Carbazole–azine based fluorescence ‘off–on’ sensor for selective detection of Cu2+ and its live cell imaging

Article abstract of DOI:10.1002/bio.3332

A new carbazole–azine based fluorescent sensor was synthesized and characterized. The selectivity of the sensor for Cu²⁺ over other counter ions in a dimethyl sulfoxide/H₂O mixture was shown through enhancement in fluorescence – an off to on transformation. The specificity of the probe towards Cu²⁺ was evident in ultraviolet/visible, fluorescence, Fourier transform infrared and mass studies. Application of the probe in the cell imaging and cytotoxicity of living cells is illustrated.

Full text of DOI:10.1002/bio.3332

Received: 7 October 2016
DOI: 10.1002/bio.3332
Revised: 4 March 2017
Accepted: 6 March 2017
R E S E A R C H A R T I C L E
Carbazole–azine based fluorescence ‘off–on’ sensor for
selective detection of Cu2+ and its live cell imaging
Denzil Britto Christopher Leslee1 Sekar Karuppannan1
Karmegam Muthu Vengaian1
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Sivaraman Gandhi2 Singaravadivel Subramanian3
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1 Department of Chemistry, Anna University –
Abstract
University College of Engineering, Dindigul,
India
A new carbazole–azine based fluorescent sensor was synthesized and characterized. The selec-
tivity of the sensor for Cu2+ over other counter ions in a dimethyl sulfoxide/H2O mixture was
shown through enhancement in fluorescence – an off to on transformation. The specificity of
the probe towards Cu2+ was evident in ultraviolet/visible, fluorescence, Fourier transform infra-
red and mass studies. Application of the probe in the cell imaging and cytotoxicity of living cells
is illustrated.
2 Institute for Stem Cell Biology and
Regenerative Medicine, Bangalore, India
3 Department of Chemistry, SSM Institute of
Engineering and Technology, Dindigul, India
Correspondence
Sekar Karuppannan, Department of Chemistry,
Anna University – University College of
Engineering, Dindigul – 624622, India.
Email: karuppannansekar@gmail.com
KEYWORDS
azine, carbazole, cell imaging, copper ion, fluorescence
Funding Information
DST‐SERB Fast Track, New Delhi, India,
Grant/Award Number: SB/FT/CS‐062/2013
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1
INTRODUCTION
distinct reaction of toxic metal ions via colorimetric quantification is
required. Thus, the development of fluorescence‐based sensors for
The design and synthesis of new chemosensors for monitoring ionic
species, especially heavy and transition metal ions, are of great interest
in chemical, biological and environmental analyses.[1–9] Copper is an
essential trace element in biological systems, although under overdose
conditions, it causes neurodegenerative diseases (e.g. Alzheimer's and
Wilson's diseases).[10–16] The toxicity of copper ions towards humans
is rather low compared with that of other heavy metals, but certain
microorganisms are affected by low concentrations of copper.[17] The
United States Environmental Protection Agency recommends an upper
limit of 1.3 mg copper in humans.[18] Because of the toxicity of copper
ions and their role as a critical catalytic co‐factor in a variety of metallo
enzymes, such as superoxide dismutase, cytochrome c oxidase and
tyrosinasemore, researchers have explored efficient recognition
metal ions has attracted much interest among the research commu-
nity.[22–27]
Cu2+ is well known among transition metals as a fluorescent
quencher due to its paramagnetic nature. It is therefore practically dif-
ficult to develop fluorescent sensors for the detection of Cu2+ [28–32]
In
.
recent years, there have been several attempts to design
chemosensors for paramagnetic metal ions. Recently, rhodamine
B‐based fluorescent probes have been reported as ‘turn‐on’ fluores-
cence, and colorimetric, sensors for Cu2+ [33–42]
In these sensors, the
.
mechanism involves opening of the spirolactam ring upon binding with
a Cu2+ ion, resulting in fluorescence enhancement. In addition, several
other chemosensors have been reported with different signaling mech-
anisms for the optical detection of paramagnetic ions based on photo‐
induced electron transfer, metal–ligand charge transfer, intramolecular
charge transfer (ICT), excimer/exciplex formation, and excited‐state
intermolecular proton transfer.[43–58]
probes for Cu2+ [19–21]
Although many analytical techniques have been
.
developed, they are time consuming and expensive, and a simple,
Abbreviations used: [CzA]2‐Cu, Carbazole–azine–copper complex; CzA,
Carbazole–azine based sensor (N,N′‐bis(9‐ethyl 3‐carbazolylmethylidene)
azine); DFT, Density functional theory; DMSO, Dimethyl sulfoxide; ESI‐MS,
Electrospray ionization mass spectrometry; FTIR, Fourier transform infrared
spectroscopy.; HOMO, Higher occupied molecular orbital; ICT, Intramolecular
charge transfer; LUMO, Lower unoccupied molecular orbital; MTT, 3‐(4,5‐
Carbazole and its derivatives possess highly conjugated systems,
engage in active ICT and are used as electron‐donating chromo-
phores.[59–63] Carbazole compounds are promising candidates for
electroluminescent and photorefractive devices because of their elec-
tron‐donating and hole‐transporting properties, and their remarkable
application as fluorescent sensors.[64–76] In the bis‐azine type ligand,
the electron density is located on the [C=N−N=C] moiety. Binding
Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide;
NMR,
Nuclear
magnetic resonance spectroscopy; PBS, Phosphate‐buffered saline; TDDFT,
Time‐dependent density functional theory; UV, Ultraviolet; Vis, Visible
Luminescence. 2017;1–7.
wileyonlinelibrary.com/journal/bio
Copyright © 2017 John Wiley & Sons, Ltd.
1

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CHRISTOPHER LESLEE ET AL.
of metal ions to the ligand affects the electron density and thus
influences the fluorescence of the ligand.[77–82] In this work, we
developed a new carbazole–azine probe, CzA, as a sensor for Cu2+
in a dimethyl sulfoxide (DMSO)/H2O mixture via enhancement of
the fluorescence – an off to on transformation.
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2.4
Cell culture and fluorescence imaging
HeLa cells were cultured as a monolayer in Eagle's minimum essential
medium, maintained at 37°C and supplemented with 10% fetal bovine
serum. Cells were treated with CzA (5 μM) in DMSO: PBS buffer and
incubated at pH 7.54 for 30 min. Thereafter, the cells are washed three
times with PBS to remove any excess probe, followed by incubation
with CuCl2 (10 μM in DMSO) for 10 min at 37°C. Cells were monitored
using Nikon fluorescence microscopy.
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2
EXPERIMENTAL
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2.1
Materials and reagents
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2.5
Synthesis
Starting materials and reagents such as N‐ethyl carbazole, dimethyl
formamide, phosphorous oxy chloride and hydrazine monohydrate
were purchased from Sigma‐Aldrich Co. Ltd. (India) and used as
received. Solvents used for synthesis and extraction were obtained
from Sisco Research Laboratories Pvt. Ltd. (India), and were distilled
and dried according to standard procedures before use. Spectroscopic
grade solvents from Central Drug House Ltd. (India) were used for
photophysical studies.
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2.5.1
Precursor 9‐ethyl‐9H‐carbazole‐3‐carbaldehyde (2)
Precursor 2 was synthesized in accordance with formerly reported
methods (Supporting Information).
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2.5.2
N,N′‐bis(9‐ethyl 3‐carbazolylmethylidene)azine
(CzA)[83–85]
Hydrazine monohydrate (0.089 g, 1.79 mmol) was added slowly with
stirring to a solution of precursor 2 (0.4 g, 17.9 mmol) in 40 ml metha-
nol. The resulting mixture was refluxed at 65°C for 12 h under a nitro-
gen atmosphere. The mixture was cooled to room temperature, and
the yellow solid appeared was filtered, washed with MeOH and Et2O,
and dried in a vacuum. N,N′‐bis(9‐ethyl 3‐carbazolylmethylidene)azine
(CzA), yellow solid, yield: 90%; m.p.: 143°C; FTIR (KBr) cm−1: 3050.83
(=C‐H), 2969.84, 2923.56, 2859.10 (N–C), 1734.66, 1607.38 (C–C
monosubstituted phenyl), 1469.49 (C = C conjugated phenyl group),
1340.28 (−C–Nstr), 1232.29, 1131.05, 1024.02 (−HC = N, out of plane
bending vibration), 885.166, 808.992 (C–H, p‐substituted benzene
ring), 741.496, 667.25, 600.717 (C–H phenyl rings). 1H NMR (DMSO‐
d6, 300 MHz), δH (ppm): 1.33 (4H, t, J = 6.9 Hz); 4.49 (2H, q,
J = 7.2 Hz); 7.26 (4H, t, J = 7.5 Hz); 7.50 (4H, t, J = 7.5 Hz); 7.65–7.74
(4H, m); 8.04 (2H, d, J = 8.7 Hz); 8.23 (2H, d, J = 7.8 Hz); 8.65 (2H, s);
8.90 (2H, s). 13C NMR (DMSO‐d6, 400 MHz), δC (ppm): 161.96,
141.73, 140.55, 126.83, 126.25, 125.54, 122.91, 122.65, 121.90,
121.12, 120.05, 110.10, 37.70, 14.23. Mass (ESI‐MS positive mode):
442.6766 (Supporting Information).
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2.2
Measurements
Melting point was determined using a hot‐plate melting point appara-
tus in an open mouth capillary and is uncorrected. 1H NMR spectra
were recorded at 300 MHz on a Bruker FT–NMR spectrometer in
DMSO‐d6 with tetramethylsilane as the internal standard. 13C NMR
spectra were recorded at 400 MHz on a Bruker FT–NMR spectrome-
ter in DMSO‐d6 with tetramethylsilane as the internal standard; any
chemical shift was measured in ppm. ESI‐MS analysis was performed
in positive ion mode on a liquid chromatography‐ion trap mass spec-
trometer (LCQ Fleet, Thermo Fisher Instruments Ltd, USA). FTIR spec-
tra were recorded with a Perkin–Elmer spectrometer equipment
(version 10.03.09) using KBr pellets. UV/Vis and fluorescence
spectroscopy measurements were carried out using a Shimadzu (model
UV1700) spectrophotometer and spectrofluorimeter (model FP‐8200),
respectively, with a quartz cell of path length 10 mm. Fluorescent
quantum yields were determined using Horiba‐FluoroMax 4 spectro-
photometer. Fluorescence imaging was performed using a Nikon con-
focal fluorescence microscope. The syntheses were dry reactions, with
magnetic stirring, performed under a nitrogen gas atmosphere. The
reactions were constantly monitored using thin‐layer chromatography.
Metal solutions were prepared from their nitrate salts. Milli‐Q water
was used throughout the analytical experiments.
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3
RESULT AND DISCUSSION
Synthesis
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3.1
The synthesis of CzA is shown in Scheme 1. Initially, compound 2 was
synthesized by Vilsmeier formylation of N‐ethyl carbazole (1). Probe
CzA was then prepared by refluxing compound 2 with hydrazine
monohydrate in the presence of MeOH. The crude products of 2 and
CzA were purified by column chromatography and dried. The purity
of these compounds was established using FTIR, 1H NMR, 13C NMR
and ESI‐MS (Fig. S1–S4).
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2.3
MTT assay
Cytotoxicity towards and the cell viability of HeLa cells were tested
using the MTT assay procedure by reduction of MTT to formazan crys-
tals. The cells and probe CzA were prepared in 96‐well plates, with
10 μl MTT solution added, and incubated at 37°C for 4 h. DMSO
(100 μl) was added to the formed formazan crystals in each well to dis-
solve the crystals, the plates were shaken for 10 min. The absorbance
was recorded at 570 and 630 nm. Percent cell viability was determined
from the formula: % survival = [live cell number (test)/live cell number
(control)] × 100.
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3.2
UV/Vis spectral studies
UV/Vis titration was undertaken to establish the specificity and the
nature of the association of CzA towards Cu2+ ion (Fig. 1). The cer-
tainty of CzA binding to Cu2+ was shown by competitive absorption

CHRISTOPHER LESLEE ET AL.
3
SCHEME 1 Synthesis of probe CzA
studies with variable metal ions in DMSO: H2O (8: 2), where the other
metal ions, Na+, K+, Ag+, Mn2+, Cd2+, Cu2+, Fe3+, Al3+, Cr3+, Pb2+, Co2+
,
Mg2+, Zn2+ and Ni2+, do not have any significant effect on the absor-
bance. Probe CzA showed strong absorption bands at λmax = 240,
298, 352 and 372 nm on addition of a solution of Cu2+ ions. A
hyperchromic shift in the absorbance was noticed, despite the other
competing metal ions (Fig. S5). Comparatively, on titration of CzA
(10 μM) with a Cu2+ ion solution ranging from 0 to 1 equiv. Cu2+
(10 μM) in the same solvent composition, a simultaneous increase in
intensity was evident. These observations suggest that the response
of probe CzA is highly Cu2+ specific with prominent enhancement in
the absorbance.
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3.3
Fluorescence spectral studies
FIGURE 2 Fluorescence emission spectrum of probe CzA (10 μM)
upon addition of Cu2+ (λex = 372 nm) (0–1 equiv.) in a DMSO: H2O
(8: 2) mixture
Emission spectral analysis of probe CzA in countering various metal
ions in DMSO: H2O (8: 2) was undertaken. The study shows that weak
fluorescent probe CzA (10 μM) resulted in emission enhancement at
556 nm by binding with Cu2+ on excitation at λex = 372 nm (Fig. 2).
There was no apparent deviation in the emission spectra of other metal
ions on encountering Cu2+ ion (Fig. S6). This fluorescence study shows
a 2: 1 stoichiometry, and selectivity towards Cu2+ in the presence of
interfering metal ions. The results coincide with the interference seen
in the absorption studies.
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3.4
Job's plots
To determine the stoichiometric binding ratio of probe CzA with Cu2+
ion, Job's plot analysis was undertaken. A plot of the absorbance at
372 nm over mole fraction was linear with maxima at 0.6 mole frac-
tions of Cu2+ ion, which indicates a 2: 1 stoichiometry; this was con-
firmed by the peak at 946.2936 [CzA]2–Cu2+ in the ESI‐MS analysis
(Fig. S8). Jobs plots from the fluorescence analysis were also in agree-
ment with the former result (Fig. S7). The association constant for the
complex of CzA with Cu2+ was 2.2 × 104 M−1 [86]
.
Emission titration of the probe with Cu2+ ions showed a progres-
sive increase in fluorescence with increasing amounts of Cu2+. After
addition of 1.2 equiv. Cu2+ ion, the mixture reaches saturation point.
In practice, the quantitative detection limit determined from the fluo-
rescence spectral changes varies linearly with the Cu2+ concentration
and was found to have a lower limit of 3.5 × 10−8 M.[87]
Fluorescence recognition by the probe of other metal ions com-
peting with Cu2+ was investigated (Fig. 3). The probe showed no
distinguishable change in fluorescence towards all other metal ions in
the presence of Cu2+, which confirms the ability of the probe to retain
its specificity for Cu2+ ion in a pool of distinct metal ions. The quantum
yields of the probe and the complex were calculated using a Horiba‐
FluoroMax 4 spectrophotometer and found to be 0.02 for probe
FIGURE 1 UV/Vis spectrum of probe CzA (10 μM) upon addition of
Cu2+ (0–1 equiv.) in a DMSO: H2O (8: 2) mixture

4
CHRISTOPHER LESLEE ET AL.
the stretching frequency of the [C–C] mono‐substituted phenyl entity
exhibits a shift from 1607.38 to 1618.96 cm−1. The intense band at
3434.6 cm−1 in [CzA]2–Cu is characteristic of OHstr, which shows the
involvement of water molecules in the binding mechanism, suggesting
a tetrahedral coordinate system for the complex; something similar
was also observed for the mass spectra of the complex (Fig. S11). A
plausible binding mechanism for CzA towards Cu2+ is shown in
Scheme 2.
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3.7
Cell imaging and cytotoxicity studies
A cell viability assay was used to study the cytotoxicity of the probe
CzA towards HeLa cells in DMSO at different doses and times. The
assay revealed that the probe is less toxic to cells and has no apparent
effect on cell viability; the solvent also had no effect on the cells. By
contrast, CzA + Cu2+ complex led to an eventual reduction in the cell
viability of HeLa cells at concentrations >20 μM. This dose‐dependent
cytotoxicity was greater at higher concentrations and resulted in
reduced cell viability. These observations are in accordance with previ-
ous reports, providing support for use of the CzA + Cu2+ complex on
cancer cells (Fig. S13 and S14).[88]
The cytotoxic assay results suggested 10 μM CzA + Cu2+ as opti-
mal for use in cell imaging studies in HeLa cells. To measure the ability
of the probe to detect Cu2+ ions in live cells using confocal microscopy,
MTT‐mediated HeLa cells were incubated with CzA (5 μM) in DMSO
for 30 min at 37°C produce a ‘off’ fluorescent image of the cell. The
cells were then treated with Cu2+ solution for 10 min at the same tem-
perature; this led to a ‘switch on’ green fluorescence in the HeLa cells
(Fig. 4), indicating the formation of the CzA + Cu2+ complex in the cell,
as seen in the optical studies. These observations suggest use of the
CzA probe for the non‐cytotoxic, biocompatible and spontaneous
detection of Cu2+ ion in living cells.
FIGURE 3 Fluorescence response of 10 μM CzA with various metal
ions. Black bars represent the addition of the corresponding metal
ion to CzA. Red bars represent the change of the emission that occurs
upon the subsequent addition of Cu2+ to the above solution
CzA; a much higher quantum yield of 0.51 was observed for CzA upon
addition of Cu2+ ion, which might be due to ICT. The electron density
in the azine unit is located on the [C=N−N=C] moiety, binding of metal
ions to the ligand affects the electron density and thus influences the
fluorescence of the ligand. All the observed photophysical properties
confirm the capability of CzA for the quantitative detection Cu2+, an
‘off–on’‐type probe.
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3.5
Factors influencing fluorescence detection
The probe acts as a rapid fluorescence detector for copper ions. On
prolonging the reaction time to 0.2 min, the emission becomes
brighter, with a sharp rise in intensity. The intensity continues to
increase with further increases in the reaction time to 1 min; becoming
constant thereafter (Fig. S9). The effect of pH on the fluorescent
detection of Cu2+ was tested. Under highly acidic conditions, the fluo-
rescence is high, but decreases as the pH of the solution is increased,
becoming very low and constant at pH 5 and beyond (Fig. S10). In
addition, the reversibility of binding between the Cu2+ ion and the
probe CzA was tested. It was found that EDTA is a proficient reagent
for the reversibility, with fluorescent on to off signaling (Fig. S11).
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3.8
Density functional theory calculations
To confirm the above results, density functional theory (DFT) studies
were undertaken to provide a theoretical basis for CzA sensor behavior
and selectivity towards Cu2+ ion. The DFT calculation was performed
using the Gaussian 09 program (B3LYP and 6‐311G/LANL2DZ
basis).[89] The optimized geometry of the probe CzA and CzA + Cu2+
confirmed 2: 1 stoichiometric binding of CzA to Cu2+ ion through
[N–Cu–N] binding (Fig. S15). The TDDFT calculation was carried out
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3.6
FTIR studies
The above results were further supported by FTIR (Fig. S12). Here, the
stretching frequency of the [N–C] vibration of the CzA probe shifts on
binding with copper ions from 2969.84, 2927.70 and 2859.10 cm−1 to
2965.98, 2923.56 and 2852.93 cm−1 respectively. The value of the
[−HC=N] out of plane bending vibration, 1232.29, 1131.05 and
1024.02 cm−1, shifts slightly to 1261.22, 1095.37 and 1025.94 cm−1
,
respectively, due to ICT towards the Cu2+ ion via lone pair electrons
in the nitrogen atom of the [C=N−N=C] entity. Because of the strong
electron‐donating capacity and ICT ability of the carbazole unit towards
the azine moiety, the band for [=C–H] of the [C=N−N=C] moiety in CzA
at 3060.83 cm−1 is not present for the [CzA]2–Cu complex. In addition,
SCHEME 2 Proposed binding mechanism of CzA towards Cu2+

CHRISTOPHER LESLEE ET AL.
5
FIGURE 4 (a) bright field image of HeLa cells. (b) fluorescence image of HeLa cells incubated with CzA (10 μM) for 2 h at 37°C. (c) fluorescence
image of CzA treated HeLa cells with Cu2+ (10 μM). (d) bright field images of HeLa cells incubated with CzA + Cu2+
from optimized figures. In the frontier molecular orbitals of probe CzA,
the HOMO spread over the π‐cloud of the carbazole fragment and
the LUMO was mounted on the azine unit. In the frontier molecular
orbitals of complex CzA + Cu2+, the HOMO was partly localized on
the π‐cloud of carbazole and azine, whereas the LUMO was towards
the Cu2+ ion. The energy gap of CzA + Cu2+ was lower than that of CzA
(Fig. 5), which promotes easy transition. Therefore, from the DFT calcu-
lation, it is clear that the probe and the complex CzA + Cu2+ are involved
FIGURE 5 Calculated (B3LYP/6‐31G*) structure for CzA and CzA + Cu2+ complex

6
CHRISTOPHER LESLEE ET AL.
[23] E. Babu, S. Singaravadivel, P. Manojkumar, S. Krishnasamy, S.
in π–π* transition from HOMO to LUMO, with greater opportunity for
ICT to govern probe CzA on approaching Cu2+ ion.
Rajagopal, Anal. Bioanal. Chem. 2013, 405, 6891.
[24] E. Babu, P. M. Mareeswaran, S. Singaravadivel, J. Bhuvaneswari, S.
Rajagopal, Spectrochim. Acta A 2014, 130, 553.
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4
CONCLUSION
[25] K. M. Vengaian, C. D. Britto, K. Sekar, G. Sivaraman, S. Singaravadivel,
RSC Adv. 2016, 6, 7668.
[26] O. Sunnapu, N. G. Kotla, B. Maddiboyina, S. Singaravadivel, G.
We have designed and synthesized a carbazole‐based azine derivative
CzA probe that was shown to be a highly selective, sensitive sensor for
Cu2+ ion. The sensitivity of probe CzA for Cu2+ was demonstrated in
living cells, and a cell toxicity assay revealed that CzA can be used
for selective imaging of Cu2+ ions in living cells.
Sivaraman, RSC Adv. 2016, 6, 656.
[27] G. Sivaraman, T. Anand, D. Chellappa, RSC Adv. 2013, 3, 17029.
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Y. S. Lee, D. G. Churchill, Inorg. Chem. 2011, 50, 5351.
C. Denzil Britto thanks the DST‐SERB for project fellowship. K. Sekar
thanks the DST‐SERB Fast Track programme for financial support
(Grant No. SB/FT/CS–062/2013), New Delhi, India.
[31] L. Tang, P. Zhou, Z. Huang, J. Zhao, M. Cai, Tetrahedron Lett. 2013, 54,
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