Imine-linked chemosensors for the detection of Zn2+ in biological samples

Article abstract of DOI:10.1039/c3ra46759g

A chemosensor 1 with a long hydrocarbon chain and polar end group is synthesized by the simple condensation reaction of a long chain amine with salicylaldehyde. A long chain hydrocarbon with a polar end group is used because of its solubility in an aqueous surfactant solution, which ensures that it can be used in a neutral water medium. The rationale for choosing an aryl aldehyde with -OH functionality is based upon the fact that a chelate ring consisting of an -OH group and an sp² nitrogen donor is always better for the selective recognition of Zn²⁺. The sensor shows selective binding to Zn²⁺ in 1% Triton-X-100 solution. Binding of Zn²⁺ by sensor 3 leads to an approximately 300% enhancement in the fluorescence intensity of the sensor, due to the combined effects of excited state intramolecular proton transfer (ESIPT) and the inhibition of the photo-induced electron transfer (PET) process by the -OH group. The fluorescence emission profiles of sensor 1 show some changes in the low and high pH ranges, however the sensor remains stable in the pH range 4-9, which makes it appropriate for use in biological fluids.

Full text of DOI:10.1039/c3ra46759g

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Imine-linked chemosensors for the detection of
Zn2+ in biological samples†
Cite this: RSC Adv., 2014, 4, 9784
Preeti Saluja,a Vimal K. Bhardwaj,a Thangarasu Pandiyan,c Simanpreet Kaur,b
b
a
*
*
Navneet Kaur and Narinder Singh
A chemosensor 1 with a long hydrocarbon chain and polar end group is synthesized by the simple
condensation reaction of a long chain amine with salicylaldehyde. A long chain hydrocarbon with a polar
end group is used because of its solubility in an aqueous surfactant solution, which ensures that it can be
used in a neutral water medium. The rationale for choosing an aryl aldehyde with –OH functionality is
based upon the fact that a chelate ring consisting of an –OH group and an sp2 nitrogen donor is always
better for the selective recognition of Zn2+. The sensor shows selective binding to Zn2+ in 1% Triton-X-100
solution. Binding of Zn2+ by sensor 3 leads to an approximately 300% enhancement in the fluorescence
intensity of the sensor, due to the combined effects of excited state intramolecular proton transfer (ESIPT)
and the inhibition of the photo-induced electron transfer (PET) process by the –OH group. The
fluorescence emission profiles of sensor 1 show some changes in the low and high pH ranges, however
the sensor remains stable in the pH range 4–9, which makes it appropriate for use in biological fluids.
Received 16th November 2013
Accepted 13th December 2013
DOI: 10.1039/c3ra46759g
www.rsc.org/advances
some success in imaging Zn2+ in living cells.6 The Zn2+ sensor
designs make use of various combinations of sp2 hybridised
Introduction
nitrogen atoms in quinoline, bipyridyl, benzimidazole and
benzothiazole as donor sites.7 These types of sensors have an
advantage, as these moieties can simultaneously act as metal
binding sites as well as uorophores.8 This duality may help to
reduce the size of the sensor and eventually reduce the burden
of long synthetic strategies. Some of these sensors operate
through a chelation enhanced uorescence (CHEF) mechanism
and exhibit enhanced uorescence intensities.9
The design and synthesis of metal chemosensors with high
selectivity and sensitivity is an active eld of supramolecular
chemistry.1 The extraordinary spectroscopic properties of uo-
rescent chemosensors make them a promising alternative to
other techniques for a variety of applications such as analytical
chemistry, medical diagnostics and materials science.2 Zinc is
the second most abundant transition metal ion in the human
body, and is an integral part of major biological processes such
as gene transcription, immune function and brain function,
etc.3 Zinc is essential for the proper functioning of cellular
metabolism, and it plays an important role in fertility in men
and women. It stimulates the catalytic activity of about 100
enzymes in vital chemical reactions in living systems.4 However,
an excess of zinc can create disorder in metabolism and can
further interfere in the absorption of iron, magnesium and
copper. Doses of zinc supplements of above 40 mg per day can
cause gastrointestinal upset, a metallic taste in the mouth,
blood in urine, and lethargy.5 Recently, many research groups
have developed a variety of uorescent sensors for Zn2+ with
The formation of a chelate ring with the participation of
donor sites from the uorophore may change the HOMO–
LUMO energy gap; in other words, metal binding close to the
uorophore generally affects internal charge transfer (ICT).10
These modulations result in with a change in the wavelength,
which is the basis of recently reported ratiometric sensors.11
Although detailed mechanistic studies are available in the
literature for Zn2+ sensors, some of these sensors have short-
comings such as poor water solubility, interference from other
metal ions, the fact that they have not been evaluated for
biocompatibility and problems monitoring the uorescence
changes at a conventional single wavelength.12 In addition to
these points, a low detection limit and wide detection range are
basic requirements for sensor development.13 A change of
substituents and their positions with respect to the sp2 nitrogen
moiety may affect the sensitivity and selectivity of the sensor.
This is the governing feature of many imine-linked chemo-
sensors and their metal complexes.14 With this intention, we
synthesized sensor 1 by a simple condensation reaction. The
long hydrocarbon chain with polar end groups may help to
aDepartment of Chemistry, Indian Institute of Technology Ropar (IIT Ropar),
Rupnagar, Punjab, 140001, India. E-mail: nsingh@iitrpr.ac.in
b
´
´
´
Facultad de Quımica, Universidad Nacional Autonoma de Mexico (UNAM), Ciudad
´
´
Universitaria, Coyoacan, 04510 Mexico D. F., Mexico
cCentre for Nanoscience and Nanotechnology (UIEAST), Panjab University,
Chandigarh, 160014, India. E-mail: navneetkaur@pu.ac.in
† Electronic supplementary information (ESI) available: NMR, IR spectra, UV-Vis
absorption, uorescence proles and graphs for the fabrication and operation of
ISE based upon 2a–c. See DOI: 10.1039/c3ra46759g
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solubilise the sensor in an aqueous surfactant solution and thus comparison between the uorescence signature of 1 with the
will ensure neutral water solubility.
uorescence proles of 1 upon addition of metal ions revealed
that Zn2+ signicantly increases the uorescence intensity of
sensor 1 (Fig. S7†). The remarkable inuence of Zn2+ on the
uorescence emission of sensor 1 indicates a strong interaction
between 1 and Zn2+. The probe 1 exhibits dual channel emission
with weak emission at lmax ¼ 355 nm and moderate emission at
lmax ¼ 440 nm in the uorescence signature of 1 recorded in
aqueous 1% Triton-X-100 solution. This is due to the existence
of 1 in both the keto and enol tautomeric forms produced
through the excited state intramolecular proton transfer (ESIPT)
mechanism, involving the –OH group and sp2 nitrogen of the
imine linkage.16 The emission at lower wavelength is assigned
to the enol form, while the keto form emits at the higher
wavelength.
To gain more insight into the modulation of the uorescence
signatures of 1 due to structural features, DFT calculations were
performed using Becke's three parameterized Lee–Yang–Parr
(B3LYP) exchange functional with 6-31G* basis sets, using
Gaussian-09 programs (Fig. 2).17 The enol form possesses the
correct symmetry to show H-bonding between the –OH group
and the sp2 hybridised nitrogen. The fully optimised structures
of 1 indicate the energy equivalence for the enol and keto states
(Table S1†). Therefore, both tautomeric forms should exist in
equilibrium and 1 may exhibit the ESIPT phenomenon.18 DFT
calculations predict the keto form of 1 to be energetically more
stable than the enol form. The observed phenomenon of uo-
rescence enhancement of 1 upon binding of Zn2+ is due to
conversion of the enol form to the keto form, and the
enhancement is due to inhibition of the PET process.19 This
change was even clearly visible when sensor 1 was viewed in the
absence or presence of Zn2+ under 365 nm UV light. A solution of
sensor 1 in aqueous 1% Triton-X-100 solution became uores-
cent when Zn2+ was added to it as shown in the inset of Fig. 3B.
Silica strips coated with the sensor and polymer coated
sensor strips dipped in Zn2+ were observed under 365 nm UV
Results and discussion
The objective of the current investigation is to develop water
soluble, biocompatible, selective and sensitive Zn2+ sensors,
and compound 1 was chosen as a model compound for this
study. The rationale for this choice is based upon the fact that a
chelate ring consisting of an –OH group and an sp2 nitrogen
donor is effective for selective recognition of Zn2+. The moieties
present in 1 may show both chromogenic and uorescent
recognition to some extent as detailed in the literature.15 The
target compound 1 was synthesized by the condensation of
oleylamine with the respective salicylaldehyde and was char-
acterized by various spectroscopic techniques (Fig. S1–S4†). Due
to the poor solubility of 1 in pure aqueous media, we used an
“aqueous friendly” version of the surfactant medium. A number
of surfactants were tried in order to dissolve 1 in an aqueous
medium, however the best solubility of this compound was
achieved with a 1% Triton-X-100 aqueous solution. Alteration of
the pH can affect the UV-Vis absorption and uorescence
proles of sensor 1, as it has an sp2 N and an –OH group that are
prone to protonation and ionization respectively with variation
in pH. The effect of the pH on sensor 1 was evaluated in 1%
Triton-X-100 in water. However, the UV-Vis absorption and
uorescence proles of sensor 1 remained almost unaltered in
the pH range 4–9 (Fig. S5†), so the sensor is suitable for use in
living systems. The effect of ionic strength on the UV-Vis
absorption and uorescence emission proles of sensor 1 was
excluded by recording its uorescence and UV-Vis spectra in the
presence of increasing concentrations of NaCl (Fig. S6†).
The literature reports highlighting the introduction of a
salicylic group in uorescent chemosensors encouraged us to
evaluate the properties of 1 as a uorescent metal chemosensor.
Sensor 1 showed an emission band centred at 440 nm when a
5 mM concentration of 1 (in aqueous 1% Triton-X-100 solution)
was excited at lex ¼ 320 nm. Fig. 1 shows the uorescence
spectra of 1 before and aer addition of 50 mM concentration of
various metal ions (Na+, K+, Ba2+, Sr2+, Ca2+, Mg2+, Mn2+, Cr3+,
Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Hg2+, and Cd2+). A
Fig. 1 Fluorescence emission spectra of 1 (5 mM) in the presence of Fig. 2 Fully optimised structures of the enol and keto forms of sensor
different metal ions (50 mM) in aqueous 1% Triton-X-100 solution 1; DFT calculations were performed at the B3LYP/6-31G* level (red,
(excitation wavelength was 320 nm).
blue and grey spheres refer to O, N and C atoms respectively).
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the uorescence intensity in the absence of Zn2+, demonstrating
the wide range of Zn2+ concentrations that can be detected by
sensor 1. The mass spectrum of 1 shows a peak at m/z value
372.4, which corresponds to [M + 1] where M is the molecular
weight of 1 (Fig. S4†). The mass spectrum of complex 1$Zn2+
shows a peak at m/z value 26ꢀ9.4, which corresponds to [M + 1]2+
,
where M is [1 + Zn2+ + NO3 + CH3CN], suggesting that a 1 : 1
complex is formed between sensor 1 and Zn2+. The observed
stoichiometry of the complex was further conrmed with a Job's
plot20 (Fig. S8†). The binding constant for the formation of the
1$Zn2+ supramolecular complex was calculated using a Benesi–
Hildebrand plot of the uorescence titration data, and it was
calculated to be 1.05 (ꢁ0.3) ꢂ 105 (Fig. S9†), revealing strong
binding between sensor 1 and Zn2+ 21 Sensor 1 was found to be
.
procient in detecting Zn2+ concentration to a level of 8 nM
(Fig. S10†). The detection limit was calculated from the uo-
rescence titration data using a known literature method, and
the details are shown in the ESI.†22
To determine the binding sites of 1 responsible for the
coordination of Zn2+, an NMR titration of 1 with successive
addition of Zn2+ was performed in a DMSO-d6–D2O (95 : 5, v/v)
solvent system (Fig. 4). It was found that the addition of 3.0
equiv. of Zn2+ led to a shi of the signals (Dd ¼ 0.22) corre-
sponding to –CH]N. On the other hand, shis were also
observed for the aromatic signals (up to Dd ¼ 0.1). These
concurrent shis led us to conclude the importance of the sp2
nitrogen of the sensor. As the titration was conducted in DMSO-
d6–D2O (95 : 5, v/v), our ability to determine the fate of the –OH
signal during the course of the titration was limited.
The application of a probe as a sensor in biological systems
requires the probe to be biocompatible. To address this, the
cytotoxicity of 1 was determined using an MTT assay with HeLa
cells cultured in a nutrient mixture. As per the standard
protocol,23 the cells were incubated at 37 ꢃC in a 5% CO2
Fig. 3 (A) UV-Vis absorption spectra of sensor 1 in the presence and
absence of Zn2+; (B) fluorescence spectra of sensor 1 upon successive
additions of Zn2+ (0–50 mM), inset of the figure shows the emission of a
solution of sensor 1 in the presence and absence of Zn2+ under
365 nm UV light; (C) silica strips (i and ii) and polymer coated silica
strips (iii and iv) dipped in sensor 1 (i–iv) and then in Zn2+ (ii and iv only)
viewed under 365 nm UV light.
light. Fig. 3C shows that the strips became uorescent in the
presence of Zn2+. The UV-Vis absorption spectrum of 1 in the
presence of Zn2+ is almost the same as that of sensor 1, as
shown in Fig. 3A.
As there is no shi in the band maxima positions, it can be
concluded that the change in uorescence due to the binding of
Zn2+ to sensor 1 is a consequence of excited state phenomena.
Sensor 1 was titrated against Zn2+ (0–50 mM) in aqueous 1%
Triton-X-100 solution with successive addition of aliquots of
Zn2+. The uorescence intensity of the emission band of 1 at 440
nm increased steadily with subsequent additions of Zn2+, as
Fig. 4 Family of partial 1H NMR spectra showing the change in the
signals of 1 upon complexation of 1 (10 mM) with Zn2+ (5 equiv.) in a
shown in Fig. 3B. The rise in uorescence intensity is 322% of DMSO-d6–D2O (95 : 5, v/v) solvent system.
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environment for 24 h. Upon addition of 1, only marginal cell
death (ꢄ97% cell survival) was observed when the experiments
were performed at the probe concentration used for recognition
studies (5 mM). To investigate the potential cytotoxic effects of 1
at higher concentrations, dose dependent studies were per-
formed. This experiment is mandatory because sometimes
higher concentrations of the probe may be needed for certain
biological applications. The results indicate that 1 is not very
toxic to HeLa cells if the probe concentration remains under 22
mM, with ꢄ85% cell survival over 24 h for a 22 mM concentration
of 1. At much higher concentrations of 1, the probe became
more toxic. For example, the IC50 value was found to be 56 mM
over 24 h. To explore the application of 1 for Zn2+ recognition in
real biological systems, we detected Zn2+ in blood serum, and
the results were validated with atomic absorption spectra. A
high Zn2+ concentration of 0.1 mM is naturally present in
blood,24 and deviation from the normal value is usually taken as
an early diagnosis of certain illnesses.25 Measurement of Zn2+ in
red blood cells (RBCs) has been reported to be able to
discriminate between hyperthyroid Grave's disease and tran-
sient thyrotoxicosis26, and it may be a predictive indicator of
sepsis syndrome in infancy.27 The literature methods devised
for the detection of Zn2+ ions in blood make use of atomic
absorption spectroscopy (AAS),28,29 such as graphite furnace
AAS,30 microwave assisted mineralization and ow injection
AAS31 and derivative microsampling ame AAS32, or the induc-
tively coupled plasma mass spectrometry (ICP-MS) method.33,34
However, these methods are time consuming and uorescence
spectroscopy offers the advantage of being a simple to use
method.
Fig. 5 Microscopic images of (A) microbe cells cultured in normal
medium, (B) microbe cells cultured in medium enriched with Zn2+, (C)
microbe cells cultured in normal medium and then treated with sensor
1, and (D) microbe cells cultured in medium enriched with Zn2+ and
treated with sensor 1. Before performing microscopy, the microbe
cells were washed with aqueous 1% Triton-X-100 solution.
This implies that Zn2+ has a strong affinity for sensor 1 and it
occupies the binding sites available in the sensor preferentially
over other cations.
The effect of the pH on the binding affinity of sensor 1 for
Zn2+ was also examined (Fig. S15†). A decrease in the pH
inhibits the binding between sensor 1 and Zn2+, however an
increase in the pH up to 8.5 has no effect on the activity of
sensor 1 for Zn2+ detection. However, at pH ¼ 10, we observed
the formation of a white ZnO precipitate.
To investigate the practical application of 1 in Zn2+ recog-
nition in biological systems, we cultured Saccharomyces cer-
evisiae in normal broth and in experimental media containing
Zn2+. The cells cultured in the media containing Zn2+ were
treated with sensor 1 dissolved in aqueous 1% Triton-X-100
solution. Before performing microscopy observations, the
microbe cells were washed with aqueous 1% Triton-X-100
solution. Microscopy images were taken of: (A) microbe cells
cultured in normal medium, (B) microbe cells cultured in
medium enriched with Zn2+, (C) microbe cells cultured in
normal medium and then treated with sensor 1, and (D)
microbe cells cultured in medium enriched with Zn2+ and
treated with sensor 1 (Fig. 5). The microscopic investigations
revealed that sensor 1 is capable of binding Zn2+ in a real
cellular medium.
Experimental section
Materials and methods
All the chemicals were purchased from commercial suppliers
and were used without further purication. H NMR and 13C
1
NMR spectra were recorded on an Avance-II (Bruker) instru-
ment, which operated at 400 MHz for 1H NMR and 100 MHz for
13C NMR. Fourier transform infrared (FT-IR) spectra of the dried
The roles of the sp2 N and –OH binding sites were estab-
lished by synthesizing compounds 2 and 3, in which the
hydroxyl group is missing in 2 and the imine linkage is missing
in 3 (Fig. S11 and 12†). Neither showed any affinity for Zn2+
(Fig. S13 and 14†), conrming that the presence of both the sp2
N and –OH binding sites is vital for the formation of a complex
with Zn2+. To check for any potential interference by any of the
tested metal ions with the detection of Zn2+ by 1, the ability of 1
to operate in solutions containing equimolar amounts of Zn2+
and other cations (10 equivalents) was tested. This experiment
(as shown in Fig. 6) eliminated the possibility of any interfer-
Fig. 6 Influence of the other tested metal ions on the sensor activity of
1 for Zn2+ binding.
ence by the other tested cations with the recognition of Zn2+
.
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compounds were measured on a Bruker Tensor 27 spectro-
photometer, using a KBr pellet technique. For cation recogni-
tion studies, the UV-Vis absorption spectra were recorded using
dilute solutions in quartz cells (1 cm path length) on a Specord
250 Plus Analytikjena spectrometer, and solid state spectra were
recorded using ne coatings of the materials between the quartz
plates. The uorescence proles of the sensor solutions were
recorded on a Perkin Elmer L55 Fluorescence spectrophotom-
eter using 1 cm path length quartz cells. The slit width for the
excitation and emission was set at 10 nm and the scan speed
was maintained at 200 scans per second throughout the
experiments. The solid-state photoluminescence (PL) study was
conducted using a setup that involved a solid sample holder.
Scanning electron microscopic studies of aqueous solutions of
the materials were carried out at a concentration of 200 mM.
SEM images were recorded with a JEOL JSM-6610LV scanning
electron microscope, which operated at 15 keV.
Synthesis of compound 3. Compound 1 (186 mg, 0.5 mmol)
was reduced with NaBH4 (76 mg, 2 mmol) in methanol (10 ml)
at 50 ꢃC for 8 h. Aer the completion of the reaction, the
methanol was evaporated and the residue was dissolved in
chloroform and washed with distilled water. The organic extract
was separated, dried and evaporated to afford a white coloured
solid compound (74% yield). 1H NMR (400 MHz, CDCl3) d
(ppm): 7.19 (t, 1H, ArH), 7.01 (d, 1H, ArH), 6.87 (d, 1H, ArH),
6.80 (t, 1H, ArH), 6.62 (br s, 1H, NH) 5.41 (m, 2H, CH]CH), 4.01
(s, 2H, N–CH2), 2.70 (t, 2H, CH2–N), 2.05 (m, 4H, C]C–CH2),
1.56 (m, 2H, CH2), 1.32 (m, 22H, CH2), 0.93 (t, 3H, CH3); 13C
NMR (100 MHz, CDCl3) d (ppm): 158.2, 129.8, 129.7, 128.6,
128.2, 122.2, 118.8, 116.3, 52.4, 48.5, 32.5, 31.8, 29.7, 29.6, 29.6,
29.5, 29.4, 29.3, 29.2, 29.1, 27.1, 27.09, 27.0, 22.6, 14.0; FTIR nmax
(KBr pellet): 3702 cmꢀ1. Anal. calcd for C25H43NO: C, 80.37; H,
11.60; N, 3.75. Found: C, 80.52; H, 11.41; N, 3.82.
Synthesis of compound 1. A solution of oleylamine (267 mg,
1 mmol) and salicylaldehyde (183 mg, 1.5 mmol) in 50 ml dry
methanol was stirred and reuxed for 4 h. Aerwards, the
solvent was evaporated to 20 ml and the solution was kept at
0 ꢃC. A yellow coloured solid separated out at low temperature.
This solid was ltered and washed with cold methanol (three
times), and the product (1) was obtained in 88% yield (327 mg).
1H NMR (400 MHz, CDCl3) d (ppm): 13.71 (br s, 1H, OH), 8.35 (s,
1H, CH]N), 7.29 (m, 2H, ArH), 6.98 (d, 1H, ArH), 6.88 (t, 1H,
ArH), 5.38 (m, 2H, CH]CH), 3.60 (t, 2H, CH2–N), 2.04 (m, 4H,
C]C–CH2), 1.71 (m, 2H, CH2), 1.33–1.28 (m, 22H, CH2), 0.89 (t,
3H, CH3); 13C NMR (100 MHz, CDCl3) d (ppm): 164.4, 161.4,
132.0, 131.1, 130.1, 129.8, 118.9, 118.4, 117.1, 59.6, 32.7, 31.9,
30.9, 29.8, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 29.1, 27.2, 22.8,
14.2; FTIR nmax (KBr pellet): 1633 cmꢀ1. Anal. calcd for
Metal recognition studies of 1–3
ꢃ
All the recognition studies were performed at 25 ꢁ 1 C, and
before recording any spectra the samples were shaken for a
sufficient time to ensure uniformity of the solutions. The cation
binding abilities of 1–3 in 1% Triton-X-100 in water were
determined by preparing standard solutions of 1–3 along with
xed amounts of the particular metal nitrate salts in 1% Triton-
X-100 in water. The cation recognition behaviours of 1–3 were
evaluated from the changes in photophysical properties of the
sensors upon addition of that metal salt. The uorescence
spectra of 1–3 were recorded with the excitation wavelengths
shown in the respective gures. For the titrations, volumetric
asks containing a standard solution of sensor 1 along with
varying amounts of a particular metal nitrate salt in 1% Triton-
X-100 in water were used. To evaluate any possible interference
by other metal ions with the detection of Zn2+, solutions were
prepared containing sensor 1 (5 mM) along with a xed
concentration of Zn2+ (5 mM) both with and without other
background cations (50 mM) in 1% Triton-X-100 in water. The
uorescence intensity of each solution was recorded. The effect
of the pH on the UV-Vis absorption and uorescence spectra of
sensor 1 was investigated by recording spectra of sensor 1 in 1%
Triton-X-100 in water with variable pH.
C
25H41NO: C, 80.80; H, 11.12; N, 3.77. Found: C, 80.54; H, 11.31;
N, 3.36; ESI-MS m/z ¼ 372.4 (M + 1) (Scheme 1).
Synthesis of compound 2. A solution of oleylamine (267 mg,
1 mmol) and benzaldehyde (159 mg, 1.5 mmol) in 50 ml dry
methanol was stirred and reuxed for 4 h. Aerwards, the
solvent was evaporated to 20 ml and the solution was kept at
ꢃ
0 C. An off-white coloured solid separated out at low temper-
ature. This solid was ltered and washed with cold methanol
(three times), and the product (2) was obtained in 82% yield
(292 mg). 1H NMR (400 MHz, CDCl3) d (ppm): 8.29 (s, 1H, CH]
N), 7.75 (m, 2H, ArH), 7.44 (m, 3H, ArH), 5.39 (m, 2H, CH]CH),
3.63 (t, 2H, CH2–N), 2.03 (m, 4H, C]C–CH2), 1.71 (m, 2H, CH2),
1.35–1.28 (m, 22H, CH2), 0.92 (t, 3H, CH3); 13C NMR (100 MHz,
CDCl3) d (ppm): 160.6, 136.4, 130.4, 129.9, 129.8, 128.5, 128.1,
61.9, 32.7, 32.0, 31.8, 31.0, 29.9, 29.7, 29.6, 29.5, 29.47, 29.4,
29.3, 29.2, 29.1, 27.4, 27.3, 22.8, 14.2; FTIR nmax (KBr pellet):
1647 cmꢀ1. Anal. calcd for C25H41N: C, 84.44; H, 11.62; N, 3.94.
Found: C, 84.62; H, 11.51; N, 3.87 (Scheme 2).
Stoichiometry determination
In order to determine the stoichiometry of the complex formed
from receptor 1 and Zn2+, solutions of 1 and Zn2+ were prepared
with ratios of 1 : Zn2+ of 1 : 9, 2 : 8, 3 : 7, 4 : 6, 5 : 5, 6 : 4, 7 : 3,
8 : 2 and 9 : 1. These solutions were kept for 1 h, and were
shaken occasionally. Fluorescence spectra were recorded
for the Zn2+ complex. A plot of [HG] vs. [H]/[H] + [G] (H ¼ host,
Scheme 1 Synthesis of sensor 1.
9788 | RSC Adv., 2014, 4, 9784–9790
Scheme 2 Chemical structures of 2 and 3.
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G ¼ guest, HG ¼ host-guest complex) was used to determine the
stoichiometry of the complex formed. The uorescence inten-
sity at 440 nm was used for the calculations. The concentration
of HG was calculated using the equation [HG] ¼ DI/Io ꢂ [H].
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Cytotoxicity of 1
The cytotoxicity of 1 was determined through an MTT assay,
using HeLa cells seeded in a 96-well at-bottomed microplate in
growth medium (100 ml) and incubated at 37 ꢃC under a 5% CO2
atmosphere for 24 h. An analysis using 1 as the test compound
and a blank analysis were performed, and 10 ml of MTT in PBS
was added to each well. The microplate was incubated at 37 ꢃC
under a 5% CO2 atmosphere for another 3 h. The medium was
then removed, and DMSO (100 ml) was added to each well. The
absorbance spectrum of each solution was measured at 570 nm.
The dose dependent cytotoxicity of 1 was determined using
different concentrations of 1 using the same conditions as
mentioned above.
Fabrication of sensor strips
Two types of strip were used: one was simply a silica strip coated
with sensor 1, and the second consisted of a silica strip rst
coated with polymer polyethylene graed maleic anhydride and
then coated with sensor 1. Both of these become uorescent
upon dipping into a solution of Zn2+.
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Conclusion
A highly selective sensor, 1, with a chelate ring consisting of
–OH and sp2 N moieties for the detection of Zn2+ in an aqueous
surfactant solution was synthesized. Sensor 1 displays an
approximately 300% increase in uorescence intensity upon
binding to Zn2+, through the collective effects of ESIPT and
inhibition of PET.
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Acknowledgements
This work was supported with a research grant (project no.:
01(2417)/10/EMR-II) from CSIR (New Delhi, India). V. K. B. is
thankful to the DST for the INSPIRE Faculty grant.
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