A coumarin based Schiff base probe for selective fluorescence detection of Al3?+ and its application in live cell imaging

Article abstract of DOI:10.1016/j.saa.2016.10.005

A new coumarin based Schiff base compound, CSB-1 has been synthesized to detect metal ion based on the chelation enhanced fluorescence (CHEF). The cation binding properties of CSB-1 was thoroughly examined in UV–vis and fluorescence spectroscopy. In fluorescence spectroscopy the compound showed high selectivity toward Al^3?+ ion and the Al^3?+ can be quantified in mixed aqueous buffer solution (MeOH: 0.01?M HEPES Buffer; 9:1; v/v) at pH?7.4 as well as in BSA media. The fluorescence intensity of CSB-1 was enhanced by ~?24 fold after addition of only five equivalents of Al^3?+. The fluorescence titration of CSB-1 with Al^3?+ in mixed aqueous buffer afforded a binding constant, K_a?=?(1.06?±?0.2)?×?10⁴?M^??1. The colour change from light yellow to colourless and the appearance of blue fluorescence, which can be observed by the naked eye, provides a real-time method for Al^3?+ sensing. Further the live cell imaging study indicated that the detection of intracellular Al^3?+ ions are also readily possible in living cell.

Full text of DOI:10.1016/j.saa.2016.10.005

A coumarin based Schiff base probe for selective fluorescence detection of
3
+
Al
and its application in live cell imaging
Bhaskar Sen, Sanjoy Kumar Sheet, Romita Thounaojam, Ramen Jama-
tia, Amarta Kumar Pal, Kripamoy Aguan, Snehadrinarayan Khatua
PII:
DOI:
Reference:
S1386-1425(16)30586-8
doi:10.1016/j.saa.2016.10.005
SAA 14704
To appear in:
Received date:
Revised date:
Accepted date:
9 June 2016
15 September 2016
8 October 2016
Please cite this article as: Bhaskar Sen, Sanjoy Kumar Sheet, Romita Thounaojam,
Ramen Jamatia, Amarta Kumar Pal, Kripamoy Aguan, Snehadrinarayan Khatua, A
coumarin based Schiff base probe for selective fluorescence detection of Al3 and its
application in live cell imaging, (2016), doi:10.1016/j.saa.2016.10.005
+
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ACCEPTED MANUSCRIPT
A Coumarin Based Schiff Base Probe for Selective
3
+
Fluorescence Detection of Al and its Application in
Live Cell Imaging
a
a
b
a
Bhaskar Sen, Sanjoy Kumar Sheet, Romita Thounaojam, Ramen Jamatia, Amarta Kumar.
a
b
a,
Pal, Kripamoy Aguan and Snehadrinarayan Khatua 
a
Centre for Advanced Studies, Department of Chemistry, North Eastern Hill University,
Shillong, Meghalaya 793022, India.
b
Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong,
Meghalaya 793022, India.
3
+
Keywords: Coumarin, Schiff Base, Fluorescence, Al Sensing, Bio-imaging
ABSTRACT:
A new coumarin based Schiff base compound, CSB-1 has been synthesized to detect metal
ion based on the chelation enhanced fluorescence (CHEF). The cation binding properties of
CSB-1 was thoroughly examined in UV-vis and fluorescence spectroscopy. In fluorescence
3
+
3+
spectroscopy the compound showed high selectivity toward Al ion and the Al can be
quantified in mixed aqueous buffer solution (MeOH: 0.01M HEPES Buffer; 9:1; v/v) at pH
7
.4 as well as in BSA media. The fluorescence intensity of CSB-1 was enhanced by ~ 24 fold
3
+
3+
after addition of only five equivalents of Al . The fluorescence titration of CSB-1 with Al
4
-1
in mixed aqueous buffer afforded a binding constant, K = (1.06 ± 0.2) × 10 M . The colour
a
change from light yellow to colourless and the appearance of blue fluorescence, which can be
3
+
observed by the naked eye, provides a real-time method for Al sensing. Further the live cell
3
+
imaging study indicated that the detection of intracellular Al ions are also readily possible
in living cell.

Corresponding author. Tel.: +91-364-272-2636; e-mail: snehadri@gmail.com; skhatua@nehu.ac.in
1

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1
. Introduction
The rational design and synthesis of fluorescent chemosensors for the selective detection
and quantification of specific cation, anion, ion-pair and biomolecule has attracted
considerable interest due to their importance in environmental and biological applications [1-
1
1]. Highly selective metal ions sensing and live cell imaging in chemistry and chemical
biology is an extremely interesting and important area of current research [12-16]. Aluminum
as the third most abundant metallic element in the lithosphere was found to be neurotoxic to
3
+
living organism. Al ion widely exists in the environment, normally in natural waters and in
most plants and can enter the human body through foods and water [17-19]. High
3
+
concentrations of Al are toxic to plants, vegetables, fish, algae, bacteria, and other species
3
+
in aquatic environment [20-22]. The excessive exposure of Al to human body is harmful as
3
+
the Al toxicity is believed to be one of the main causes of neurological disorders such as
Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), chronic renal
failure, bone softening and gastrointestinal problems etc. [23-24]. Accordingly, the
3
+
development of Al -specific molecular probes have been of considerable interest in the
3
+
biological and analytical chemical sciences [25-30]. The detection and quantification of Al
3
+
has been problematic because of the strong hydration of Al in aqueous media which leads
to its weak coordination ability in comparison to transition metals [31-35]. Therefore, it is
3
+
challenging to design a highly selective and sensitive fluorescence probe for Al in aqueous
2
+
2+
2+
2+
2+
media as the other interfering ions such as Zn , Cu , Cd , Hg and Pb are very much
present in biological system and in environmental sample. [36-38]. Due to the hard nature,
3
+
3+
Al prefers to bind N and O donor ligands and, therefore, most of the reported Al probes
are based on the N and O donor ligand [39-42].
.
2

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Schiff base derivatives having N and O donor site and a fluorescent moiety are appealing
tools for designing optical probes for metal ions [43-46]. It is already established that Schiff
base alone is weekly or non-fluorescent as the fluorescence is quenched due to the free
rotation around C=N bond [47]. In the recent past, numerous research papers described the
specific detection of various metal ions through the chelation of Schiff base probes which
obstruct the above C=N isomerization mechanism [47-49]. Coumarin is a well-known
chromophore used in fluorescence sensing of cation, anion and bio-relevant species [50-54].
Coumarin derivatives have been used extensively due to their tunable photophysical
properties and substantial fluorescent behavior in the visible region. The coumarin derived
Schiff base probe containing an O or N donor can bind the metal ion as well as act as a
signaling unit.
Herein, we report the synthesis and characterization of coumarin based Schiff base, CSB-1
3
+
and the Al ion sensing in mixed aqueous buffer as well as BSA media through the chelation
enhanced fluorescence (CHEF). Further, the imaging experiments using live HeLa cell and
3
+
detection of intracellular Al ion in living cell are also discussed herein.
2
. Experimental
2
.1 Materials and methods
All commercially available chemicals were used without further purification except 3-
Amino-4-hydroxycoumarin which was synthesized according to the reported
procedure [55]. The IR spectra were measured on a Perkin-Elmer FT- IR spectrometer
−
1
with KBr pellets in the range of 4000–400 cm . Nuclear magnetic resonance spectra
were recorded on Bruker Avance II (400MHz) spectrometer using the residual protic
solvent resonance as the internal standard and chemical shifts were expressed in ppm.
ESI-MS was performed with Waters ZQ-4000 and QTOF Micro YA 263 mass
3

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spectrometer. Elemental analysis was carried out in PerkinElmer 2500 series II
elemental analyzer. The UV–vis absorption spectra were recorded using a Perkin-
Elmer Lambda 25 UV–vis scanning spectrophotometer. Fluorescence spectra were
obtained with a Hitachi F-4500 spectrophotometer with quartz cuvette (path length = 1
cm).
2
.2 A general method for the UV-vis and fluorescence experiments
The different metal ions sensing by coumarin Schiff base compound, CSB-1 was
investigated using UV–vis and fluorescence spectrophotometer in mixed aqueous buffer
MeOH : 0.01 M HEPES, 9:1 v/v, 7.4 pH) solution at room temperature. A stock solution of
CSB-1 (1 mM) was prepared in methanol and diluted to 10 and  using 0.01M
(
3
+
HEPES buffer and methanol for Al sensing in fluorescence and UV-vis spectroscopy,
respectively. Stock solutions of various cations (10 mM) were prepared using their
perchlorate salt in Millipore water. An excitation wavelength at  = 395 nm was used for all
fluorescence experiments and the excitation and the emission slits were set to 2.5 mm.
2
.3 Binding constant calculation
The binding constant of the metal complex formed in solution has been estimated by using
the standard Benesi-Hildebrand (B-H) equation 1.
1
/(I - I ) = 1/{K (I - I )C} + 1/(I - I0)
(1)
0
a
max
0
max
I is the fluorescence intensity of free CSB-1 at emission maximum ( = 461 nm), I is the
0
em
observed fluorescence intensity at that 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 em = 461 nm during titration with varying metal ion concentration, K is the
a
4

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binding constant and was determined from the slope of the linear plot, and C is the
3
+
concentration of the Al ion added during titration experiment.
2
.4 Quantum Yield calculation
The fluorescence quantum yield was determined using fluorescein as reference with a known
3
+
Ф of 0.91 in 0.1M NaOH [56]. The compounds (free CSB-1 and in the presence of Al ) and
R
reference were excited in such a way that the absorbance should nearly equal (~0.1). The
quantum yield is calculated according to the following equation:
-
A
R
-A
S
2
S
2
Ф = Ф × (1-10 )/(1-10 ) × I /I × η / η
R
(2)
S
R
S
R
Here, Ф and Ф are the fluorescence quantum yield of the sample and reference,
S
R
respectively. I and I are the area under emission spectra of the sample and the reference,
S
R
respectively. A and A are the absorbance of the sample and the reference solution at the
S
R
excitation wavelength, respectively. η and η are the values of refractive index for the
S
R
respective solvent used for the sample and reference.
2
.5 Determination of detection limit
3
+
The limit of detection (DL) of CSB-1 for Al was calculated based on the fluorescence
titration data and determined from the following equation:
DL = 3σ/K
Where σ is the standard deviation of the blank solution; K is the slope of the calibration
curve.
(3)
2
.6 Methods for cell imaging
A fluorescence Inverted microscope (Leica DMI4000B) was used to visualize the
fluorescence of the cells following the addition of the respective compound with 20X
5

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objective lens. Fluorescence detection was carried out using excitation filter BP 340-380 for
complex CSB-1. The HeLa cells were cultured in DMEM media containing low glucose
(
Invitrogen) with 10% FBS (Invitrogen) at 37°C in 5% CO incubator chamber. For imaging
2
experiments, cells were seeded into 6 well plates. Cells were washed with PBS (phosphate
buffer saline) after 24 hours of incubation and fresh 500 μL of PBS were added in two
successive wells. One of the well was treated with only CSB-1 (10 M) and incubated for 20
3
+
min. Other well was initially treated with Al (30 M) and incubated for 10 min. After
incubation, the cells were washed with PBS buffer, treated with CSB-1 (10 M) and
3
+
incubated for another 10 min. After incubation, both the Al treated and untreated cells were
washed with PBS buffer twice and observed under fluorescence microscope at bright field
and blue emission channel at excitation of 340-380 nm.
2
.7 Theoretical study
The electronic properties of the CSB-1 and Al -CSB-1 complex has been investigated by
3
+
means of density functional theory (DFT) calculations. All calculations have been performed
using the B3LYP exchange correlation functional [57-59] as implemented in the Gaussian
0
3(G03) program package. The 6-31G+ (d, p) basis set was assigned for the elements.
2
.8 Synthesis of CSB-1[(E)-3-(2-hydroxybenzylideneamino)-4-hydroxy-2H-chromen-2-
one]
Compound CSB-1 was synthesized by an extremely facile procedure as illustrated in
Scheme 1. The solution (10 ml) of salicylaldehyde (0.049 ml, 0.455 mmol) in methanol was
added dropwise to the solution (20 ml) of 3-amino-4-hydroxycoumarin (75.0 mg, 0.423
mmol) in methanol over a period of 15 minutes with continuous stirring. The solution was
stirred at room temperature. The pH of the solution was maintained at ~8.0 by the drop wise
addition of dilute methanolic NaOH solution. The solution turned into yellow after few
6

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minutes which indicated the formation of the intended product. The stirring was continued
further for 12 h and the volume of the solution was reduced under rotary evaporator. A
yellow solid compound was obtained and washed thoroughly with chloroform and diethyl
ether to remove unreacted excess salicylaldehyde. Finally it was dried under vacuum to get
1
solid yellow CSB-1. Yield: 115 mg (96 %). H NMR (DMSO-d , 400 MHz,): δ(ppm) =
6
3
3
1
7
5.66 (s, 1H, O-Hcoumarin), 9.97 (s, H ), 7.91 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H),
11
3
13
.25 (d, J = 8.0, 1H), 7.20-7.10 (m, 3H), 6.76-6.74 (m, 2H). C NMR (DMSO-d , 100
6
MHz): δ(ppm) = 169.9, 161.1, 161.0, 152.6, 151.2, 130.4, 129.6, 129.3, 125.0, 123.5, 122.2,
+
1
21.3, 117.2, 116.6, 115.6, 107.2. ESI-MS [C H NO + H] : calcd., m/z = 282.07; found
16
11
4
+
m/z, 282.05 and ESI-MS [C H NO + Na ]: calcd., m/z = 304.06; found, m/z = 304.13. C,
16
11
4
-1
H, N analysis: Calcd. (%) for C H NO (Mw = 281.07 g mol ): C 68.32, H 3.94, and N
1
6
11
4
-1
4
1
.98; found: C 68.31, H 3.96, and N 4.99. FTIR (KBr disk) (νmax /cm ): 3435, 1664, 1611,
537, 1464, 1417, 1363, 1203, 1144, 1085, 751, 728, 468.
3
. Results and discussion
The coumarin based Schiff base compounds, CSB-1 was synthesized by reacting
salicylaldehyde and 3-amino-4-hydroxycoumarin in methanol and isolated as a bright yellow
solid with a high yield (Scheme 1). The CSB-1 was fully characterized by elemental analysis,
1
13
H and C NMR spectroscopy and ESI-MS spectrometry. The photophysical properties were
1
13
determined by UV-vis and fluorescence spectroscopy. The H and C spectra of CSB-1
recorded in DMSO-d at room temperature clearly showed all expected resonances for the
6
Schiff base probe (Figure S1, S2 in SI). For CSB-1, the ESI-MS clearly displayed peaks at
m/z = 282.05 (calcd. 282.07) and 304.13 (calcd. 304.06) that are assigned to [C H NO +
16
11
4
+
+
H ] and [C H NO + Na ], respectively (Figure S3 in SI).
16
11
4
7

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of probe, CSB-1. Reagents and conditions: (i) Glacial acetic acid, conc.
HNO , stirring, 100 °C, 15 min. (ii) 5% Pd/C, 1% HCl in MeOH, H , 5h. (iii) MeOH, NaOH
3
2
solution, pH ~8.0, stirring, 12h. Compound 1 and 2 were synthesized according to the
reported published procedure [55].
3
.1 Sensing study in UV-vis and fluorescence spectroscopic channel
The UV-vis spectrum of CSB-1 in mixed aqueous buffer (0.01M HEPES buffer : methanol
-1
-1
1
:9 ; v/v; pH 7.4) at room temperature displayed bands at 326 nm (ε = 9900 M cm ), 339
-1
-1
-1
-1
-1
-1
nm (ε = 11000 M cm ), 395 nm (ε = 11000 M cm ), 415 nm (ε = 10000 M cm ) and
-1
-1
4
55 nm (ε = 4500 M cm ), assigned to intraligand (IL) π-π* and n-π* transitions. The effect
of the different cations (present as perchlorate salts) was examined in a mixed aqueous buffer
(0.01M HEPES buffer: methanol 1:9 v/v; pH 7.4) at room temperature. As shown in figure
+
+
2+
+
3+
+
2+
1
a, metal ions e.g. Na , K , Ca , Ag , Fe , H and Hg did not show any response whereas
2
+
2+
2+
2+
2+
2+
2+
3+
3+
other metals ions e.g. Cu , Ni , Co , Mn , Zn , Cd , Pb , Cr and Al altered the n-π*
3
+
transition band. The addition of Al caused a decrease in the absorbance of the peak at 455
nm along with the growth of two peaks at 395 nm and 415 nm (Figure 1b). The decrease and
3
+
increase in absorbance at 455 nm, 395 nm and 415 nm indicate chelation of Al . But other
8

ACCEPTED MANUSCRIPT
cations also altered the n-π* transition band remarkably. Therefore, the UV-vis spectroscopic
3
+
channel is not beneficial to sense Al selectively.
Figure 1: (a) UV−vis absorption spectra of CSB-1(50 M) with various cations in mixed
aqueous buffer (0.01M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature. (b)
3
+
Absorption titration of CSB-1 (50 M) with Al (0-300 M) in mixed aqueous buffer at pH
.4.
The compound CSB-1 itself is weakly fluorescent under neutral aqueous conditions
0.01M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature. It exhibits very weak
7
(
fluorescence (Ф = 0.026) at λem = 461 nm when excited at λex = 395 nm. The weak
fluorescence of CSB-1 is due to the C=N isomerization with the 2-hydroxybenzylidene
3
+
amino group (Scheme 2) [60-61]. Upon addition of only 5 equiv. of Al to mixed aqueous
buffer solution of CSB-1, however, a significant enhancement of the emission was
documented at 461 nm (Ф = 0.731). The bright blue luminescence may readily be observed
by the naked eye using a hand-held UV light (Figure 2a inset). The fluorescence intensity was
+
+
2+
not notably influenced by the addition of other competitive metal ions, such as Na , K , Ca ,
+
2+
2+
2+
2+
2+
2+
2+
2+
+
3+
3+
2+
Ag , Cu , Ni , Co , Mn , Zn , Cd , Hg , Mg , H , Fe , Cr and Pb . It is assumed
3
+
that the coordination of the Al ion to the O and N center of the CSB-1, the C=N
isomerization with the 2-hydroxybenzylidene amino group is inhibited and as a consequence
9

ACCEPTED MANUSCRIPT
the fluorescence is enhanced (Scheme 2). The fluorescence titration of CSB-1 (10 μM) with
3
+
0
-6.0 equiv. of Al shows a gradual red shift and a 24-fold enhancement of the initially weak
fluorescence band at λmax 453 to 461 nm (Figure 2b).The Benesi-Hilderbrand (B-H) plot
3
+
shows a linear fitting and confirms the 1:1 stoichiometry of Al to CSB-1 (Figure 3a).
3
+
Scheme 2: Schematic illustration of Al sensing by inhibiting of C=N isomerization.
Figure 2: (a) Cation selectivity by CSB-1 (10 M) in fluorescence spectroscopic channel in
aqueous buffer (0.01M HEPES buffer: methanol 1:9; v/v; pH 7.4) at room temperature.(inset)
3
+
Naked eye fluorescence colour change of CSB-1 in addition of Al . (b) Fluorescence
3
+
titration of CSB-1 (10 M) with Al (0-60 M) in aqueous buffer at pH 7.4.
3+
The B-H plot from the fluorescence titration of CSB-1 with Al in mixed aqueous buffer
4
-1
afforded a binding constant, K = (1.06 ± 0.2) × 10 M . During the fluorescence titration the
a
3
+
emission intensity of CSB-1 is linearly increased at a lower concentration of Al and the
3
+
calculated detection limit of Al was as low as 1.34 M (Figure S4 in SI) in a mixed aqueous
1
0

ACCEPTED MANUSCRIPT
3
+
buffer solution which is lower than many other reported Al probes and the maximum
permissible level stipulated by WHO (7.41 M) [62-67]. A competition experiments with
other metal ions were carried out with the fluorescence spectroscopy to check for possible
3
+
interferences in Al sensing. The addition of other competitive metal ions (100 equiv.) to
3
+
CSB-1 (10 M) did not influence the fluorescence intensity. Consequently, Al (5 equiv.) in
a 1:100 mixture of CSB-1 and other competitive cations was readily detected by a clear
3
+
fluorescence enhancement at 461 nm (Figure 3b). However, the Al sensing was slightly
3
+
3+
interfered by Fe and Cr ions.
Figure 3. (a) Benesi-Hildebrand (B-H) plot from the fluorescence titration of CSB-1 with
3
+
3+
Al (b) Selectivity studies of CSB-1 (50 M) with Al (5 equiv.) in the presence of
competing cations (100 equiv.) in mixed aqueous buffer solution.
1
1

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3+
3
.1.1. Effect of pH in Al detection and reversibility study
3
+
The effect of the pH in Al detection was examined in fluorescence spectroscopy. The CSB-
1
shows weak fluorescence over the pH range of 3.0-11.0 (Figure 4). On the other hand, upon
3
+
the addition of only 2.0 equiv. of Al , a significant increase in the fluorescence intensity is
3
+
observed in the pH range of 7.0–11.0, and this endorses its possibility for Al detection
under physiological conditions (Figure 4).
It has been already established that pyrophosphates (PPi) and EDTA can form stable
3
+
3+
complexes with Al [68-69]. To check the stability of the Al -CSB-1 complex, PPi / EDTA
3
+
3+
was used to remove Al from the Al -CSB-1 complex. When incremental amount of PPi or
3
+
EDTA was added to the Al -CSB-1 ensemble solution, the intensity of the emission band at
61 nm was decreased very slowly. The emission intensity was decreased by ~ 8 fold after
4
addition of 87.5 equiv. of PPi (Figure S5). In a separate titration experiment, the emission
intensity was decreased by ~ 3.6 fold after addition of 880 equiv. of EDTA (Figure S6).
3
+
3+
Therefore, the Al -CSB-1 complex is very stable and the Al displacement by PPi / EDTA
is inefficient.
Figure 4. The pH effect on fluorescence responses of CSB-1 (10 M) in the absence (black)
3
+
and presence (red) of Al (5 equiv.). Excitation and emission was monitored at  = 395 nm
and 461 nm respectively.
1
2

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3
+
3
.1.2. Detection of Al in BSA media
Bovine serum albumin (BSA) is a hydrophobic protein and it contains specific metal ion
binding sites. For the practical applicability of the probe, CSB-1 and to detect and quantify
3
+
Al ions in the biological medium we examined the photophysical properties of the probe in
the presence of bovine serum albumin (BSA). The CSB-1 is weakly fluorescent in BSA
media as well as in the mixed HEPES buffer. The fluorescence intensity of the CSB-1 at 469
3+
nm is enhanced by ~12 fold upon addition of 5 equiv. of Al and the band is blue shifted to
59 nm in BSA medium (Figure 5a). Whereas, in the mixed HEPES buffer media the
fluorescence intensity
4
3
+
Figure 5. (a) Fluorescence detection of Al in mixed HEPES buffer (pH 7.4) and BSA
3
+
medium. (b) Fluorescence titration of CSB-1 (10 M) with Al (0-20 M) in BSA medium.
3
+
(
inset) plot of fluorescence intensity at 459 nm as a function of Al concentration.
1
3

ACCEPTED MANUSCRIPT
3
+
of the CSB-1 at 461 nm is enhanced by ~24 fold upon addition of 5 equiv. of Al . The
3
+
fluorescence titration with Al in BSA medium reveals a gradual blue shift of the emission
band to 459 nm and a concomitant intensity enhancement. No significant enhancement is
3
+
observed after addition of 2.0 equiv. of Al (Figure 5b). The plot of the emission intensity at
3
+
3+
4
59 nm vs concentration of Al shows linearity at low concentration and supports that Al
3
+
quantification is possible in BSA medium particularly, assessing the presence of Al in
blood plasma (Figure 5b inset).
3
+
3
.1.3 Evidence for Al binding
3
+ 1
To gain insight into the interaction between CSB-1 and Al , H NMR titration experiment
3
+
was carried out in DMSO-d (Figure 6). After the addition of only 0.5 equiv. of Al , the
6
signals of the hydroxyl protons of the free CSB-1 at  (ppm) = 15.66 disappeared completely
due to the instantaneous deprotonation of the phenolic -OH groups and the coordination of
3
+
CSB-1 with Al . The imine proton, H of CSB-1 is highly affected during the complexation
1
1
process of CSB-1 with metal ions and shows a gradual upfield shift from  (ppm) = 9.97 to
3
+
1
9
.62 upon addition of increasing amounts of Al (0 - 2 equiv.). The H NMR titration result
3
+
supported the binding of Al with CSB-1 through a chelation reaction via phenolic -O and
the imine -N atoms.
1
3+
Figure 6. H NMR titration of CSB-1 with Al in DMSO-d .
6
1
4

ACCEPTED MANUSCRIPT
3
+
The ESI-MS spectrum clearly indicated the formation of the Al -CSB-1 complex upon
3+
treatment of CSB-1 with excess Al . A major peak is observed at m/z = 338.20
+
corresponding to the [Al(CSB-1)(MeOH)] . The other peak is observed at m/z 306.15
+
3+
indicating to the [Al(CSB-1)] and thereby confirming 1:1 stoichiometry of CSB-1 to Al
binding (Figure S7 in SI).
3
.2 Probable binding mode and DFT study
3
+
To further understand the electronic structure of the CSB-1 and the Al -CSB-1 complex, we
carried out density functional theory (DFT) calculations using the B3LYP/6-31G+ (d, p)
basis set in the Gaussian 03 program. The ESI-MS studies confirms that a solvent methanol
3
+
is coordinated with the Al -CSB-1 complex. The formation of tetra coordinated Aluminum
complex except for Al-salen complex is quite unusual, so it expected that a water molecule is
3+
also coordinated to the Aluminum in Al -CSB-1 complex as a fifth ligand. In the figure 7,
the energy minimized structure and highest occupied molecular orbital (HOMO) and the
3
+
lowest unoccupied molecular orbital (LUMO) of the CSB-1 and the Al -CSB-1 complex is
3
+
shown. The optimized structure of the Al -CSB-1 complex clearly shows that the central
3
+
Al adopts a distorted square pyramidal geometry with an axial aqua ligand. Both the
HOMO and LUMO is stabilized after the complex formation. The HOMO to LUMO energy
3
+
gap in the free CSB-1 (E = 3.63 eV) is lowered considerably upon the Al -CSB-1 complex
(E = 2.28 eV) complex formation. The DFT calculation shows that the total energy of CSB-
3
+
3+
-
1
(ECSB-1 = -26451.40 eV) is significantly higher than the formed Al -CSB-1 complex (E
Al
3
+
=
-38244.84 eV) which suggests the higher stability of the Al -CSB-1 complex than the
CSB-1
free CSB-1.
1
5

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3
+
Figure 7. Energy optimized structure of (left) CSB-1 and (right) [Al -CSB-1] and the
3
+
corresponding molecular orbital of CSB-1 and [Al -CSB-1].
3
.3 Live cell imaging
3
+
Due to the high selectivity and sensitivity of CSB-1 toward Al in the mixed aqueous
buffer, we further tried to find out the biological activity of our compound. Therefore, the
3
+
living cell imaging experiment was done in HeLa cell using CSB-1 for monitoring Al ions
in biological systems. Using the fluorescence microscopy, an imaging application of CSB-1
3
+
for Al in biological samples was developed. As shown in figure 8, the intracellular imaging
performances of CSB-1 on HeLa cells with the help of fluorescence microscopy displayed no
significant intracellular fluorescence when treated with only 10 μM of CSB-1 (Figure 8a and
3+
8
d) and incubated for 20 min at 37°C. In contrast, cells which were treated with both the Al
(30 μM) and CSB-1, a bright blue intracellular fluorescence was observed without celluar
alteration of morphology (Figure 8b and 8e) after 20 mins incubation. The bright blue
fluorescence image of live HeLa cells indicates that the CSB-1 pass through the cell
1
6

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3
+
membrane, diffuse well within the cell and form stable complex with added Al . Thus,
above results clearly documents the applicability of CSB-1 as a fluorescent chemosensor to
3
+
detect intracellular Al ions by fluorescence OFF-ON mechanism.
Figure 8: Images of HeLa cells: (a) bright field image of HeLa cells incubated with CSB-1
(10 μM) for 20 min; (d) fluorescence image of (a); (b and c) bright field image of HeLa cells
3
+
incubated with Al (30 μM) for 10 min. and the CSB-1 (10 μM) for additional 10 min; (e)
fluorescence image of (b) and (f) overlay image of (c) and (e).
4
. Conclusions
We have successfully developed a new Schiff base compound, CSB-1 from 3-amino-4-
3
+
hydroxy-2H-chromen-2-one and salicylaldehyde to detect Al ion based on the chelation
enhance fluorescence (CHEF) in mixed aqueous buffer media. Fluorescence intensity
3
+
enhancement of 24-fold upon addition of 50 μM Al to CSB-1 (10 μM) is observed. The
3
+
possible fluorescent species of CSB-1 with Al were deduced according to the ESI-MS and
1
3+
H NMR spectroscopy. The probable working mechanism is based on the Al -induced
3
+
formation of a 1 : 1 Al -CSB-1 complex, which inhibits the C=N isomerization process and
produces a chelation-enhanced fluorescence (CHEF) effect. The fluorescence “Turn–on”
1
7

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3
+
3+
sensing phenomenon caused by Al is irreversible and the formed Al -CSB-1 complex is
highly stable which is supported by fluorescence spectroscopy and DFT study. The detection
3
+
limits for Al was found to be as low as 1.34 M. More importantly the developed
3
+
chemosensor can also detect Al in the intracellular region of live human cervical cancer
cells.
Acknowledgements
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
Mass data. B.S and S.K.S thank North Eastern Hill University and RGNF SC for their
research fellowship respectively. Also we would like to thank the reviewers for their critical
comments and suggestions.
Supplementary Material
1
13
Supplementary data (UV-vis, fluorescence, H, C NMR and MS spectra of the synthesized
compound) associated with this article can be found, in the online version.
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Graphical abstract
A Coumarin Based Schiff Base Probe for Selective
3+
Fluorescence Detection of Al and its Application in Live
Cell Imaging
a
a
b
a
Bhaskar Sen, Sanjoy Kumar. Sheet, Romita Thounaojam, Ramen Jamatia, Amarta Kumar.
a
b
a,*
Pal, Kripamoy Aguan and Snehadrinarayan Khatua
2
3

ACCEPTED MANUSCRIPT
Highlight
3+
.
Fluorescence “Turn ON” Al sensing in mixed aqueous buffer solution based on CHEF effect
is presented.
3
+
.
.
The sensor is competent to quantify Al at its low concentration (detection limit = 1.34 M).
Compound diffuses well within the cell and appropriately detect the low amount extracellular
3
+
Al ions.
2
4