Naked-eye fluorescent sensor for Cu(II) based on indole conjugate BODIPY dye

Article abstract of DOI:10.1016/j.poly.2016.05.056

Graphical abstract A novel indole mono-styryl BODIPY was synthesised and characterized for the colorimetric recognition of Cu²⁺. It was also revealed from this study 3 can passively diffuse through cell membrane and stain both bacterial and mammalian cells even in very low concentrations. BODIPY-indole 3 retains inside the cells in cytometry and can be further modified for other applications.

Full text of DOI:10.1016/j.poly.2016.05.056

Accepted Manuscript
Naked-Eye Fluorescent Sensor for Cu (II) Based on Indole Conjugate BODIPY
Dye
Sureyya O. Tümay, Elif Okutan, Ibrahim F. Sengul, Emrah Özcan, Hakan
Kandemir, Tuğrul Doruk, Metin Çetin, Bünyemin Çoşut
PII:
S0277-5387(16)30209-1
DOI:
http://dx.doi.org/10.1016/j.poly.2016.05.056
POLY 12030
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
4 March 2016
29 May 2016
Please cite this article as: S.O. Tümay, E. Okutan, I.F. Sengul, E. Özcan, H. Kandemir, T. Doruk, M. Çetin, B.
Çoşut, Naked-Eye Fluorescent Sensor for Cu (II) Based on Indole Conjugate BODIPY Dye, Polyhedron (2016),
doi: http://dx.doi.org/10.1016/j.poly.2016.05.056
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Naked-Eye Fluorescent Sensor for Cu (II) Based on Indole
Conjugate BODIPY Dye
Sureyya O. Tümaya , Elif Okutana, Ibrahim F. Sengula, Emrah Özcana, Hakan Kandemirb,
Tuğrul Dorukc, Metin Çetinc, Bünyemin Çoşuta*
Contribution from:
a Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze, Kocaeli,
Turkey
b Department of Chemistry, Faculty of Art and Science, Namık Kemal University, Tekirdag,
Turkey
c Department of Molecular Biology and Genetics, Faculty of Science, Gebze Technical
University, Gebze, Kocaeli, Turkey
*Author for correspondence:
Dr. Bünyemin Çoşut, Department of Chemistry, Gebze Technical University, P.O.Box: 141,
Gebze 41400, Kocaeli, Turkey
Tel:
00 90 262 6053015
00 90 262 6053105
bc@gtu.edu.tr
Fax:
e-mail:

Abstract
A novel on/off fluorescent indole\ BODIPY- based Cu2+ chemosensor (3) was
synthesized by Knoevenagel condensation of BODIPY derivative 1 with 2-methyl-indole-3-
carbaldehyde 2. The identity of compound 3 was confirmed by 1H NMR, 13C NMR, FT-IR,
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and single crystal
x-ray diffraction techniques. Fluorescent chemosensor (3) is found to be highly selective and
sensitive for detection of Cu2+ with a color change from purple to yellow. The optical sensor
features for the Cu2+ complex of 3 were investigated by UV−vis and fluorescence
spectroscopy. The addition of Cu2+ caused quenching of fluorescence intensity and the
detection limit was calculated to be 0.124 µM. Also, the stoichiometry ratio of 3+Cu2+ was
obtained 2:1 by Job’s plot. Live cell image, flow cytometry and cytotoxic properties of
compound 3 were examined.
Keywords: Borondipyrromethenes, Indole, Cu2+ ion sensor, Fluorescence, Chemosensor,
2

1. Introduction
The design and construction of fluorescent chemosensors with high selectivity and sensitivity
through metal ions such as iron, aluminium and copper has been the subject of intense study
because of their potential application in supramolecular chemistry, organic chemistry, drug
delivery, biological chemistry and environmental research [1-3]. Also, they have a significant
sensitivity and selectivity for detecting metal ions in both aqueous and non-aqueous media [4-
7] many chemosensors have been developed specific for Ca2+, Hg2+, Cu2+, Zn2+ or other
transition metals [8-13]. The selective recognition and sensing of soft metal ions is quite
important as these cations play an important role in various biological processes, including
iron absorption, haemopoiesis, various enzyme-catalyzed and redox reactions [14]. Among
these transition metals, copper is known as third abundant transition metal element in human
body and generally found as Cu (II) in natural water [15]. It is one of the most essential trace
elements of importance for both physical and mental health and serves as a key co-factor for a
wide variety of enzymes in living organism [16-19]. Copper overload can lead to vomiting,
lethargy, increased blood pressure and respiratory rates, acute haemolytic anaemia, liver
damage, neurotoxicity and neurodegenerative disease [20-21]. Furthermore, it can disrupt
natural ecosystems due to their adverse effects on microorganism [22]. Recently, some fully
water soluble fluorescence probes have been reported here represents the first example of cell-
elective imaging. [23-25]. Consequently, much attention has been drawn to the development
of highly selective fluorescent chemosensor for Cu2+ to satisfy the biological and
environmental applications [26]. The limit for copper in drinking water, as set by the US
Environmental Protection Agency (EPA) is 1.3 ppm (20 µM). Also, the average concentration
of blood copper in the normal group is 5.7-23.6 µM.
Among the fluorescent sensors, boradiazaindacene (BODIPY) is one of the most important
highly fluorescent dyes and extensively studied due to its excellent photo physical properties
3

such as high fluorescence quantum yield, large extinction, high photo-stability, long
absorption and fluorescence wavelengths [27]. These dyes are widely used as optical
chemosensor, fluorescent bio-labelling reagents, light harvesting materials and photodynamic
therapy reagents have been broadly investigated [28-31]. In particular, BODIPY-based
metallic cation sensors have attracted great deal of attention [32]. BODIPY based fluorescent
chemosensor derivatives for selective detection of metal ions such as Fe3+, Al3+ and Cu2+ was
recently reported [33-34].
Indole is an aromatic heterocycle that is widely distributed in nature [35]. There has been a
significant recent interest in synthesizing indole and its derivatives because of the utility of
these compounds as fluorescence probe and also displayed wide range of biological activity
such as anti-fungal, anti-inflammatory and anti-tumor properties [38-40].
Herein, we reported the facile synthesis of a new fluorescence chemosensor based on indole-
conjugate mono-stryl BODIPY 3 which was served as reliable, more sensitive, more selective
and more soluble for Cu2+ sensor. Compound 3 and Cu2+ ion complex were detected by UV-
visible absorption and fluorescence emission methods. Also, properties of compound 3 in live
cells, flow cytometry and bacterial and mammalian cells were investigated.
2. Experimental
2.1. Materials
The deuterated solvent (CDCl3) for NMR spectroscopy, silica gel, dichloromethane and metal
chlorides were provided from Merck. Following chemicals were obtained from Sigma
Aldrich;
2,4-dimethylpyrrole,
trifluoroacetic
acid,
2,3-Dichloro-5,6-dicyano-1,4-
benzoquinone, trimethylamine, Boron trifluoride diethyl etherate, glacial acetic acid,
piperidine, 2-methylindole, phosphorus(V)oxychloride, N,N-dimethylformamide, benzene and
4

1,8,9-Anthracenetriol for the MALDI matrix was obtained from Fluka. All other chemicals
used for the synthesis were reagent grade unless otherwise specified.
2.2. Equipment
Electronic absorption spectra were recorded with a Shimadzu 2101 UV spectrophotometer in
the UV-visible region. Fluorescence excitation and emission spectra were recorded on a
Varian Eclipse spectrofluorometer using 1 cm pathlength cuvettes at room temperature. The
fluorescence lifetimes were obtained using Horiba- Jobin-Yvon-SPEX Fluorolog 3-2iHR
instrument with Fluoro Hub-B Single Photon Counting Controller at an excitation wavelength
of 470 nm. Signal acquisition was performed using a TCSPC module. Mass spectra were
acquired in linear modes with average of 50 shots on a Bruker Daltonics Microflex mass
spectrometer (Bremen, Germany) equipped with a nitrogen UV-Laser operating at 337 nm. 1H
and 13C NMR spectra were recorded in CDCl3 solutions on a Varian 500 MHz spectrometer.
Analytical thin layer chromatography (TLC) was performed on silica gel plates (Merck,
Kieselgel 60 Å, 0.25 mm thickness) with F254 indicator. Column chromatography was
performed on silica gel (Merck, Kieselgel 60 Å, 230-400 mesh). Suction column
chromatography was performed on silica gel (Merck, Kieselgel 60 Å, 70-230 mesh).
2.3. X-ray data collection and structure refinement
Unit cell measurements and intensity data collection were performed on an Bruker APEX II
QUAZAR three-circle diffractometer using monochromatized Mo Kα X-radiation (λ =
0.71073 Å). Indexing was performed using APEX2 [41]. Data integration and reduction were
carried out with SAINT V8.34A [42]. Absorption correction was performed by multi-scan
method implemented in SADABS V2014/5 [43]. The structures were solved and refined using
the Bruker SHELXTL Software Package [44]. All non-hydrogen atoms were refined
anisotropically using all reflections with I > 2σ(I). The C-bound H atoms were positioned
5

geometrically and refined using a riding mode. The N-bound H atoms were located from the
difference Fourier map and restrained to be 0.89 Å from N atom using DFIX and their
position were constrained to refine on their parent N atoms with Uiso(H)= 1.2Ueq(N).
Crystallographic data and refinement details of the data collection are given in Table 1. The
final geometrical calculations and the molecular drawings were carried out by Platon (version
1.17) and Mercury CSD (version 3.5.1) program [45-46].
2.4. Live Cell Imaging and Flow Cytometry
Bacterial and mammalian cells were analysed by Nikon Eclipse 80i fluorescence microscopy
utilizing TritC filter (Ex:540/25 nm and Em:605/55 nm) and mammalian cells were analysed
by BD Accuri C6 flow cytometer. For bacterial imaging Bacillus thuringiensis (Bt) vegetative
cells and for mammalian cell imaging HuH7 cells were stained by 0.1 µM of compound 3 for
15 minutes. Flow cytometry analysis of HuH7 performed after staining of cells with 0.1 and 5
µM of compound 3 for 15 min.
2.5. Cytotoxicity Assays
Bacterial toxicity of 0.1 µM of compound 3 was analysed against 4 different bacterial cells
namely, Pseudomonas Aeruginosa, Staphylococcus Aureus, Bacillus Cereus, Escherichia
Coli. Colony forming units calculated and compared to DMSO treated control bacterial cells
after 24 hours incubation. The viability of HuH7 cells were examined by The CellTiter 96®
Aqueous One Solution Cell Proliferation Assay (Promega, USA) in 96-well culture plate
format. HUH7 cells were treated with DMSO control and various concentrations (0.1, 0.5, 1,
5, 100 µM) of compound 3. Absorbance was recorded at 490 nm using Varioskan™ Flash
Multimode Reader (Thermo Scientific, USA).
6

3. Synthesis
Compound 1 was prepared with a minor revision by the procedure described previously [47].
The synthesis method of compound 2 was as follows based on literature [48] (Scheme 1).
3.1. Synthesis of compound 3
A 100 mL round bottomed flask was charged with benzene (50 mL) and purged with argon
for 15 min. To a solution of BODIPY 1 (100 mg, 0.3 mmol) and 2-methyl-indole-3-
carbaldehyde 2 (48 mg, 0.3 mmol) in benzene was added piperidine (0.2 mL) and glacial
acetic acid (0.2 mL). The solution was refluxed in a Dean-Stark apparatus for 6h. The
resultant was concentrated by evaporation, and the mixture was then extracted several times
with dichloromethane/water. The combined extracts were dried over Na2SO4, evaporated
under reduced pressure and purified by silica gel column chromatography using
dichloromethane-hexane (5:2) as eluent to afford the compound 3, yield: 70 mg, 50%.
Spectral data of 3: (Found: C 74.89, H 5.57, N 8.98 %, C29H26BF2N3 (465) requires C 74.85,
H 5.63, N 9.03%). 1H NMR (500 MHz, CDCl3) δ 8.18 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.71
(d, J = 16.2 Hz, 1H), 7.48 (d, J = 6.3 Hz, 3H), 7.45 (s, 1H), 7.33 (d, J = 6.9 Hz, 2H), 7.20 (t, J
= 7.5 Hz, 1H), 6.64 (s, 1H), 5.97 (s, 1H), 2.62 (s, 3H), 2.56 (s, 3H), 1.44 (s, 3H), 1.38 (s,
3H).13C NMR (126 MHz, CDCl3) δ 130.39, 129.10, 128.86, 128.67, 128.64, 123.04, 123.03,
122.68, 122.63, 122.56, 122.52, 121.68, 121.64, 120.46, 120.44, 120.37, 110.72, 110.70,
110.16, 110.14, 110.13, 110.07, 48.00, 47.64, 29.88, 29.86, 14.87, 14.36, 12.87. IR (ATR)
ʋmax (cm-1): 3378 (secondary NH), 2955 (aromatic CH), 2918 (aliphatic CH), 2852 (aliphatic
CH), 1605 (aromatic C=C), 1539 (aromatic C=C), 1503 (B-F), 1455 (aliphatic CH), 1370,
(aliphatic CH), 1291 (C-N), 1194, 1146. MS (MALDI-TOF) m/z (%): 465 [M]+, 446 [M-F]+.
4. Results and Discussion
7

4.1. Synthesis and Structural Characterization
The synthetic route adopted here to prepare BODIPY-indole 3 is outlined in Scheme 1.
Compound 3 was simply prepared by Knoevenagel condensation of a BODIPY derivative 1
with 2-methyl-indole-3-carbaldehyde 2 in a Dean–Stark apparatus using standard conditions
in a moderate yield. The targeted mono-styryl BODIPY-indole compound 3 were easily
separated from the side products via silica gel column chromatography. The structure of 3 was
confirmed by FT-IR, 1H, 13C NMR, MALDI- MS and elemental analyses and the results were
consistent with the assigned formulations. The mass spectrum of compound 3 was obtained
by MALDI-TOF MS using dithranol as a MALDI matrix, and the spectrum revealed the peak
groups representing the molecular ions at 465 Da and molecular ion rupture flour at 446 Da
1
(Fig S1). The H NMR of compound 3 showed three sets of methyl protons of BODIPY and
one for indole unit appeared ~ 1.4 ppm and ~ 2.6 ppm, respectively. The β- pyrrolic signals of
the compound 3 were noted as sharp singlets at 5.97 and 6.64 ppm. For the mono-styryl 3, one
of the β- pyrrolic protons is shifted to 6.64 ppm as the other remains at ~ 6 ppm. Aromatic
protons of BODIPY and indole units were located at ~7-8 ppm region. In addition, the
observed 16 Hz proton-proton coupling constant at 7.71 ppm proved an E conformation of the
double bonds as expected. The N-H proton appeared as a singlet at 8.18 ppm (Fig S2). The
solid state structure of the molecule 3 was further confirmed by X-ray crystallography (Fig.
1). Unit 3 crystallizes in the triclinic space group P-1 in the asymmetric unit cell (Table 1).
8

O
H
(a) TFA, DCM
(b) DDQ
(c) NEt3, BF3 OEt2
+
N
F
N
F
N
B
H
(1)
O
H
POCl3, DMF
reflux, 6h
N
N
H
H
(2)
O
Acetic acid
Piperidine
Benzene
H
+
Dean-Stark
N
N
F
N
F
N
F
N
F
H
B
B
2
( )
(1)
3
( )
NH
Scheme 1. Chemical structure and synthetic pathway of BODIPY-indole compound (3)
9

Scheme 2. Proposed binding mechanism of 3 with Cu2+
Fig. 1. The asymmetric unit of compound 3. Displacement ellipsoids are drawn at the 30%
probability level. H-atoms are shown as small spheres of arbitrary radii.
10

Table 1. Crystal data and refinement parameters for 3.
Empirical Formula
Formula weight (g. mol-1)
Temperature (K)
Wavelength (Å)
Crystal system
Space group
C29H26N3BF2
465.34
293(2)
0.71073
Triclinic
P -1
9.9821(5)
a (Å)
11.4520(5)
12.5022(5)
85.206(3)
b (Å)
c (Å)
α(°)
β(°)
67.687(3)
67.139(3)
γ(°)
0.072 x 0.144 x 0.305
1214.89(10)
2
Crystal size (mm)
V (Å3)
Z
ρcalcd (g. cm−3)
µ (mm−1)
1.272
0.085
488
F(000)
3.42 – 25.68
-12/12, -13/13, -15/15
16342
θ range for data collection (°)
h/k/l
Reflections collected
Independent reflections
Absorption correction
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Largest diff. peak and hole (e.Å−3)
4603 [R(int) = 0.1028]
Multi-scan
4603 / 0 / 324
1.008
R1= 0.0587, wR2= 0.1263
R1= 0.1242, wR2= 0.1528
0.281 and -0.270
11

4.2. Spectral Studies
The UV-Vis spectroscopic experiments of BODIPY-indole 3 were measured in acetonitrile
with dilute solutions of 5x10-6 mol.dm-3 in the presence of 1 equivalent of various metal
cations (Li+, Na+, K+, Cs+ ,Mg2+, Ca2+, Ba2+, Hg2+ , Pb2+, Mn2+, Cd2+, Ag+, Ni2+, Cu2+, Zn2+,
Co2+, Al3+, Cr3+, Fe3+) as shown in Fig. 2. Compound 3 exhibited an absorption band at ~ 593
nm, characteristic for the mono-styryl BODIPY (Fig S4a) [49]. Upon addition of 1 equivalent
of various metal ions, only Cu2+ induces the peak decreased which means 3 had higher
binding affinity towards Cu2+ than other surveyed metal ions as a result of Pearson's Hard-
Soft Acid-Base classification system in which Cu2+ acid and R2NH bases are in the borderline
[50].
Fig. 2. UV–Vis absorption change profiles of compound 3 (5.0 µM) in CH3CN with various
metal ions (5.0 µM).
In order to obtain more information about binding mechanism of compound 3 towards Cu2+,
spectrometric titration experiments were performed in the presence of Cu2+. While the
sequential addition of Cu2+ ions from 0 to 2 equivalents to the solution of compound 3
12

showed a gradual increase in absorbance at 506 nm, the other band at 593 nm showed a
decrease in absorbance signal (Fig. 3). Consequently, 3 could serve as a sensitive fluorescent
chemosensor for Cu2+ cations. The changes in the absorption curve of 3 could be assigned to a
charge transfer transition occurring between the ligand and metal ion.
Fig. 3. UV–Vis absorption spectrum of 3 (5.0 µM) with gradual addition of Cu2+ in CH3CN
solution.
The fluorescence sensing of BODIPY-indole 3 was investigated on the aforementioned metal
cations groups. The emission spectrum of 3 displayed a strong emission band at 635 nm when
excited at 570 nm (Fig S4b). Except for Cu2+, all tested metal ions caused minor changes in
the emission band at 630 nm (characteristic for mono-styryl Bodipy emission) as shown in
13

Fig. 4. Upon addition of 1 equivalent of Cu2+ to the solution of 3, remarkable decreasing of
the fluorescent intensity of 3 is observed.
Fig. 4. Fluorescence responses of 3 (5.0 µM) in CH3CN solution upon the addition of 1
equivalent of various metal ions (Excitation wavelength= 570 nm).
Furthermore, competition ion studies were also performed for 3 in the presence of Cu2+ at 5
µM that mixed with 5 µM of the tested metal cations such as Li+, Na+, K+, Cs+, Mg2+, Ca2+,
Ba2+, Hg2+, Pb2+, Mn2+, Cd2+, Ag+, Ni2+, Cu2+, Zn2+, Co2+, Al3+, Cr3+ and Fe3+ (Fig. 5). It was
clearly found that the interference of other ions was insignificant during the detection of Cu2+.
Fluorescence titration experiments were also carried out in order to understand the binding
mode of BODIPY-indole 3 with Cu2+ (Fig. 6). The fluorescence titration profile of compound
3 with Cu2+ in the range of 0 to 1.1 equivalents showed the Cu2+ detection limit. Time
resolved fluorescence studies (using a 674 nm laser source and monitoring 630 nm as λems
)
revealed a single exponential decay for 3 emissions and the lifetime was found to be 3.806
14

( 0.006) ns for molecule 3 and 0.261 ( 0.009) ns for molecule 3+Cu2+. Changes in the
lifetime from τ = 3.806 ns to τ = 0.261 ns clearly showed binding of Cu2+ to 3. The detection
limit of 3 for Cu2+ ion by fluorescence changes was calculated on the basis of 3σ /K [51-52]
and found to be 0.124 µM pointing the high detection sensitivity (Fig. 7). These results
suggested that 3 can be used for the selective detection of Cu2+ in acetonitrile solution.
Fig. 5. Metal ions selectivity of molecule 3 (5.0 µM) in CH3CN solution. The red bars
represent the fluorescent intensity of only 3 and the blue bars represent the fluorescent
intensity of 3 and various ions (Excitation wavelength= 570 nm).
15

Fig. 6. Fluorescence titration of compound 3 (5.0 µM) with various concentration of Cu2+ in
CH3CN solution (Excitation wavelength= 570 nm). Inset: Fluorescence decay profile of
compound 3 in the presence and absence of Cu2+ using laser excitation source of 674 nm.
Fig. 7. Fluorescence intensity of 3 versus Cu2+ ions. [compound 3] = 5 µM
16

Job’s plot analyses were to use for investigating the stoichiometry between compound 3 and
Cu2+ (Fig. 8). A Job’s plot of 3 and Cu2+ indicated that the target complex exhibited a 2:1
ligand–metal ratio with a good linear relationship. This 2:1 stoichiometry was further
confirmed by the MALDI-MS data with a peak of [3+Cu]+ at m/z 993.6 (Fig. S5). The FT-IR
spectra of the synthesized molecule 3 and target complex (molecule 3-Cu2+) are also given in
(Fig. S6). PET is often the cause of fluorescence quenching, when the PET process is
followed by a nonluminescent process returning to the ground state [53]. The intense
fluorescence of 3 is quenched by oxidative PET from the excited-state fluorophore to the
indole moiety coordinated to Cu2+. Cu2+ formed the most Stable 2:1 complex with 3 (Scheme
2). The structure of the 3 was confirmed by IR and the spectrum illustrated the N-H stretching
band of indole secondary amine at ~3400 cm-1 and the aromatic C–H stretching bands were
~2900 cm-1. After the complexation between compound 3 and Cu2+; stretching vibration of
secondary amine changed and shows two streches at 3440 cm-1 and 3340 cm-1 due to the
complexation effect.
17

Fig. 8. The Job’s plot for compound 3 and Cu 2+ (Excitation wavelength= 570 nm).
As shown in Fig. 9, the association constant K of the targeted complex was then calculated to
be 1.53x108 M-1 by the Benesi–Hildebrand equation.
Fig. 9. Plot of 1/F-F0 against 3/[ Cu 2+] for compound 3 in CH3CN solution.
Moreover, 3 can detect Cu2+ in visual, and the visible color change can be easily observed by
the addition of Cu2+. In the presence of Cu2+, a colour change of 3 was observed from purple
to yellow followed by green under both sunlight and UV light (Fig. 10). Precision of the
sensor is an important analytical parameter for sensor application. Therefore, ten
measurements were performed for 5x10-6 M of Cu2+ under the same conditions. The relative
standard deviation (RSD %) was calculated as 2.03 %. As a result, high reproducibility can
provided for selected sensor.
18

Fig. 10. Solution colour of molecule 3 exposed to various types of cations under sunlight (top)
and UV light (bottom).
The photo stability is preferred for many fuorescent chemosensor dyes. The photo stability of
3 in the absence and presence of Cu2+ ion was investigated for 30 minutes in acetonitrile
solution (Fig. 11). The fluorescence intensity of 3 and 3-Cu2+ remain the same as initial value.
Consequently, it was found that compound 3 and compound 3-Cu2+ complexes have excellent
photo stability.
19

Fig. 11. The photo stability of 3 and 3-Cu2+. The change of fluorescence intensity with
irradiation time for molecule 3 and compound 3-Cu2+.
4.3. Biological properties
BODIPY-indole 3 (0.1 µM) was used to explore the cellular staining of mammalian and
bacterial cells. After 15 minutes incubation, both mammalian cells (Fig. 12) and bacterial
cells (Fig 13) were stained efficiently. According to literature, it is quite important to stain
cell without any damage for live cell imaging applications [54]. The fluorescence properties
of 3 was analysed using flow cytometry and the results showed brighter fluorescence signals
which were obtained from the stained cells in regard to increasing doses of 3 (0.1 and 5 µM)
(Fig. 14). The toxicity of 3 was examined using bacterial and mammalian cell cultures. In this
regard, bacterial cells were subjected to 100 µM of 3 and no toxic effect was observed (Fig.
15a). HuH7 cells were subjected to various concentrations (0.1, 0.5, 1, 5, 100 µM) of
compound 3 and cytotoxicity was very low when compared to DMSO control cells (Fig.
15b). In the light of these findings, molecule 3 can serve as a biocompatible fluorescent agent
20

and stain bacterial/mammalian cells with low concentrations and it also showed promising
potential for flow cytometry applications.
A
B
Fig. 12. Fluorescent image of 3 in mammalian cells. HuH7 cells were treated with 0.1 µM of
compound 3 and incubated for 15 minutes. A. Bright field image B. TritC filtered image.
21

A
B
Fig. 13. Fluorescent image of molecule 3 in bacterial cells. Bacillus thuringiensis cells were
treated with 0.1 µM of 3 and incubated for 15 minutes. A. Bright field image. B. TritC filtered
image.
Fig. 14. Flow cytometry analysis of fluorescent 3 stained mammalian cells. HuH7 cells were
stained with 0.1 µM and 5 µM of compound 3. Overlay histogram of cells, unstained, stained
22

with both 0.1 µM of and 5 µM of 3. Cell excited with red laser and data obtained from FL4:
675/25 nm filter.
23

A
B
Fig. 15. Cytotoxic effects of 3 on bacterial and mammalian cells. A. Bacterial cells were
treated with 100 µM of compound 3 and incubated for 24 hours. Viable cells count compared
with DMSO treated control cells and calculated as cfu/ml. B. MTS assay results of HuH7
cells which were treated with 0.1, 0.5, 1, 5, 100 µM of compound 3 and incubated for 24
hours. Percentage of viable cells compared with DMSO treated control cells (set at 100%).
Bars represent the corresponding standard deviations (n=3).
24

5. Conclusion
In conclusion, a novel indole mono-styryl BODIPY was successfully synthesised and
characterized for the recognition of Cu2+. It was found that 3 showed a remarkable selective
and sensitive response towards Cu2+ by using UV-vis and fluorescence methods. According to
Job’s plot, binding stoichiometry was found by fluorescence method as 2:1 3+Cu2+. The
detection limit of compound 3 was found to be 0.124 µM for Cu2+. It was also revealed from
this study 3 can passively diffuse through cell membrane and stain both bacterial and
mammalian cells even in very low concentrations. BODIPY-indole 3 retains inside the cells in
cytometry and can be further modified for other applications.
Acknowledgment
The CCDC number 1412690 contains the supplementary crystallographic data (CIF) for this
article. These data can be obtained free of charge from the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (Fax: +44-1223-336-033; e-mail: deposit@ccdc.cam. ac.uk or
http://www.ccdc.cam.ac.uk).
References
[1] Gou C, Qin SH, Wu HQ, Wang Y, Luo J, Liu XY. A highly selective chemosensor for
Cu2+ and Al3+ in two different ways based on Salicylaldehyde Schiff. Inorg Chem Commun
2011; 14:1622-25.
[2] Ghosh K, Saha I. Selective sensing of Zn(II) ion by a simple anthracene-based tripodal
chemosensor. Tetrahedron Lett 2010; 51:4995-99.
[3] Azadbakht R, Keypour H. A new Schiff base system bearing two naphthalene groups as
fluorescent chemodosimeter for Zn2+ ion and its logic gate behaviour. Spectrochim Acta A
2012; 85:293-97.
25

[4] Wang LN, Qin WW, Liu WS. A sensitive Schiff-base fluorescent indicator for the
detection of Zn2+. Inorg Chem Commun 2010; 13:1122-25.
[5] Bhalla V, Tejpal R, Kumar M. Terphenyl based fluorescent chemosensor for Cu2+ and F−
ions employing excited state intramolecular proton transfer. Tetrahedron Lett 2011; 67:1266-
71.
[6] Udhayakumari D, Saravanamoorthy S, Ashok M, Velmathi S. Simple imine linked
colorimetric and fluorescent receptor for sensing Zn2+ ions in aqueous medium based on
inhibition of ESIPT mechanism. Tetrahedron Lett 2011; 52: 4631-35.
[7] Jiang JQ, Gou C, Luo J, Yi CL, Liu XY. A novel highly selective colorimetric sensor for
Ni(II) ion using coumarin derivatives. Inorg Chem Commun 2012; 15:12-15.
[8] Delavaux NB, Maynadié J, Lavabre D, Fery FS. Ferrocenyl Compound as a
Multiresponsive Calcium Chemosensor with Remarkable Fluorescence Properties in CH3CN.
Inorg Chem 2006; 45:5691-02.
[9] An JM, Yang ZY, Yan MH, Li TR. A novel off–on fluorescence chemosensor for Ca2+
based on Rhodamine–Coumarin Schiff base derivative. J Lumin 2013; 139:79-83.
[10] Singh TS, Paul PC, Pramanik HA. Fluorescent chemosensor based on sensitive Schiff
base for selective detection of Zn2+. Spectrochim Acta A 2014; 121:520-26.
[11] Cao W, Zheng XJ, Sun JP, Wong WT, Fang DC, Zhang JX, Jin LP. A Highly Selective
Chemosensor for Al(III) and Zn(II) and Its Coordination with Metal Ions. Inorg Chem 2014;
53:3012-21.
[12] Mahato P, Saha S, Das A. Rare Example of TICT Based Optical Responses for the
Specific Recognition of Cr3+ by a 2,2′:6′,2″-Terpyridine Derivative and Demonstration of
Multiple Logic Operations. J Phys Chem C 2012; 116:17448-57.
26

[13] Dong M, Ma TH, Zhang AJ, Dong YM, Wang YW, Peng Y. A series of highly sensitive
and selective fluorescent and colorimetric “off-on” chemosensors for Cu (II) based on
rhodamine derivatives. Dyes Pigm 2010; 87:164-72.
[14] Malvankar PL, Shinde VM. Ion-pair extraction and determination of copper (II) and
zinc(II) in environmental and pharmaceutical samples. Analyts 1991; 116: 1081-84.
[15] Li Q, Gou Y, Shao S. A BODIPY based fluorescent chemosensor for Cu(II) ions and
homocysteine/cysteine. Sens Actuators B 2012; 171:872-77.
[16] Narayanaswamy N, Govindaraju T. Aldazine-based colorimetric sensors for Cu2+ and
Fe3+. Sens Actuators B 2012; 161:304-10.
[17] Maria JCM, Colin F, Barry HW, Graham HRC. A new use for an old molecule: N-
phenyl-2-(2-hydroxynaphthalen-1-ylmethylene)hydrazinecarbothioamide as a ratiometric
‘Off–On’ fluorescent probe for iron. Tetrahedron Lett 2012; 53;670-73.
[18] Sahin AC, Tokgoz I. A novel solidified floating organic drop microextraction method for
preconcentration and determination of copper ions by flow injection flame atomic absorption
spectrometry. Anal Chim Acta 2010; 667:83-87.
[19] Linder MC, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin
Nutr 1996; 63:797-811.
[20] Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene
for Menkes disease and evidence that it encodes a copper−transporting ATPase. Nat Genet
1993; 3:7-13.
[21] Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a
−type ATPase similar to the Menkes gene. Nat
putative copper transporting P
5:327-37.
Genet 1993;
[22] Kramer R. Fluorescent Chemosensors for Cu2+ Ions: Fast, Selective, and Highly
Sensitive. Angew Chem Int Ed 1998; 37:772-73.
27

[23] Kai BL,Zang Y, Wang H, Li J, Chen GR, James TD, He XP and Tian H. Hepatoma-
selective imaging of heavy metal ions using a ‘clicked’ galactosylrhodamine probe. Chem.
Commun. 2014; 50:11735-37
[24] Shi DT, Zhou D, Zang Y, Li J, Chen GR, James TD, He XP and Tian H. Selective
fluorogenic imaging of hepatocellular H2S by a galactosyl azidonaphthalimide probe
Chem. Commun. 2015; 51: 3653-55
[25] Dong L, Zang Y, Zhou D, He XP, Chen GR, James TD, Li J, and Tian H. Glycosylation
enhances the aqueous sensitivity and lowers the cytotoxicity of a naphthalimide zinc ion
fluorescence probe Chem. Commun. 2015; 51: 11852-55
[26] Liu WY, Li HY, Lv HS, Zhao BX, Miao JY. A rhodamine chromene-based turn-on
fluorescence probe for selectively imaging Cu2+ in living cell. Spectros Chim Acta A Mol
Biomol Spectros 2012; 95:658-63.
[27] Ulrich G, Ziessel R, Harriman A. The Chemistry of Fluorescent Bodipy Dyes:
Versatility Unsurpassed. Angew Chem Int Ed 2008; 47:1184-1201.
[28] Cheng T, Xu Y, Zhang S, Zhu W, Qian X, Duan L. A Highly Sensitive and Selective
OFF-ON Fluorescent Sensor for Cadmium in Aqueous Solution and Living Cell. J Am Chem
Soc 2008; 130:16160-61.
[29] Yee M, Fas SC, Stohlmeyer MM, Wandless TJ, Cimprich KA. Cell-permeable, Activity-
based Probe for Protein and Lipid Kinases. J Biol Chem 2005; 280:29053–59.
[30] Goeb S, Ziessel R. Convenient Synthesis of Green Diisoindolodithienylpyrromethene−
Dialkynyl Borane Dyes. Org Lett 2007; 9:737-40.
[31] Gorman A, Killoran J, O’Shera C, Kenna T, Gallagher WM, O’Shea DF. In Vitro
Demonstration of the Heavy-Atom Effect for Photodynamic Therapy. J Am Chem Soc 2004;
126:10619-31.
28

[32] Zhang X, Xiao Y, Qian X. A Ratiometric Fluorescent Probe Based on FRET for Imaging
Hg+2 Ions in Living Cells. Angew Chem Int Ed 2008; 47:8025-29.
[33] Kursunlu AN, Guler E, Ucan HI, Boyle RW. A novel Bodipy-Dipyrrin fluorescent probe:
Synthesis and recognition behaviour towards Fe (II) and Zn (II). Dyes Pigm 2012; 94:496-
502.
[34] Kursunlu AN. A fluorescent “turn on” chemosensor based on Bodipy–anthraquinone for
Al(III) ions: synthesis and complexation/spectroscopic studies. RSC Adv 2015; 5:41025-32.
[35] Garg NK, Caspi DD, Stoltz BM. Development of an Enantiodivergent Strategy for the
Total Synthesis of (+)- and (−)-Dragmacidin F from a Single Enantiomer of Quinic Acid. J
Am Chem Soc 2005; 127:5970-78.
[36] Sakemi S, Sun HH. Nortopsentins A, B, and C. Cytotoxic and antifungal
imidazolediylbis[indoles] from the sponge Spongosorites ruetzleri. J Org Chem 1991; 56:
4304-07.
[37] Lum GM, Aisenbrey GA, Dunn MJ, Berl T, Schrier RW, McDonald KM. In vivo effect
of indomethacin to potentiate the renal medullary cyclic AMP response to vasopressin. J Clin
Invest 1977; 59:8-13.
[38] Ahmad A, Sakr WA, Rahman KM. Anticancer properties of indole compounds:
mechanism of apoptosis induction and role in chemotherapy. Current drug targets 2010;
11:652-66.
[39] Johnson IS, Cullinan GJ, Boder GB, Grindey CB, Laguzza BC. Structural modifications
of the vinca alkaloids. Cancer Treat Rev 1987; 14:407-10.
[40] Eftink MR, Ghira CA. Indole fluorescence quenching studies on proteins and model
systems: use of the inefficient quencher succinimide. Biochemistry 1984; 23:3891-99.
[41]Bruker (2014) APEX2, version 2014.11-0, Bruker AXS Inc., Madison, Wisconsin, USA
29