscopy. In pri-
mary stage, HeLa cells incubated with the receptors (DPSAD
and IPSAD) did not show any fluorescence image (Figs. 14
and Fig. 15). After incubation of the receptor treated cells with
Zn2+ ions, which shows the blue fluorescence observed and
identified by the fluorescence microscope. From the observed
results of fluorescence image shown that the fluorescence sig-
nals are localized on the intracellular region, which
representing a good cell membrane permeability of
chemosensors DPSAD and IPSAD. The blue fluorescence
from the intracellular region proves that the receptors
(DPSAD and IPSAD) are convincing for imaging Zn2+ in
living cells. All these result shows that DPSAD and IPSAD
are biocompatible in nature and it could be applied for detect-
ing Zn2+ ions in cells quickly.
Acknowledgments The authors acknowledged to the Department of
Science and Technology, SERB, Extramural Major Research Project
(Grant No. EMR/2015/000969), Council of Scientific and Industrial
Research (CSIR), HRDG, No. 01(2901)/17/EMR-II, New Delhi,
Department of Science and Technology DST/TM/CERI/C130(G), New
Delhi, India for financial support, and also we thank to DST-FIST, DST-
PURSE and UPE for providing instrumental facilities.
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