Highly selective chemosensor for nano molar detection of Cu2+ ion by fluorescent turn-on response and its application in living cells

Article abstract of DOI:10.1016/j.dyepig.2014.01.001

A new, simple and symmetric thiophene based Schiff base 1 as highly selective and fluorescent sensor for Cu²⁺ ion detection in aqueous medium over other metal ions has been synthesized. Structure of the receptor 1 was confirmed by single crystal X-ray diffraction and other spectroscopic techniques. Absorption and emission spectra were used to study the cation sensing properties. Receptor 1 displays colorimetric response as yellow to colorless and fluorescence turn-on property in presence of Cu ²⁺ ion. Receptor 1- Cu²⁺ complex show 35 folds higher fluorescence response than that of receptor 1. Binding constant for receptor 1- Cu²⁺ complex was found as 4.23 × 10⁵ with the detection limit of 0.418 nM. Further the recognition of Cu²⁺ ion in the living cell was achieved using fluoresce microscope. Theoretical calculations were done to support the above findings.

Full text of DOI:10.1016/j.dyepig.2014.01.001

Dyes and Pigments 104 (2014) 116e122
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Highly selective chemosensor for nano molar detection of Cu2þ ion
by fluorescent turn-on response and its application in living cells
,
b
Sivalingam Suganya a, Sivan Velmathi a , Davoodbasha MubarakAli
*
a Organic and Polymer Synthesis Laboratory, Department of Chemistry, National Institute of Technology, Tiruchirappalli 620 015, India
b Central Inter-Disciplinary Research Facility (CIDRF), Mahatma Gandhi Medical College and Research Institute Campus, Puducherry 607402, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new, simple and symmetric thiophene based Schiff base 1 as highly selective and fluorescent sensor for
Cu2þ ion detection in aqueous medium over other metal ions has been synthesized. Structure of the
receptor 1 was confirmed by single crystal X-ray diffraction and other spectroscopic techniques. Ab-
sorption and emission spectra were used to study the cation sensing properties. Receptor 1 displays
colorimetric response as yellow to colorless and fluorescence “turn-on” property in presence of Cu2þ ion.
Receptor 1- Cu2þ complex show 35 folds higher fluorescence response than that of receptor 1. Binding
constant for receptor 1- Cu2þ complex was found as 4.23 ꢀ 105 with the detection limit of 0.418 nM.
Further the recognition of Cu2þ ion in the living cell was achieved using fluoresce microscope. Theo-
retical calculations were done to support the above findings.
Received 25 October 2013
Received in revised form
28 December 2013
Accepted 3 January 2014
Available online 12 January 2014
Keywords:
Schiff base
Fluorescence sensor
Cu2þ ion
Ó 2014 Elsevier Ltd. All rights reserved.
Nano molar
Bio imaging
DFT studies
1. Introduction
it can be visualized via fluorescence imaging [15e17]. In addition,
performing an aqueous mediated [18e20] cation sensor only shows
To device fluorescent probe for the detection of biologically
significant metal ions in the living cells or tissue is of current in-
terest and important target in the field of cation sensors [1e3].
Generally, the receptor-cation interaction will be in the form of
coordination bond, electrostatic and Vander walls interaction via
electron or charge transfer mechanism. Recently many efforts have
been taken for the recognition of transition metal ions [4e10] due
to their own importance in the environmental and biological
concern. Copper [11e14] was found as the third most essential
element for plants, animals and human beings due to its involve-
ment in the biological process like electron transfer, enzyme hy-
drolysis and being an efficient catalyst for redox process. The
American Medical Association suggests the normal human body
should contain 1.2e1.3 mg/day of copper. When the concentration
of Cu2þ ion crosses its limit in living organism, it will produce
reactive organic species (ROS) and consequently causes Alz-
heimer’s, Parkinson’s, and Wilson diseases.
precise output in biological application. Due to the paramagnetic
nature of Cu2þ ion, it is used as fluorescence quencher [21,22].
However fluorescence enhancement will give promising results
than quenching in case of guestehost interaction and can be easily
visualized in the bio imaging of living cells and drug delivery to the
targeted cells. Recently rhodamine based compounds were re-
ported as Cu2þ ion sensor with good fluorescence enhancement
[23e25]. Hence to synthesize a selective and high fluorescent Cu2þ
ion sensor in aqueous medium has gained additional importance
for biological applications.
In this article, for the first time we are reporting the synthesis
of thiophene based Schiff base (receptor 1) in single step and
studied its cation sensing properties. Receptor 1 showed good
selectivity and sensitivity towards Cu2þ ion over other cations in
aqueous medium. Weak fluorescence of the Receptor 1 is due to
the effect of photo induced electro transfer mechanism through
the lone pair of electrons present on the nitrogen of imine and
amine group [26,27]. When Cu2þ is binding with receptor 1, lone
pair of electrons accompanies the vacant orbital of Cu2þ ion which
inhibits PET mechanism and exhibits blue emission with large
fluorescence intensity at 430 nm. Receptor 1 showed higher
binding constant and detection limit towards Cu2þ ion in aqueous
medium. Quantum yield calculation of receptor 1 implies that the
To monitor the presence of excess Cu in cells and tissues of the
human body, an efficient fluorescence sensor has to be devised and
* Corresponding author. Tel.: þ91 431 2503640, þ91 09486067404; fax: þ91 431
2500133.
E-mail addresses: velmathis@nitt.edu, svelmathi@hotmail.com (S. Velmathi).
0143-7208/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2014.01.001

S. Suganya et al. / Dyes and Pigments 104 (2014) 116e122
117
method. Yield: 70%. M.pt: 220 ꢂC. IR (cmꢁ1, KBr): 3366, 3450, 1609,
1593, 1488, 1457, 1284, 1050. 1H NMR (400 MHz DMSO-d6): 8.82
(CH]N,1H, s), 7.66 (AreH, 1H, s), 7.16e7.18 (AreH, 1H,d), 6.96e7.01
(AreH,1H, t), 6.71e6.74 (AreH, 1H, d), 6.56e6.59 (AreH, 1H, t), 5.20
(NH2, 2H, s).13C NMR (100 MHz DMSO-d6): 149.84, 146.59, 144.63,
134.70, 133.21, 128.58, 117.47, 116.73, 115.36. HRMS: 321.1170
(found), 321.1168 (calculated).
Scheme 1. Synthesis of receptor 1.
emission after the detection of Cu2þ is 35 fold higher than before
detection. Hence the utility of the receptor 1 was demonstrated for
the recognition of Cu2þ ion in the living cells using confocal mi-
croscope. DFT calculations also carried out to prove the Cu2þ ion
binding with receptor 1.
3. Results and discussion
Receptor 1 was synthesized in a single step (Scheme 1) and the
structure was confirmed by spectroscopic (Figure S1), HRMS
(Figure S2) and crystallographic method (Fig. 1). The single crystal
XRD analysis tells the receptor 1 is monoclinic system with C2/c
space group. The CCDC reference number is 942484e942485. All
the other crystallographic data and packing diagram of receptor 1 is
given in Figures S5 and S6.
2. Experimental section
2.1. Materials and instruments used
Solvents such as acetonitrile (ACN), ethanol (EtOH) and reagents
used for spectroscopic experiments, synthesis were received
commercially and used as such without any further purification. All
metal salts as chloride, nitrate, acetate, sulfate were purchased and
used for all colorimetric and spectroscopic titrations. Molecular
weight of the receptor 1 was taken by Orbitrap Q exactive mass
spectrometer. 1H and 13C NMR spectra were obtained on a BRUKER
AV III-400 MHz Spectrometer using DMSO-d6 as solvent. The
structure of the receptor 1 was analyzed by X-ray diffraction
method using Enraf Nonius CAD4-MV31 Bruker Kappa APEXII in-
strument. IR spectra were recorded on NICOLET IS5 instrument
using KBr plates. UVevis spectra were recorded on a Shimadzu UV-
3.1. Colorimetric and UVevis analysis
Receptor 1 was treated with various cations like Cr3þ, Mn2þ
,
Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ, Hg2þ, Pb2þ, and Sn2þ to study its
sensitivity and selectivity towards particular metal ion over other
cations. Among all cations, selectively Cu2þ ion showed colori-
metric response as yellow to colorless and effective fluorescence
“turn-on” under UV light with the addition of 0.8 equiv (Fig. 2).
Other metal ions did not show significant color changes even with
excess addition. Simultaneously, colorimetric result was further
confirmed by UVevis and fluorescence spectroscopy for the addi-
tional evidence of Cu2þ ion detection. In UVevis titrations also,
even with the addition of 0.8 equiv. of Cu2þ ion a new absorption
band at 370 nm was appeared with higher intensity (Fig. 3a).
Likewise, in fluorescence titrations maximum emission was
observed at 430 nm in presence of 0.8 equiv. of Cu2þ ion (Fig. 3b).
Additionally, 5 equiv. of Cr3þ, Sn2þ ions showed slight fluorescence
emission at 465 nm with 35 nm red shift and all other cations did
not show any significant color and optical changes even with excess
addition.
2600 Spectrophotometer with
a
quartz cuvette (path
length ¼ 1 cm) at room temperature (r.t). Fluorescence spectra
were recorded on Shimadzu RF-5301 PC spectrophotometer. A
2.5 ꢀ 10ꢁ5 M solution of the receptor 1 in ACN and 1.5 ꢀ 10ꢁ3 M of
all cations in H2O were prepared and used for spectroscopic titra-
tions. Bio imaging studies of Escherichia coli MTCC 2939 cell was
carried out using confocal fluorescence microscope (Carl Zeiss,
Germany).
2.2. Experimental procedure for the synthesis of the receptor 1
In order to calculate the binding constant and detection limit
values, spectroscopic titrations were carried out with gradual
addition (0e0.8 equiv.). In UVevis experiments, for each addition of
Cu2þ ion to receptor 1, the band in the visible region at 448 nm
decreased and a new band was appeared at 370 nm with a blue
shift of 78 nm (Fig. 4). This color and optical changes is due to the
formation of a coordination bond between receptor 1 and Cu2þ ion
via the transformation of lone pair of electrons of imine and amine
into the vacant orbital of Cu2þ ion. Ultimately the conjugation of
Receptor 1 was prepared by reacting 1 mmol of 2, 5- thiophene
dicarboxaldehyde with 2 mmol of o-phenylene diamine in EtOH
medium. A drop of acetic acid was added to the reaction mixture
and allowed to reflux for 3 h at 70 ꢂC. After cooling to r.t the pre-
cipitate formed was filtered, washed with EtOH and dried in a
vacuum oven (Scheme 1). Orange color crystal of receptor 1 was
obtained using dichloromethane as solvent by slow evaporation
Fig. 1. ORTEP diagram of receptor 1.

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S. Suganya et al. / Dyes and Pigments 104 (2014) 116e122
Fig. 2. Color change normal (top) and under UV light 365 nm (bottom) observed upon
the addition of (0e5 equiv.) of various cations (1.5
ꢀ
10ꢁ3 M) to receptor
1
(2.5 ꢀ 10ꢁ5 M) (from left to the right: R, R þ Cr3þ, R þ Mn2þ, R þ Fe3þ, R þ Co2þ
,
R þ Ni2þ, R þ Cu2þ (0.8 equiv.), R þ Zn2þ, R þ Cd2þ, R þ Sn2þ, R þ Hg2þ, R þ Pb2þ). (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
free receptor 1 is disturbed when Cu2þ ion bind with receptor 1,
resulting a new band in the UV region.
Fig. 4. Absorption spectra of the receptor 1 in ACN recorded (2.5 ꢀ 10ꢁ5 M in ACN)
with gradual addition of (0e0.8 eq.) of Cu2þ ion (1.5 ꢀ 10ꢁ3 M in H2O).
3.2. Fluorescence titration
As like UVevis titration, receptor 1 reached maximum fluores-
cence intensity in the blue region at 430 nm (Fig. 5) with the gradual
titration of Cu2þ ion (0e0.8 equiv.). Also slight fluorescence
enhancement with 35 nm red shift at 465 nm for Cr3þ, Sn2þ ions was
Fig. 5. Emission spectra of the receptor 1 in ACN recorded (2.5 ꢀ 10ꢁ5 M in ACN) with
gradual addition of (0e0.8 eq.) of Cu2þ ion (1.5 ꢀ 10ꢁ3 M in H2O). Inset: fluorescence
image and linear increase in the formation of receptor 1- Cu2þ complex.
Fig. 6. Color Changes normal (top) and under UV light 365 nm (bottom) observed
upon the addition of various cations to receptor 1-Cu2þ complex. (From left to the
Fig. 3. (a) Absorption change (b) Emission change observed upon the addition of (0e
5 equiv.) of various cations (1.5 ꢀ 10ꢁ3 M) to receptor 1 (2.5 ꢀ 10ꢁ5 M) (from left to the
right: R, R þ Cr3þ, R þ Mn2þ, R þ Fe3þ, R þ Co2þ, R þ Ni2þ, R þ Cu2þ (0.8 equiv.),
R þ Zn2þ, R þ Cd2þ, R þ Sn2þ, R þ Hg2þ, R þ Pb2þ).
right: R, ReCu2þþCr3þ, ReCu2þþMn2þ, ReCu2þþ Fe3þ, ReCu2þþCo2þ, ReCu2þþ Ni2þ
,
ReCu2þþZn2þ
,
ReCu2þþCd2þ
,
ReCu2þþHg2þ
,
ReCu2þþPb2þ
,
ReCu2þþ Sn2þ). (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)

S. Suganya et al. / Dyes and Pigments 104 (2014) 116e122
119
photo induced electrons are blocked, hence the PET is inhibited and
leads to increase in the fluorescence intensity at 430 nm. To examine
the interference of other metal ions in the Cu2þ ion detection, the
competitive experiment was performed. Five equiv. of all the metal
ions were added to the Cu2þ ion complex of receptor 1 solution, but
no changes were observed in the color of the receptor 1-Cu2þ
complex. This result is an added evidence for the high selectivity of
the receptor 1 and can be used as selective Cu2þ ion sensor even in
presence of other cations without any interference (Fig. 6).
In order to find out the effect of counter anions in the Cu2þ ion
sensing mechanism of receptor 1, colorimetric and fluorescence
titrations were carried out with different copper sources like CuCl2,
Cu(NO3)2, CuSO4, Cu(OAc)2 in aqueous medium. Colorimetric re-
sults showed, the yellow color of receptor 1 was completely turned
into colorless only with 0.8 equiv. of CuCl2 and slight color change
was observed with CuSO4 whereas other copper salts induced no
change. The same outcome was reflected in fluorescence titration
also, maximum emission was observed with 0.8 equiv. of CuCl2 and
CuSO4. Remaining copper sources did not show significant changes
in colorimetric and emission spectrum (Fig. 7). Particular sensing of
CuCl2 indicates that the coordination nature of Cu2þ and counter
anion Clꢁ is favorable for the Cu2þ ion detection by receptor 1 over
other sources. Such discriminate interaction of the receptor 1 to-
ward copper chloride finds important usefulness in chemo-sensor
application due to its highly selective binding to a single ionic
copper compound. Such a peculiar response to single copper
compound, as far as we are aware, has been reported elsewhere.
Both binding constant and binding mode were calculated using
the measurement of absorbance. Binding constant (Ka) was
Fig. 7. Emission spectra of the receptor 1 recorded (2.5 ꢀ 10ꢁ5 M in ACN) after the
addition of different copper salts (1.5 ꢀ 10ꢁ3 M in H2O). Inset: colorimetric changes
(From left to the right: R, R þ CuCl2, R þ Cu(NO3)2, R þ CuSO4, R þ Cu(OAc)2).
observed in the gradual addition of (0e5 equiv.) of Cr3þ, Sn2þ ions
(Figure S4). This large fluorescence enhancement at 430 nm of re-
ceptor 1-Cu2þ complex is highly indicating the inhibition of photo
induced electron transfer mechanism. The weak fluorescence of
receptor 1 is due to the transfer of lone pair electron and delocal-
ization of electron via PET to thiophene ring. The moment receptor 1
binds with Cu2þ ion, the transformation of delocalized electrons and
Fig. 8. (a) Binding mode and (b) Job’s plot of receptor 1 with Cu2þ ion.

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S. Suganya et al. / Dyes and Pigments 104 (2014) 116e122
estimated using BenesieHildebrand plot and the plot was done by
absorbance changes of consequent titration (1/A ꢁ A0) against 1/
[Cu2þ]. The magnitude of Ka was calculated from the intercept and
slope of the straight line, estimated value is 4.23ꢀ105. Job’s plot
showed maximum mole fraction at 0.5 indicates the receptor binds
with Cu2þ in 1:1 stoichiometry (Fig. 8a and b). The ESI-mass
spectrum also support 1:1 binding mode and the molecular ion
peak at 478.9022 suggests the complex formation as [R1CuCl2].H2O
and a base peak at 434.2151 suggest the removal of one chlorine ion
and complex as [R1CuCl] þ.H2O. FTIR spectra also sustain the
binding of Cu with receptor 1, which was confirmed with the sig-
nificant shift. The peaks corresponding to NH2 and CH]N at
3366 cmꢁ1 and 1609 cmꢁ1 shifted into 3157 cmꢁ1 and 1628 cmꢁ1
respectively. In addition to that, a strong and broad band at
3424 cmꢁ1 was observed which suggests that H2O molecule was
coordinated in the complex. Fluorescence intensity was used to
calculate quantum yield, showed 35 fold higher for receptor 1- Cu2þ
complex (4 ¼ 0.598) than that of receptor 1 alone (4 ¼ 0.017).
Detection limit of Cu2þ ion by receptor 1 was calculated from the
plot of fluorescence intensity vs concentration of Cu2þ ion [25].
From the plot, detection limit was calculated using the formula 3s/
m. Where
s the standard deviation and ‘m’ is the slope of the
straight line and it was found as 0.418 ꢀ 10ꢁ9 M. Thus the receptor 1
can be utilized as an efficient Cu2þ ion sensor at nano molar level.
3.3. Bio imaging of living cells
To assess the potential application of receptor 1 for an inevitable
applications in bio-labeling and bio-imaging work in imaging
research. It is revealed that E. coli, known to uptake copper ions in
their microenvironment [28e30]. A bacterium, E. coli MTCC 2939
was chosen for the study and was exposed to copper ions in the
concentration of 15
with receptor 1(25
m
m
M. The bacterial cells were then incubated
M) at 37 ꢂC for 30 min and observed under
fluorescence microscope. For the control, bacterial cells were
incubated separately with copper ions and receptor 1 to under-
stand the unique fluorescence property of receptor 1. In this study
we observed that the cells contain copper ions along with receptor
1 showed high fluorescence, whereas in cells with copper ions and
receptor 1 alone not shown any fluorescence (Fig. 9aec). Such
fluorescence image indicates that the ability of the receptor 1 to-
wards Cu2þ ion recognition in the living cells and has the good
permeability, which is inevitable for bio-imaging experiments.
3.4. Theoretical calculations
Fig. 9. Bio-imaging of copper ions in E. coli MTCC 2939 at 37 ꢂC under fluorescence
microscope. E. coli MTCC 2939 cells incubated with receptor 1 and (a) presence, (b)
absence of copper ions and (c) E. coli MTCC 2939 cells alone under dark field
microscope.
For the complete understanding of the receptor 1 and its copper
complex, DFT calculation and structure optimization were done
using Gaussian09 program package [31] and the energies were
compared with experimental observation. The Density Functional
Fig. 10. Optimized structures of receptor 1 and Cu(II)-receptor 1 complex.

S. Suganya et al. / Dyes and Pigments 104 (2014) 116e122
121
Fig. 11. (a) Energy levels of various HOMO and LUMO’s of receptor 1 (b) Energy levels of various HOMO and LUMOþ1 of Cu(II)-receptor 1 complex.
Theory (DFT) based Beck-3 Lee Young Parr (B3LYP)/6-31G(d,p)
model chemistry was employed for L1 and effective core potential
(ECP) based LANL2DZ basis set was incorporated to Cu complex.
Fig. 10 represent the optimized energy structure of the receptor 1
and its Cu(II) complex. The Cu2þ ion is coordinated with the two
nitrogen atom of the receptor 1 with a bond distance of 2.0804 A0
and 2.0642 A0. The calculated energy value from the HOMO-LUMO
excitation reveals that the predominant absorption peak for re-
ceptor 1 observed at 453 nm is nearly close with the experimentally
observed value at 448 nm. However, the energy of its receptor 1-
Cu2þ complex was calculated from HOMO-LUMOþ1 excitation. In
this case, main absorption peak at 448 nm was blue shifted to
370 nm which is also in well hold up with the theoretically pre-
dicted lmax at 363 nm (Fig. 11). Table 1 clearly explains that the
addition of copper ion blocks the conjugation of receptor 1 via the
acceptance of lone pair of electrons present on nitrogen atom and
leads to higher energy.
aqueous medium was achieved by colorimetric, UVevis and fluo-
rescence spectroscopic methods with distinct color and optical
changes without any interference even in presence of other metal
ions. The application of receptor 1 for the Cu2þ ion recognition was
successfully demonstrated in the living cells by means of receptor 1
as bio probe. This bio sensing of Cu2þ ion was confirmed by imaging
the fluorescence nature of the E. coli cells in presence of receptor 1-
Cu2þ complex using fluorescence microscope. Receptor 1 was
found to be able to act as a nano molar (0.423 nm) Cu2þ ion sensor
with the binding constant of 4.23 ꢀ 105. DFT calculations also done
to support the Cu2þ ion binding with receptor 1 and closely
matched with the experimental observations.
Acknowledgment
One of the authors S.V thanks DRDO for a major sponsored
Project ERIP/ER/1006004/M/01/1333 dated 23-05-2011 for finan-
cial support.
4. Conclusion
A new, selective aqueous mediated Cu2þ sensor 1 was success-
fully synthesized in single step and well characterized with single
crystal XRD. The selective sensing of receptor 1 towards Cu2þ ion in
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.01.001.
Table 1
Calculated energies of receptor 1 and its copper complex.
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