Coumarin based hydrazone as an ICT-based fluorescence chemosensor for the detection of Cu2+ ions and the application in HeLa cells

Article abstract of DOI:10.1016/j.saa.2019.02.020

We have constructed a new coumarin based fluorescence probe BENZPYR with ICT character through condensation of N, N-diethylamino-3-acetyl coumarin with 2-hydrazinobenzothiazole. The absorbance and fluorescence spectral characteristics of BENZPYR revealed that the chemosensor can specifically detect for Cu²⁺ ions over other different metal ions and the lowest limit of detection was found in nano molar range. The turn off sensor of BENZPYR is related to chelation enhanced quenching (CHEQ) and intramolecular charge transfer (ICT) processes were serve as excellent fluorescent detection of Cu²⁺ ions in DMF medium. Fluorescence microscopy experiments revealed that probe BENZPYR may have application as a fluorophore to detect the Cu²⁺ in living cells. The simulated DFT analysis of electronic and structural properties and also UV–vis absorption spectra are in well accordance with the experimental UV–vis absorption spectra.

Full text of DOI:10.1016/j.saa.2019.02.020

Accepted Manuscript
Coumarin based hydrazone as an ICT-based fluorescence
chemosensor for the detection of Cu2+ ions and the application in
HeLa cells
Kailasam
Balasubramanian Murugesapandian, Ramasamy Shankar, Gandhi
Sivaraman, Malaichamy Ilanchelian, Subramaniam
Parameswaran Rajendran
Saravana
Mani,
Ramar
Rajamanikandan,
PII:
S1386-1425(19)30131-3
DOI:
Reference:
https://doi.org/10.1016/j.saa.2019.02.020
SAA 16796
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 6 December 2018
Revised date:
Accepted
date:
4 February 2019
10 February 2019
Please cite this article as: K.S. Mani, R. Rajamanikandan, B. Murugesapandian, et al.,
Coumarin based hydrazone as an ICT-based fluorescence chemosensor for the detection of
Cu2+ ions and the application in HeLa cells, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.02.020
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ACCEPTED MANUSCRIPT
Coumarin based hydrazone as an ICT –based fluorescence chemosensor for
the detection of Cu2+ ions and the application in HeLa cells
Kailasam Saravana Mania*, Ramar Rajamanikandana, Balasubramanian Murugesapandiana,
Ramasamy Shankarb, Gandhi Sivaramanc, Malaichamy Ilancheliana, Subramaniam
Parameswaran Rajendrana
aDepartment of Chemistry, Bharathiar University, Tamil Nadu, India
b Department of Physics, Bharathiar University, Tamil Nadu, India
cInstitute for stem cell biology and regenerative medicine, National centre for Biological sciences,
Bangalore, India.
Corresponding author: chemsaran.mani@gmail.com
Abstract
We have constructed a new coumarin based fluorescence probe BENZPYR with ICT
character through condensation of N, N-diethylamino-3-acetyl coumarin with 2-
hydrazinobenzothiazole. The absorbance and fluorescence spectral characteristics of BENZPYR
revealed that the chemosensor can specifically detect for Cu2+ ions over other different metal ions
and the lowest limit of detection was found in nano molar range. The turn off sensor of BENZPYR
is related to chelation enhanced quenching (CHEQ) and intramolecular charge transfer (ICT)
processes were serve as excellent fluorescent detection of Cu2+ ions in DMF medium. Fluorescence
microscopy experiments revealed that probe BENZPYR may have application as a fluorophore to
detect the Cu2+ in living cells. The simulated DFT analysis of electronic and structural properties
and also UV−vis absorption spectra are in well accordance with the experimental UV−vis
absorption spectra.
Keywords
Coumarin based hydrazone, Cu2+ detection, Colorimetric and Fluorimetric detection, DFT
calculations, Fluorescence bioimaging.
Introduction
In recent years, the synthesized fluorescent chemosensors are having the ability to detect
cations with good biocompatibility because of their impending application value in biological
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medicine and environmental conservation studies [1]. The transition metal ions are earned
enormous anxiety about their toxicity, because they can lead to severe environmental and health
issues [2, 3]. Among different transition metal ions, copper is a necessary trace element that plays
a vital role in environmental, biological and chemical systems [4]. In living system, copper is
usually present at small levels but it turn into toxic when the level of Cu2+ions exceeds the cellular
requirements. Whereas, excess growth of Cu2+ in the neuronal cytoplasm can lead to oxidative
stress and neurological disorders as well as Alzheimer’s, Parkinson’s and Wilson’s diseases [5].
Moreover, copper can be a serious pollution source in environment because it has been used much
in industry and agriculture [6, 7].
Of other techniques, fluorescent probes emerged significant attention due to their great
advantages including high specificity, selectivity, low detection limit, sensitivity and concurrent
monitoring with instant response [8, 9]. Till date, so many fluorescent chemosensors for the
detection of Cu2+ have been described and even enlarged to applications in biological and
environmental fields [10-14]. Detection of Cu2+ ions based on simple fluorescent probes presents
inherent advantages over traditional other techniques, such as considerable selectivity and
sensitivity, consistency, simplicity compared to other traditional analytical methods [15].
Nevertheless, most of the currently reported Cu2+ selective probes have drawbacks, such as multi-
step synthesis, cross sensitivity towards other metal ions and lack of bio-compatibility [16].
Significant efforts have been made to synthesize fluorescent probes which are sensitive, selective
and suited to high-resolution bioimaging for monitoring biological processes [17]. Still great
accomplishments in the field of colorimetric and/or fluorescent probes for the detection of Cu2+
have been reported [18-20], there is still a thrust to develop a novel chemosensor with superior
properties.
In the continuation of our research towards the synthesis of quinoline and coumarin type
of heterocycles with widened biological and photophysical properties [21] herein, we designed a
coumarin hydrazone based fluorescent chemosensor (E)-3-(1-(2-(benzo[d]thiazol-2-
yl)hydrazono)ethyl)-7-(diethylamino)-2H-chromen-2-one (BENZPYR) through condensation
reaction between N,N’-diethylamino-3-acetyl coumarin with 2-hydrazino benzothiazole .
Interestingly, BENZPYR probe can highly recognize Cu2+ ions over other interfering metal ions
through absorbance and fluorescence emission spectral studies. The colorimetric response after
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the addition of Cu2+ ions in the probe from light yellow to wine red in color will be easily visualized
through naked eye. It is may be due to intramolecular charge transfer and d-d transitions of metal
ions. The decrease in the fluorescence spectral features is also attributed to CHEQ pathway and
lowest detection limit is also calculated in nano molar range. The binding stoichiometry between
the synthesized probe and Cu2+ ions is found to be 1:1 and it will be confirmed through Job’s plot,
DFT theoretical and mass spectral analysis. In addition, the present BENZPYR probe is also
applied for the detection of Cu2+ ions in living cells.
EXPERIMENTAL
Materials
All the reagents for the synthesis of BENZPYR was obtained from commercial suppliers.
Solvents were purified by previously reported procedure. 1H and 13C NMR spectral studies were
1
13
taken from Bruker avance (400 MHz for H and 100 MHz for C) in DMSO-d6 solvent and
chemical shift values were expressed in parts per million using TMS as an internal standard. The
reaction path was tracked by TLC on silica gel coated F254 Merck plates. Elemental analyses were
examined by using Perkin Elmer 2400 series II Elemental CHNS analyzer. ESI-MS were recorded
on a Thermo Scientific High resolution Magnetic Sector MS DFS by negative ion electrospray
ionization (ESI) method.
Synthesis of BENZPYR
Sensor BENZPYR was synthesized by the condensation reaction of
N, N’-diethylamino-3-acetyl coumarin with 2-hydrazino benzothiazole in ethanol with the addition
of few drops of Con. sulphuric acid under stirring at room temperature. After the completion of
reaction monitored by TLC, the precipitate were filtered off and washed with cold methanol. The
crude product was recrystalized using chloroform: methanol solvents in 1:1 ratio.
3-(1-(2-(benzo[d]thiazol-2-yl)hydrazono)ethyl)-7-(diethylamino)-2H-chromen-2-one;
Orange solid; mp; 163-165 °C Yield 85 %; FT-IR (KBr, νmax, cm-1): 1712, 1608, 1506; 1H NMR
(400 MHz, CDCl3) δ ppm: 8.07(s, 1H), 7.74(d, J = 8.0 Hz, 1H), 7.58(d, J = 8.8 Hz, 2H), 7.39(d,
J = 7.6 Hz, 1H), 7.33(t, J = 7.6 Hz, 1H), 7.14(t, J = 7.6 Hz, 1H), 6.76(dd, J = 2.0 Hz, J = 2.0 Hz,
1H), 6.56(s, 1H), 3.46(t, J = 6.8 Hz, 4H), 2.32(s, 3H), 1.15(t, J = 6.8 Hz, 6H); 13C NMR(100 MHz,
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CDCl3)δ ppm: 167.3, 160.0, 156.4, 151.0, 142.0, 130.3, 127.3, 126.2, 121.9, 117.5, 115.6, 109.5,
107.8, 96.1, 55.9, 44.2, 18.4, 16.5, 12.2; HRMS (TOF MS ES+) m/z 407.1569; Anal. Calcd. for
C22H22N4O2S; C, 65.00; H, 5.46; N, 13.78; S, 7.89; Found; C, 65.2; H, 5.45; N, 13.76; S, 7.88.
General methods
Emission and absorption analysis
UV-vis absorption spectral measurements were performed by JASCO V-630 UV-visible
spectrophotometer with 1.0 cm quartz cuvettes used. Steady state fluorescence spectral
measurements were employed with JASCO FP-6600 spectrofluorometer attached with a 1cm
quartz cuvette.
Fluorescence bio- imaging for living cells
HeLa cells were developed in customized Eagle's medium connected with 10% FBS (fetal
bovine serum) at 37 °C. HeLa cells were incubated with BENZPYR (5.0 µM) in HEPES buffer,
pH = 7.54, holding 1% DMSO as co-solvent. The incubated cells were swabbed with HEPES
buffer for more to eradicate excess of BENZPYR in the extracellular medium. HeLa cells were
pictured via fluorescence microscopic analysis. After washing with the cells with HEPES buffer
for more times to remove the excess of the BENZPYR in the cells, the BENZPYR affected HeLa
cells were further incubated in the presence of 10.0 µM CuNO3 for 10 min at 37 ºC and finally
captured with fluorescence microscope.
Results and Discussion
Sensor BENZPYR was synthesized by the condensation reaction of N,
N’-diethylamino-3-acetyl coumarin with 2-hydrazino benzothiazole with the addition of few drops
of Con. sulphuric acid under stirring at room temperature (Scheme-1). Probe BENZPYR was
1
unambiguously identified by H and 13C NMR, FT-IR, ESI–MS spectrometry and elemental
1
analysis. In H NMR spectrum, the characteristic peak at δ 7.80 due to 3rd position of coumarin
ring and disappearance of –C=O and appearance of –C=N at δ 160.216 attested the formation of
BENZPYR. The molecular ion peak at m/z 407.1569 (M+H)+ further attested the formation of
chemoreceptor BENZPYR.
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Scheme 1. Synthetic procedure for the formation of probe BENZPYR
Absorption spectroscopic studies
To examine the photophysical properties of BENZPYR in the presence and absence of
Cu2+ ions, UV-vis titration studies were employed (Figure 1). In the absence of metal ions,
BENZPYR exhibits a broad absorbance bands at 297 nm and 419 nm in DMF medium. Upon
gradual addition of Cu2+ ions to a solution of BENZPYR the absorption band at 297 nm and
419 nm gradually decreases and a new band at 503 nm, whereas, the color of the solution also
changes from yellow to wine red in color. The formation of new peak at 503 nm corresponds to
coordination of Cu2+ towards probe BENZPYR due to intra-ligand transitions and d-d transitions.
Similar kinds of results have been reported in the several organic moieties in the presence of Cu2+
ions [22, 23]. Hence, in this analysis it is assumed that intra molecular charge transfer is the crucial
role for the alterations in UV-vis spectra of BENZPYR with Cu2+ ions. The TD-DFT calculations
have been carried out with B3LYP/LANL2DZ and M06-2X/ LANL2DZ level of theories and the
corresponding absorption peaks at 300nm and 400 nm are comparable with the experimental
values Figure S11 & S12(ESI). Moreover, addition of Cu2+ has given a new absorption peak near
500 nm, shows that the experimental results are in good agreement with the theoretical values.
Both levels of theories are reliable to the experimental results, but the M06-2X level of theory is
more reliable with the experimental results when the presence of Cu2+ metal ion. Similarly, B3LYP
is more reliable to the experimental results without Cu2+.
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Figure 1. (A) UV–vis absorption spectral changes of BENZPYR in DMF with the increasing
concentration of Cu2+ ions, [Cu2+ ions] = 0 – 48 M (each addition 4 M)
Fluorescence spectral studies
To appraise the binding capability of BENZPYR with Cu2+, the emission spectral
measurements of BENZPYR without and with increasing concentrations of Cu2+ ions were
investigated in DMF medium and corresponding emission spectral data were given in Figure 2.
As evident from Figure 2, before the addition of Cu2+ ions, emission spectra of probe BENZPYR
illustrates an emission maximum at 536 nm upon the excitation wavelength at 420 nm. Upon the
addition of Cu2+ ions to BENZPYR solution, emission intensity (572 nm) gradually decreased and
red shift from 536 nm to 572 nm clearly depicts that coordination of Cu2+ to the receptor
BENZPYR, whereas, color also changes from yellow to wine red. The observed emission
quenching is due to that intra molecular charge transfer and chelating enhanced emission
quenching (CHEQ) mechanism. These results were well consistent with the previously reported
literature [24]. Based on emission quenching experiments concentrations of Cu2+ ions were
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calculated. A good linear relationship was obtained in the relative emission intensity against 3 M
to 48 M of Cu2+ ions (R=0.9916) and the lowest detection limit was estimated as 40 nM (S/N=3).
(Figure. 3). We made a comparison table between our synthesized for the detection of Cu2+ ions
with currently reported Cu2+ sensors (Table 1(ESI)). The main significance from the table reveals
that, our present fluorescent receptor is one step facile synthesis with readily available chemicals,
applicability for naked eye detection in the visible region.
Figure 2. Emission spectral results of BENZPYR in DMF with the increasing concentrations
of Cu2+ ions λex = 420 nm. [Cu2+ ions] = 0-48 M (each addition 3 M). (a) BENZPYR
and (b) BENZPYR after the addition of Cu2+ ions.
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Figure 3. Calibration plot for relative emission intensity of (BENZPYR) versus Cu2+ ions
Emission quenching mechanism
The fluorescence quenching of BENZPYR by Cu2+ ions take place either static or
collisional quenching in nature. To establish the probable quenching tactic of BENZPYR with Cu2+
ions, the relevant fluorescence spectral observations were explored by classical Stern-Volmer
equations (Eqn. 1)
F0
[ ]
= 1 + 퐾푠푣 Q … … … (1)
F
whereas, F0 and F are the emission intensities with and without quencher, respectively. Ksv is the
Stern-Volmer quenching constant and [Q] is the concentration of quencher.
The Stern-Volmer plot of BENZPYR with different amounts of Cu2+ ions are presented in Figure
S6 (ESI). As depicted in Figure S6, the plot of F0/F vs [Cu2+ ions] illustrated a fine linearity in the
investigated concentrations of Cu2+ ions ranges from 3 M to 39 M. The good linear Stern-
Volmer plot is investigative of single class of quenching either ground state complex formation or
collisional quenching in nature. The KSV value calculated from the slope of the linear Stern-Volmer
plot is 4.09 × 104 dm3 mol-1. The obtained Ksv value clearly suggested that the presence of strong
emission quenching between the BENZPYR and Cu2+ ions.
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Determination of binding constant and quantum yield
Data from emission quenching studies were utilized to estimate the binding constant (Kb)
value by using the following equation (Eqn.2)
F0 − F
log [
] = log K푏 + n log [푄] … … … (2)
F0
where, F0, F, and [Q] are the same as in (Eq. (1)), n is the number of binding sites and Kb
is the binding constant. Kb and n can be computed from the intercept and slope obtained through
plotting log[F−F0/F0] against log[Q] (Figure S7(ESI)). The binding affinity constant (Kb) was
determined as 4.89 × 105 dm3 mol−1. The calculated binding constant value recommended that
there is a strong binding force between BENZPYR and Cu2+ ions.
The fluorescence quantum yields (ΦF) of the BENZPYR in the absence and presence of
Cu2+ ions were calculated using the following Eaton equation (Eqn.3)
2
퐹
푛
푠푎푚푝푙푒
퐴
푠푎푚푝푙푒
휑퐹(푋) = 휑퐹 푠푡푎푛푑푎푟푑
(
푠푡푎푛푑푎푟푑) (퐹
) (푛
) … … …(3)
퐴
푠푎푚푝푙푒
푠푡푎푛푑푎푟푑
푠푡푎푛푑푎푟푑
where Fsample and Fstandard (Quinine sulfate as a reference standard, 0.54 in 0.1 M H2SO4)
[25-27] are the measured fluorescence for the sample and standard, respectively, Astandard and
Asample are the measured absorbances, nsample and nstandard are the refractive indexes of the solvent
used for the sample and standard, respectively, and 휑푠푡푎푛푑푎푟푑 is the fluorescence quantum yield of the
standard. According to the emission and absorbance data, fluorescence quantum yield values of
BENZPYR in the absence and presence of Cu2+ ions were estimated as 0.413 and 0.027, respectively.
The acquired high fluorescence quantum yield value recommends that the highly fluorescence nature
of synthesized compound BENZPYR. Upon the addition of Cu2+ ions to BENZPYR reduces the
emission intensity due to intra molecular charge transfer between them and thereby the quantum yield
also decreased.
Selectivity and analytical application of the probe
To examine the selective determination capability of BENZPYR toward Cu2+ ions,
relevant other metal ions were tested. Emission spectral changes are examined
from the BENZPYR in the presence of different potential interfering metal ions such as, Ag+,
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Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Fe3+, Hg2+, Mn2+, Ni2+, Pb2+ and Zn2+ were represented in Figure 4.
As described in Figure 4, the relative emission intensity changes in the presence of different
interferents added into BENZPYR and distinguished larger relative emission intensity changes
confirmed the outstanding selectivity of the present BENZPYR probe. In addition, naked eye
colorimetric and fluorescence color changes of probe BENZPYR in the presence of various
Figure 4. (A) Emission spectral results of BENZPYR in DMF with solution of different
potential interfering metal ions. (B) and (C). Colorimetric and Fluorimetric response
of BENZPYR in DMF with same environmental relevant metal ions
metal ions were also investigated. Except Cu2+ ions, the probe didn’t show any apparent
color change in the solution of Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Fe3+, Hg2+, Mn2+, Ni2+, Pb2+ and
Zn2+ metal ions. Cu2+ ions only stimulated the BENZPYR color from yellow to wine red, while,
other metal ions didn’t affect the yellow color under day light and UV-lamp fluorescence color of
BENZPYR probe. The obtained experiment high specificity is ascribed to the strong binding
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interaction and ICT process between the BENZPYR and Cu2+ ions. From the above experiments,
we suggested that BENZPYR probe is extremely specific and high responsive towards Cu2+ ions.
To understand the binding nature of Cu2+ with BENZPYR, the continuous variation method (Job’s
plot), in which the intensity of the band was plotted against the mole fraction of BENZPYR
(Figure S8 ESI). These observed results suggested that the formation of
1 : 1 stoichiometry between BENZPYR and Cu2+ during their complexation. This stoichiometry
was further confirmed by mass spectroscopy m/z value of 512.1672 (Figure S4 ESI). This
stoichiometry was further confirmed by mass spectroscopy m/z value of 532.0758 (Figure S5 ESI)
(Scheme-2).
Scheme-2: Plausible sensing mechanism for Cu2+ ions
Due to the super selectivity of this probe towards Cu2+ ions, BENZPYR probe is further
applied in real water samples such as tap and drinking water samples. Tap and drinking water
samples were taken from our lab and our department (Department of Chemistry, Bharathiar
University), respectively. For the real water sample analysis standard addition procedure was
followed by this method. Moreover, spiked and found amount of Cu2+ ions in real water samples
were listed in Table S2(ESI). The recovery amount of Cu2+ ions is designated that the proposed
assay for Cu2+ ions is of good accuracy and reasonable.
DFT theoretical calculations
To gain better insight into the electronic structure, geometry of BENZPYR and
BENZPYR with Cu2+ metal complex is optimized with two different DFT functional such as
B3LYP and M06-2x functional [28] with high level basis sets of LANL2DZ were employed for
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the predicting the electronic properties. The optimized electronic structures of BENZPYR and
BENPYR-Cu2+ complex revealed as 1:1 stoichiometry. From the obtained geometry of the above
complex, three coordination bonds were formed between metal ion (Cu2+) with BENZPYR, the
first coordination bond has occurred in coumarin 2nd position of oxygen atom(O) with 2.408 Å and
second coordinate bond on benzothiazole group of nitrogen atom (N) with coordination distance
of 2.07 Å and other bond at nitrogen atom (N) in hydrazine group with coordination distance of
2.27 Å (Figure S9(ESI)). Moreover, planarity and other structural parameters of the BENZPYR
and BENZPYR complex are found to be similar with minimal structural fluctuations has been
observed at above level of theories.
LUMO=-1.930eV
LUMO= -1.319eV
ΔE=3.370 eV
ΔE=5.116 eV
HOMO=-6.435eV
BENZPYR + Cu2+
HOMO=-5.300eV
BENZPYR
Figure 5. HOMO-LUMO energy levels and the interfacial plots of the orbitals for BENZPYR
and BENZPYR-Cu2+ complex calculated from B3LYP and M062x level of theories where
calculated electronic properties are same for both level of theories.
In order to acquire more insight into optical response of BENZPYR and BENZPYR metal
complex, we further explored the frontier molecular orbital analysis at B3LYP and M062x level
of theories. As can be seen from Figure 5, the band energy gap between HOMO (-5.300 eV) and
LUMO (-1.930 eV) of BENZPYR was found to be 3.370 eV. Therefore, due to the formation of
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BENZPYR-Cu2+ complex, the band energy gap between the HOMO (-6.435 eV) and LUMO (-
1.139 eV) of BENZPYR-Cu2+ complex was increased to 5.116 eV at above level of theories. The
energy gap between the HOMO and LUMO of BENZPYR-Cu2+ complex was greater than that of
BENZPYR at above level of theories. It is documented that, the HOMO has the ability to donate
electrons, while LUMO detract electron. A molecule with high HOMO energy will be more
unstable and thus more reactive. The above DFT based explanations have further confirmed that
the BENZPYR-Cu2+ complex patterned with the 1:1 stoichiometry. Moreover, the interaction
energy of the above mentioned complex are found to be
-10.41 and
-11.922 Kcal/mol at B3LYP/lanl2dz and M06-2x/ lanl2dz level of theories respectively.
Molecular Electrostatic Potential (MEP)
Molecular electrostatic potential surfaces (MEP) are helpful to visualizing relative polarity
and reactive active sites of molecules [29, 30]. MEP diagram Figure S10(ESI) highlighted blue
color for positive regions and red color for negative regions where these negative regions are
positioned around the electronegative N and O atoms to draw electron deficient Cu2+. Cu2+ was
tightly encapsulated into the cavity of BENZPYR by the strong electrostatic attraction with two
N and one O atom due to size and shape matching. This strong interaction pulls the charge towards
it around the attraction region and it causes the increase in charge around the region up to -0.23e.
Fluorescence cellular bioimaging
By taking into consideration the excellent sensing activity of BENZPYR towards Cu2+
ions, we make use of BENZPYR for fluorescence bioimaging of Cu2+ ions into HeLa(Cervical
cancer cells) by using fluorescence microscopic analysis. The HeLa cells were incubated with
BENZPYR (5.0 µM) in HEPES buffer, pH = 7.54, containing 1% DMSO as co-solvent, after
incubation the cells are cleansed with HEPES buffer more times to eradicate excess of BENZPYR
present in the extracellular media. The cells were captured through the fluorescence microscopic
analysis.
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Figure 6. Bright field and fluorescence image of HeLa cells. (A and D) Bright field images of
BENZPYR and BENZPYR + Cu2+ treated HeLa cells. (B and E) Fluorescence images of
HeLa cells incubated with BENZPYR (5 µM) for 30 minutes and subsequent addition of
(5 µM) CuNO3 for 10 minutes at 37 °C. (C and F) Overlay images of A, B, D and E
respectively, λex = 419 nm.
After washing with the cells with HEPES buffer for more times to remove the excess of
the BENZPYR in the cells, the cells were further incubated with CuNO3 ((10.0 µM) in H2O) for
10 min at 37 ºC and captured with fluorescent microscope (Figure 6.). From Figure 6, the result
indicating that the probe BENZPYR is biocompatible and appropriate for fluorescence bioimaging
in living cells with low toxicity. As evidenced from Figure 6, the cells displayed bright green
fluorescence upon incubation only with BENZPYR (5.0 µM). After successive incubation with
Cu2+ ions, the green fluorescence of the cells will be gradually disappeared. These results revealed
that, BENZPYR has good cell-membrane permeability and can be used as a marker for sensing
Cu2+ in living cells.
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Conclusion
In summary, a new coumarin based fluorescent chemosensor BENZPYR was designed
successfully and was found to have a strong and selective detection towards Cu2+ compared to
other metal ions in DMF medium. On addition of Cu2+ ions in BENZPYR, the color of the solution
changed from yellow to wine red attributed to metal to ligand energy transfer and d-d transition of
metal ions. The limit of detection (LOD) is found to be as low as 40 nM, which is superior to most
reported fluorescent probes for the detection of Cu2+ ions. Most significantly, fluorescence cell
imaging experiments revealed that the probe BENZPYR has good cell-membrane permeability
and can be used as a marker for sensing Cu2+ in living cells. We believe that in future, our
constructed new sensor BENZPYR can produce numerous significant opportunities in the
synthesis of novel essential fluorescent sensors with additional usage in biological organisms.
Acknowledgement
Author KSM would like to thank SAIF, CUSAT, Cochin for NMR spectral analysis and
Department of Chemistry, Pondicherry University for mass spectral analysis. B.M. thanks SERB,
New Delhi, India (YSS/2015/000037), for financial support and UGC, New Delhi, for a start-up
grant and UGC FRP faculty award (F.4-5(94-FRP)/2014(BSR).
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Coumarin based hydrazone as an ICT –based fluorescence chemosensor for
the detection of Cu2+ ions and the application in HeLa cells
Kailasam Saravana Mania*, Ramar Rajamanikandana, Balasubramanian Murugesapandiana,
Ramasamy Shankarb, Gandhi Sivaramanc
Malaichamy Ilancheliana, Subramaniam Parameswaran Rajendrana
aDepartment of Chemistry, Bharathiar University, Tamil Nadu, India
b Department of Physics, Bharathiar University, Tamil Nadu, India
cInstitute for stem cell biology and regenerative medicine, National centre for Biological sciences,
Bangalore, India.
Corresponding author: chemsaran.mani@gmail.com
GRAPHICAL ABSTRACT
HeLa cells
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Coumarin based hydrazone as an ICT –based fluorescence chemosensor for
the detection of Cu2+ ions and the application in HeLacells
Kailasam Saravana Mania*, Ramar Rajamanikandana, Balasubramanian Murugesapandiana
Ramasamy Shankarb, Gandhi Sivaramanc
Malaichamy Ilancheliana, Subramaniam Parameswaran Rajendrana
aDepartment of Chemistry, Bharathiar University, Tamil Nadu, India
b Department of Physics, Bharathiar University, Tamil Nadu, India
cInstitute for stem cell biology and regenerative medicine, National centre for Biological sciences,
Bangalore, India.
Corresponding author: chemsaran.mani@gmail.com
Research Highlights
 Developed a new coumarin based fluorescence chemosensor BENZPYR with strong ICT.
 Highly sensitive and selective detection of Cu2+ ions through fluorescence quenching
 Lowest limit of detection for Cu2+ ions are calculated as 40 nM.
 1:1 Binding stoichiometry between BENZPYR and BENZPYR + Cu2+ ions.
 Detection of Cu2+ ions through bioimaging in HeLa cells.
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