Terpyridine derivatives as turn-on fluorescence chemosensors for the selective and sensitive detection of Zn2+ ions in solution and in live cells

Article abstract of DOI:10.1039/c8pp00186c

A terpyridine based compound L1 was designed and synthesized as an off-on chemosensor for the detection of Zn²⁺. Chemosensor L1 showed excellent selectivity and sensitivity toward Zn²⁺ by exhibiting a large fluorescence enhancement (～51-fold) at 370 nm whereas other competitive metal ions did not show any noticeable change in the emission spectra of chemosensor L1. The chemosensor (L1) was shown to detect Zn²⁺ ions down to 9.76 μM at pH 7.4. However, chemosensor L1 binds Zn²⁺ in a 1:2 ratio (receptor:metal) with an association constant of 1.85 × 10⁴ (R² = 0.993) and this 1:2 stoichiometric fashion is established on the basis of a Job plot and mass spectroscopy. DFT/TD-DFT calculations were carried out to understand the binding nature, coordination features and electronic properties of L1 and the L1-2Zn²⁺ complex. In addition, this turn-on fluorescence probe was effectively used to image intracellular Zn²⁺ ions in cultured MDA-MB-468 cells.

Full text of DOI:10.1039/c8pp00186c

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Photochem. Photobiol. Sci., 2018, DOI: 10.1039/C8PP00186C.
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Terpyridine derivative as “turn-on” fluorescent chemosensor for
the selective and sensitive detection of Zn2+ ions in solution as
well as in live cells
Received 00th January 20xx,
Accepted 00th January 20xx
Tripti Mandal,a Anowar Hossain,a Anamika Dhara,a Abdulla Al Masum,b Saugata Konar,c Saikat
Kumar Manna,*d Saikat Kumar Seth,e Sudipta Pathak*d and Subrata Mukhopadhyaya
DOI: 10.1039/x0xx00000x
www.rsc.org/
A terpyridine based compound L1 was designed and synthesized as “off–on” chemosensor for Zn2+. Chemosensor L1
showed excellent selectivity and sensitivity toward Zn2+ by exhibiting a large fluorescence enhancement (~ 51-fold) at 370
nm whereas other competitive metal ions did not show any noticeable change in the emission spectra of chemosensor L1.
The chemosensor (L1) was shown to detect Zn2+ ion down to 9.76 µM at pH 7.4. However, chemosensor L1 binds Zn2+ in a
1:2 ratio (receptor : metal) with an association constant 1.85 × 104 (R2 = 0.993) and this 1:2 stoichiometric fashion is
established on the basis of Job’s plot and mass spectroscopy. DFT/TDDFT calculations were carried out to understand the
binding nature, coordination feature and electronic properties of L1 and L1-2Zn2+ complex. In addition, this “turn-on”
fluorescent probe was effectively used to image intracellular Zn2+ ions in cultured MDA-MB-468 cells.
above physiological importance of zinc and its biomedical
significance, there is a growing interest in developing a noninvasive
and sensitive method to trace and visualize free zinc ions. As the
Introduction
One of the contemporary thrust areas where scientific community
finds great interest is the detection and determination of transition
metal ions at low concentration level due to their biological,
analytical, medicinal and environmental importance.1,2 Among
these metal ions, Zn2+ ion is one of the most abundant d-block
transition metals and essential trace element in human body with
the average human being composed of 2–3 g and its concentration
as high as 10 μM in serum.3,4 Zn2+ plays vital roles in many necessary
physiological processes such as enzyme regulation, brain function,
protein synthesis, neurotransmission, metalloenzymes, cellular
apoptosis, DNA repair, gene expression and mammalian
reproduction.5-7 In spite of its essential role in living organisms,
anomalous zinc metabolism is associated with a variety of severe
neurological diseases such as Alzheimer’s, Parkinson’s, Wilson’s
diseases, amyotropic lateral sclerosis (ALS), ischemia and
epilepsy.8,9 Moreover, zinc is a food waste and agricultural product
in the environment, considerable concentration of which cause
phytotoxic effects on soil microbes.10 Therefore, recognizing the
zinc ion itself is magnetically or spectroscopically silent because of
its 3d104S0 electron configuration, hence the development of
simple, rapid, proficient, highly sensitive and selective zinc
fluorescent chemosensors is of high importance not only for the
elementary research but also for biological application in living
cells.11 Up to now, several fluorescent and colorimetric
chemosensors for zinc ions have been designed and reported.12,13
However, it is still an enormous challenge to develop fluorescent
chemosensors that can selectively detect Zn2+ ions without any
interference of other transition metal ions, mainly Cd2+, because
both Zn2+ and Cd2+ are in the same group of the periodic table and
have comparable properties. Consequently, they often induce
similar spectroscopic changes when interact with fluorescent
chemosensors.14 Besides most of the reported zinc sensors are
synthesized following multistep processes which are quite time
consuming and not economical (Table S3†).15 Thus, there is an
immense need for the development of sensitive and selective Zn2+
sensors that can be prepared easily and economically.
It is eminent that 2,2′:6′,2′′-terpyridine and its derivatives represent
a significant class of ligands in the areas of supramolecular,
materials science and coordination chemistry16 due to their strong
binding ability towards various transition metal ions with their
properly arranged ring nitrogen’s. Such type of strong chelating
properties of terpyridine make them more useful building blocks for
various technological applications such as in organic light-emitting
devices and probes, dye sensitized solar cells, mixed-valence
chemistry as well as in two-photon luminescent systems and
electrochromic.17
a.Department of Chemistry, Jadavpur University, Kolkata 700032, India.
b.Department of Life science
700032, India.
& Bio-technology, Jadavpur University, Kolkata
c. Department of Chemistry, Bhawanipur Education Society College, Kolkata 700
020, India.
d.Department of Chemistry, Haldia Government College, Debhog, Purba Medinipur,
West Benga 721657, India.
e. Department of Physics, Jadavpur University, Kolkata 700032, India.
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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With all the above mentioned criterion in mind, herein we report Anal. Calcd for C36H24N6: C, 79.98; H, 4.47; N, 15.55. Found C, 79.87;
three terpyridine based fluorescent ‘Off-On’ probes, 4′,4′′′′-(1,4- H, 4.42; N, 15.64.; ES + TOF MS: m/z 541.1332 [M+H]+ (calc. For
+
2+
Phenylene)bis(2,2′:6′,2′′-terpyridine) (L1); 4',4''''-(1,3-Phenylene)bis- C36H25N6 541.2136), 271.0494 [M + 2H]2+ (calc. for C28H26N6
2,2':6',2''-terpyridine (L2) and 2,4,6-Tris(2-pyridyl)-s-triazine (L3) 271.1104).
which can selectively detect Zn2+ ion in semi aqueous solution.
Preparation of (4',4''''-(1,3-Phenylene)bis-2,2':6',2''-terpyridine)
Among them L1 was found to be the most potential turn-on
fluorescence probe for Zn2+. To know the binding nature and
coordination feature of the complex (L1-2Zn2+), we performed DFT
computational studies using Gaussian software. Moreover, we have
studied fluorescence based bio-imaging experiments in MDA-MB-
468 cells using L1.
(L2):
Acetylpyridine (3.65 g, 30 mmol, 4 equivalents) was added to the
ethanolic solution (100 mL) of isophthalaldehyde (1.0 g, 7.5 mmol, 1
equivalent) at 25 oC. 10 mL of 1 molar aqueous solution of NaOH
followed by 40 mL aqueous NH3 (35%) solution was poured into the
reaction mixture. The reaction mixture was then stirred for 10
hours. After that a yellowish solid formed which was collected by
filtration and washed with ethanol for several times and dried in air.
Yellow solid (2.37 g, 58%); M.P. > 300 oC; IR (KBr): (cm-1) 1673, 1601,
Experimental
Instrumentation and materials
1
1584, 1567; H NMR (300 MHz, CDCl3) δ in ppm: 8.84-8.38 (m, 8H),
1H NMR spectra was recorded on a Brucker 300 MHz instrument.
For NMR spectra, CDCl3 was used as solvent using TMS as an
internal standard. Chemical shifts are expressed in ppm (δ) units.
Mass spectra were carried out using a HRMS QTOF Micro YA263
mass spectrometer. UV-visible and fluorescence spectra
measurements were performed on a JASCO V530 and a PerkinElmer
LS-55 spectrofluorimeter respectively. Chemicals used for the
synthesis of L1 and L2 were purchased from Sigma Aldrich Chemical
Co. (USA) and used without further purification. Fluorescence probe
L3 is commercially available in Sigma Aldrich Chemical Co. (USA)
(Scheme 1). Salts of different cations and solvents were purchased
from a commercial supplier, stored in desiccators under vacuum
containing self-indicating silica, and used without any further
purification. The following abbreviations are used to describe spin
8.38-8.02 (m, 2H), 7.99-7.66 (m, 8H), 7.55-7.25 (m, 5H), 7.00-6.89
(m, 1H); Anal. Calcd for C36H24N6: C, 79.98; H, 4.47; N, 15.55. Found
C, 79.83; H, 4.51; N, 15.67.; ES + TOF MS: m/z 541.1523 [M+H]+
(calc. For C36H25N6+ 541.2136).
Computational studies
All geometries for L1 and L1-2Zn2+ were optimized by density
functional theory (DFT) calculations using the Gaussian09 (B3LYP/6-
311G(d,p)) software package.18
Calculations for detection limit
The detection limit (DL) of L1 for Zn2+ were determined from the
following equation19
:
DL = K*Sb1/S, Where K = 2 or 3 (we take 2 in this case); Sb1 is the
standard deviation of the blank solution; S is the slope of the
calibration curve.
1
multiplicities in H NMR spectra: s = singlet; d = doublet; t = triplet;
m = multiplet.
Binding constants calculation using fluorescence titration data
General method of UV−Vis and fluorescence spectral studies
The association constant and stoichiometry for the formation of the
respective complexes were evaluated using the Benesi−Hildebrand
(B−H) plot (eq 1).20
For UV-vis and fluorescence titrations, stock solution of sensor L1,
L2 and L3 (4 × 10−5 M) were prepared in CH3CN/H2O (9:1, v/v, 10
mM HEPES buffer, pH 7.4) solution. Solutions of 4 × 10−4 M salts of
the respective cations were prepared in Millipore water. In titration
experiments, solutions of every chemosensors (4 × 10−5 M) were
filled each time in a quartz optical cell of 1.00 cm optical path
length, and the metal ions stock solutions were added into the
quartz optical cell gradually by using a micropipette. Spectral data
were recorded at 1 min after the addition of the ions.
1/(I – I0 ) = 1/K(Imax – I0 )[Mn+] + 1/(Imax – I0 )
(1)
where I0, Imax, and I represent the emission intensity of free L1, the
maximum emission intensity observed in the presence of added
metal ion at 370 nm for Zn2+ (λext = 312 nm), and the emission
intensity at a certain concentration of the metal ion added,
respectively.
Preparation of 4′,4′′′′-(1,4-Phenylene)bis(2,2′:6′,2′′-terpyridine) [L1]
Cell imaging experiments
Acetylpyridine (3.65 g, 30 mmol, 4 equivalents) was added to the
ethanolic solution (100 mL) of terephthalaldehyde (1.0 g, 7.5 mmol,
1 equivalent). 10 mL of 1 molar aqueous solution of NaOH was
poured into the reaction mixture at 25 oC, followed by 40 mL
aqueous NH3 (35%) solution. The reaction mixture was then stirred
at 25 oC for 10 hours. A yellow solid formed which was collected by
filtration and washed with ethanol for several times and dried in air.
The desired product was obtained as a yellow solid. Unlike reported
literature this reaction was carried out in single step with
satisfactory yield and acceptable purity. Yellow solid (2.51 g, 62%);
M.P. > 300 oC; IR (KBr): (cm-1) 1667, 1602, 1566; 1H NMR (300 MHz,
CDCl3) δ in ppm: 8.80 (s, 4H), 8.76 (d, J=3.76 Hz, 4H), 8.69 (d, J=7.0
Hz, 4H), 8.06 (s, 4H), 7.90 (t, J=7.1 Hz, 4H), 7.37 (t, J=4.2 Hz, 4H);
Minimum Inhibitory Concentration (MIC):
Test tube containing LB broth was taken and single colony of the
organism was inoculated. The tube was kept for overnight
incubation at 37 °C. The following day two test tubes each
containing LB broth was taken for the organism. 50 µl of the
overnight grown culture was inoculated in both the test tubes and
incubated at 37 °C for 3-4 hrs. OD at 600 nm was taken from each
of these tubes and recorded as control for future reference. After
that different dilution of the probe L1 was prepared (i.e. stock, half
dilution and one-fourth dilution). Next 50 µl of the probe L1 with
different dilution was added in each of the tubes respectively.
These tubes were kept for overnight incubation at 37 °C. Another
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set of test tubes containing only the broth and probe L was kept for toward Zn2+ over the other ions was investigated by UV-vis and
overnight incubation. OD at 600 nm was taken from each of these fluorescence spectroscopy. All the spectroscopic studies were
tubes.
carried out in CH3CN/H2O (9:1, v/v, 10 mM HEPES buffer, pH 7.4)
solution by adding aliquots of different metal cations dissolved in
water.
Cell Survivability Assay
The inhibition of cell growth was measured by MTT assay. In brief,
cells were seeded in 96 well plates at 1 × 104 cells per well and
exposed to probe L1 at different concentrations for 24 hrs. After
incubation, cells were washed twice with 1X PBS and incubated
with MTT solution (450 μg/ ml) for 3–4 hrs at 37 °C. The resulting
formazan crystals were dissolved in a MTT solubilization buffer and
the absorbance were measured at 570 nm by using a microplate
reader (Biotek, USA). Each point was assessed in triplicate.
Cellular Imaging
For confocal imaging studies, 1 x 104 MDA-MB 468 cells in 1000 μL
of medium, were seeded on sterile 35 mm glass bottom culture dish
(ibidi GmbH, Germany), and incubated at 37 °C in a CO2 incubator
for 10 hours. Then cells were washed with 500 μL DMEM followed
by incubation with probe L1 (10 µM) dissolved in 500 μL DMEM at
37 °C for 30 minutes in a CO2 incubator and observed under an
Olympus IX81 microscope equipped with a FV1000 confocal system
using 1003 oil immersion Plan Apo (N.A. 1.45) objectives. Images
obtained through section scanning were analyzed by Olympus
Fluoview (version 3.1a; Tokyo, Japan) with excitation at 350 nm
monochromatic laser beam, and emission spectra were integrated
over the range 470 nm (single channel).
Fig. 1 (a) Changes in UV–vis spectra of probe L1 (4 × 10−5 M) upon
gradual addition of Zn2+ (c = 4 × 10−4 M) in CH3CN/H2O solution (9:1,
v/v, 10 mM HEPES buffer, pH 7.4).
The UV-vis spectrum of sensor L1 in CH3CN/H2O solution (9:1, v/v,
10 mM HEPES buffer, pH 7.4) exhibited one strong band at 290 nm
which may be attributed to the π–π* transitions of the terpyridine
unit.22
With gradual addition of Zn2+ to the solution of L1, the absorbance
at 290 nm gradually decreased with concomitant formation of a
new peak at 327 nm and also an isosbestic point at 312 nm
observed (Fig. 1). The peaks at 312 nm and 327 nm might be
attributable to the formation of terpyridine bounded zinc complex
in sensor L1. By contrast, no significant absorption spectral change
Results and discussion
Synthesis
was observed for other cations such as Li+, K+, Na+, Mg2+, Ca2+, Sr2+
,
The chemosensor molecule L1 and L2 were synthesized following a
modified literature method21 as shown in Scheme 1.
Ba2+ , Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+, Pb2+, Hg2+, Pd2+, Al3+ and Ag+,
under similar experimental conditions (Fig. S6†).
CHO
CHO
N
N
N
N
The fluorescence emission spectrum from 336 to 500 nm was
observed by exciting sensor L1 at 312 nm. As shown in figure 2a,
the free sensor L1 (Quantum yield ϕ = 0.0005) (Table S4†) showed
no fluorescence in CH3CN/H2O solution (9:1, v/v, 10 mM HEPES
buffer, pH 7.4) but on incremental addition of Zn2+, the fluorescence
intensity at 370 nm increased progressively and reached saturation
with ~ 51-fold enhancement (Quantum yield ϕ = 0.0254) (Table S4†)
in integrated intensity. In presence of Zn2+ ion, the fluorescence
intensity of sensor L1 increased in a Zn2+ concentration-dependent
way as shown in Figure 2b, accompanied with an apparent emission
color change from colorless to sky blue. Hence, compound L1 can
be considered as being a real fluorescent off–on chemosensor. Such
type of fluorescence enhancement by Zn2+ might be due to the
(i) Aq. NaOH
+
N
N
CH3
(ii) NH4OH/ EtOH
r.t., 10h
N
O
L1
N
N
N
OHC
CHO
(i) Aq. NaOH
+
N
CH3
N
N
(ii) NH4OH /EtOH
r.t 10h
N
O
N
L2
N
N
N
N
efficient coordination of Zn2+
, causing the chelation-enhanced
N
L3
fluorescence (CHEF) effect which attribute to the energy
minimization due to ligand to metal charge transfer.
Scheme 1 Synthetic route for chemosensors L1 and L2 and structure
of chemosensor L3.
From the fluorescence titration profile, the association constant of
L1 with Zn2+ ion has been determined to be 1.85 × 104 (R2 = 0.993),
from the Benesi–Hildebrand equation.20 The observed high Ka value
clearly points out strong binding affinity of Zn2+ to probe L1. The
detection limit19 was evaluated to be 9.76 μM, which is below the
Zn2+ concentration of the WHO guidelines for drinking water,23
UV-vis and fluorescence studies
The sensing behaviour of compound L1 toward different metal
cations such as Li+, K+, Na+, Mg2+, Ca2+, Sr2+, Ba2+ , Mn2+, Fe2+, Co2+,
Ni2+, Cu2+, Cd2+, Pb2+, Hg2+, Pd2+, Al3+, Ag+ and its superior selectivity
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suggestive of L1 is potentially useful for quantitative determination
fold for L2 and only ~ 7 fold for L3) in the emission compared to L1
(~ 51-fold), suggesting a sensitive detection of Zn2+ only by L1
compared to other two control molecules .
of Zn2+ in environment and also monitoring zinc ion in living cells.
(a)
Zn2+
L1
(b)
Fig. 3 Fluorescence responses of L1 to different competitive metal
ions in CH3CN/H2O solution (9:1, v/v, 10 mM HEPES buffer, pH 7.4).
Zn2+
N
N
N
N
N
N
Fig. 2 (a) Fluorescence emission changes of L1 (4 × 10−5 M) with the
continuous addition of Zn2+ (c = 4 × 10−4 M) in CH3CN/H2O solution
(9:1, v/v, 10 mM HEPES buffer, pH 7.4). λex = 312 nm. Inset: Visual
emission color changes of L1 in presence of Zn2+. (b) Change of
Zn2+
emission intensity at 370 nm with incremental addition of Zn2+ (λex
312 nm).
=
N
N
N
N
N
N
Zn2+
L1ꢀ2Zn2+
Moreover, a careful Job’s plot analysis using fluorescence titration
experiment suggested a 1:2 binding stoichiometry between L1 and
Zn2+ (Fig. S9†). In addition, the formation of 1:2 complex (receptor:
metal) was further confirmed through mass spectral analysis of the
L1-Zn2+ complex, in which the peaks at m/z 243.0867, assignable to
[L1+2Zn2+ + OH-+CH3CN]3+ was clearly observed (Fig. S3†).
L1
Scheme 2 Probable binding modes of L1 and Zn2+ in solution phase.
Theoretical study
To gain insight into the geometry of probe L1 and L1-Zn2+ and the
optical response of L1 towards Zn2+
, we performed density
An imperative feature of probe L1 is its high selectivity toward zinc
ion over the other competitive metal ions. As shown in figure 3, the
initial fluorescence intensity of probe L1 did not changes
significantly upon addition of 10 equivalent of different
comparative metal cations such as Li+, K+, Na+, Mg2+, Ca2+, Sr2+, Ba2+,
Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Cd2+, Pb2+, Hg2+, Pd2+, Al3+ and Ag+ to the
semi aqueous solution of L1, but in presence of zinc ion L1 showed
a large fluorescence intensity enhancement at 370 nm. Only in
presence of Zn2+, sensor L1 showed a visual emission color change
during the above process from non fluorescent to sky blue. Thus,
the probe L1 can be used as a highly selective and specific
chemosensor for zinc ions over other competing metal ions in
several environmental and biological systems. Such type of high
selectivity of L1 for Zn2+ is mainly due to the high affinity of Zn2+ for
three nitrogen atoms of the terpyridine moieties.
functional theory (DFT) and time-dependent-density functional
theory (TD-DFT) calculations with the B3LYP/6-311+G(d,p) method
basis set using the Gaussian 09 program.18 The optimized
geometries along with the highest occupied molecular orbital
(HOMO) and the lowest unoccupied molecular orbital (LUMO) of L1
and L1-2Zn2+ complex are presented in Fig. 4. From these HOMO
and LUMO energy diagrams, we found that the energy gap for the
L1-2Zn2+ (90.90 kcal/mole) was reduced as compared to the probe
L1 alone (108.80 kcal/mole) (Table S1†), indicating probe L1 formed
a stable di-nuclear zinc complex as shown in Fig. 5. Initial peak at
290 nm of L1 in absorbance spectra is due to HOMO-LUMO
transition. In case of Zn-complex bathochromic shift (312 nm) in
absorbance spectra was observed due to minimization of energy,
owing to Ligand to metal charge transfer. As shown in fig. 5, π-
electron cloud in HOMO is mainly located at center of L1-2Zn2+
complex, which is spread out to all over the molecule in LUMO,
Similar fluorescence studies were carried out in presence of Zn2+
with the other two control molecules L2 and L3 (Fig. S13 & S14†)
and found very small changes in fluorescence intensity (only ~ 4
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indicating LMCT phenomenon leads to the bathochromic shift and concentration up to 40 µM can be employed for the fluorescence
enhancement in fluorescence intensity.
imaging studies of L1 and L1-2Zn2+ in living cells. Hence to assess
the efficacy of probe L1 as a chemosensor for intracellular detection
of Zn2+ by confocal microscopy, MDA-MB-468 cells were treated
with 20 µM ZnCl2 for 1 h followed by incubation with 10 µM probe
solution to encourage the formation of L1-2Zn2+. From this
fluorescence microscopic studies it was found that either probe L1
or ZnCl2 alone failed to display any fluorescence in MDA-MB-468
cells. However, upon incubation with ZnCl2 followed by probe L1,
distinctive and fluorescence turn-on blue fluorescence was
observed inside the cells, which could be recognized to the
formation of the intracellular di-nuclear zinc complex, as observed
earlier in solution studies. Moreover, bright-field images of cells did
not demonstrate any gross morphological changes, signifying that
the MDA-MB-468 cells were viable throughout the bio-imaging
experiments. These results indicate that probe L1 is cell membrane
permeable and can be employed for sensing intracellular zinc ions
in living cells.
(a)
(b)
Fig. 4 Calculated energy-minimized structure of (a) L1 and (b) L1-
2Zn2+ complex.
(A)
(B)
(C)
Fig. 6 Confocal microscopic image of L1-treated MDA-MB-468 cells
before (A) and after (B &C) incubation with ZnCl2. (A & C) represent
the dark field image; (B) represent the bright-field image.
Conclusions
In conclusion, we have designed and synthesized a highly selective
and sensitive terpyridine based fluorescent “turn-on” chemosensor
L1 for Zn2+ ions among the other biologically important eighteen
different metal ions studied in CH3CN/H2O solution (9:1, v/v, 10 mM
HEPES buffer, pH 7.4). We have simultaneously studied
fluorescence sensing behaviour of two other terpyridine based
chemosensors (L2 and L3) towards Zn2+ but they are not so
effective. Unlike most of the fluorescence probes, our reported
chemosensor L1 can be synthesized in single step using low cost
and commercially available starting materials with satisfactory
yield. Entire reaction can be carried out in one pot and at room
temperature. It can selectively detect Zn2+ ion down to 9.76 µM
which is below the WHO guidelines for drinking water (76 μM) and
the whole process is economically favorable. The sensor L1 shows
an obvious fluorescent enhancement (~ 51-fold) upon complexation
with Zn2+ in semi-aqueous solution with a fluorescent color change
from dark to sky blue. The selectivity and sensitivity of L1 was
demonstrated on the basis of fluorescence, absorption, and ESI
mass spectrometry data. The association constant for L1 with Zn2+ is
in the order of 104 M−1 based on fluorescence spectral studies,
signifying a strong binding for Zn2+. Moreover, the Zn2+ binding
nature of L1 have been studied by computational calculations at the
DFT level and TDDFT calculations were also performed to show the
electronic properties of L1 and their corresponding zinc complexes.
L1
L1ꢀ2Zn2+
Fig. 5 HOMO and LUMO distributions of L1 and L1-2Zn2+ complex.
Now, TDDFT calculations have been carried out by TDDFT/CPCM
method in acetonitrile solvent. Calculated wavelengths and
oscillator frequencies from TDDFT calculations are listed in
Table S2 (ESI†) and from this data we found that the calculated
vertical electronic excitations well matched with the experimentally
observed bands both for the L1 and L1-2Zn2+ complex.
Cell imaging study
In order to demonstrate the prospective biological application of
sensor L1, fluorescence based bio-imaging experiments were
carried out using MDA-MB-468 cells. However, to achieve this goal,
it was essential to initially assess the cytotoxic effect of probe L1 on
live cells. The well-established MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay, which is based on
mitochondrial dehydrogenase activity of viable cells, showed that
neither L1 nor its zinc complex was capable to reveal any effect on
the viability of MDA-MB-468 cells (Fig. S11 & S12†). The results
obtained from the in vitro cytotoxic assay suggested that probe
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From these theoretical studies, we have found noteworthy
similarities with the experimental results. Furthermore, the cell
imaging experiments showed that L1 was cell permeable and shows
large fluorescence enhancement at pH 7.4. Thus it can be applied
for imaging and monitoring zinc ions in living cells.
Conflicts of interest
There is no conflict to declare.
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Acknowledgements
TM and AH thankfully acknowledge the UGC, New Delhi for
senior research fellowships. SP and SKM thank Haldia
Government College for laboratory facility. AD is grateful to
UGC, India for DSK Postdoctoral fellowship.
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Table of Contents
Terpyridine derivative as “turn-on” fluorescent chemosensor for the selective
and sensitive detection of Zn2+ ions in solution as well as in live cells
Tripti Mandal,a Anowar Hossain,a Anamika Dhara,a Abdulla Al Masum,b Saugata Konar,c Saikat Kumar Manna,*d
Saikat Kumar Seth,e Sudipta Pathak*d and Subrata Mukhopadhyaya
Zn2+