Highly selective light-up Al3+ sensing by a coumarin based Schiff base probe: Subsequent phosphate sensing DNA binding and live cell imaging

Article abstract of DOI:10.1016/j.jphotochem.2016.08.019

A coumarin based simple fluorescent “turn-on” probe, 4-Hydroxy-3-[(2-hydroxy-5-methoxybenzylidene)-amino]-chromen-2-one (HMC), to detect metal ion based on the chelation enhanced fluorescence (CHEF), was synthesized by condensing 3-amino-4-hydroxycoumarin and 2-hydroxy-5-methoxybenzaldehyde. It was found to be highly selective and sensitive sensor for Al³⁺ in 90% aqueous methanol (MeOH: 0.01?M HEPES Buffer; 9:1; v/v) at pH 7.4 in fluorescence spectroscopy. Fluorescence intensity enhancement along with 10?nm blue shift was observed only in the presence of Al³⁺. The HMC binds Al³⁺ in 1:1 stoichiometry with a binding constant, K_a?=?(9.9?±?0.04)?×?10³?M^?1 and the detection limit was calculated to be as low as 1.62?μM. The binding mode of interaction with Al³⁺ and the chelate complex formation was supported with the help of a ¹H NMR spectroscopy titration, ESI–MS and also by theoretical studies. Moreover, the HMC·Al³⁺ ensemble subsequently detected the biologically important phosphate ions and nucleotides via fluorescence quenching. The live cell imaging study indicated that HMC is highly efficient in the detection of exogenous Al³⁺ in living cell.

Full text of DOI:10.1016/j.jphotochem.2016.08.019

Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Highly selective light-up Al3+ sensing by a coumarin based Schiff base
probe: Subsequent phosphate sensing DNA binding and live cell
imaging
Sanjoy Kumar Sheeta, Bhaskar Sena, Romita Thounaojamb, Kripamoy Aguanb,
Snehadrinarayan Khatuaa,
*
a
Centre for Advanced Studies, Department of Chemistry, North Eastern Hill University, Shillong, Meghalaya 793022, India
Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong, Meghalaya 793022, India
b
A R T I C L E I N F O
A B S T R A C T
Article history:
A coumarin based simple fluorescent “turn-on” probe, 4-Hydroxy-3-[(2-hydroxy-5-methoxybenzyli-
dene)-amino]-chromen-2-one (HMC), to detect metal ion based on the chelation enhanced fluorescence
(CHEF), was synthesized by condensing 3-amino-4-hydroxycoumarin and 2-hydroxy-5-methoxyben-
zaldehyde. It was found to be highly selective and sensitive sensor for Al3+ in 90% aqueous methanol
(MeOH: 0.01 M HEPES Buffer; 9:1; v/v) at pH 7.4 in fluorescence spectroscopy. Fluorescence intensity
enhancement along with 10 nm blue shift was observed only in the presence of Al3+. The HMC binds Al3+
in 1:1 stoichiometry with a binding constant, Ka = (9.9 ꢀ 0.04) ꢁ 103 Mꢂ1 and the detection limit was
Received 20 June 2016
Received in revised form 12 August 2016
Accepted 16 August 2016
Available online 17 August 2016
Keywords:
Coumarin
Schiff base
Al3+ sensing
CHEF
calculated to be as low as 1.62
formation was supported with the help of a 1H NMR spectroscopy titration, ESI–MS and also by
theoretical studies. Moreover, the HMC
Al3+ ensemble subsequently detected the biologically important
m
M. The binding mode of interaction with Al3+ and the chelate complex
ꢃ
Phosphate ion
Cell imaging
phosphate ions and nucleotides via fluorescence quenching. The live cell imaging study indicated that
HMC is highly efficient in the detection of exogenous Al3+ in living cell.
ã 2016 Elsevier B.V. All rights reserved.
1. Introduction
solutions and adjuvant in vaccines for human health [20,21].
Therefore, effective detection of Al3+ has become very essential to
In recent years, considerable effort has been paid to develop
highly selective and sensitive fluorescent probes for metal ions
detection in environment and in the field of biology [1–8]. The
fluorescent sensors are highly valuable due to their operational
simplicity, high sensitivity and instant response [9–11]. In the
Earth’s crust aluminium is the one of the most abundant metal.
Excessive exposure of the human body to aluminium is known to
have a negative effect on human health [12–14]. Its severe toxicity
can lead to Alzheimer’s disease, chronic renal failure, bone
softening, damage of kidney and Parkinson’s disease [15–17].
The World Health Organization (WHO) reported aluminium as a
source of food pollution and limited the aluminium concentration
regulate its concentration level in environment and to prevent its
direct impact in human health. The coordination ability of Al3+ is
less than the transition metals and the lack of photophysical
properties makes it difficult to detect spectroscopically. Al3+ is a
hard-acid and it prefers hard-base donor sites for coordination, so
the Schiff base probes with N and O donor centre are considered to
be very good ligands for Al3+ detection. It is already established
that, due to the free rotation around C N bond, the free Schiff
¼
bases are non-fluorescent or weakly fluorescent in solution. In the
recent past, numerous research papers described the fluorescence
“turn-on” detection of various metal ions through the chelation of
Schiff base probes by inhibiting the C N isomerisation [22–32].
¼
to 200
m
g Lꢂ1 (7.41
mM) in drinking water [18,19]. Despite of this
Coumarin derivatives have been extensively explored due to their
tunable photophysical properties and substantial fluorescent
behaviour in the visible region [33–35].
large amount, the aluminium containing compounds are frequent-
ly used as pharmaceutical drugs such as antiperspirants, antiseptic
Recently, fluorescent metal complexes have been used exten-
sively as secondary sensor for phosphate ions (pyrophosphate; PPi/
3ꢂ
HP2O7
and H2PO4ꢂ), as the metal ions provide supportive
* Corresponding author.
binding site for the phosphate ions [36–38]. Pyrophosphate can be
E-mail addresses: skhatua@nehu.ac.in, snehadri@gmail.com (S. Khatua).
http://dx.doi.org/10.1016/j.jphotochem.2016.08.019
1010-6030/ã 2016 Elsevier B.V. All rights reserved.

102
S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
biologically important target as it is the product of ATP hydrolysis
under cellular condition. Moreover, pyrophosphate has a close
connection with cancer, regulation of fibrin function and
chondrocalcinosis or calcium pyrophosphate dehydrates (CPPD)
crystal deposition [39–42]. Hence, the selective detection and
quantification of phosphate ions have attracted the growing
attention due to their involvement in various cellular processes
such as DNA sequencing and DNA replication catalyzed by DNA
polymerase [43–47].
of metal ions (10 mM) were prepared using their perchlorate salt
in Millipore water. Thereafter, HMC and metal ions with desired
concentrations were mixed and measured by UV–vis and
fluorescence spectroscopy. Stock solutions of anions (10 mM)
were prepared using their tetra-n-butyl ammonium salt in
Millipore water. The HMC
ꢃ
Al3+ ensemble was prepared by mixing
Al3+ ensemble and
of HMC with Al3+ (1:5). Subsequently, HMC
ꢃ
anions with desired concentrations were mixed and measured
by fluorescence spectroscopy. The 1H NMR spectroscopic titra-
tions were performed by treating HMC solution in DMSO-d6
(14 mM) with Al3+ in DMSO-d6 (70 mM) at varying equivalents
(0.5, 1.0 and 2.0). Calf thymus DNA (ctDNA) stock solution was
prepared by diluting ctDNA with Tris-HCl buffer (pH = 7.4), and
kept at 4 ꢄC for less than a week. The UV–vis absorbance at
260 and 280 nm of the ctDNA solution in the Tris-HCl buffer gave
the ratio 1.75-1.90, signifying that the DNA is sufficiently protein
free. The concentration of ctDNA was measured from its
absorbance at 260 nm with a molar extinction coefficient of
6600 Mꢂ1 cmꢂ1 [49].
Herein, we report the synthesis and characterization of
coumarin based Schiff base compound, HMC [48] and selective
fluorescence light-up sensing of Al3+ in physiological condition
ꢂ
through CHEF effect. The selective detection of the HP2O7 (PPi)
and H2PO4ꢂ ions over other competitive anions is accomplished by
the resultant probe-metal ensemble under the same physiological
condition. Further, the possible interaction of the HMC
Al3+
ꢃ
ensemble with ctDNA is studied through a systematic fluorescence
turn-off response. Moreover, the live cell imaging experiments
using HeLa cell and detection of exogenous Al3+ ion in living cell is
also discussed.
2.3. Synthesis of 3-Amino-4-hydroxycoumarin (A)
2. Experimental
3-Amino-4-hydroxycoumarin (A) was synthesised according to
the modified reported procedure [50]. See Supplementary material
for the synthesis of A.
2.1. Chemicals and instruments
All chemicals were commercially available and used without
further purification. The IR spectrum was collected on a Perkin-
Elmer FT- IR spectrometer with KBr pellets in the range 4000–
2.4. Synthesis of coumarin Schiff base probe(HMC)
400 cmꢂ1
.
Nuclear magnetic resonance (NMR) spectra were
The HMC was synthesized by an extremely facile procedure as
illustrated in Scheme 1, and was isolated in high yield. The
methanolic solution (20 ml) of A (84 mg, 0.48 mmol) was added
dropwise to the methanolic solution (10 ml) of 2-hydroxy-5-
methoxy benzaldehyde (73 mg, 0.48 mmol) over a period of 15 min
with continuous stirring. The solution was stirred at room
temperature. The pH of the solution was maintained at ꢅ8.0 by
the drop wise addition of aqueous NaOH solution. The solution
turned in to orange after 20 min which indicated the formation of
the intended product. The stirring was continued to further 12 h to
yield an orange precipitate. The volume of the solution was
reduced under rotary evaporator. The solid was filtered, washed
with CHCl3 and finally dried under vacuum which yielded 83%
orange solid HMC (0.12 g, 0.40 mmol). 1H NMR (DMSO-d6,
recorded on Bruker Avance II (400 MHz) spectrometer using the
residual protic solvent resonance as the internal standard and
chemical shifts were expressed in ppm. ESI–MS was performed
with a QTOF Micro YA 263 mass spectrometer in methanol. The
UV–vis absorption spectra were recorded using a Perkin-Elmer
Lambda 25 spectrophotometer. Fluorescence spectra were
obtained with a Hitachi F-4500 spectrophotometer with quartz
cuvette (path length = 1 cm). Digital photographs were taken by
Nikon D90 camera.
2.2. General methods
The interaction between coumarin Schiff base and different
metal cations was investigated by UV–vis and fluorescence
spectroscopy in 90% aqueous methanol (MeOH: 0.01 M HEPES,
9:1 v/v, 7.4 pH) solution at room temperature. The stock solution
of HMC was prepared at a concentration of 1.0 mM and diluted to
400 MHz):
d
(ppm) = 14.90 (s, 1H, OꢂꢂHcoumarin), 9.95 (s 1H, H11),
7.90 (d, J = 7.2 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 7.16-7.13 (m, 2H), 6.84
(s,1H, H13), 6.72-6.67 (m, 2H), 3.71 (s, 3H, H18); 13C NMR (DMSO-d6,
100 MHz): d(ppm) = 179.4, 170.3, 163.8, 162.2, 160.9, 160.3, 140.4,
50
m
M and 10
m
M using 0.01 M HEPES buffer and methanol for
134.7, 132.7, 132.1 130.1 126.7, 126.6, 125.4 123.3, 116.4 65.0; MS
fluorescence and UV–vis studies, respectively. The stock solutions
(ESI–MS; negative mode): (m/z, %): 310.08 [(C17H13NO5 ꢂ H)ꢂ,
OH
OH
OH
O
Glacial acetic acid
72% HNO3
Conc. H2SO4
Stirring, rt, 5h
NO2
NH2
O
Pd/C
NaBH4
Stirring, rt, 6h
O
O
O
O
B (80%)
A
(74%)
16
13
OHC
HO
OCH3
HO
15
OH
17
MeOH, NaOH
Stirring, rt, 12h
5
8
4
11
10
9
N
O
6
7
12
14
C
H
OCH3
3
18
2
O
(83%)
1
HMC
Scheme 1. Synthetic scheme of HMC.

S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
103
100%]; calcd. m/z, 310.07; Elemental analysis calcd. (%) for
methoxybenzaldehyde in aqueous methanol at pH ꢅ8 and isolated
as an orange solid with high yield (83%). The HMC was fully
characterized by various spectroscopic techniques namely, 1H and
13C NMR and ESI–MS spectrometry. The photophysical properties
were determined by UV–vis and fluorescence spectroscopy. The 1H
and 13C spectrum of HMC, recorded in DMSO-d6 at room
temperature, clearly showed all expected resonances for the Schiff
base probe (Fig. S3, S4). 1H NMR spectrum showed four singlets at
C
17H13NO5 (MW = 311.29): C 65.59, H 4.21, N 4.50; found: C
66.52, H 4.39, N4.48; FTIR in KBr disk (nmax/cmꢂ1): 3468, 2985,
1677, 1625, 1446, 1384, 1246, 1039, 748.
2.5. Binding constant calculation
The binding constant of the metal complex formed in solution
was estimated by using the standard Benesi-Hildebrand (B-H)
Eq. (1).
d
= 14.89, 9.94, 6.94 and 3.72 ppm which were assigned to the
coumarin ꢂꢂOH, imine proton (H11), aldehyde proton (H13) and
ꢂꢂOCH3 (H18), respectively. The ESI–MS in negative mode displayed
peak at m/z = 310.08 (calcd. 310.07) and was assigned to
[C17H12NO5]ꢂ(Fig. S5).
1/(I ꢂ I0) = 1/{Ka(Imax ꢂ I0)C} + 1/(Imax ꢂ I0)
(1)
I0 is the fluorescence intensity of free HMC at emission maximum
(lem = 510 nm), I is the observed fluorescence intensity at that
3.2. Sensing study by HMC in UV–vis and fluorescence spectroscopy
particular wavelength in the presence of a certain concentration of
the metal ion (C), Imax is the maximum fluorescence intensity value
that was obtained at lem = 510 nm during titration with varying
metal ion concentration, Ka is the binding constant and was
determined from the slope of the linear plot, and C is the
concentration of the Al3+ ion added during titration experiment.
The effect of the various cations such as Na+, K+, Mg2+, Ca2+
,
Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Cd2+, Hg2+ and Al3+ (as their
perchlorate salts) on the UV–vis spectra of HMC was studied in
90% aqueous methanol (0.01 M HEPES buffer: methanol 1:9; v/v;
pH 7.4) at room temperature (Fig. S6). All the cations including Al3+
altered the n-
p* transition band remarkably. The UV–vis titration
2.6. Determination of detection limit
of HMC (10
m
M) with increasing amount of Al3+ (0–3.5 equiv.) in
90% aqueous methanol (Fig.1) displayed a clear isosbestic points at
410 nm and 450 nm. Bands at 370 nm and 484 nm gradually
decreased along with appearance of a band at 425 nm with the
addition of Al3+. The decrease and increase in absorption bands
indicates the chelation of Al3+ with HMC. The UV–vis study is not
practically useful for selective sensing of Al3+. Consequently, a
systematic fluorescence experiments was performed to ensure the
Al3+ selectivity of HMC in the presence of various cations.
The fluorescence spectroscopic study was used to explore the
cation selectivity of HMC in the presence of various metal ions such
The limit of detection (DL) of HMC was calculated based on the
fluorescence titration data and determined from the following
equation:
DL = 3
s
/K
(2)
Where
s
is the standard deviation of the blank solution; K is the
slope of the calibration curve.
2.7. Methods of cell imaging study
as Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Cd2+
,
A fluorescence Inverted microscope (Leica DMI4000B) was used
to visualize the fluorescence of the cells followed by the addition of
the respective compound with 10ꢁ–20ꢁ objective lens. At first,
HeLa cells were cultured in DMEM media containing low glucose
(Invitrogen) with 10% FBS (Invitrogen) at 37 ꢄC in 5% CO2 incubator
chamber. Cells were seeded into 24 well plates for the imaging
experiment. After 24 h of cell growth, cells were washed with PBS
Hg2+ and Al3+. Probe HMC exhibited a weak emission peak at
520 nm upon excitation at 410 nm in 90% aqueous methanol. This
weak fluorescence of HMC is due to the C N isomerization with
¼
the 2-hydroxy-5-methoxy-benzylidene amino group. Upon the
addition of 5.0 equiv. of various metal ions in 90% aqueous
methanol, the fluorescence intensity of HMC increased by ꢅ7-fold
only in the presence of Al3+ and it showed a bright green
fluorescence under UV light (Fig. 2). A complex was formed due to
(phosphate buffer saline) and fresh 500
two successive wells. One of the well was treated with only HMC
(10 M) and kept in incubator for 30 min. Other well was initially
treated with Al3+ (30
M), incubated for 10 min and subsequently
washed twice with PBS buffer. Afterwards, free HMC (10 M) was
ml of PBS were added in
m
m
m
added and incubated for further 20 min. After the incubation, cells
were washed with PBS and observed under fluorescent microscope
at bright field and at excitation of BP 450–490 nm.
2.8. Theoretical study
The electronic properties of the HMC and HMC
Al3+ complex
ꢃ
were investigated by means of density functional theory (DFT)
calculations. All calculations were performed using the B3LYP
exchange correlation functional [51,52] as implemented in the
Gaussian 03(G03) program package. The 6–31G+ (d, p) basis set was
assigned for all the elements.
3. Results and discussion
3.1. Synthesis and characterization
The synthetic route of coumarin based Schiff base probe,
HMC is shown in Scheme 1. The HMC was synthesized easily by
stirring 3-amino-4-hydroxycoumarin (A) and 2-hydroxy-5-
Fig. 1. UV–vis titration of HMC (10 m
M) with Al3+ (0–3.5 equiv.) in 90% aqueous
methanol (0.01 M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature.
(Inset) Absorbance at 370 nm and 425 nm.

104
S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
Fig. 2. Fluorescence spectra of HMC (50 m
M) upon addition of 2 equiv. of various metal ions (Al3+, Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Cd2+, and Hg2+) in
90% aqueous methanol (0.01 M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature. (lex = 410 nm; lem = 510 nm). (inset) Naked eye fluorescence colour change of
HMC in addition of Al3+
.
Scheme 2. Binding mode of Al3+ with the probe HMC and the effect of H2PO4ꢂ/PPi on HMC Al3+ ensemble.
ꢃ
the interaction of Al3+ with the probe HMC through the
deprotonation of hydroxyl groups, which was further corroborated
by 1H NMR spectroscopic titration experiment. Like Al3+
check the practical applicability of HMC as highly selective Al3+
sensor. The fluorescence intensity of HMC was recorded upon
addition of 2 equiv. of Al3+ in a mixed 90% aqueous methanol in the
selective various CHEF based probes [22–32], the C
¼
N isomer-
presence of other competitive cations (Na+, K+, Mg2+, Ca2+, Mn2+
,
isation in HMC is inhibited due to this complexation which results
in rigidity in the molecular assembly and fluorescence enhance-
ment (Scheme 2). To understand the binding mechanism more
precisely, the fluorescence titration experiment was carried out in
90% aqueous methanol. During titration, the emission band of HMC
Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Cd2+ and Hg2+) in large excess (30
equiv.). Addition of other competitive cations did not influence the
fluorescence intensity of HMC, while 2 equiv. of Al3+ was readily
detected by the probe through an obvious enhancement in
fluorescence intensity (Fig. 4). For the biological application of
the probe, the pH effect on the fluorescent detection of Al3+ by
HMC was examined under different pH conditions (Fig. S8). The
HMC has weak fluorescence over the pH range of 3–10. In contrast,
upon the addition of Al3+, a significant increase in the fluorescence
intensity was found in the pH range of 6.5–10.0. This study
illustrated that HMC can readily detect the Al3+at wide pH range
from 6.5 to 10.
(50 mM) at 520 nm gradually increased and blue shifted to 510 nm
with the addition of increasing amount of Al3+ (0–2.0 equiv.).
(Fig. 3a). The BenesiꢂHildebrand (BꢂH) plot shows a linear fitting
and confirms the 1:1 stoichiometry of binding of Al3+ to HMC
(Fig. 3b), which is supported afterwards by ESI mass spectrum.
From the fluorescence titration data, the binding constants of the
HMC
ꢃ
Al3+ complex were determined as Ka = (9.9 ꢀ 0.04) ꢁ 103 Mꢂ1
on the basis of Benesi–Hildebrand equation. The plot of fluorescent
intensity of HMC with concentration of Al3+ showed a good linear
3.3. Evidence of Al3+ binding with HMC
relationship in lower concentration of Al3+ (0–30
mM) and the
detection limit of HMC for Al3+ was calculated as low as 1.62
mM
To understand the complexation behaviour of HMC with Al3+
,
(Fig. S7) in a 90% aqueous methanol.
A competition experiments with other possible interfering
metal ions were carried out in the fluorescence spectroscopy to
1H NMR spectroscopic titration experiments were carried out in
DMSO-d6. Significant spectral changes were observed upon
gradual addition of increasing amount of Al3+ (0–2.0 equiv.) to

S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
105
HMC (Fig. 5). Upon addition of 0.5 equivalent of Al3+, the signals of
the hydroxyl protons of the free HMC at (ppm) = 14.89
d
disappeared completely due to the deprotonation of the hydroxyl
groups and the coordination to Al3+. In contrast, upon incremental
addition of Al3+ the signal of imine proton (H11) gradually shifted
upfield from
d (ppm) = 9.94 to 9.61. These observations suggest
strong binding of HMC with Al3+ and formation of rigid complex via
chelation with HMC through the phenolate oxygen and imine
nitrogen atoms.
ESI–MS spectral analysis clearly indicates the formation of 1:1
HMC
Al3+ complex upon treatment of HMC with excess Al3+
ꢃ
(Fig. S9). The molecular ion peaks at m/z = 368.34 and 336.30
corresponds to the formation of complexes [Al(HMC)(MeOH)]+ and
[Al(HMC)]+, respectively.
3.4. Anion detection studies of HMC
spectroscopy
ꢃ
Al3+ensemble in fluorescence
It is well known that the phosphate ions (H2PO4ꢂ and HP2O7ꢂ or
PPi) interact with Al3+ strongly and form stable complexes [53,54].
Therefore the HMC
ions in fluorescence spectroscopy. The selectivity and sensitivity of
HMC
Al3+ ensemble toward phosphate ions were investigated in
ꢃ
Al3+ complex may be used to detect phosphate
ꢃ
fluorescence spectroscopy in the presence of various anions
including Fꢂ, Clꢂ, Brꢂ, Iꢂ, HOꢂ, AcOꢂ, NO3ꢂ, HSO4ꢂ, CNꢂ, H2PO4
,
ꢂ
PPi. Fluorescent intensity of HMC
ꢃ
Al3+ quenched ꢅ82% only in
ꢂ
presence of PPi in 90% aqueous methanol whereas H2PO4
quenched the intensity by ꢅ58% (Fig. 6). Quenching of fluorescent
intensity indicates the weakening of HMC and Al3+ binding, which
allowing the C
Upon the addition of increasing amount of PPi (0–1.0 equiv.) to
the HMC
Al3+ ensemble, the fluorescent intensity of the band at
¼N isomerization.
ꢃ
510 nm gradually decreased with 10 nm red shift (Fig. 7a) in 90%
aqueous methanol. Linear fitting in BꢂH plot analysis indicates the
1:1 binding stoichiometry of PPi to HMC
Al3+ ensemble. From the
ꢃ
fluorescence titration profiles, the binding constant of PPi to
Fig. 3. (a) Fluorescence titration spectra of HMC (50
90% aqueous methanol at pH 7.4. (lex = 410 nm; lem = 510 nm). (b) BꢂH plot from
fluorescence titration of HMC with Al3+
m
M) with Al3+ (0–2.0 equiv.) in
HMC
ꢃ
Al3+ was determined as (1.24 ꢀ 0.16) ꢁ 104 Mꢂ1 on the basis of
Benesi-Hildebrand equation (Fig. 7b). Furthermore, quenched
.
fluorescence of HMC
ꢃ
Al3+ ensemble was restored to ꢅ72% upon
addition of Al3+ once again to the mixture of HMC Al3+ and PPi
ꢃ
Fig. 4. Selectivity study of HMC with Al3+ in presence of various cations in 90% aqueous methanol (0.01 M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature.
Colour code: Blue bar stand for intensity of HMC in presence competitive cations (30 equiv.); red bar stand for intensity of HMC in the presence competitive cations and Al3+ (2
equiv.). Number code: (1) HMC, (2) Na+, (3) K+, (4) Mg2+, (5) Ca2+, (6) Mn2+, (7) Co2+, (8) Ni2+, (9) Cu2+, (10) Zn2+, (11) Pb2+, (12) Ag+, (13) Cd2+ and (14) Hg2+. (lex = 410 nm;
lem = 510 nm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

106
S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
Fig. 5. Partial 1H NMR spectra of (a) HMC only and HMC in the presence of (b) 0.5 equiv., (c) 1.0 equiv., and (c) 2.0 equiv. of Al3+ in DMSO-d6.
Fig. 6. Fluorescence spectra of HMC
90% aqueous methanol (0.01 M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature. (lex = 410 nm; lem = 510 nm).
ꢃ
Al3+ (50 M) ensemble upon addition of 2 equiv. of various metal anions (Fꢂ, Clꢂ, Brꢂ, Iꢂ, HOꢂ, AcOꢂ, NO3ꢂ, HSO4ꢂ, CNꢂ, H2PO4ꢂ, PPi) in
m
(Fig. S10). The probe HMC found to be reversible towards Al3+ and
PPi up to two cycles (Fig. S11).
competitive study found to be promising for selective detection of
PPi by HMC
Al3+ ensemble in 90% aqueous methanol pH 7.4.
ꢃ
From the fluorescence titration result it is observed that the
intensity of HMC
concentration range of PPi (0 ꢂ 21
was calculated to be as low as 0.2
methanol. To establish the high selectivity of HMC
ꢃ
Al3+ ensemble linearly decreases at lower
3.5. DNA binding studies
m
M) and the detection limit
M (Fig. 8a) in 90% aqueous
Al3+ ensemble
m
The DNA binding studies with metal complexes are immensely
important for design and development of new DNA targeted drugs
[55,56]. Metal complexes can bind to DNA by electrostatic
interaction, groove binding and intercalative binding [57]. Herein,
the phosphate ions interact with the Al3+ centre and able to remove
ꢃ
toward phosphate ions in the presence of various competitive
anions an interference study was carried out. Addition of other
competitive anions to the HMC
Al3+ ensemble in 90% aqueous
ꢃ
methanol did not affect the fluorescence intensity,ꢂ while in
addition of only 2.0 equivalents of PPi and H2PO4 even in
presence of large excess (20.0 equiv.) of other competitive anions
significantly quenched the fluorescence intensity (Fig. 8b). The
the Al3+ from the HMC
isomerization is allowed in the free HMC and fluorescence is
ꢃ
Al3+ complex. Accordingly, the C
N
¼
quenched. The phosphate ions are backbone of DNA hence the
interaction of ctDNA with HMC
Al3+ ensemble tested in
ꢃ

S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
107
Fig. 7. (a) Fluorescence titration spectra of HMC
of PPi. (b) BꢂH plot from fluorescence titration of HMC
ꢃ
Al3+ ensemble with gradual addition of PPi (0–1.0 equiv.). (Inset) Plot of fluorescence intensity at 510 nm versus concentration
ꢃ
Al3+ ensemble with PPi. (lex = 410 nm; lem = 510 nm).
fluorescence spectroscopy. Affinity of HMC
ꢃ
Al3+ ensemble toward
ensemble with ctDNA calculated by the BꢂH plot analysis was
found to be (4.24 ꢀ 0.2) ꢁ 103 Mꢂ1 (Fig. 9b).
ctDNA was tracked through the change in emission intensity
during fluorescence titration. Incremental addition of ctDNA (0–
2.0 equiv.) to the 90% aqueous methanol of highly fluorescent
3.6. Probable binding mode and DFT study
HMC
ꢃ
Al3+ ensemble resulted ꢅ62% quenching of fluorescence
(Fig. 9a). It is probably due to the interaction and removal of
Toward a further understanding of the electronic structures of
coordinated Al3+ from the HMC Al3+ by the phosphate groups
ꢃ
the HMC and the HMC
Al3+ complex, density functional theory
ꢃ
present in ctDNA. The Plot of fluorescence intensity at 520 nm
against the concentration of ctDNA shows good linearity at lower
concentration of ctDNA (Fig. S12) which allows the quantitative
(DFT) calculations were carried out by using the B3LYP/6-31G+
(d, p) basis set in the Gaussian 03 program. From the ESI–MS
studies it is confirmed that a methanol is coordinated with the
detection of ctDNA. The binding constant (Ka) for HMC
Al3+
ꢃ
HMC
Al3+complex. As the formation of tetra coordinated
ꢃ

108
S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
Fig. 8. (a) The linear relation of the fluorescence intensity of HMC
with PPi in the presence of various anions in 90% aqueous methanol (0.01 M HEPES buffer: methanol 1:9; v/v; pH 7.4). Colour code: Green bar stands for intensity of HMC in
ꢃ
Al3+ ensemble with concentration of PPi (0–21
m
M) at 520 nm. (b) Selectivity study of HMC
ꢃ
Al3+ ensemble
Al3+
presence competitive anions (20 equiv.), Red bar stands for intensity of HMC
ꢃ
Al3+ ensemble in presence competitive anions and PPi (2 equiv.). Number code: (1) HMC
ꢃ
ensemble, (2) Fꢂ, (3) Clꢂ, (4) Brꢂ, (5) Iꢂ, (6) HOꢂ, (7) AcOꢂ, (8) NO3ꢂ, (9) HSO4ꢂ, (10) CNꢂ, (11) H2PO4ꢂ. (lex = 410 nm; lem = 520 nm). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Aluminum complex except for Al-salen complex is quite unusual
so it is expected that the Aluminum complex is formed with a
water molecule as a fifth ligand. The energy minimised structure
and highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) of the HMC and the
monitoring Al3+ ions in vivo. Using the fluorescence microscopy
an imaging application of HMC for Al3+ in biological samples was
developed. As shown in Fig. 11, the intracellular imaging
behaviours of HMC on HeLa cells with the help of fluorescence
microscopy displayed no fluorescence when treated with only
complex, HMC
of the HMC
Al3+ complex, the aqua ligand is axially coordinated to
the central Al3+ which adopts a distorted square pyramidal
geometry. The HOMO to LUMO energy gap ( E = 3.13 eV) in the
HMC
Al3+ complex is lowered significantly than that of free HMC
E = 3.83 eV). Both the HOMO and the LUMO is stabilized after the
complex formation. The total energy of HMC is decreased
ꢃ
Al3+ is shown in Fig. 10. In the optimized structure
10 mM HMC (Fig. 11a and b). While a bright green fluorescence was
ꢃ
observed in cells which were treated with both the Al3+ and HMC
without alteration of morphology (Fig. 11d and e).The figure
displayed the uptake of HMC probe in living HeLa cells and it forms
the complex inside the cell with externally added Al3+and
visualized in terms of emission of the green colour. green
fluorescence image of live HeLa cells indicates that HMC can
easily permeate through the cell membrane, diffuse well within
the cell and can appropriately detect the low amount intracellular
Al3+ ions by forming stable complex.
D
ꢃ
(
D
significantly (EHMC = ꢂ29565.74 eV and EHMC Al3+ = ꢂ41360.30 eV)
ꢃ
upon complexation with Al3+ which suggests that the formed
HMC
Al3+ complex is highly stable.
ꢃ
3.7. Live cell imaging study
4. Conclusions
In the above-mentioned studies, it is demonstrated that the
HMC has high affinity to coordinate Al3+, hence the living cell
imaging experiment was done in HeLa cell using HMC for
We have developed a new Schiff base compound, HMC
from amine substituted hydroxyl coumarin and 2-hydroxy-5-
methoxybenzaldehyde for selective and sensitive detection of

S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
109
Fig. 9. (a) Fluorescence titration spectra of HMC
ensemble with ctDNA. (lex = 410 nm; lem = 520 nm).
ꢃ
Al3+ ensemble with incremental addition of ctDNA (0–2.0 equiv.). (b) The BꢂH plot from fluorescence titration of HMC
ꢃ
Al3+
Al3+ through CHEF mechanism. The probe shows excellent
fluorescence selectivity toward Al3+ ion in 90% aqueous methanol
without any interference of other cations. High binding affinity of
cells and rapidly sense Al3+ in the intracellular region of human
cervical cancer cells.
HMC with Al3+ formed 1:1 HMC
ꢃ
Al3+ complex which inhibits the
Acknowledgements
C N isomerization and generates chelation-enhanced fluores-
¼
cence (CHEF) effect. This complexation was confirmed with the
assist of ESI–MS, 1H NMR spectroscopy and finally through
theoretical calculation. Anion selectivity studies with the
We thank DST SERB (SB/FT/CS/115/2012) for the financial
assistance and Sophisticated Analytical and Instrumentation
Facility (SAIF), North Eastern Hill University for NMR and MS
data. S.K.S and B.S thank RGNF and North Eastern Hill University for
their research fellowship. Finally, we thank the reviewers for their
valuable comments and suggestions.
HMC
the complex is dissociated easily in the presence of the
pyrophosphate ion, which makes the HMC
Al3+ complex an
efficient probe for PPi. The interaction of nucleotides with
ꢃ
Al3+ complex in the fluorescence spectroscopy revealed that
ꢃ
HMC
ꢃ
Al3+ complex has been established which shows fluores-
Appendix A. Supplementary data
cence quenching due to strong attraction of Al3+ to phosphate ions.
The usefulness of HMC as a sensor for intracellular detection of
Al3+ by fluorescence microscopy was also studied. Importantly the
developed CHEF based sensor could readily permeate into HeLa
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
jphotochem.2016.08.019.

110
S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
Fig. 10. Energy optimized structure of (left) the probe HMC and (right) [HMC
ꢃ
Al3+] and the corresponding molecular orbital of HMC and HMC Al3+
.
ꢃ
Fig.11. Images of HeLa cells: (a) bright field image of HeLa cells incubated with HMC 10 mM) for 30 min; (b) and (c) fluorescence image and overlay image of (a). (d) bright field
image of HeLa cells incubated with HMC (10 m
M) and Al3+ for 30 min; (e) and (f) fluorescence image and overlay image of (d).
Recently, fluorescent metal complexes have been used extensively as secondary sensor for phosphate ions (pyrophosphate; PPi/HP2O73ꢂ and H2PO4ꢂ), as the metal ions
provide supportive binding site for the phosphate ions [36–38]. Pyrophosphate can be biologically important target as it is the product of ATP hydrolysis under cellular
condition. Moreover, pyrophosphate has a close connection with cancer, regulation of fibrin function and chondrocalcinosis or calcium pyrophosphate dehydrates (CPPD)
crystal deposition [39–42]. Hence, the selective detection and quantification of phosphate ions have attracted the growing attention due to their involvement in various
cellular processes such as DNA sequencing and DNA replication catalyzed by DNA polymerase [43–47].

S.K. Sheet et al. / Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 101–111
111
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