A novel chemosensor with visible light excitability for sensing Zn 2+ in physiological medium and in HeLa cells

Article abstract of DOI:10.1039/c4ob00653d

In the present study a novel imine-hydrazone based fluorescent chemosensor (L₁) for efficient and selective sensing of Zn²⁺ over other biologically important metal ions under physiological conditions is reported. An enhancement in fluorescence emission intensity of the developed probe with a red shift of ～25 nm was observed for Zn²⁺, whereas other metal ions failed to reveal any significant change in the emission spectra. Interestingly, the receptor functioned under completely physiological conditions (99.7% HEPES buffer) and has visible light excitability. Sensing of Zn²⁺ was investigated in detail by absorption spectroscopy, emission spectroscopy, DFT calculation, ¹H-NMR titration experiment and ESI-MS experiment. The association constant between L₁ and Zn²⁺ was found to be 5.58 × 10⁵ M^-1. The receptor could detect as low as 69 ppb Zn²⁺. Sensing of Zn²⁺ is proposed through switch-on of intramolecular charge transfer (ICT) and chelation enhanced fluorescence (CHEF) processes after the introduction of Zn²⁺ into the free ligand. The developed receptor was non-toxic and rendered intracellular sensing of Zn²⁺ in HeLa cells through fluorescence imaging studies. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00653d

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A novel chemosensor with visible light excitability
for sensing Zn2+ in physiological medium and in
HeLa cells†
Cite this: DOI: 10.1039/c4ob00653d
Barun Kumar Datta,a Durairaj Thiyagarajan,b Soham Samanta,a Aiyagari Ramesh*b
and Gopal Das*a
In the present study a novel imine-hydrazone based fluorescent chemosensor (L1) for efficient and selec-
tive sensing of Zn2+ over other biologically important metal ions under physiological conditions is
reported. An enhancement in fluorescence emission intensity of the developed probe with a red shift of
∼25 nm was observed for Zn2+, whereas other metal ions failed to reveal any significant change in the
emission spectra. Interestingly, the receptor functioned under completely physiological conditions (99.7%
HEPES buffer) and has visible light excitability. Sensing of Zn2+ was investigated in detail by absorption
1
spectroscopy, emission spectroscopy, DFT calculation, H-NMR titration experiment and ESI-MS experi-
ment. The association constant between L1 and Zn2+ was found to be 5.58 × 105 M−1. The receptor
could detect as low as 69 ppb Zn2+. Sensing of Zn2+ is proposed through switch-on of intramolecular
charge transfer (ICT) and chelation enhanced fluorescence (CHEF) processes after the introduction of
Zn2+ into the free ligand. The developed receptor was non-toxic and rendered intracellular sensing of
Zn2+ in HeLa cells through fluorescence imaging studies.
Received 27th March 2014,
Accepted 13th May 2014
DOI: 10.1039/c4ob00653d
www.rsc.org/obc
Imaging of intracellular Zn2+ with fluorescent chemosen-
sors has been established to be an efficient tool to provide
Introduction
Detection and imaging of biologically relevant target mole- chronological–spatial evidence on Zn2+ homeostasis in live
cules through fluorescence in living systems has emerged as cells.5 In general, most fluorogenic chemosensors depend on
an area of intensive interest in the chemistry–biology interface the changes of emission intensity (quenching, enhancement,
owing to its significant biomedical implications.1 Among the or ratiometric) of the chromophore units which act as signal-
many biologically important metal ions, Zn(II) is known to play ling units for the sensing ability.6 Even though sensing of ana-
crucial roles in the activity of various enzymes and is also a key lytes in solution can be correlated with the systematic
component of some transcription factors, gene expression, variations in emission intensity, the detection of analytes in
apoptosis, neurotransmission and so forth.2 Perturbations in cells is fraught with the challenge of specific sensing in a
zinc homeostasis are of great concern, resulting in disorders complex milieu. For example, during imaging of cells using
such as impaired cognition, immune dysfunction, type 2 dia- fluorescence microscopy, quenching may occur due to the
betes mellitus (T2DM), Alzheimer’s disease, epilepsy, ischemic photobleaching7 and/or aggregation8 to several degrees in
stroke, age-related macular degeneration (AMD), and infantile different cellular environments. Furthermore, live cell imaging
diarrhoea.3 Being a harmful pollutant, zinc is also important with a probe within biological cells may result in hetero-
for environmental safety.4
geneous labelling of the sensor, which can lead to varying
emission intensities at different spatial positions.9 The afore-
mentioned anomaly presents a great challenge towards analys-
ing signal changes due to the recognition of the analytes with
those arising from variations in probe concentration within
cells. Therefore, it is significant to design new sensors which
undergo profound spectroscopic changes like spectral shifts,
upon binding with the analyte like Zn2+. Fluorescent probes
are designed on the basis of inter-charge transfer (ICT),10
photoinduced electron transfer (PET),11 chelation-enhanced
fluorescence (CHEF),12 metal–ligand charge transfer (MLCT),13
aDepartment of Chemistry, Indian Institute of Technology Guwahati,
Guwahati 781039, India. E-mail: gdas@iitg.ernet.in; Fax: +91 361 2582349;
Tel: +91 361 2582313
bDepartment of Biotechnology, Indian Institute of Technology Guwahati,
Guwahati 781039, India. E-mail: aramesh@iitg.ernet.in; Fax: +91 361 2582249;
Tel: +91 361 2582205
†Electronic supplementary information (ESI) available: H-NMR data, 13C-NMR
data, ESI-MS spectra, 1H-NMR titration data, Benesi Hildebrand plot, Job’s plot
and tables of DFT calculations. See DOI: 10.1039/c4ob00653d
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excimer/exciplex formation,14 imine isomerization,15 inter-
molecular hydrogen bonding,16,14e excited-state intramolecular
proton transfer,17 displacement approach,18,23 and fluore-
scence resonance energy transfer.19 CHEF is an important
process by which fluorescent probes detect analytes.12 When
the CHEF process is in the switch-on condition, the conju-
gation increases drastically, which leads to an enhancement in
the emission and also a red shift is expected. So, the fluoro-
phore probe senses a metal ion with enhancement in the emis-
sion intensity accompanied by a red shift.
Scheme 1 Synthesis of L1 and L2.
Considering the physiological and biomedical significance
of zinc, there is overriding interest in the development of selec-
tive and sensitive zinc sensors. Zn2+ ions do not respond to recorded in parts per million (ppm) on the scale. The follow-
common analytical techniques like Mossbauer, NMR, and elec- ing abbreviations are used to describe spin multiplicities in 1H
tron paramagnetic resonance (EPR) as compared to other tran- NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet.
sition metal ions like Fe2+, Mn2+ and Cu2+. Thus it is difficult
Synthesis of the receptor
to trace zinc ions in highly complex biological systems. So far,
a number of fluorescent chemosensors for selective detection The synthetic routes of the two compounds L1 and L2 are
of Zn(II) have been reported with some success in biological depicted in Scheme 1.
applications,20 but most of the reported fluorescent as well as
Synthesis of picolinic hydrazide
colorimetric sensors for Zn2+ work in either purely organic
solutions or a mixture of organic and aqueous solutions.21 Picolinic acid was dissolved in ethanol and under ice cold con-
This restricts their applications in a physiological environ- ditions thionyl chloride was added dropwise over a period of
ment. Hence there is a critical demand to develop fluorescent 30 min with constant stirring. After 1 hour stirring, the hazy
probes for Zn2+ that are based on easy synthesis, render visible mixture was refluxed overnight. After evaporation of the
light excitation, and exhibit large red-shifts in emission for solvent, water was added and the pH was adjusted to 8.0 by
Zn2+ sensing, and their sensing capability is immune from any the addition of sodium bicarbonate. Subsequently the mixture
pH interference in the physiological range.
was extracted with ethyl acetate (3 × 50 ml). The organic layer
In our continuous endeavour at sensing various analytes,22 was dried over sodium sulphate and evaporation of the solvent
herein we report the synthesis, characterization and sensing gave the ethyl ester of picolinic acid as a colourless liquid.
behaviour of a novel fluorescent chemosensor towards Zn2+ This ester was used without further purification. This ester
with visible light excitability and the ability to sense the metal was then treated with excess hydrazine monohydrate in
under completely physiological conditions. The recognition of ethanol. The mixture was heated to reflux overnight. After
Zn2+ by our receptor L1 has been investigated by absorption evaporation of the solvent and the excess hydrazine under
spectroscopy, emission spectroscopy, DFT calculation, ESI-MS reduced pressure, a white solid was obtained, which was dried
experiment and 1H-NMR titration. Detection of intracellular under vacuum and was used in the next step without further
zinc in live HeLa cells by fluorescence imaging is also demon- purification.
strated in the study.
Synthesis of L1
2,6-Diformyl-4-methylphenol (1 mmol) was dissolved in
ethanol. Picolinic hydrazide (2 mmol) was added to the above
Experimental section
General information and materials
solution and the mixture was refluxed for 4 hours to give a
yellow crystalline product. Yield 72%. 1H NMR [400 MHz,
All the materials used for the synthesis were purchased from DMSO-d6, δ (ppm)]: 11.47 (2H, s), 11.26 (1H, s), 7.84 (2H, s),
commercial suppliers and used without further purification. 7.67 (2H, d, J = 4.4 Hz), 7.08 (2H, d, J = 7.6 Hz), 7.01 (2H, t, J =
2,6-Diformyl-4-methylphenol (DFMP) was prepared by modifi- 7.8 Hz), 6.62 (2H, t, J = 5.8 Hz), 6.48 (2H, s), 1.25 (3H, s). 13C
cation of the literature method.23 Absorption spectra were NMR [100 MHz, DMSO-d6, δ (ppm)]: 160.8, 155.0, 149.3, 148.8,
recorded on a Perkin-Elmer Lambda-750 UV-vis spectropho- 147.8, 138.2, 130.8, 128.4, 127.3, 122.9, 120.1, 20.0. ESI-MS
tometer using 10 mm path length quartz cuvettes in the range (positive mode, m/z) calculated for C21H19N6O3 [L1 + H] =
of 250–700 nm wavelength. Fluorescence measurements were 403.1519, found 403.1546.
conducted on a Fluoromax-4 spectrofluorometer using 10 mm
path length quartz cuvettes with a slit width of 5 nm at 298 K.
Synthesis of L2
All the mass spectra were obtained using an Agilent Techno- Isophthalaldehyde (1 mmol) was dissolved in ethanol, picoli-
logies 6520 Accurate mass spectrometer. NMR spectra were nic hydrazide (2 mmol) was added to the above solution and
recorded either on a Varian FT-400 MHz instrument or on a the mixture was refluxed for 4 hours to give a white crystalline
Bruker 600 MHz instrument. The chemical shifts were product. Yield 75%. 1H NMR [400 MHz, CDCl3, δ (ppm)]: 11.09
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(2H, s), 8.60 (2H, d, J = 8 Hz), 8.32 (4H, d, J = 8.4 Hz), 8.17 (1H, Detection limit for Zn2+ ions
s), 7.94–7.89 (4H, m, J = 3.6 Hz), 7.52–7.47 (3H, s). 13C NMR
The detection limit was calculated on the basis of the fluore-
scence titration. The fluorescence emission spectrum of L1 was
measured 10 times, and the standard deviation of blank
measurement was achieved. To gain the slope, the ratio of the
emission intensity at 560 nm was plotted against the concen-
tration of Zn2+. The detection limit was calculated using the
following equation:
[100 MHz, CDCl3, SiMe4, δ (ppm)]: 160.4, 149.2, 148.3, 148.1,
137.8, 134.4, 129.6, 129.3, 127.7, 127.0, 123.1. ESI-MS (positive
mode, m/z) calculated for C20H17N6O2 [L2 + H] = 373.1413,
found 373.1426.
UV-visible and fluorescence spectroscopic studies
Stock solutions of various ions (1 × 10−3 mol L−1) were pre-
pared in deionized water. Perchlorate, chloride or nitrate salts
of metal ions were used to prepare metal stock solutions. A
stock solution of L1 (1 × 10−3 mol L−1) was prepared in DMSO.
The solution of L1 was then diluted to 1 × 10−6 mol L−1 with
aqueous HEPES buffer (1 mM, pH 7.4). All the spectroscopic
experiments were performed in aqueous HEPES buffer
medium (1 mM, pH 7.4) containing 0.33% of DMSO. In titra-
tion experiments, a solution of L1 (1 × 10−6 mol L−1) was filled
in a quartz optical cell of 1.0 cm optical path length, and the
ion stock solutions were added gradually to achieve a concen-
tration of 1 × 10−5 mol L−1. In selectivity experiments, the test
samples were prepared by interacting appropriate amounts of
the cations stock into 2 mL of L1 solution (2 × 10−5 mol L−1).
For all the samples, the spectra were recorded following 1 min
of the addition of the ions. For fluorescence measurements,
the excitation wavelength was set at 470 nm and emission was
recorded from 480 nm to 700 nm. The selective binding of L1
with Zn2+ among all other metal ions was also studied by
fluorescence emission spectroscopy of the solution of L1
(10.0 × 10−6 mol L−1) in the absence and presence of an excess
(10 eq.) of each of the metal ions in the mixed solvent.
Detection limit ¼ 3σ=k
ð2Þ
where σ is the standard deviation of blank measurement,
and k is the slope between the ratio of emission intensity
versus [Zn2+].
Detection of Zn2+ in HeLa cells by fluorescence microscope
analysis
HeLa cells (human cervical carcinoma cells) were initially
cultured in a 25 cm2 tissue culture flask containing DMEM
medium supplemented with 10% FBS, penicillin (100 µg mL−1
)
and streptomycin (100 µg mL−1) in a CO2 incubator. Prior to
cell imaging studies, HeLa cells were seeded into a 6 well plate
and grown in DMEM medium at 37 °C till 80% confluency in a
CO2 incubator. Subsequently, the cells were washed thrice with
sterile phosphate buffered saline (PBS), incubated with 25 μM
L1 in DMEM at 37 °C for 1 h in a CO2 incubator and their
images were acquired using
a fluorescence microscope
(Eclipse Ti-U, Nikon, USA) with a filter that allowed green light
emission. The cells were further washed with sterile PBS in
order to remove excess L1 and then incubated for 1 h with
50 μM Zn(ClO4)2 prepared in sterile PBS. The images of the
cells were again acquired with a fluorescence microscope as
mentioned earlier.
Evaluation of the apparent binding constant for the formation
of L1·2Zn2+
A stock solution of Zn(ClO4)2, with a concentration of 0.5 ×
10−3 mol L−1, in an aqueous HEPES buffer (pH 7.4) solution
was used. Receptor L1 with an effective concentration of 10.0 ×
10−6 mol L−1 in the aforementioned HEPES buffer medium
was used for the emission titration studies. The effective Zn2+
concentration was varied between 0 and 10 × 10−5 mol L−1 for
this titration. The solution pH was adjusted to 7.4 using an
aqueous HEPES buffer solution having an effective concen-
tration of 1.0 mM.
Results and discussion
Sensing of Zn2+
Absorption spectroscopic studies. All the spectroscopic
studies were performed in HEPES buffer medium (1 mM,
pH = 7.4) containing 0.33% of DMSO.
The ligand L1 exhibited two characteristic absorption peaks
at 305 nm (ε = 4 × 104 M−1 cm−1) and 367 nm (ε = 1.8 ×
104 M−1 cm−1), which may be attributed to the π–π* transitions
and the long conjugation present in the free ligand system.
Upon addition of 10 equivalents of different metal ions (Na+,
K+, Mg2+, Ca2+, Co2+, Ni2+, Cu2+, Cd2+, Ag+, Pb2+, Hg2+, Al3+,
Cr3+, Fe3+, and Zn2+), Zn2+ promoted a prominent change in
The apparent binding constants for the formation of the
respective complexes were evaluated using the Benesi–Hilde-
brand (B–H) plot (eqn (1)).24
1=ðI ꢀ I0Þ ¼ 1=fKðImax ꢀ I0ÞCg þ 1=ðImax ꢀ I0Þ
ð1Þ
where I0 is the emission intensity of L1 at λ = 550 nm, I is the the absorption spectra of L1 (Fig. 1a). Amongst other metals,
observed emission intensity at that particular wavelength in only Cd2+ caused a nominal change in the absorption spectra
the presence of a certain concentration of the metal ion (C), of L1. However, this change was distinctly less in magnitude as
Imax is the maximum emission intensity value that was compared to the change observed with Zn2+ (Fig. 1a). Upon the
obtained at λ = 550 nm during titration with varying metal ion addition of Zn2+ ions (10 eq.), the absorption peak at 305 nm
concentrations, K is the apparent binding constant (M−1) and (ε = 1.8 × 104 M−1 cm−1) decreased and shifted to 326 nm and
was determined from the slope of the linear plot, and C is the a new peak emerged at 450 nm (ε = 0.98 × 104 M−1 cm−1). On
concentration of Zn2+ ions added during titration studies.
the other hand, with the addition of Cd2+ the absorption at
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Fig. 1 Changes in absorption spectroscopy of L1 (25 μM) with (a)
various metal ions (10 eq.) and (b) with incremental addition of Zn2+
(0 μM–60 μM). Inset: plot of absorbance at 450 nm vs. [Zn2+].
305 nm (ε = 3 × 104 M−1 cm−1) decreased, akin to that observed Fig. 3 Normalized fluorescence responses of L1 (10 μM) at 550 nm in
with Zn2+, while a new peak appeared at 436 nm (ε = 0.24 ×
the presence of various cations in the mixed solvent. The red bars rep-
resent the emission intensities of L1 in the presence of cations of interest
104 M−1 cm−1). Incremental addition of Zn2+ to L1 resulted in
(5.0 eq.). The blue bars represent the change of emission upon sub-
three isosbestic points at 329 nm, 352 nm and 396 nm, indi-
cating the transition of the free ligand to a metal complex
(Fig. 1b). A visual colour change was observed during the
above process from colourless to yellow (Fig. S13†). From the
absorption titration experiment it was also witnessed that the
change in the absorption spectra became minimum after the
addition of two equivalents of metal ions (Fig. 1b, inset),
which suggested a 1 : 2 binding stoichiometry between L1 and
Zn2+. Essentially, the absorption spectrum indicated that the
receptor exhibited high selectivity towards Zn2+ with a mar-
ginal response to Cd2+.
sequent addition of Zn2+ to the above solution. Display error bars with
5% values.
ment in the emission intensity of the free ligand with a pro-
gressive red shift (Fig. 2b). After the addition of two
equivalents of Zn2+ ions, the change in the emission spectra
became minimal, supporting the result obtained from UV-Vis
experiments (Fig. 2b). Fluorescence quantum yields of the
Zn2+ complex were calculated to be 18 times higher than that
of the probe alone (0.16 and 0.009, respectively). A change in
the fluorescence emission from colourless to yellow was
observed under a UV-lamp after the addition of Zn2+ to the
receptor solution (Fig. 2b, inset). A careful analysis of Job’s
plot (Fig. S11†) obtained from the fluorescence titration experi-
ment established a 1 : 2 binding stoichiometry between L1
and Zn2+. The association constant and the detection limit of
L1 for Zn2+ were calculated to be 5.58 × 105 M−1 (Fig. S12†) and
approximately 69 ppb respectively.
The amplification of fluorescence intensity of the receptor
L1 by Zn2+ was also confirmed by screening with competing
metal cations of interest (Fig. 3). Except for Zn2+, only Cd2+
among the verified cations triggered the emission enhance-
ment, but it was negligible as compared to that of Zn2+. It is to
be noted that even highly abundant intracellular alkali metal
ions (Na+ and K+) and alkaline earth metal ions (Mg2+ and
Ca2+) did not affect the emission spectra of L1 even in the pres-
ence of excess amounts. The effect of different counteranions
of zinc salt was also investigated under the same conditions
using different zinc salts like Zn (ClO4)2, Zn (OAc)2, Zn (NO3)2,
ZnCl2, ZnBr2 and ZnSO4. From the experiment it was found
that the counteranions of zinc did not influence the recog-
nition process of Zn2+ by L1.
Fluorescence spectroscopic studies of L in the presence of
1
metal ions. With visible light excitability, when the receptor
(L1) was excited at 470 nm, a weak emission peak was observed
at ∼530 nm. A set of different metal ions (Na+, K+, Mg2+, Ca2+,
Co2+, Ni2+, Cu2+, Cd2+, Ag+, Pb2+, Hg2+, Al3+, Cr3+, Fe3+, and
Zn2+) were used to ascertain the selectivity of L1. Interestingly
only Zn2+ caused a drastic enhancement in the emission inten-
sity of L1 with a prominent peak obtained at 555 nm while
Cd2+ causes a very small enhancement in the emission inten-
sity (Fig. 2a). This observation clearly demonstrated high
selectivity of L1 towards Zn2+ over other metal ions including
Cd2+. During titration with Zn2+, L1 displayed a linear enhance-
pH dependent study. The fluorescence properties of the
receptor L1 was checked at different pH. At lower pH (2–6), the
receptor displayed blue fluorescence whereas at higher pH (9
and above) the receptor exhibited a yellow fluorescence with
lower intensity. Possibly at lower pH, protonation or hydrolysis
of the imine bond may have resulted in a blue fluorescence.
Fig. 2 Changes in emission spectra of L1 (25 μM) in the presence of (a)
various metal ions (10 eq.) and (b) with incremental addition of Zn2+
(0 μM–60 μM). Inset: change in colour under a UV lamp (λem = 365 nm)
upon addition of Zn2+ to L1.
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On the other hand, at higher pH, deprotonation of the
hydroxyl group perhaps leads to a weak yellow fluorescence.
Interestingly, it was observed that the ligand has stable fluo-
rescence within the pH range of 6.5–9.0. This observation
suggested that the ligand could render pH-independent fluo-
rescence measurement in a physiological microenvironment.
The high selectivity of L1 towards Zn2+ over other biologically
important metal ions, its sensing capability under physiologi-
cal conditions and a lower detection limit enhance the analyti-
cal merit of the developed receptor (L1) for the recognition of Scheme 2 Proposed mechanism of sensing.
intracellular Zn2+.
1H NMR titration. We were unsuccessful in getting a single
crystal of the Zn2+ complex of L1 suitable for X-ray diffraction
even after several attempts. Therefore, in order to understand
the mode of interaction between L1 and Zn2+ or the structural
changes in L1 after interacting with Zn2+, we acquired 1H-NMR
interacting with Zn2+ perhaps results in the enhancement of
the fluorescence intensity of the free receptor along with a red
shift of ∼25 nm. The proposed CHEF mechanism was verified
by conducting control experiments with the receptor L2, which
lacked the hydroxyl group. As anticipated, L2 did not show any
change in its emission spectra after interaction with Zn2+
owing to the lack of chelation of Zn2+ and a CHEF effect
thereof. This result strongly suggested that the di-imine moiety
with the hydroxyl group at the core was essentially implicated
in the Zn2+ sensing phenomenon.
Further, the above premise of our proposed sensing mecha-
nism was corroborated by the theoretical calculations of L1
and its Zn complex. To understand the relationship between
the structural changes of L1 and its complex with Zn2+ and the
optical response of L1 to Zn2+, we carried out density func-
tional theory (DFT) calculations with the B3LYP/6-31+G(d,p)
method basis set using the Gaussian 03 program. The opti-
mized geometries along with the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO) of L1 and its Zn2+ complex are presented in Fig. 3.
These HOMO and LUMO energy diagrams revealed that the
energy gap for the di-nuclear zinc complex of L1 (two zinc ions
are linked through the oxo-bridge) was reduced as compared
to the ligand alone (Fig. 4(a)). This perhaps led to the occur-
rence of a red shifted (25 nm) fluorescence emission band
upon chelation of the ligand with two Zn2+ ions in the pro-
1
data. H-NMR titration of the receptor L1 with Zn(ClO4)2 was
performed in DMSO-d6. During 1H-NMR titration of L1 with
Zn2+ significant spectral changes of L1 were observed.
Reduction in the intensity of the hydroxyl group (–OH) indi-
cated deprotonation as a result of interaction with Zn2+ and
after the addition of two equivalents of Zn2+ ions, the hydroxyl
group peak was obliterated. As illustrated in Fig. S14,† upfield
shifts were observed in the case of the pyridyl hydrogen atoms.
The Schiff base hydrogen atom and the hydrogen atoms on
the DFMP ring also underwent upfield shifts, whereas very
little change was observed for the amide proton (–NH) as com-
pared to the other protons within the system. Collectively
1H-NMR titration strongly suggested the formation of a
hydroxo bridged complex between L1 and Zn2+ resulting in the
deprotonation of the hydroxyl group and the pyridyl nitrogen
atom coordinating to Zn2+ causing shifts to the pyridyl hydro-
gen atoms.
ESI-MS experiment. The binding mode between L1 and Zn2+
was also investigated by ESI-MS. A peak at 869.8240 signifies
the mass of the ensemble (L1 + 2Zn + 3ClO4 + CH3CN)
C23H21Cl3N7O15Zn2 and another peak at 403.1546 represents
the mass of the free receptor. So, the result from ESI-MS experi-
ments also supports the results obtained from UV-Vis and
emission spectroscopy.
Mechanism of Zn2+ sensing. The enhancement in the fluo-
rescence intensity of the receptor L1 after interacting with Zn2+
may be attributed to the CHEF and ICT processes. Firstly, the
low fluorescence of L1 may be due to the free rotation of the
imine (–CvN) bond. As illustrated in Scheme 2, in the pres-
ence of Zn2+, chelation of the metal ion with the –OH group
and with the two imine N atoms ensues. Consequently, the
free rotation around the imine bond gets restricted and the
system becomes more rigid. Furthermore, chelation of Zn2+ by
the hydroxide group and the Schiff base N atoms leads to the
formation of a hydroxo bridged binuclear zinc complex in
which the conjugation increases drastically, resulting in a
CHEF effect. On the other hand, due to the binding of Zn2+
the ICT is facilitated over the π-system. This also caused
sufficient enhancement in the fluorescence. The CHEF
Fig. 4 (a) Energy diagrams of HOMO and LUMO orbitals of L1 and the
L1–Zn complex calculated at the DFT level using a B3LYP/6-31+G(d,p)
phenomenon in conjunction with the ICT process upon basis set. (b) Optimized structure of L1 and its Zn2+ complex.
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posed fashion. Selected orbitals and their corresponding ener- treated with Zn(ClO4)2, a distinct and strong turn-on green
gies for both L1 and its Zn complex were provided in ESI fluorescence was observed which could be attributed to the
(Tables S1 and S2 in ESI†) which might have been playing a formation of the intracellular L1–Zn complex. This observation
vital role in the optical spectral outcome.
is in good agreement with the results observed earlier in solu-
Cellular sensing of Zn2+. Owing to the promising response tion studies. It may be mentioned here that bright-field
of L1 towards Zn2+ and its intense emission in the visible images of treated HeLa cells revealed the characteristic mor-
region, it was envisaged that compound L1 could be used for phological attribute of HeLa cells, which also suggested that
detection of intracellular Zn2+ by fluorescence-based imaging the cells were viable. The fluorescence microscopic analysis
of live cells. However, to accomplish this target, it was impera- strongly suggested that compound L1 could readily traverse the
tive to initially evaluate the cytotoxic potential of compound L1 membrane barrier, pervade into HeLa cells, and rapidly sense
on live cells. The well-established MTT (3-(4,5-dimethylthiazol- intracellular Zn2+.
2-yl)-2,5-diphenyltetrazolium bromide) assay, which is based
on mitochondrial dehydrogenase activity of viable cells, illus-
trated that neither L1 nor its zinc complex was able to exhibit Conclusion
any effect on the viability of HeLa cells (human cervical carci-
noma cells), even at concentrations as high as 50 μM
(Fig. S15†). The results obtained from the in vitro cytotoxic
assay were encouraging and suggested that a ligand concen-
tration up to 50 μM can perhaps be employed for the fluo-
rescence imaging studies of L1 and the L1–Zn complex in live
In brief, we have synthesized and demonstrated the sensing
potential of an efficient receptor (L1) for Zn2+ under physiologi-
cal conditions (99.7% HEPES buffer, pH = 7.4). The ligand L1
can be excited in visible light and has high selectivity towards
Zn2+ over other biologically significant metal ions, even in the
presence of higher concentration of competing metal ions.
cells. Hence, to evaluate the efficiency of compound L1 as a
The judicious choice of a DFMP core in the receptor rendered
chemosensor for intracellular detection of Zn2+ by fluo-
a strong CHEF-based turn-on fluorescence following chelation
rescence microscopy, HeLa cells were treated with 25 μM L1
of Zn2+ by the hydroxide group and the Schiff base N atoms.
solution for 1 h followed by incubation with 50 μM Zn(ClO4)2
Theoretical calculations of L1 and L1–Zn ensemble also
to promote the formation of L1–Zn complex. Fluorescence
microscope analysis revealed that compound L1 alone failed to
exhibit any fluorescence in HeLa cells (Fig. 5). However, when
support our proposed mechanism. Due to the high selectivity
of L1 towards Zn2+ in exclusively physiological medium and its
non-cytotoxic nature, the newly developed receptor was suc-
cessful in sensing intracellular Zn2+, wherein immensely
intense turn-on green fluorescence was manifested in HeLa
cells. The non-cytotoxic behaviour of L1 and its capability of
sensing intracellular Zn2+ are well suited for future in vivo
biomedical applications of the sensor.
Acknowledgements
The authors acknowledge CSIR (01/2727/13/EMR-II), Science &
Engineering Research Board (SR/S1/OC-62/2011) and Depart-
ment of Biotechnology (BT/01/NE/PS/08), New Delhi, India for
financial support and the Central Instruments Facility, IIT
Guwahati for providing instrument facilities. B.K.D., D.T., S.S.
and C.K. acknowledge IIT Guwahati for research fellowships.
Special thanks are due to Moumita Pait for her kind
cooperation.
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