An azine based sensor for selective detection of Cu2 + ions and its copper complex for sensing of phosphate ions in physiological conditions and in living cells

Article abstract of DOI:10.1016/j.saa.2017.09.072

A simple and cost effective unsymmetrical azine based Schiff base, 5-diethylamino-2-[(2-hydroxy-benzylidene)hydrazonomethyl]-phenol (1) was synthesized which selectively detect Cu^{2 +} ions in the presence of other competitive ions through “naked eye” in physiological conditions (EtOH–buffer (1:1, v/v, HEPES 10 mM, pH = 7.4)). The presence of Cu^{2 +} induce color change from light yellow green to yellow with the appearance of a new band at 450 nm in UV–Vis spectra of Schiff base 1. The fluorescence of Schiff base 1 (10 μM) was quenched completely in the presence of 2.7 equiv. of Cu^{2 +} ions. Sub-micromolar limit of detection (LOD = 3.4 × 10^{? 7} M), efficient Stern–Volmer quenching constant (K_SV = 1.8 × 10⁵ L mol^{? 1}) and strong binding constant (log K_b = 5.92) has been determined with the help of fluorescence titration profile. Further, 1 ? Cu^{2 +} complex was employed for the detection of phosphate ions (PO₄^{3 ?}, HPO₄^{2 ?} and H₂PO₄^?) at micromolar concentrations in EtOH–buffer of pH 7.4 based on fluorescence recovery due to the binding of Cu^{2 +} with phosphate ions. Solubility at low concentration in aqueous medium, longer excitation (406 nm) and emission wavelength (537 nm), and biocompatibility of Schiff base 1 formulates its use in live cell imaging.

Full text of DOI:10.1016/j.saa.2017.09.072

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
An azine based sensor for selective detection of Cu2+ ions and its copper
complex for sensing of phosphate ions in physiological conditions and in
living cells
Karishma Tiwari a, Sumit Kumar a, Vipan Kumar a, Jeevanjot Kaur b, Saroj Arora b, Rakesh Kumar Mahajan a,
⁎
a
Department of Chemistry, UGC-Centre for Advanced Studies, Guru Nanak Dev University, Amritsar 143005, India
b
Department of Botany, Guru Nanak Dev University, Amritsar 143005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2017
Received in revised form 14 September 2017
Accepted 28 September 2017
Available online 29 September 2017
A simple and cost effective unsymmetrical azine based Schiff base, 5-diethylamino-2-[(2-hydroxy-benzylidene)
hydrazonomethyl]-phenol (1) was synthesized which selectively detect Cu2+ ions in the presence of other compet-
itive ions through “naked eye” in physiological conditions (EtOH–buffer (1:1, v/v, HEPES 10 mM, pH = 7.4)). The
presence of Cu2+ induce color change from light yellow green to yellow with the appearance of a new band at
450 nm in UV–Vis spectra of Schiff base 1. The fluorescence of Schiff base 1 (10 μM) was quenched completely in
the presence of 2.7 equiv. of Cu2+ ions. Sub-micromolar limit of detection (LOD = 3.4 × 10−7 M), efficient Stern–
Volmer quenching constant (KSV = 1.8 × 105 L mol−1) and strong binding constant (log Kb = 5.92) has been deter-
mined with the help of fluorescence titration profile. Further, 1 − Cu2+ complex was employed for the detection of
phosphate ions (PO34−, HPO42− and H2PO−4 ) at micromolar concentrations in EtOH–buffer of pH 7.4 based on
fluorescence recovery due to the binding of Cu2+ with phosphate ions. Solubility at low concentration in aqueous
medium, longer excitation (406 nm) and emission wavelength (537 nm), and biocompatibility of Schiff base 1
formulates its use in live cell imaging.
Keywords:
Schiff base
Copper
Phosphorus
UV–Vis
Fluorescence
Live cell imaging
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
always interfering in the detection of ions due to their high degree of hy-
dration in water [11,12]. Hence, synthesis of a novel receptor that can de-
Copper is one of the most essential trace elements of importance for
both physical and mental health and serve as a key factor for a wide
variety of enzymes in living organisms [1]. However, its excess amount
may lead to vomiting, lethargy, increased blood pressure and respiratory
rates, acute hemolytic anemia, liver damage, neurotoxicity and neurode-
generative diseases [2,3]. Phosphate ions play essential roles in genetic in-
formation storage, gene regulation, energy transduction, signaling
processing and muscle contraction [4,5]. Phosphate is a key unit of
DNA, RNA and many chemotherapeutic and antiviral drugs [6,7]. In con-
trast, the over-use of phosphate can lead to excessive algal growth,
followed by decomposition and depletion of dissolved oxygen, and final-
ly, the eutrophication of aquatic ecosystems [8]. Hence, much attention
has been paid to the development of highly selective and sensitive copper
and phosphate sensors for biological and environmental applications.
Although, a number of papers have been published for the detection
of copper and phosphate ions, however, there is still an urgent need for
simple, biocompatible and cost effective synthetic compounds which
may detect copper and phosphate ions with high affinity in competitive
aqueous media at physiological conditions [9,10]. Aqueous medium is
tect these ions selectively and sensitively in aqueous solution will be
attention-grabbing due to their biological application in live cell imaging.
There are numerous reports in literature enlightening the photo-
chemical behavior and sensing applications of azine based Schiff
bases. For example, aldazine based chromo-fluorogenic sensor for fluo-
ride ions in DMSO was developed by Li et al. [13]. Guchhait et al. have
investigated spectral properties and sensing ability towards protic envi-
ronment of a simple azine Schiff base [14]. This sensor is found to be
nonfluorescent in aprotic solvents (MeCN and Dioxane) and become
highly fluorescent in protic solvents (MeOH and H2O) due to the forma-
tion of intermolecular hydrogen bonding between sensor and protic sol-
vents. However, most of the reported azine based Schiff bases are
symmetrical type (RHC_N-N_CHR) and shows aggregation induced
emission (AIE) in aqueous solution [15]. A very small amount of re-
search work in literature for unsymmetrical azine based Schiff bases
(RHC_N-N_CHR1) motivate us to synthesize these Schiff bases [16].
The designing and synthesis of novel unsymmetrical azine based Schiff
base will be interesting because introduction of asymmetry may tune
the photochemical properties of synthesized Schiff base.
In this work unsymmetrical azine based Schiff base, 5-diethylamino-
2-[(2-hydroxy-benzylidene)hydrazonomethyl]-phenol (1) was synthe-
sized in excellent yield from inexpensive and biocompatible starting
⁎
Corresponding author.
E-mail address: rakesh_chem@yahoo.com (R.K. Mahajan).
https://doi.org/10.1016/j.saa.2017.09.072
1386-1425/© 2017 Elsevier B.V. All rights reserved.

K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
17
Scheme 1. Synthesis of Schiff base 1.
materials having 2-hydroxysalicylaldehyde as main component. The
Schiff base 1 possess both charge transfer and proton transfer group
and was found to be highly fluorescent in both organic and aqueous
solution. Coupled excited state intramolecular proton transfer (ESIPT)
and intramolecular charge transfer (ICT) have been widely explored in
the molecular systems possessing both charge transfer and proton
transfer groups. However, there are a very small number of reports in
literature, where one process is suppressed by other viz. Guchhait
et al. have observed the ICT suppressed ESIPT single fluorescence at
527 nm for 4-(diethylamino)-2-hydroxybenzaldehyde [17]. In the
case of Schiff base 1, ICT is suppressed and only ESIPT is observed
because uneven intramolecular charge transfer facilitates ESIPT.
Most of the reported azine based Schiff base chromo- fluorogenic
sensors for Cu2+ suffer from the interferences caused by Fe3+, poor
water solubility, use of organic solvents and poor detection limit [18,
19]. However, this chromo-fluorogenic Schiff base (1), synthesized by
simple condensation reactions, is very efficient in selective detection
of Cu2+ in EtOH–buffer solution (1:1, v/v, HEPES 10 mM, pH = 7.4)
with LOD = 3.4 × 10−7 M. Further, 1 − Cu2+ complex was also utilized
for sensing of phosphate ions in the same aqueous solution. Logic gate
were constructed successfully by taking two input Cu2+ and EDTA in
aqueous solution and a number of reversible cycles were observed
upon alternative addition of Cu2+ and EDTA in EtOH–buffer (1:1, v/v,
HEPES 10 mM, pH = 7.4) solution of Schiff base 1. To further evaluate
the biological application of Schiff base 1, fluorescence has also been
employed for detection of these ions in living cells.
Chemicals, USA. Di sodium hydrogen phosphate dehydrate, Potassium
dihydrogen orthophosphate, Aluminium(III) nitrate, tetrasodium EDTA
(ethylene diamine tetra acetic acid) and all the solvents were obtained
from Merck Chemicals, India. Absolute ethanol (Emsure grade) was
purchased from Merck Germany. All the solvents were used after
checking their purity thoroughly by UV–Vis and fluorescence spectral
techniques. Double distilled water was prepared in lab using double
distillation unit.
2.2. Synthesis of Schiff Base 1
The Schiff base, 5-diethylamino-2-[(2-hydroxy-benzylidene)
hydrazonomethyl]-phenol (1) was synthesized by two step condensation
reactions (Scheme 1). Synthetic methodology involved initial stirring of
hydrazine hydrate with 2-hydroxybenzaldehyde in dry methanol at
room temperature for 30 min to yield 2-hydrazonomethyl phenol (SA).
The heating of SA with 4-diethylamino-2-hdroxybenzaldehyde in ethanol
at 50 °C afforded the corresponding 5-diethylamino-2-[(2-hydroxy-
benzylidene)hydrazonomethyl]-phenol (1) in excellent yield.
2.2.1. Analytical Data
C18H21N3O2: Physical state: Pale yellow solid, Yield 88%; M.p. 160 °C;
found % for C, 69.51; H, 6.73; N, 13.55, Calc: C, 69.43; H, 6.80, N, 13.49; 1H
NMR (300 MHz, CDCl3) δ 1.20 (t, J = 6.9 Hz,\\CH3); 3.40 (q, J = 7.2 Hz,
4H, –CH2); 6.21 (d, J = 2.4 Hz, 1H, Ar\\H); 6.27 (dd, J = 8.7, 2.7 Hz, 1H,
Ar\\H); 6.93 (dt, J = 7.8, 1.2 Hz, 1H, Ar\\H); 7.00 (d, J = 8.1 Hz, 1H,
Ar\\H); 7.12 (d, J = 8.7 Hz, 1H, Ar\\H); 7.29–7.35 (m, 2H, Ar\\H);
8.52 (s, 1H, Iminic-H); 8.60 (s, 1H, Iminic-H), 11.64 (s, 1H,\\OH, ex-
2. Experimental
2.1. Materials and Methods
changeable with D2O), 11.66 (s, 1H, –OH, exchangeable with D2O) 13
C
NMR (75 MHz, CDCl3) δ 12.55, 44.47, 97.80, 104.30, 106.38, 116.91,
117.99, 119.59, 131.91, 132.51, 133.95, 152.00, 159.50, 161.16, 161.97,
163.91; HRMS Calc. [M + H]+ 312.16 found 311.38 (100% abundance).
All analytical reagent grade chemicals were obtained from the com-
mercial sources. Perchlorate salts of cations, tertabutylammonium salts
of anions and HEPES buffer were purchased from Sigma-Aldrich
Fig. 1. Changes in UV–Vis spectra (a) and fluorescence spectra (b) of Schiff base 1 (30 μM) in different solvents with varying polarities and hydrogen bonding abilities.

18
K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
Table 1
where, A and Amin are absorbance/fluorescence of Schiff base 1 at partic-
ular wavelength, in the presence and absence of guest ions and Amax is
the maximum absorbance/fluorescence in the presence of guest ions.
The Stern–Volmer Eq. (2) was applied for the calculation of quenching
constant [21].
UV–Vis absorption and fluorescence spectral maxima of Schiff base 1 in various solvents,
and solvent polarity parameters. Where, α is solvent hydrogen-bond donor acidity, β is
solvent hydrogen-bond acceptor basicity and π* is a measure of the solvent dipolarity/
polarizability.
Solvents
Schiff base 1
abs (nm)
ET(30)a αb
βb
π*b
h
i
Io=I ¼ 1 þ KSV Cu2þ
ð2Þ
λ
λ
em nm (intensity)
ex = 406 nm
λ
Toluene
Diethyl ether 394, 410
Ethyl acetate 403
DMSO
Acetonitrile
Ethanol
Methanol
Ethanediol
Water
400, 416
461 (999), 536 (2530)
457 (700), 531 (862)
33.9
34.6
0.0
0.0
0.0
0.0
0.13 0.54
0.47 0.27
0.45 0.55
0.76 1.00
where, Io and I are the fluorescence intensity in the absence and pres-
ence of Cu2+ ions. KSV is the Stern–Volmer quenching constant and
[Cu2+] is the concentration of copper ions. Cu2+ binds to Schiff base 1
and forms 1 − Cu2+ complex, accordingly, the binding number of
Cu2+ in Schiff base 1 was calculated by using double–logarithmic Eq.
(3) [21].
465 (1156), 530 (1079) 38.1
496 (2191), 530 (1901) 45.1
488 (2106), 526 (1866) 45.9
409
406
0.19 0.40 0.75
0.86 0.75 0.54
0.98 0.66 0.60
0.90 0.52 0.92
1.17 0.47 1.09
406
404
412
464 (414), 536 (2688)
467 (269), 535 (1505)
472 (513), 542 (3166)
51.9
55.5
56.3
414, 449
484 (1167), 542 (2023) 63.1
h
i
logðIo–I=IÞ ¼ log Kb þ n log Cu2þ
ð3Þ
a
Values obtained from the literature [27], given in kilocalories per mole.
Values obtained from the literature [28,29].
b
where, n is the binding number, and log Kb is the binding constant.
The limit of detection (LOD) was determined from a plot of absor-
bance/fluorescence intensity as a function of the concentration of the
added ions. To determine the S/N ratio, the absorbance/fluorescence in-
tensity of receptor without ions was measured by 10 times and the stan-
dard deviation of blank measurements was determined. The detection
was calculated as three times the standard deviation from the blank
measurement (in the absence of ions) divided by the slope of calibration
plot between ion concentration and absorbance/fluorescence intensity
[22].
2.3. Instrumentation
1H and 13C NMR spectra were recorded in CDCl3 on a JEOL AL-300
FT-NMR multinuclear spectrometer. Chemical shifts were reported in
parts per million (ppm) using tetramethylsilane (TMS) as an internal
standard. UV–Vis spectra were recorded by Shimadzu spectrophotome-
ter, model, Pharmaspec UV–1800. Fluorescence spectra were recorded
on a Hitachi, F-4600 fluorescence spectrophotometer. Time resolved
fluorescence was performed with a HORIBA time resolved fluorescence
spectrophotometer. Mass spectrometric analysis was carried out on a
Water-Q-Tof micromass equipment using ESI as the source.
Quantum yield was calculated by using following equation:
ꢂ
ꢃ
Q ¼ QR ðI=IRÞ ꢂ ðODR=ODÞ ꢂ n2=nR
ð4Þ
2
2.4. General Methods
where, Q is fluorescence quantum yield, I is the integrated fluorescence
intensity, n is the refractive index of solvent, and OD is the optical den-
sity (absorption). The subscript R refers to the reference quinine sulfate
of known quantum yield (0.54 in 0.1 M H2SO4). The excitation wave-
length was set at 358 nm which was the UV–Vis crossing point obtained
by plotting UV–Vis of Schiff base 1 in absolute ethanol and quinine
sulfate in 0.1 M H2SO4 (OD was kept at 0.1 at 358 nm).
UV –Vis and fluorescence experiments were performed at room
temperature. For UV –Vis and fluorescence titration 10 μM solution of
Schiff base 1 in ethanol/ethanol-buffer solution (1:1, v/v, HEPES
10 mM, pH = 7.4) was used. 10 mM HEPES buffer was prepared in
double distilled water. The solutions of metal perchlorate and
tetrabutylammonimum anion were prepared in ethanol –water (1:1,
v/v). Donor-receptor binding ratio was determined by Job's plots,
while binding constant (log K) of the donor-receptor adduct was
analyzed by linear fitting of UV –Vis/fluorescence titration curve in the
following equation (Eq. (1)) [20].
2.5. Details of Biological Study
2.5.1. Materials
Dulbecco Modified Eagle Medium (DMEM), trypsin and DMSO were
purchased from Himedia, India. Fetal Bovine Serum (FBS) from Biological
Industries Ltd. Israel. Antibiotic solution (Penicillin 1000 IU and
ꢀ
ꢁ
log½
A
̶
Amin=Amax
̶
Aꢀ ¼ log K þ log Gnꢁ
ð1Þ
Fig. 2. (a) UV–Vis absorption, fluorescence excitation and fluorescnce emission spectra, and (b) Fluorescence spectra at different excitation wavelengths of Schiff base 1 in ethanol.

K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
19
Fig. 3. 3D spectra contour plot and corresponding three dimensional projections and (b) Time resolved fluorescence spectra of Schiff base 1 in ethanol (λexc = 377 nm and λem = 464 nm &
536 nm).
Streptomycin 10 mg/mL and Gentamycin), MTT(3–(4,5–
dimethylthiazol–2–yl)–2,5-diphenyltetrazoliumbromidedye) were ob-
tained Sigma-Aldrich Chemicals, USA.
2.5.2. Cell Culture
A549 cancerous cells were cultured in DMEM solution which was
prepared by mixing of 2.70 g DMEM, 0.3 g NaHCO3, 0.24 g Penicillin,
0.1 g Streptomycin, 500 μL Gentamycin, 20 mL FBS, 180 mL autoclaved
water. The cells were incubated in a humidified atmosphere at 37 °C
in 5% CO2. Stock solution of Schiff base 1 was prepared in DMSO
which was further diluted with DMEM.
2.5.3. MTT Assay
For cytotoxic analysis A549 cancerous cells were seeded at the den-
sity of 5 × 103 cells per well. Further, cells were incubated in the pres-
ence of different concentrations of Schiff base 1 (5 μM, 10 μM, 20 μM
and 50 μM) for 24 h at 37 °C in 5% CO2. After treatment, 100 μL of
0.5 mg/mL MTT in incomplete growth medium was added and the
plate was re-incubated for 2–4 h. Supernatant was discarded and
water insoluble formazan crystals were dissolved in 100 μL of DMSO
by mixing for 10 min. The optical density (O.D.) was taken at 595 nm
at Biotech Synergy HT MultiMode Microplate reader.
2.5.4. Cell Imaging
A549 cells (105 cells/mL/well) were seeded in a six well tissue cul-
ture plate over coverslip in each well and allowed to adhere overnight.
Thereafter, the cells were incubated with 5 μM Schiff base 1 for 2 h. For
detection of Cu2+ and PO34−, pre-treated cells were treated for 30 min
with Cu2+. The cells, pre-treated with Schiff base 1 + Cu2+ were then
Fig. 4. Changes in UV–Vis spectra of Schiff base 1 (30 μM) upon addition of 300 μM of
various ions (Li+, Na+, Al3+, Pb2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Hg2+, Br−,Cl−
HSO4−, F−, SCN−, CH3COO−, CN−, H2PO4−, HPO42− and PO43−) in ethanol.
,

20
K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
Fig. 5. (a) Changes in UV–Vis spectra of Schiff base 1 (10 μM) upon gradual addition of Cu2+ (0–39.7 μM) in ethanol–buffer solution (1: 1, v/v, 10 mM HEPES, pH = 7.4) (b) Limit of
detection (LOD) curve plot. The error bars represent the standard deviation of three independent measurements.
treated subsequently with Cu2+ and PO34− for 30 min each. For cell im-
aging PO34− was taken as representative ions among other phosphate
ions. After treatment, cells were washed thoroughly with chilled phos-
phate buffer saline (PBS) and fixed with chilled 4% paraformaldehyde
for 20 min and images were recorded on Nikon Air Laser Scanning
Confocal Microscope System for analyzing the fluorescence changes.
color, visible to naked eye and displayed an absorption band in the
range 400–449 nm in all the solvents (Fig. 1, Table 1). The high absor-
bance for the band in the region 400–449 nm indicates it may be π →
π* type transition. The UV–Vis spectra showed less pronounced changes
in wavelength position (394–416 nm) upon varying the polarity or
protocity of solvents except in case of water showing a red shifted
split band at 414–449 nm.
3. Results and Discussion
The fluorescence spectra were recorded in different solvents by
exciting the Schiff base 1 at its λmax absorption 406 nm and the results
are displayed in Fig. 1. The fluorescence spectra showed two well
resolved bands in all the solvents, shorter wavelength fluorescence
(B band) in the range 464–494 nm and longer wavelength fluorescence
(A band) in the range 520–542 nm (Table 1). The intensity ratio of IA/IB
is found to vary significantly in different solvents. In aprotic solvents
(toluene) and polar protic solvents (ethanol, methanol, water and
ethanediol) band A is found to be more intense than band B. However,
in DMSO, acetonirile and ethyl acetate, band B becomes more intense.
Diethyl ether showed equal abundance of both the bands in solution.
The variation in intensity, IA/IB may be due to the differential stabiliza-
tion of both the emission bands by different solvents at excited state.
The shorter wavelength fluorescence was found to red shifted in
DMSO, acetonirile, water and ethanediol at 496, 488, 484 and 472 nm
respectively. Although, longer wavelength fluorescence (530–536)
showed less pronounced changes at band position in most of the tested
solvents, however, in water and ethanediol red shifted band at 542 nm
and in acetonitrile blue shifted band at 526 nm was observed.
3.1. Synthesis and Structural Characterization of Schiff Base 1
The
receptor,
5-diethylamino-2-[(2-hydroxy-benzylidene)
hydrazonomethyl]-phenol (1) was obtained with a high yield by the
reaction between 2-hydrazonomethyl phenol and 2–4-diethylamino-2-
hdroxybenzaldehyde in ethanol in 1:1 molar ratio. Schiff base 1 is highly
stable pale-yellow solid and was well characterized by using physico-
chemical and spectroscopic tools viz. NMR and mass spectra. Since Schiff
base 1 contain hydroxyl groups it may show unusual properties in solu-
tion phase due to the existence of O\\H· ··N or O···H\\N type hydrogen
bonds and tautomerism between enol and keto forms (Fig. S1). Therefore,
it is important to investigate its possible form in solution state. The 1H
NMR spectrum showed two sharp singlet, at δ = 11.66 ppm and
11.64 ppm these are assigned to two −OH proton, indicating existence
of Schiff base 1 in enol form in CDCl3 solution (Fig. S2). Two singlet ob-
served for two NCH_N\\at 8.52 ppm and 8.60 ppm, clearly proves the
formation of Schiff base 1. In 13C NMR spectrum two imine carbons also
showed an expected chemical shift value of δ = 159.50 ppm and
161.16 ppm (Fig. S3). Peak at m/z of 312.1705 in HRMS corresponding
to [M + H]+ also validate the molecular structure of Schiff base 1
(Fig. S4).
3.2.2. Nature of UV–Vis Bands
Since in Schiff base 1 proton transfer groups are present, therefore
this molecule may show keto–enol tautomerization at ground state.
Ethanol has been assigned as representative solvent for recording the
spectra of various reference compounds to go insight the UV–Vis spectra
of Schiff base 1.The UV–Vis spectra of salicylaldehyde exhibited
two bands at 324 nm and 255 nm (Fig. S5). The low energy band at
324 nm may originate from the transition S0 → S1 (π → π*) of the intra-
molecular hydrogen bonded salicylaldehyde and high energy band may
assigned to the S0 → S2 transition. Monohydrazone of salicylaldehyde,
3.2. UV–Vis and Fluorescence Studies
3.2.1. Effects of Solvents on UV–Vis and Fluorescence Spectra of Schiff Base 1
The UV–Vis and or fluorescence spectra of Schiff base 1 (30 μM)
were examined at room temperature in a series of polar, nonpolar,
protic and aprotic solvents. Schiff base 1 exhibited yellowish green
Scheme 2. Schematic diagram representing binding interactions of Schiff base 1 with Cu2+
.

K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
21
show blue shift due to the intermolecular hydrogen bonding between
amine nitrogen and water molecule. However, in water it is showing a
red shifted band at 414–449 nm this may be explained as: there are
three hydrogen bond acceptor (HBA) site in Schiff base 1 viz. nitrogen
atom of \\NEt2 and \\C_N group, and oxygen atoms of hydroxyl
groups. Charge density at oxygen atoms is higher compared to\\NEt2
owing to the presence of nearby hydrogen bond acceptor imine group.
In the ground state\\NEt2 group is expected to be co-planar with aro-
matic ring and therefore it can delocalize its charge density with the π
cloud of the aromatic ring. This may decrease the charge density at
\\NEt2 group, however, increases the same at oxygen atoms of hydrox-
yl groups through resonance effect. Therefore, the water, strongest HBD
(hydrogen bond donor) type solvent prefers oxygen atoms for strong
intermolecular hydrogen bonding [24]. Consequently, the Schiff base 1
is energetically more stabilized and shows red shifted UV–Vis spectra
in water.
Fig. 6. Changes in fluorescence spectra of 30 μM Schiff base 1 upon addition of 2 equiv. of
3.2.3. Nature of Fluorescence Bands
various ions (from left, 1, Li+, Na+, Al3+, Pb2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Hg2+
,
Both charge transfer and proton transfer moieties are present in
Schiff base 1, therefore, LE (local emission), ICT (intramolecular charge
transfer) and PT (proton transfer) can be observed in this molecule.
The observation of two different fluorescence bands for a molecule is ei-
ther due to two different ground state species or one single species that
undergoes a excited state reaction (for example, proton transfer and
twisted intramolecular charge transfer (TICT) etc.). The fluorescence
spectra of Schiff base 1 in representative solvent, ethanol exhibited
two bands at 536 and 464 nm. The fluorescence excitation spectra mon-
itored either at 536 or 464 nm shows similar band at 412 nm except for
the difference in intensity and well agree with its absorption band at
406 nm Fig. 2. These results provide evidences for the origin of both
fluorescence bands through excitation of the same ground state. To con-
firm this result 3D fluorescence spectral studies were performed and
only one contour was observed in ethanol. The corresponding excitation
shows a band at 400 nm, and the fluorescence displayed two bands at
538 and 464 nm (Fig. 3). The excitation and 3D fluorescence demon-
strate the presence of only one species in ground state. Moreover, exci-
tation independent fluorescence also validates the same (Fig. 2). Since
dual fluorescence is not because of two different species in ground
state, there may be possibility of excited state reaction upon excitation
of Schiff base 1. Time-resolved fluorescence measurement has been per-
formed to investigate the excited state reaction. The decay curve at
464 nm and 536 nm could not well fitted by mono, bi and multi expo-
nential decay pattern with acceptable χ2 values (χ2 b 1.2) (Fig. 3) indi-
cating existence of more than one species at excited state. The excited
Br−,Cl, HSO−4 , F−, SCN−, CH3COO−, CN−, H2PO4−, HPO42− and PO34−) in ethanol-buffer
solution (1:1, v/v, 10 mM HEPES, pH = 7.4), λexc = 406 nm and λem = 537 nm. Where,
Io & I is the fluorescence intensity at λmax = 537 in the absence and presence of ions,
respectively.
SA (318 & 272 nm) and dihydrazone (357 & 293 nm) of salicylaldehyde
(SAA) showed bands for enol form only and no keto form (generally ob-
served at higher wavelength N 400 nm) is found to be present. Ziolek
et al. also observed the similar results for SAA and explained that the
keto–enol equilibrium cannot be formed at ground state due to the
strong intramolecular hydrogen bonding in SAA [23]. Unsymmetrical
introduction of charge transfer moiety in SAA (Schiff base 1) showed
drastically red shifted broad absorption band at 406 nm as mentioned
earlier. Now to know whether this band in Schiff base 1 is keto/enol
type UV–Vis spectra in various solvents were examined carefully. The
insignificant polarity and protocity dependency of this band insist us
to assign it as enolic band. Moreover, 1H NMR of Schiff base 1 showed
the presence of only enol form in CDCl3 solution. Therefore, we have
also recorded UV–Vis spectra of Schiff base 1 in CDCl3 and found a
very similar split band at 402 and 414 nm (Fig. 1). These findings clearly
proofs that this is the enol form which is found to exist in different
solvents. The absence of keto tautomer in solvents shows that the enol
tautomer is energetically much stabilized than keto tautomer and
hence, keto-enol tautomerization could not observed in the ground state.
Now if this band is due to intramolecular charge transfer from
diethyl amino group to imine group then in protic solvents it must
Fig. 7. Reversibility of UV–Vis response of Schiff base 1in ethanol–buffer solution (1:1, v/v, 10 mM HEPES, pH = 7.4) with alternate addition of Cu2+ and EDTA (left). Diagram representing
molecular logic functions, IMP (410 nm) and INH (450 nm) for Schiff base 1 in the presence of Cu2+ and or EDTA (right).

22
K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
Table 2
from light yellow green to dark yellow visible to naked eye. Further, sol-
vent optimization studies helps in finding the suitable ethanol–buffer
solution (1:1, v/v, HEPES 10 mM, pH = 7.4) for selective and efficient
detection of Cu2+ in polar protic solvents. Similar to ethanolic solution,
upon addition of Cu2+ in ethanol–buffer solution (1:1, v/v, 10 mM, pH
= 7.4), a new band is observed at 450 nm with the disappearance of
410 nm absorption band (Fig. 5). The appearance of new band may
be due the deprotonation and binding of Cu2+ with Schiff base 1
(Scheme 2) [26]. Interestingly, presence of Al3+ and Fe3+ do not show
significant changes in UV–Vis spectra of 30 μM ethanol–buffer solution
(1:1, v/v, 10 mM, pH = 7.4) of Schiff base 1 which may be due to the
high degree of solvation of these ions in aqueous solution (Fig. S6).
The fluorescence changes of Schiff base 1 with aforementioned ions
were investigated in ethanol–buffer solution (1:1, v/v, HEPES 10 mM,
pH = 7.4). The Schiff base 1 (30 μM) displayed a very intense fluores-
cence band at 537 nm and a weak intense band at 464 nm when excited
at 406 nm. Upon addition of 2 equiv. of various ions, incredible fluores-
cence quenching at 537 and 464 nm was observed only in the presence
of Cu2+ ions. Other tested ions do not show significant changes in the
fluorescence intensity (Fig. 6).
Truth table for 10 μM Schiff base1 in ethanol–buffer solution (1:1, v/v, 10 mM HEPES, pH
= 7.4) with 10 equiv. Cu2+ and 10 equiv. EDTA.
Input
Cu2+
Output
EDTA
IMP
INH
Absorbance at 410 nm
Absorbance at 450 nm
0
1
0
1
0
0
1
1
1
0
1
1
0
1
0
0
state reaction that is going on upon excitation may be ESIPT this is be-
cause the band at 536 nm was found to be polarity and protocity inde-
pendent. Charge transfer bands are generally found to be polarity and
protocity dependent. Hence, the fluorescence at 464 and 536 nm of
Schiff base 1 in ethanol may be assigned as LE and ESIPT fluorescence,
respectively.
Although, Schiff base 1 posses both proton transfer and charge trans-
fer groups, however, only ESIPT is observed this may be due to the
suppression of ICT by ESIPT. There are various reports in literature
explaining coupled ESIPT and ICT reaction in those molecular systems
which have both charge transfer and proton transfer groups [25]. How-
ever, there are very few reports for these molecular systems, where fluo-
rescence is observed either by ICT or ESIPT. Up to my knowledge till date
there is only one report explaining ICT suppressed ESIPT single fluores-
cence at 527 nm for 4-(diethylamino)-2-hydroxybenzaldehyde [17].
Quantum yield for Schiff base 1 was calculated in ethanol (0.033), aceto-
nitrile (0.017) and in toluene (0.030) by taking quinine sulfate as
reference.
Hydrolysis of Schiff bases in the presence of even small amount of
water in protic solvents is the major concern of issue. Therefore, the
temporal absorption changes for the Schiff base 1 in the ethanol–
water (1:1, v/v) were measured and found that hydrolysis does not
occur (Fig. S7). These results strengthen the stability of Schiff base 1 in
ethanol–water (1:1, v/v) for performing spectral studies in this solution
mixture.
3.2.5. Colorimetric Sensing of Cu2+ Ions
To study the binding interactions of Schiff base 1 (10 μM) with Cu2+
ions, UV–Vis titration was performed in ethanol (Fig. S8). With the ad-
dition of incremental amount of Cu2+ ions (0–44.4 μM), the absorption
band at 406 nm diminished gradually with the appearance of newly
growing band centered at 455 nm, corresponding to a colorimetric
change from light yellow green to yellow. A clear isosbestic point at
430 nm indicates that only two species are in equilibrium. Job's plot in-
dicates the formation of 1:1, 1 − Cu2+ complex (Fig. S9). With the help
of titration profile association constant (log K) and limit of detection
(LOD = 3σ / slope) has been calculated to be 6.710 and 8.5 × 10−7 M,
respectively (Figs. S8 & S10). To study the interference of other poten-
tially competitive ions on 1 − Cu2+ complexation, UV–Vis competition
experiments was performed in the presence of 10 equiv. of Cu2+ mixed
with 10 equiv. of other ions. Unfortunately, a few ions are found to inter-
fere in the detection of Cu2+ in ethanol (Fig. S11).
3.2.4. Effects of Ions on UV–Vis and Fluorescence Spectra of Schiff Base 1
Insignificant changes are observed in UV–Vis spectra of Schiff base 1
upon varying the polarity and protocity of solvents. However, interac-
tions with different ions may bring some interesting findings. Therefore,
the molecular interactions of Schiff base 1 with Li+, Na+, Al3+, Pb2+
,
Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Hg2+, Br−, Cl−, HSO4−, F−, SCN−,
CH3COO−, CN−, H2PO4−, HPO24− and PO34−were studied initially in eth-
anol and the results are depicted in Fig. 4. Interestingly, it was found that
upon addition of various ions (300 μM) in 30 μM Schiff base 1, drastic
changes are observed only in the presence of Cu2+, Al3+ and Fe3+. In
the presence of Al3+ and Fe3+ the band at 406 nm is found to be red
shifted at 420 nm and 414 nm respectively. A new band at 445 nm is
also found to develop in the presence of Al3+. Addition of 300 μM of
Cu2+ results in appearance of a new band at 455 nm with color change
Fig. 8. (a) Fluorescence titration spectra of Schiff base 1 (10 μM) upon incremental addition of 2.7 equiv. of Cu2+ in ethanol-buffer solution (1:1, v/v, 10 mM HEPES, pH = 7.4), λexc
=
406 nm and λem = 537 nm. Inset: Changes in the fluorescence intensity at 541 nm with incremental addition of Cu2+(b) Detection limit curve plot. The error bars represent the
standard deviation of three independent measurements.

K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
23
binding affinity towards Cu2+ and forms highly stable EDTA–Cu2+
complex. The reversibility remains static with respect to color change
and absorbance even after several cycles with the sequentially alterna-
tive addition of Cu2+ and EDTA (Fig. 7). These results indicate that Schiff
base 1 can be employed as a reversible colorimetric sensor for Cu2+ ions
in ethanol–buffer solution (1:1, v/v, HEPES 10 mM, pH = 7.4).
The change in absorbance at 410 and 450 nm upon addition of Cu2+
and or EDTA can be used to construct IMPLICATION (IMP) and INHIBIT
(INH) logic functions (Fig. 7). An IMP logic gate was constructed by
monitoring absorbance at 410 nm and with two inputs (EDTA and
Cu2+). The absorbance at 410 nm was low (off state) than that of
threshold value only in the presence of Cu2+ (0, 1). However, the high
absorbance (on state) was observed in the absence (0, 0) and presence
(1, 1) of both inputs and also EDTA alone (Table 2). An INH logic gate,
demonstrating noncumulative behavior was constructed by using
similar chemical inputs Cu2+ and EDTA, and absorbance at 450 nm as
an output signal. In the presence of Cu2+ alone (1, 0), the absorbance
at 450 nm was high enough (on state). In the presence of EDTA
(0, 1) or both (1, 1), the absorbance was low (off state).
Fig. 9. Bar diagram representing selectivity of Schiff base 1 for Cu2+ in ethanol–buffer
solution (1:1, v/v, 10 mM HEPES, pH 7.4). The maroon bars represent the
=
fluorescence of solution of 10 μM Schiff base1 and 10 equiv. of Cu2+. The green bars
show the fluorescence change upon addition of 10 equiv. of the corresponding ions
(from left, Li+, Na+, Al3+, Pb2+, Fe2+, Fe3+, Co2+, Ni2+, Zn2+, Hg2+, Br−, Cl−, HSO4−, F−
, SCN−, CH3COO−, CN−, H2PO4−, HPO42− and PO34−) to the mixture solution of 10 μM
Schiff base 1 + 10 equiv. of Cu2+. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
3.2.6. Fluorogenic Sensing of Cu2+ Ions
Since we are interested in the detection of Cu2+ ions in aqueous
solution thus all the fluorescence studies was further conducted in
ethanol–buffer solution (1:1, v/v, HEPES 10 mM, pH = 7.4) only. In
order to understand the binding interactions of Schiff base 1 with
Cu2+, fluorescence titration experiments were performed at r.t.
(Fig. 8). The fluorescence intensity at both the wavelength decreases
with concomitant addition of Cu2+ ions (0–27.7 μM) in 10 μM Schiff
base 1, and almost complete quenching was observed upon addition
of 2.7 equiv. of Cu2+. The concurrent decrease in intensity at both
emission bands also proofs the existence of single species in solution.
Fluorescence is quenched because the presence of Cu2+ ions which
have empty d orbital (d9), leads to faster forbidden intersystem crossing
(ISC) from S1 to T1 state of fluorophore, that is deactivated by bimolec-
ular non–radiative processes (paramagnetic effect).
Further, colorimetric LOD, 1.7 × 10−6 M for Cu2+ ions was calculated
in 10 μM ethanol–buffer solution (1:1, v/v, HEPES 10 mM, pH = 7.4) of
Schiff base 1 with the help of UV–Vis titration profile (Fig. 5). Upon vary-
ing the concentration of Cu2+ ions (0–39.7 μM), a new band was ap-
peared at 450 nm with an isosbestic point at 428 nm. The competition
experiment for the selectivity of Cu2+ ions showed insignificant interfer-
ence by any other ions unusual to ethanolic solution (Fig. S12). The reason
may be due to the higher binding affinity of Schiff base 1 towards Cu2+
ions than that of other ions in aqueous solution. The calculated LOD in
both the solution is much lower than that (3.0 × 10−5 M) recommended
by WHO.
The reversibility of Schiff base 1 towards Cu2+ was examined by the
addition of 100 μM EDTA in a mixture solution of 10 μM Schiff base 1 and
100 μM Cu2+. Addition of EDTA brings back the absorption spectrum
(λmax = 410 nm) as well as yellow green color of Schiff base 1. The pres-
ence of EDTA recovers the absorption of Schiff base 1 because it has high
Fluorescence quenching mechanism may be static or dynamic type.
Static quenching corresponds to change in UV–Vis spectra of host in the
presence of guest molecule. In the case of Schiff base 1, addition of Cu2+
ions results in appearance of a new band, as discussed earlier, validating
Fig. 10. Changes in fluorescence intensity of mixture solution, 10 μM Schiff base 1 + 10 equiv. Cu2+ upon addition of different phosphate ions, PO43− (a), HPO42− (b) and H2PO4− (c), and
their corresponding detection limit curve plot. The error bars represent the standard deviation of three independent measurements.

24
K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
the static type quenching. The UV–Vis changes evidenced formation of 1
− Cu2+ ground state complex, so it is concluded that the fluorescence
quenching of Schiff base 1 induced by Cu2+ ions is primarily due to
the complex formation. Quenching constant 1.8 × 105 L Mol−1 was
calculated by the use of Stern–Volmer equation (Fig. S13). The LOD of
Schiff base 1 for Cu2+ from fluorescence titration curve was determined
to be 3.4 × 10−7 M, indicating high detection sensitivity (Fig. 8). Binding
number of Cu2+ with Schiff base 1 was obtained by using double–
logarithmic equation. Log [Io–I/I] is a function of log [Cu2+], a straight
line is obtained with slope n (binding number). From this linear regres-
sion plot (R2 = 0.9979), value of n is found to be very close to 1, indicat-
ing 1:1 stoichiometry between Schiff base 1 and Cu2+. Simultaneously,
association constant, log Kb (5.92) was calculated by using the same
equation indicating high binding affinity between Schiff base 1 and
Cu2+ (Fig. S13). Furthermore, competition experiments were also
performed for Schiff base 1 at 10 μM in the presence of 100 μM of
Cu2+ with 10 equiv. of aforementioned ions in ethanol–buffer solution
(1:1, v/v, HEPES 10 mM, pH = 7.4) (Fig. 9). Although, the various ions
did not show significant changes in the fluorescence intensity of 1 −
Cu2+ complex, however, the presence of 10 equiv. of phosphate ions
(PO34−, HPO42− and H2PO4−) causes fluorescence enhancement at 537
and 464 nm bands.
3.3. Fluorogenic Sensing of Phosphate Ions
The above results indicate that 1 − Cu2+ complex could be used as a
receptor for the sensing of phosphate ions. The 1 − Cu2+ was found to
unable to distinguish between three types of tested phosphate ions at
physiological pH. Upon addition of incremental concentration of PO43−
(0–180 equiv.), HPO24− (0–110 equiv.) and H2PO−4 (0–110 equiv.) in
ethanol–buffer solution (1:1, v/v, HEPES 10 mM, pH = 7.4) of 1 −
Cu2+ (10 μM + 100 μM Cu2+), the fluorescence intensity increases
gradually (Fig. 10). From the corresponding fluorescence titration pro-
file LOD for PO34−, HPO42− and H2PO−4 were calculated to be 1.1 μM,
2.6 μM and 1.3 μM respectively (Fig. 10). The fluorescence recovery
may be due to the release of Schiff base 1 from 1 − Cu2+ complex
owing to chelation of phosphate with Cu2+. Binding constants (logK)
for phosphate ions was calculated by applying Eq. (1) and was found
to be 4.59, 5.19 and 5.58 for PO34−, HPO42− and H2PO4− respectively.
Other anions do not show significant fluorescence enhancement
Fig. 11. Living cell imaging, bright field image (A) and fluorescence images (A1 and A2) of A549 cells incubated with Schiff base 1. bright field image (B) and fluorescence images (B1 and
B2) of A549 cells incubated with Schiff base 1 and Cu2+. Bright field image (C) and fluorescence images of A549 cells incubated with Schiff base 1 + Cu2+ + PO34−
.

K. Tiwari et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 191 (2018) 16–26
25
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comparing with phosphate may be because of high basicity of phos-
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Acknowledgements
This work was financially supported by DST (Project No. SR/S1/PC-
02/2011) and UGC (Project No. F. No. 42-278/2013 (SR)), New Delhi,
India. K.T. is thankful to UGC, New Delhi, India for providing D.S. Kothari
fellowship. We also thank, Dr. Vandana Bhalla, GNDU, Amritsar for
allowing access to time resolved fluorescence measurement facilities
and for valuable discussions.
(b) USEPA, Appendix B to Part 136-Definition and Procedure for the Determination
of the Method Detection Limit-Revision 1.11, Federal Register 49 (209), 43430,
October 26, 1984 Also Referred to as “40 CFR Part136”.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
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Jeevanjot Kaur has obtained her M.Phil degree in Botany in 2013 from Guru Nanak Dev
solvatochromism and their use as solvatochromic switches and as probes for the inves-
tigation of preferential solvation in solvent mixtures, J. Org. Chem. 77 (2012)
10668–10679.
University, Amritsar, India; presently working as Research Scholar at Department of Bo-
tanical and Environmental Sciences, Guru Nanak Dev University, Amritsar. Her current
field of interest is evaluating the bioactivities of natural compounds.
[29] M.J. Kamlet, J.-L.M. Abboud, M.H. Abraham, R.W. Taft, Linear Solvation Energy Rela-
tionships. 23. A comprehensive collection of the solvatochromic parameters, π*, α,
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J. Org. Chem. 48 (1983) 2877–−2887.
Saroj Arora has done Ph.D. in Botany from Guru Nanak Dev University, Amritsar and later
joined as Professor in the same institute. Her area of specialization includes, Environmen-
tal Toxicology and she is widely recognized for her insights into the bioprotective activities
of plant based compounds with particular emphasis on antimutagenicity and anticancer
research. Her research has identified number of compounds from different plant parts
and showed their mechanism of action. She has published N130 research papers in reput-
ed international and national journals. She has successfully completed a number of re-
search projects funded by various National bodies. She is currently supervising research
projects that aim to establish structure activity relation of bioactive molecules with respect
to their basic molecule functional groups and stereochemistry.
Karishma Tiwari has obtained her Ph. D. degree in Inorganic Chemistry in 2014 from Ba-
naras Hindu University, Varanasi, India; presently working as DSK-PDF in the Department
of Chemistry, Guru Nanak Dev University under the supervision of Prof. Rakesh K.
Mahajan. Her research interest mainly focused on chemical sensing.
Sumit Kumar has obtained his M.Sc. degree from Guru Nanak Dev University, Amritsar.
He joined Dr. Vipan's research group as junior research fellow for pursuing his doctoral de-
gree in year 2015. His research interests are mainly focused on the synthesis and bio-
evaluation of isatin/nitroimidazole based molecular conjugates targeted against tropical
infections viz. Malaria and Trichomoniasis.
Rakesh Kumar Mahajan received his master's degree in 1977 and his Ph.D. in 1982 in the
field of analytical chemistry. Presently, he is working as a Dean, colleges in Guru Nanak
Dev University, Amritsar, India. His research interests include chemical sensors, surfactant
chemistry, and development of new electro-analytical methods of analysis. He has pub-
lished N160 research papers in different international journals of repute.
Vipan Kumar (Ph.D.) has been working as an Assistant Professor in the Department of
Chemistry, Guru Nanak Dev University, Amritsar since 2009. He obtained his Ph.D with
Prof. Mohinder P Mahajan, in the Department of Applied Chemistry, GNDU. In 2007, he
moved to the University of Cape Town (UCT), South Africa to pursue his postdoctoral stud-
ies with Prof. Kelly Chibale. In 2015, he was awarded research fellowship from Carl
Tryggers research foundation, Sweden to carry out research at University of Umea,
Sweden. His research interests include the development of diverse synthetic protocols
for synthesis of novel molecular frameworks targeting tropical infections. He has also been
engaged in the utilization of β-lactam synthon protocols for the synthesis of functionally
decorated and biologically relevant heterocycles with medicinal potential.