A new selective chromogenic and turn-on fluorogenic probe for copper(ii) in solution and vero cells: Recognition of sulphide by [CuL]

Article abstract of DOI:10.1039/c4dt03969f

A new coumarin-appended thioimidazole-linked imine conjugate, viz. L has been synthesized and characterized. L has been found to recognize Cu²⁺ selectively among a wide range of biologically relevant metal ions. The chemosensing behavior of L has been demonstrated through fluorescence, absorption, visual fluorescence color changes, ESI-MS and ¹H NMR titrations. The chemosensor L showed selectivity toward Cu²⁺ by switch on fluorescence among the 18 metal ions studied with a detection limit of 1.53 μM. The complex formed between L and Cu²⁺ is found to be 1:1 on the basis of absorption and fluorescence titrations and was confirmed by ESI-MS. DFT and TDDFT calculations were performed in order to demonstrate the structure of L and [CuL] and the electronic properties of chemosensor L and its copper complex. This highly fluorescent [CuL] complex has been used to recognize sulphide selectively among the other allied anions. Microstructural features of L and its Cu²⁺ complex have been investigated by SEM imaging (scanning electron microscopy). The biological applications of L were evaluated in Vero cells and it was found to exhibit low cytotoxicity and good membrane permeability for the detection of Cu²⁺. This journal is

Full text of DOI:10.1039/c4dt03969f

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ARTICLE TYPE
A New Selective Chromogenic and Turn-On Fluorogenic Probe for
Copper (II) in Solution and Vero Cells: Recognition of Sulphide by
[CuL]
Ajit Kumar Mahapatra* ,† Sanchita Mondal,† Saikat Kumar Manna, † Kalipada Maiti, † Rajkishor Maji, †
‡
§
§
5
Md. Raihan Uddin, ‡ Sukhendu Mandal, Deblina Sarkar, Tapan Kumar Mondal and Dilip Kumar
Maiti ɠ
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A new coumarin-appended thioimidazole-linked imine conjugate, viz. L has been synthesized and characterized. The L has been found to
10 recognize Cu2+ selectively among a wide range of biologically relevant metal ions. The chemosensing behavior of the L has been
1
demonstrated through fluorescence, absorption, visual fluorescent color changes, ESI-MS and H NMR titrations. The chemosensor L
showed selectivity toward Cu2+ by switch on fluorescence among the 18 metal ions studied with a detection limit of 1.53 µM. The
complex formed between L and Cu2+ is found to be 1:1 on the basis of absorption and fluorescence titrations and was confirmed by ESI-
MS. DFT and TDDFT calculations were performed in order to demonstrate the structure of L and [CuL] and electronic properties of
15 chemosensor L and their copper complex. This highly fluorescent [CuL] complex has been used to recognize sulphide selectively among
the other allied anions. Microstructural features of L and its Cu2+ complex have been investigated by SEM imaging (scanning electron
microscopy). The biological applications of L were evaluated in Vero cells and it was found to exhibit low cytotoxicity and good
membrane permeability for the detection of Cu2+.
emission. Therefore several ON-OFF fluorescent probes for Cu2+
20 Introduction
have been reported.12 Recently, many fluorescent chemosensors
Copper is a essential trace element, the third most abundant (after
45 for Cu2+- selective detection were reported and have been used
with some success in biological applications.13 However, some of
them have shortcomings for practical application such as cross-
sensitivities toward other metal cations, low water solubility, a
narrow pH span, slow response, a low fluorescence quantum
50 yield in aqueous media, and cytotoxicities of ligand. To prevent
fluorescence quenching by Cu2+ and to achieve a practical “off-
on” imaging with a strong fluorescent response upon binding the
analyte, we devised a sensor system that separates the sensing
and the signaling events. Two different fluorogenic ligands
55 capable of metal chelation are employed whereby one of the
ligands binds Cu2+, while the other produces a fluorescent signal
to report the binding event. Although the designed chemosensor
is more effective in sensing Cu2+, whereas other important cations
such as Au3+, Pd2+ and Zn2+ ions exhibited little bit fluorescence
60 turn ON response.
Fe2+ and Zn2+) in humans, and is present at low levels in a variety
of cells and tissues with the highest concentrations in the liver,1
and in the brain.2 Copper is a redox-active nutrient that is needed
25 at unusually high bodily levels for normal brain function.3 Owing
to the large oxygen capacity and oxidative metabolism of brain
tissue, neurons and glia alike require copper for the basic
respiratory and antioxidant enzymes cytochrome c coxidase
(CcO) and Cu/Zn superoxide dismutase (SOD1), respectively. In
30 addition, copper is a necessary cofactor for many brain-specific
enzymes that control the homeostasis of neurotransmitters,
neuropeptides, and dietary amines. However, it is toxic at higher
concentration level, for example, the accumulation of Cu2+ in the
liver and kidney may cause gastrointestinal, Wilson disease,4
35 amyotrophic lateralsclerosis,5 Menkes syndrome,6 Alzheimer’s
disease,7 hypoglycemia, dyslexia and infant liver damage.8,9
Therefore, the development of molecular turn-on fluorescent
probes for fast detection of Cu2+ in water or physiological
samples is of toxicological and environmental concern.10
40 However, fluorescence detection of Cu2+ by a turn-on response is
particularly difficult due to its paramagnetic nature,11 as unpaired
electrons in close proximity to fluorescent dyes tend to quench
Sulphide anion, as a toxic, hazardous and traditional pollutant, is
widely spread in the environment. It is produced naturally and as
a result of human activity. Natural sources include non-specific
and anaerobic bacteria reduction of sulfates and sulfur containing
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DOI: 10.1039/C4DT03969F
amino acids in meat proteins. Sulphide anion is found naturally in
crude petroleum, volcanic eruption, and hot springs. It is released
from stagnant or polluted waters and manure or coal pits.
Sulphide anion may be produced by a variety of commercial
thiazolylamine derivative (2) by condensation reaction with
thiourea as shown in Scheme 1.24
The final chemosensor molecule L has been synthesized by the
condensation of 2 with 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-
5
methods as a byproduct in industrial processes, for example, 60 benzo[ij]quinolizine-9-carboxaldehyde (b) in ethanolic medium
conversion into sulfur and sulfuric acid, dyes and cosmetic
manufacturing, production of an agricultural disinfectant, paper
and wood pulp, etc.14 Therefore, it is possible to bioconcentrate
and biomagnify in the food-chain. So exposure to a high level of
with 72% yield (Scheme 1).25 All the intermediates and the final
chemosensor molecule were characterized by various analytical
and spectral techniques. The structure of
L was well
characterized by 1H NMR, 13C NMR, and mass spectra (Fig. S3-
10 sulphide can lead to irritation in mucous membranes, 65 S5, ESI†). The molecular structure of the intermediate
1
unconsciousness, and respiratory paralysis.15 Once sulphide anion
is protonated, it becomes even more toxic. Therefore, the
detection of sulphide anion has become very important from an
thiazolylamine compound 2 has been established by H NMR,
ESI-MS as well as single-crystal XRD analysis (Fig. S1, S2 and
S8, ESI†). Single crystals of 2 suitable for X-ray diffraction study
were obtained by slow diffusion of methanol into the solution of
industrial, environmental, and biological point of view.16
A
15 variety of detection techniques have been developed for the 70 2 in chloroform and the data were collected26 (Table S4, ESI†).
determination of sulphide anion,17 Among them, sulphide anion
sensing by fluorescence spectrometry has received much
importance due to its high sensitivity and easy detection.18
However, the development of fluorescent sensors for anions in
UV-vis and fluorescence spectral behavior of L. The sensitivity
of L toward different metal ions and their preferential selectivity
toward Cu2+ over the other ions has been studied by fluorescence
20 aqueous media is still a challenging task because of the strong 75 and absorption titrations.
hydration nature of anions, which weakens the interactions of the
(a)
chemosensors with the target anions.19 This could be avoided by
using the metal displacement approach, which is based on the
stability constant of the complex formed by metal-anion affinity
25 is larger than that of the complex of metal and its chemosensor.20
Sulphide is known to react with copper ions to make a very stable
80
CuS form with a very low solubility product constant Ksp
=
6.3×10−36 that of cyanide one (3.2×10-20). Recently, Nagano and
Zeng groups reported new approaches for the detection of
30 sulphide in live biological systems through the development of
Cu2+ complex for chemoselective sulphide-responsive fluorescent
sensors.21 Recently, we presented a displacement-based sensing
method by using metal-based ensembles for other anion
recognition or sensing.22,23 From this idea, we have successfully
35 developed an ensemble of fluorescence and colorimetric sulphide
chemosensors based on the traditional Cu2+ chemosensors.
(b)
85
Results and discussion
90
The fluorescent chemosensor molecule (L) has been synthesized
by going through two consecutive steps starting from 3-
40 bromoacetylcoumarin (1) followed by the preparation of its
Fig. 1 (a) UV−vis absorption titration spectra of spectra of L (c =
4×10−5 M) in aq. CH3CN (CH3CN/H2O = 7:3 v/v, 10 mM HEPES
O
S
95 buffer, pH = 7.4) upon addition of Cu2+ (c = 2×10−4 M). Inset:
The inset shows the relative absorption intensity (A/A0) as a
function of [Cu2+]/[L], and the photographs showed the color
change of L in the presence of Cu2+. (b) Competitive absorption
spectra of L in the presence of different metal ions (perchlorate,
100 chloride, or nitrate salts of Hg2+, Cd2+, Fe2+, Co2+, Ni2+, Mn2+,
Zn2+, Au3+, Cu2+, Ag+, Pd2+, Cr3+, Al3+, Pb2+, Ca2+, Mg2+, Na+,
and K+) in aq. CH3CN (CH3CN:H2O = 7:3 v/v, 10 mM HEPES
buffer, pH = 7.4).
Br
NH2
(a)
N
O
O
O
O
1
2
45
(b)
S
N
N
N
OH
O
O
50
L
105 The UV-vis absorption spectra of chemosensor L in aqueous
acetonitrile (CH3CN:H2O = 7:3 v/v, 10 mM HEPES buffer, pH =
7.4) are dominated by two absorption bands at 278 and 358 nm,
respectively, and a low energy (LE) band centered at 446 nm
Scheme 1 Synthesis of L. Reagents and conditions: (a) thiourea,
dry ethanol, reflux, 12 h; (b) 8-hydroxy-2,3,6,7-tetrahydro-
1H,5H-benzo[ij]quinolizine-9-carboxaldehyde,
55 reflux, 6 h.
dry
ethanol,
2
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DOI: 10.1039/C4DT03969F
attributed to the iminothiazole moiety. During the titration, the
well known that Cu2+ is a paramagnetic ion with an unfilled d-
shell and could strongly quench the emission of the fluorophore
near it via electron or energy transfer. But in our case, the
concentration of L was kept constant at 40 µM and the mole ratio
of Cu2+ was varied. The absorbance of 278, 358, and 446 nm
bands is found to decrease upon addition of Cu2+ (Fig. 1a), 60 enhancement of emission of L after binding of Cu2+ ion is quite
5
indicating the interaction of Cu2+ with the iminothiazole/aromatic
moiety. The low energy (LE) band at 446 nm was gradually
decreases, upon addition of Cu2+ ions, which is responsible for
the change of color from light yellow to colorless. This fact can
interesting and mentionable along with other few related existing
systems.29
To our opinion, the switch on fluorescence emission is expected
due to the chelation of Cu2+ through deprotonated phenolic
be used for a ‘naked-eye’ detection of Cu2+ ions. However the 65 oxygen, the imine and thiazole N atoms, and the lactone carbonyl
10 absorption titration carried out with all the other metal ions
showed no significant change, indicating their noninteractive
nature with L (Fig. 1b). The binding affinities of Cu2+ toward L
have also been calculated from the Benesi−Hildebrand equation
oxygen. Such chelation leads to suppression of C=N bond
isomerization to attain conjugated coplanar structure as well as
ESIPT and fluorescence enhancement takes place as
a
consequence.30 Further, chelation imparts rigidity to the system
using absorption data and found to have the association constants 70 and the metal-fluorophore interaction is modulated, which are the
15 of 2.28×104 M−1, respectively (Fig. S14A, ESI†).
underlying cause of chelation enhanced fluorescence (CHEF).31
A plot of the relative fluorescence intensity (I/I0) vs [Cu2+]/[L]
mole ratio (Fig. 2a, inset) gives sigmoidal plot and exhibits
midpoint ratio of 1:1 stoichiometric complex for L to Cu2+. The
The chemosensor L exhibits weak fluorescence emission at 500
nm owing to the isomerization of the imine C=N bond as well as
excited-state intramolecular proton transfer (ESIPT) from the
salicyl −OH to the imine nitrogen when excited at 446 nm in 75 binding affinities of Cu2+ toward L have been calculated from the
20 CH3CN-H2O mixture (7:3 v/v in 10 mM HEPES buffer at pH =
7.4) which has been well documented in literature pertaining to
Schiff base molecular systems.27-28
Benesi- Hildebrand equation and found to have association
constant 3.33×104 M-1 (Fig. S14B, ESI†).
(a)
Cu2+
(a)
80
85
90
95
25
30
)
)
)
I
I
)
)
(
)
I
)
)
I
)
I
I
I
I
(
I
(
a
K
I
I
I
I
(
b
C
P
I
I
(
g
(
(
(
(
a
e
d
g
n
N
M
F
Z
C
A
(b)
(b)
35
40
)
)
)
)
I
I
(
I
)
)
I
)
I
I
I
I
I
I
l
C
I
(
I
(
I
(
(
a
(
n
I
o
C
(
u
d
b
P
M
N
C
A
Fig. 2 (a) Fluorescence emission spectra obtained during the 100 Fig. 3 (a) Histogram showing the relative fluorescence response
45 titration of L (c = 1×10−5 M) with Cu2+ (c = 1×10−4 M) in
aqueous CH3CN (CH3CN:H2O = 7:3 v/v, 10 mM HEPES buffer,
pH = 7.4), λext = 446 nm. Inset: The inset shows the relative
fluorescence intensity (I/I0) as a function of [Cu2+]/[L ] mole
of various metal ions (c = 1×10−4 M) with L (c = 1×10−5 M) in
aq. CH3CN (CH3CN:H2O = 7:3 v/v, 10 mM HEPES buffer, pH =
7.4), Inset: Fluorescence photograph of L and L+Cu2+. (b)
Fluorescence response of L (c = 1×10-5 M) to addition of 3 equiv.
ratio, and fluorescence emission color changes of the receptor L 105 Cu2+ (c = 1× 10-4 M) and 10 equiv. of other metal ions (c = 1×10-4
50 solution on addition of Cu2+ ions. (b) Change of emission
M) [the black bar portion] and to the mixture of 10 equiv. of other
divalent metal ions with addition of 3 equiv. Cu2+ [the red bar
portion].
intensity at 500 nm with incremental addition of Cu2+ ions [λext
=
446nm].
Titration of L with Cu2+ results in the enhancement of the
55 fluorescence intensity at 488 nm as a function of [Cu2+] (Fig. 2a)
and the intensity saturates at >1 equiv. In this connection, it is
110 The observed high Ka value clearly indicates the strong affinity of
Cu2+ to L. Titration of L (500 nm) with 3 equiv. Cu2+ results in
the sizable enhancement of fluorescence intensity at 488 nm as a
function of the added Cu2+ concentration [(Icomplex/Ifree ligand) ~ 18-
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fold] (Fig. 2a). Under UV light, the solutions of L is weak
fluorescent, whereas in the presence of Cu2+ an intense green
intensity of the probe L in the absence or presence of Cu2+. The
results showed that fluorescence intensity (I488 nm) of L showed
color fluorescent was observed (Fig. 2a, inset) and no such 60 no apparent changes in the pH range from 6.0 to 9.0 no matter
fluorescent color was observed in case of the other metal ions
(Fig. S13, ESI†). Thus, Cu2+ can easily be differentiated by its
fluorescent color change from the other metal ions (Fig. S13,
ESI†).
with or without Cu2+, indicating that L is stable in this pH range
and its response towards Cu2+ was also almost invariable in this
pH range (Fig. S12, ESI†).
5
Further, the stoichiometry of the complex formed between L and 65 1H NMR Titration Experiment. To know the mode of
Cu2+ has been derived to be 1:1 based on basis of a Job’s plot
complexation of L with Cu2+, we carried out 1H NMR titration in
CDCl3. During the 1H NMR titration of L with Cu2+, the addition
of 1 equiv. of Cu2+ into the solution of L in CDCl3 provided the
1H NMR spectrum to be very broad due to the complexation of
10 (Fig. S9, ESI†). The formation of a 1:1 binding mode of the
sensor with Cu2+ was also confirmed by the ESI-MS, where the
spectrum obtained for the in situ complex results in a molecular
ion peak at m/z = 524.7646 (calc. 524.09) (Fig. S6, ESI†). The 70 paramagnetic Cu2+ to the L. The proton NMR signals
+ +
mass spectrum is assignable to the mass of [L+Cu2++NH4 ] ,
15 supports the presence of copper. Therefore, we suggest that probe
L coordinates with Cu2+ with 1:1 stoichiometry.
corresponding to the thiazole (Hc) and imine proton (Hb)
experiences considerable broadening owing to the binding of this
region with paramagnetic Cu2+ (Fig. 4).
Competitive metal ion titrations. In order to check the practical
utility of Cu2+ recognition by L, competitive titrations were
carried out in the presence of other biologically and ecologically
20 relevant metal ions. Fluorescence spectra (Fig. 3b) were recorded
for the titration of L against Cu2+ in the presence of 10 equiv. of
other different metal ions, viz. Au3+, Ag+, Hg2+, Fe3+, Pd2+, Zn2+,
Cd2+, Ni2+, Co2+, Mn2+, Cr3+, Al3+, Pb2+, Ca2+, Mg2+, Na+, and K+.
In the fluorescence titration, the result found, none of these metal
25 ions significantly affect the emission intensity of L upon the
addition of Cu2+, to have only marginal changes in the emission
intensity, suggesting that none of these ions interfere in the
fluorescence emission of the Cu2+ complex (Fig. 3b). Therefore, it
can be concluded that L recognizes Cu2+ even in the presence of
30 other metal ions. In the other fluorescence titrations were carried
out in the same medium with 17 other different metal ions, viz.
Au3+, Ag+, Hg2+, Fe3+, Pd2+, Zn2+, Cd2+, Ni2+, Co2+, Mn2+, Cr3+,
Al3+, Pb2+, Ca2+, Mg2+, Na+, and K+, found no significant
fluorescence enhancement except Au3+, Pd2+ and Zn2+ revealed
35 relatively insignificant fluorescence ON responses in this region,
manifesting the pronounced OFF–ON type of Cu2+ selectivity of
L (Fig. 3a). Therefore, L is selective to Cu2+ among the 18 ions
studied. Visual fluorescent color change experiments have been
carried out to look at the behavior of L in the presence of various
40 metal ions. The solution of L with > 1 equiv of Cu2+ ions
converted the visual emission color change from faint yellow to
bright green when excited with a hand-held 365 nm UV lamp,
which is otherwise not present in case of the other metal ions
studied (Fig. S13, ESI†). Therefore, Cu2+ can easily be
45 differentiated by visual color change among the other metal ions.
The sensitivity of L for Cu2+ has been further evaluated by
measuring the lowest concentration that can be determined. The
fluorescence titration carried out between L and [Cu2+] by
maintaining a 1:1 ratio gives a value of 1.53 µM suggesting its
50 applicability to detect Cu2+ ions in aqueous medium at
physiological conditions.
Hb
75
Ha
Hc
Hd
He
Hh
Hf
Hg
Hi
80
(e) L+2equiv. Cu2+ +Na2S
(d) L+2equiv. Cu2+
85
(c) L+1 equiv. Cu2+
(b) L+0.5 equiv. Cu2+
90
Hf
Hd
Ha
Hg
Hc
Hh
Hb
He
Hi
(a) L+0 equiv. Cu2+
Fig. 4 Partial 1H NMR spectra (400 MHz) of L in CDCl3 at 25 °C
95 and the corresponding changes after the gradual addition of
different equivalents of copper perchlorate in MeOH- d4 from (a)
L, (b) L + 0.5 equiv. Cu2+, (c) L + 1.0 equiv. Cu2+, (d) L + 2.0
equiv. Cu2+ and (e) L + 2.0 equiv. Cu2+ + Na2S.
However, treatment of the [CuL] system with sulphide ions
100 afforded a well resolved spectrum, which was almost identical
with the 1H NMR spectrum of L itself (Fig. S3, ESI†). To further
clarify the spectrum, the product of [CuL+S2-] was isolated by a
1
silica gel column and was then subjected to H NMR analysis.
The H NMR of the resulting product is essentially identical to
1
To be useful in biological applications, it is necessary for a
fluorescent probe to operate over a suitable range of pH,
especially at physiological pH. A series of buffers with pH values
55 ranging from 1 to 13 was prepared by mixing sodium hydroxide
solution and hydrochloric acid in HEPES buffer. Thus, we
proceeded to investigate the effect of pH on the fluorescence
105 that of free L.
Density Functional Theory (DFT) Calculations. To verify the
configuration of [CuL] and to investigate the electronic structure
4
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and the electronic transitions of the π-conjugated copper 45 distorted geometries about the copper centers that deviate from
complex, DFT geometry optimizations followed by TD-DFT
calculations at the B3LYP level32 were carried out. All element
except Cu were assigned 6-31+G(d) basis set. The LANL2DZ
basis set with effective core potential (ECP) set of Hay and Wadt
[4] was used for Cu. The optimized geometries of L and [CuL]
complex are shown in (Fig. 5) the complex shows that the Cu2+
ion binds to L very well through four coordination sites, and the
whole molecular system forms a nearly planar structure.
both the tetrahedral as well as that of the square planar.
The optimized complex of Cu2+ with L associated with four
bonds ( N29−Cu, N36−Cu, O43−Cu and O27−Cu ) to the central ion
with their distances of 2.04, 2.01, 2.17 and 1.87 Å respectively
50 (Table S1, ESI†).33 The geometry about Cu2+ is significantly
distorted by exhibiting coordination angles in the range
65.59−120.970. The spatial distributions and orbital energies of
HOMO and LUMO of L and [CuL] were also determined (Fig.
6a).
5
10 The geometry optimizations for L and [CuL] complex were done
in a cascade fashion starting from semiempirical PM2 followed
by ab initio HF to DFT B3LYP by using various basis sets, viz.,
PM2 → HF/STO-3G → HF/3-21G → HF/ 6-31G → B3LYP/ 6-
31G(d,p). For [CuL] complex, a starting model was generated by
15 taking the DFT optimized L and placing the Cu2+ ion well in the
core of the coumarin lactonecarbonyl O, enamine N, phenolic OH
and / thiazole N as donors moieties at a non-interacting distance.
This model was then optimized initially using HF/3-21G level of
calculations, and the output structure from this was taken as input
20 for DFT calculations performed using B3LYP with LANL2DZ
relativistic pseudopotentials basis set for Cu2+ and 6-31+G(d,p)
for all other atoms in the complex.
(a)
55
LUMO
LUMO
2.88 ev
HOMO
3.23 ev
HOMO
60
L-Cu2+
L
S35
(a)
O27
C32
C3
C6
C31
N29
C28
C8
C7
(b)
C33
C39
C1
C5
C40
C45
LUMO+2
C4
N36
C37
C38
N18
C49
C47
25
C9
C10
C42
O43
C2
LUMO+1
LUMO
O44
C11
C46
C12
S35
C31
(b)
65
C32
N29
C28
C9
C10
C
33C39
C8
2
C11
.
0
C45
C40
4
N36
C37
A
HOMO
C12
C2
C4
C49
C47
C5
C7
C6
C38
O43
Cu53
O27
C42
O44
N18
C1
30
C46
2.17 Ao
HOMO-1
HOMO-2
C3
1.87 Ao
Fig. 5 B3LYP Optimized structure of (a) L by DFT/B3LYP/6-
31+G(d) method (b) L-Cu2+ complex by DFT/UB3LYP method.
70
The geometry optimization resulted in the conformational
CuL
L
Fig. 6 (a) HOMO and LUMO distributions of L and L−Cu2+
35 changes at the two sides of thioimidazole ring i.e.; coumarin and
aminophenol parts to accommodate copper ion. The
conformational changes can be estimated through the torsional
angles of the two sides (Table S1, ESI†) and the structure given
in (Fig. 5). In the progress, the lactone oxygen of the coumarin
40 moiety and phenolate oxygen of the amiophenol part turned to the
same direction form a binding core. In the CuL optimized
structure, the copper is bound by the two nitrogen atoms (imine
and thioimidazole) and two oxygen atoms (coumarin lactone and
phenolate oxygens) in the binding core (N2O2) leads to highly
complex. (b) Molecular orbital plots of L and L−Cu2+.
The π electrons on the relevant HOMOs and LUMOs of [CuL]
75 complex
are
essentially
distributed
in
the
entire
coumarinthioimidazole−aminophenol backbone. By contrast, in
the case of L, the π electrons on HOMO and HOMO−1 are
primarily resided on the electron-donating aminophenol moiety,
whereas those on LUMO, HOMO−2 and LUMO +2 are mainly
80 located on the electron withdrawing coumarin moiety (Fig. 6b).
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DOI: 10.1039/C4DT03969F
This indicates that
L bears efficient electron transfer from the
(a)
aminophenol moiety to the coumarin parts, thus rendering the
fluorescence relatively weak (ϕ = 0.020). By contrast, the nearly
complete overlap of electrons on the transition orbitals may
induce the strong fluorescence emission for [CuL] complex (ϕ =
0.62).34 Moreover, the HOMO−LUMO energy gap of complex
+
2
-
2
5
become much smaller relative to that of probe
between HOMO and LUMO in the probe and [CuL] complex
were 3.23 eV and 2.88 eV respectively (Fig. 6a).
L. The energy gaps
L
50
10 SEM imaging studies. To understand the aggregation property
of the and [CuL], and its effect on their chemo-sensing
property, scanning electron microscope (SEM) imaging were
carried out. The and [CuL] are possessing charge transfer,
(b)
L
L
hydrogen bonding, π- π stacking, van der Waals force of
15 attraction which enforce the compounds to form sheet-like 2D
nanostructure and globular material respectively (Fig. 7a and b).
Exact mechanism aggregation to construct uniform globular
material is unknown to us. However, in presence of Cu2+-ion a
strong complexation with the ligand along with charge transfer is
20 expected. This SEM imaging study supports the unprecedented
55
60
Fig. 8 (a) Fluorescence spectra of
L
(c = 1×10−5 M) with 3 equiv.
chemosensing nature of
cell imaging.
L and [CuL] which can be utilized for
of Cu2+ upon addition of sodium sulphide (c = 4×10−4 M) in aq
CH3CN (CH3CN:H2O = 7:3 v/v, 10 mM HEPES buffer, pH = 7.4)
and Photographs of
L L
+Cu2+, +Cu2++S2- in color changes. (b)
65 Fluorescence intensity (I) vs [S2-] mole ratio plot at 488 nm, [λext
= 446nm].
(a)
25
a
70
b
(b)
30
Fig. 7 (a) Monolayer SEM image of
−Cu2+ complex.
L and (b) SEM image of
L
75
Fluorescence and UV-Vis spectroscopic studies of [CuL]
complex in presence of S2-. Since detects Cu2+ selectively, the
L
35 in situ prepared [CuL] complex has been used for its anion
recognition studies. Owing to the strong affinity of Cu2+ towards
sulphide the highly fluorescent [CuL] complex has been studied
for their secondary sensing property toward selective anion.35 The
-
r
-
-
-
-
-
c
-
2
S
4
3
O
B
A
O
l
N
2
O
C
Fig. 9 (a) Competitive fluorescence spectra of CuL (1.0×10–5 M)
in the presence of tetrabutyl ammonium salt of different anions
80 (4.0×10–4 M) (F−, Br−, S2−, I−, CN-, SO4 2−, SCN−, AcO−, Cl−,
H2PO4 , PO43−, NO3 , ClO4 and HSO4 .) in CH3CN-H2O(7:3
v/v, pH = 7.4). (b) The anion sensitivity profile for CuL: the
change in the emission intensity of CuL (1.0×10–5 M) in presence
of 4.5 equivalents of S2- ion, and 10 equivalents of other
85 interfering anions in aq. CH3CN (CH3CN:H2O=7:3 v/v, 10 mM
HEPES buffer, pH = 7.4).
chemosensing ensemble was prepared in situ by mixing
L and
40 Cu2+ in 1:1 ratio and it have been used for the anion recognition
−
−
–
−
studies by different anions, viz., F−, Br−, S2−, I−, CN-, SO4
,
2−
−
−
–
−
SCN−, AcO−, Cl−, H2PO4 , PO43−, NO3 , ClO4 and HSO4 . All
the anion titrations exhibited no significant change in the
fluorescence intensity except sulphide and cyanide.
45
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DOI: 10.1039/C4DT03969F
During the titration of [CuL] with sulphide, the emission band
observed at 488 nm is quenched (Fig. 8a) gradually to reach an
intensity that is similar to that of . This is exactly reverse to
Sulphide selective signaling was not affected by the presence of
other common interfering anions. Competition experiments of the
L
[
CuL] system revealed that the S2− induced fluorescence turn-off
what happens when
L
is titrated with Cu2+, indicating the removal
was retained in the presence of 100 equiv. of other common,
. This is 50 coexisting anions. Thus, the ensemble seems to be useful for
selectively sensing S2− even involving relevant anions.
5
of Cu2+ by sulphide and thereby the releasing the free
L
true even with cyanide (Fig. S16, ESI†).
Thus the [CuL] complex acts as a secondary recognition
ensemble toward sulphide, and cyanide. The minimum
concentration of sulphide that can be detected by [CuL] has been
Reusability and Reversibility of the Receptor L. The
reversibility is an important aspect of any receptor to be
employed as a chemical sensor for detection of specific metal
55 ions. To examine whether the process is reversible, an excess
amount of S2− was added into the solution of [CuL] ensemble.
10 found to be 11.4 µM (Fig. S15B, ESI†).
In order to support the results obtained from the fluorescence
studies, the removal of Cu2+ by sulphide ion, similar absorption
titrations were carried out. During the titration of the [CuL] by
CN− (Fig. 10a), the low energy absorption band arising from the
15 precursor complex observed at 446 nm have been progressively
increases and gradual addition of sulphide two bands, one at 278
nm and the other at 358 nm, which correspond to the free
Cu2+
S2-
L (O F F )+
C u S
2 +
L+Cu2++S2-
L ( O F F )
L + C u
ON
Scheme 2 Schematic Presentation for Fluorescence Quenching.
Cu2+
Cu2+
Cu2+
receptor
10a).
L
, and the absorbance of these bands increases (Fig.
60
F
F
F
F
F
F
L
(a)
O
O
O
20
S2-
S2-
S2-
Fig. 11 Fluorescence experiment showing on-off reversible visual
65 fluorescent color changes after each addition of Cu2+ and S2–
sequentially.
Isomerization inhibited
(b)
25
S
S
Isomerization
Cu2+
×
N
N
N
N
S2-
O
O
O
O
Cu2+
N
OH
N
O
ClO-
4
Weakly fluorescent
Strongly fluorescent
70
Scheme 3 Proposed mechanism for the fluorescence changes of
upon addition of Cu2+.
L
30
Fig. 10 (a) Changes in the absorption spectra of
L−Cu complex
The bright green fluorescence immediately turned off (Fig. 8a,
(1.0×10–5 M) in presence of S2-, CN- (4.0×10–4 M ) in aq. CH3CN
(CH3CN:H2O = 7:3 v/v, 10 mM HEPES buffer, pH = 7.4). (b)
Competitive UV−vis titration spectra of CuL (1.0×10–5 M) in the
inset). The reusability of [CuL] in sensing S2− has been
75 demonstrated by carrying out four alternate cycles of titration of
CuL] with S2− followed by Cu2+. Titration of [CuL] with S2−
[
35 presence of tetrabutyl ammonium salt of different anions
(4.0×10–4 M) (F−, Br−, S2−, I−, CN-, SO4 2−, SCN−, AcO−, Cl−,
shows significant switch off fluorescence, and the fluorescence is
regained when Cu2+ is added to result in the switch on mode.
Three such consecutive cycles are shown in Fig. 11, and the
−
−
–
−
H2PO4 , PO43−, NO3 , ClO4 and HSO4 .) in CH3CN-H2O(7:3
V/V, pH = 7.4).
80 results clearly demonstrate the reversible property of
Cu2+ followed by S2− / CN− (Fig. S16, ESI†).
L in sensing
All this suggests the disruption of the [CuL] complex followed
40 by the removal of Cu2+ by the sulphide as well as cyanide ions to
result in free receptor, it has been noted that the changes observed
for different bands during the titration between the [CuL] and S2−
Detection and imaging in living cells. The desirable features of
chemosensor such as high sensitivity with turn-on
L
a
fluorescence, fast response, reversibility, working well at
85 physiological pH, and high selectivity encouraged us to further
evaluate the potential of the sensor for imaging Cu2+ in living
is exactly reverse to that observed during the titration of
L with
Cu2+ (Fig. 1a). This suggests a displacement mechanism during
45 the titration of S2−. Similar observations were noticed with CN−.
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cells. Here Vero cells (very thin endothelial cell) were used as
Conclusion
models (Experimental section). Before microscopic imaging, all
the solutions were aspirated and mounted on slides in a mounting
medium containing DAPI (1 ꢀg /mL) to label the cell nuclei and
stored in dark before microscopic images are acquired. The Vero
The iminothioimidazole-phenolic-coumarin based probe
L
was
50 synthesized and characterized. The probe
L was found to be
sensitive and selective fluoroionophore of Cu2+ among the
eighteen different metal ions studied in HEPES buffer medium.
The selectivity and sensitivity were demonstrated on the basis of
5
cells incubated with only sensor
L (1 µM) exhibited very weak
intracellular fluorescence. Again, the cells were pre-treated with
Cu2+ in the growth medium for 30 min. The cells were then
washed with PBS to remove the remaining Cu2+ and further
1
fluorescence, absorption, and H NMR spectroscopy, ESI mass
55 spectrometry, and visual fluorescent color changes. Interaction of
Cu2+ with
L enhances the fluorescence emission and induces a
10 incubated with probe
L
for 30 min.
turn on response in electronic and fluorescence spectra in the
visible region. Thus, these receptors could be used as a dual
probe for visual detection through change in color and
60 fluorescence. Fluorescence and absorption spectroscopy provides
information for the formation of a 1:1 complex between Cu2+ and
(A)
(E)
(I)
(B)
(F)
(J)
(C)
(D)
(H)
(L)
L
, and ESI-MS confirms the fact. The association constant, Ka for
with Cu2+ is in the order of 104 M−1 based on fluorescence as
(G)
L
well as absorption studies, suggesting a strong binding for Cu2+.
65 The receptor
detection limit of 1.53 µM 0.1 ppm Cu2+. The Cu2+ binding
characteristics of has been studied by computational
calculations at DFT level and TDDFT calculations were carried
out to demonstrate the electronic properties of and the
70 corresponding copper complex. The in situ prepared copper
complex of , viz.,
−Cu, was able to detect S2– exactly reverse
manner to what happens when Cu2+ is added to
in fluorescence
spectroscopy. To know the supramolecular microstructural
features of and −Cu complex, SEM studies were carried out
75 in which the spherically shaped particles of were found
L has been shown to be sensitive with a minimum
15
(K)
L
L
L
L
L
Fig. 12 Confocal microscopic images of probe in Vero 76 cells
20 pretreated with Cu2+: (A) bright field image of the cells of
controlled set. (B) only Cu2+ at 1.0×10-4 M concentration, nuclei
L
L
L
counterstained with DAPI (1 ꢀg/mL). (C) only Cu2+ at 1.0×10-4
M
enlarged as well as aggregated in the complex. Nonfluorescent
images were observed when Vero cells were incubated with
concentration. (D) overlay of (B and C). (E) bright field image of
the cells stained with probe
at concentration 1.0×10-6 M. (F)
L
probe
in Vero cells in the presence of Cu2+. Hence, these results clearly
80 indicate that the probe
is effective intracellular Cu2+ imaging
L alone. However, strong green fluorescence was observed
25 image scan of DAPI for E. (G) with probe
L at concentration
1.0×10-6 M detected on λext = 450 nm, λem = 519 nm. (H) overlaid
L
image of F and G. (I) bright field image of the cells treated with
agents with cell permeability.
1.0×10-4 M Cu2+, 1.0×10-6 M probe
L and Na2S at concentration
4.0×10-5 M (J) cells of I scanned for DAPI (K) cells of I scanned
30 at λext = 473, λem = 519 (L) overlay of J and K. All images were
acquired with a 60x objective lens. Scale bar represents 20 ꢀm.
Experimental Section
General Information and Materials. All the solvents were of
analytic grade. All cationic compounds such as perchlorate of
85 Hg2+, Cd2+, Fe2+, Co2+, Ni2+, Mn2+, Zn2+, Au3+, Cu(ClO4)2, nitrate
of Ag+, Pb2+, chlorides of Cr3+, Al3+, and Pb2+, were purchased
from Sigma−Aldrich Chemical Co., stored in a desiccators under
vacuum containing self-indicating silica, and used without any
further purification. Solvents were dried according to standard
90 procedures. Unless stated otherwise, commercial grade chemicals
were used without further purification. Elix Millipore water was
used throughout all experiments. All reactions were magnetically
stirred and monitored by thin-layer chromatography (TLC) using
Spectrochem GF254 silica gel coated plates. Column
95 chromatography was performed with activated neutral aluminum
oxide. FTIR spectra were recorded as KBr pellets using a JASCO
Vero cells incubated with chemosensor
fluorescence, whereas a bright green fluorescence signal was
L exhibited very weak
observed in the cells stained with both the chemosensor
L and
35 Cu2+, which in good agreement with the fluorescence turn-on
profile of the sensor in the presence of Cu2+ in the solution (Fig.
12[E-H]). Moreover, bright green colored fluorescence cells
obtained from the incubation of the receptor
L followed by
treatment with Cu2+ became invisible in fluorescence upon
40 addition of Na2S (40 ꢀM) (Fig. 12[I-L]). The results establish that
sensor
L
is cell membrane permeable and capable of sensing Cu2+
in living cells. To the best of our knowledge, this is the first
known thiazole-coumarin-based conjugate in the literature which
has been demonstrated to recognize Cu2+ in live cells. Cells were
45 intact and showed healthy spread and adherent morphology
1
FTIR spectrometer (model FTIR-460 plus). The H NMR spectra
were recorded on Bruker 400 MHz spectrometer. Mass spectra
were carried out using a Waters QTOF Micro YA 263 mass
100 spectrometer. The 1H NMR chemical shift values are expressed in
ppm (δ) relative to CHCl3 (δ = 7.26 ppm). UV−vis and
during and after the labeling process with chemosensor
indicating an absence of cytotoxic effects.
L,
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fluorescence spectra measurements were performed on a JASCO
Millipore water. All experiments were carried out in CH3CN/H2O
V530 and a Photon Technology International (PTI-LPS-220B)
spectrofluorimeter, respectively. The following abbreviations are
used to describe spin multiplicities in 1H NMR spectra: s =
singlet; d = doublet; t = triplet; m = multiplet.
55 solution (CH3CN/H2O = 7:3 v/v, 10 mM HEPES buffer, pH =
7.4). In titration experiments, each time a 1×10−5 M solution of
L
was filled in a quartz optical cell of 1 cm optical path length, and
the ion stock solutions were added into the quartz optical cell
gradually by using a micropipet. Spectral data were recorded at 1
60 min after the addition of the ions. In selectivity experiments, the
test samples were prepared by placing appropriate amounts of the
5
Synthesis and Characterization of L: Synthesis of (2): A
mixture of compound 1 (3-bromoacetyl coumarin) (0.50 g, 1.87
mmol) and thiourea (0.168 g, 2.21 mmol) in 15ml absolute
ethanol was refluxed for 12 h. After the completion of the
anions/cations stock into 2 mL of solution of
L
(1×10−5 M). For
fluorescence measurements, excitation was provided at 446 nm,
and emission was collected from 470 to 610 nm.
10 reaction (monitored by TLC) the solvent was evaporated and the
reaction mixture was poured into ice-water, and powdered
product was extracted with CHCl3. The organic layer was washed
with saturated aqueous solution of NaCl, dried over anhydrous
MgSO4 and evaporated to give a yellow solid which was
15 crystallized from MeOH/CHCl3 (1:1) solution to give compound
65 Finding the Detection Limit. The detection limit was calculated
on the basis of the fluorescence titration (Figure S13, Supporting
Information). The fluorescence emission spectrum of
L was
measured 10 times, and the standard deviation of blank
measurement was achieved. To gain the slope, the ratio of the
70 fluorescence intensity at 488 nm was plotted as a concentration of
Cu2+. So the detection limit was calculated with the following
equation.
1
2 in 69% yield; M.P. > 250˚C. H NMR (400 MHz, d6-DMSO,
Si(CH3)4, J (Hz), δ (ppm)): 8.50 (1H, s), 7.83(1H, d, J=7.76 Hz),
7.60 (1H, t, J=7.82 Hz), 7.50 (1H, s), 7.43 (1H, d, J=8.28 Hz),
7.37 (1H, t, J=7.52 Hz), 7.16 (2H, s). TOF MS ES+, m/z =
20 245.0093, [M+H]+, calc. for C12H8N2O2S =244.26.
Detection limit =3Sb1/S
(1)
Synthesis of chemosensor L. To a mixture of compound 2
where Sbl is the standard deviation of blank measurement and S
75 is the slope of the calibration curve.
(0.112g,
0.4608mmol)
and
2,3,6,7-tetrahydro-1H,5H-
benzo[ij]quinolizine-9-carboxaldehyde (3) (0.10g, 0.4608mmol)
in dry EtOH (10ml) 2-3 drops of AcOH was added; then the
25 reaction mixture was refluxed for 4-6 hours. After the completion
of the reaction the solvent was evaporated and the deep yellow
solid precipiteted was filtered through suction, washed with
milipore water and thereafter washed several times with EtOH
Computational Studies. All geometries for
L and L
-Cu2+ were
optimized by density functional theory (DFT) calculations were
performed with Gaussian09 program package,36 with the aid of
the GaussView visualization program.
and dried in air. The deep yellow compound
L was collected in
80 Cell Line and Cell Culture. Vero cell (very thin endothelial cell)
(Vero 76, ATCC No CRL-1587) lines were prepared from
continuous culture in Dulbecco’s modified Eagle’s medium
(DMEM, Sigma Chemical Co., St. Louis, MO) supplemented
with 10% fetal bovine serum (Invitrogen), penicillin (100
85 ꢀg/mL), and streptomycin (100 ꢀg/mL). The Vero 76 were
obtained from the American Type Culture Collection (Rockville,
MD) and maintained in DMEM containing 10% (v/v) fetal
bovine serum and antibiotics in a CO2 incubator. Cells were
initially propagated in 75 cm2 polystyrene, filter-capped tissue
90 culture flask in an atmosphere of 5% CO2 and 95% air at 37°C in
CO2 incubator. When the cells reached the logarithmic phase, the
cell density was adjusted to 1.0×105 per/well in culture media.
The cells were then used to inoculate in a glass bottom dish, with
1.0 mL (1.0×104 cells) of cell suspension in each dish. After cell
95 adhesion, culture medium was removed. The cell layer was rinsed
twice with phosphate buffered saline (PBS), and then treated
according to the experimental need.
1
30 72% yield; M.P. > 250˚C. H NMR (CDCl3, 400 MHz) δ (ppm):
12.91 (1H, s, -OH), 8.84 (1H, s, -CH=N), 8.72 (1H, s), 8.20 (1H,
s), 7.63 (1H, d, J = 6.8 Hz), 7.52 (1H, t, J=7.2 Hz) , 7.36 (1H, d,
J=8.2 Hz), 7.30 (1H, t, J=7.7 Hz), 6.85 (1H, s), 3.31-3.26 (4H, q,
J=8Hz), 2.75-2.68 (4H, m), 1.95 (4H, t, J=5.6). 13C NMR (CDCl3,
35 500 MHz) δ (ppm): 19.2, 19.9, 20.9, 21.8, 26.4, 31.1, 33.0, 49.1,
49.4, 105.2, 107.0, 113.2, 113.3, 115.5, 116.1, 118.8, 120.2,
123.7, 127.5, 130.5, 138.4, 144.7, 147.7, 152.1, 162.2; LCMS
ES+, m/z = 444.2, [M+H]+, calcd for C25H21N3O3S = 443.13.
UV−Vis Spectral Studies. A stock solution of the probe
L
40 (4.0×10−5 M) was prepared in CH3CN/H2O (7:3 v/ v). Solutions
of 2.0×10−4 M salts of the respective cation were prepared in
Millipore water. All experiments were carried out in CH3CN/H2O
solution (CH3CN/H2O = 7:3 v/v, 10 mM HEPES buffer, pH =
7.4). In titration experiments, each time a 4×10−5 M solution of
L
45 was filled in a quartz optical cell of 1 cm optical path length, and
the ion stock solutions were added into the quartz optical cell
gradually by using a micropipette. Spectral data were recorded at
1 min after the addition of the ions. In selectivity experiments, the
test samples were prepared by placing appropriate amounts of the
Cell Imaging Study. For confocal imaging studies Vero cells,
1×104 cells in 1000 ꢀL of medium, were seeded on sterile 35 mm
100 covered Petridis, glass bottom culture dish (ibidi GmbH,
Germany), and incubated at 37°C in a CO2 incubator for 10
hours. Then cells were washed with 500 ꢀL DMEM followed by
incubation with 1.0 x 10-4 M Cu(ClO4)2 dissolved in 500 ꢀL
DMEM at 37°C for 1 h in a CO2 incubator and observed under an
50 anions/cations stock into 2 mL of solution of
L
(4×10−5 M).
Fluorescence Spectral Studies. A stock solution of the probe
L
(1.0×10−5 M) was prepared in CH3CN/H2O (7:3 v/ v). Solutions
of 1.0×10−4 M salts of the respective cation were prepared in
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Olympus IX81 microscope equipped with a FV1000 confocal
55 Acknowledment
system using 1003 oil immersion Plan Apo (N.A. 1.45)
objectives. Images obtained through section scanning were
analyzed by Olympus Fluoview (version 3.1a; Tokyo, Japan)
with excitation at 446 nm monochromatic laser beam, and
emission spectra were integrated over the range 470-600 nm
(single channel). The cells were again washed thrice with
phosphate buffered saline PBS (pH 7.4) to remove any free
We
thank
the
DST-West
Bengal
[Project
no.
124(Sanc.)/ST/P/S&T/9G-17/2012] for financial support. SM and
SKM thanks to the UGC, New Delhi for a fellowship.
5
Notes and references
60 †Department of Chemistry, Indian Institute of Engineering Science and
Technology, Shibpur, Howrah – 711103, India.
Cu(ClO4)2 and incubated in PBS containing probe
L to a final
10 concentrations of 1.0 x 10-6 M, incubated for 10 min followed by
washing with PBS three times to remove excess probe outside the
cells and images were captured. In a separate culture dish
undergoing the same treatment the cells were then treated with
4.0×10-5 M of Na2S solution for 1 h; the cells were washed with
15 PBS three times to remove free compound and ions before
analysis. In separate culture dish the cells were similarly treated
‡ Department of Microbiology, Ballygunge Science College, Kolkata-
700019.
§Department of Chemistry, Jadavpur University, Kolkata-700032, India.
65 ɠ Department of Chemistry, University of Calcutta, Kolkata-700009,
India.
with 1.0×10-6 M probe
L, incubated for 10 min, washed thrice
*
Corresponding author: Email: akmahapatra@rediffmail.com, Fax:
with PBS and the image was captured to get any possible
background fluorescence. According to the need of the
20 experiment we follow similar procedures to label the cell nuclei
by treatment with DAPI (1 ꢀg/mL) followed by three times wash
with PBS and subsequently image was captured with excitation
wavelength of laser was 473 nm, and emission was 519 nm. For
all images, the confocal microscope settings, such as transmission
25 density, and scan speed, were held constant to compare the
relative intensity of intracellular fluorescence. Before
microscopic imaging, all the solutions were aspirated and
mounted on slides in a mounting medium containing DAPI (1 ꢀg
/mL) to label the cell nuclei and stored in dark before
30 microscopic images are acquired.
+913326684564
† Electronic Supplementary Information (ESI) available: [details of any
70 supplementary information available should be included here]. See
DOI: 10.1039/b000000x/.
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Cytotoxicity Assay. The cytotoxic effects of probe
L, Cu(ClO4)2,
and
L
─Cu2+complex were determined by an MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
following the manufacturer’s instruction (MTT 2003, Sigma-
35 Aldrich, MO). Vero cells were cultured into 96-well plates (104
cells per well) for 24 h. After overnight incubation, the medium
was removed, and various concentrations of
L
L, Cu(ClO4)2, and
─Cu2+ complex (0, 5, 25, 50, 75, and 100 ꢀM) made in DMEM
were added to the cells and incubated for 24 h. Control
40 experiments were set with DMSO, cells without any treatment
and cell-free medium were also included in the study. Following
incubation, the growth medium was removed, and fresh DMEM
containing MTT solution was added. The plate was incubated for
3−4 h at 37°C. Subsequently, the supernatant was removed, the
45 insoluble colored formazan product was solubilized in DMSO,
and its absorbance was measured in a microplate reader (Perkin-
Elmer) at 570 nm. The assay was performed in triplicate for each
concentration of
L, Cu(ClO4)2, and L
─Cu2+. The OD value of
wells containing only DMEM medium was subtracted from all
50 readings to get rid of the background influence. The cell viability
was calculated by the following formula: (mean OD in treated
wells / mean OD in control wells) x 100.
13 H. S. Jung, P. S. Kwon, J. W. Lee, J. I. Kim, C. S. Hong, J. W. Kim, S.
Yan, J. Y. Lee, J. H. Lee, T. Joo and J. S. Kim, J. Am. Chem. Soc., 2009,
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