Sensors and Actuators B 238 (2017) 903–907

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Bis-pyridobenzene as a fluorescence light-up sensor for Hg2+ Ion in

water
Ujjal Ghosh a , Subhendu Sekhar Bag a,b,∗ , Chandan Mukherjee a,b,∗
a
Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
b
Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India

a r t i c l e i n f o a b s t r a c t

Article history: Heavy metal ion such as mercury is a highly toxic in all its oxidation states in the environment. Mercury
Received 3 March 2016 exposure causes a variety of adverse environmental and health problems, such as brain damage, neuro-
Received in revised form 22 July 2016 logical disorder, damage of immune system etc. Mercuric ions (Hg2+ ) being highly water-soluble are easily
Accepted 25 July 2016
bioavailable for humans and animals through ingestion leading to many of the physiological problems.
Available online 27 July 2016
Thus, continued alarm over mercury pollution has prompted increasing interest in the design of new
fluorescent probe for mercury detection in biological and environmental samples. Therefore, we synthe-
Keywords:
size meta-di-4-methylpyridyl benzene (m-DPB) core as a possible fluorescent probe for Hg2+ detection.
Hg2+ sensor
Fluorescence
The probe shows a dual fluorescence emission at long wavelength region (350, 425 nm) indicating its
m-di-4-Pyridyl benzene (m-DPB) potential use as a probe in aqueous medium which could be utilized for sensing of metal ions. Herein,
Synthesis we report the synthesis of that new and novel fluorescence probe and its use in switch-on sensing of
Light-up sensor Hg2+ ion. The probe is very simple in design and is found to be highly sensitive for sensing Hg2+ ion in
Water media the presence of other interfering metal cations with a high binding constant for 1:1 probe-Hg2+ com-
plexation and high sensitivity (1 ␮M) in water. All the observations are evident from the absorption and
fluorescence spectroscopic studies. A theoretical calculation and NMR titration suggest that the probe
binds Hg2+ through the coordination with two pyridyl nitrogens.
© 2016 Published by Elsevier B.V.

1. Introduction complexation properties of mercury still present plenty opportuni-

The mercury, being the most toxic heavy metal element, can lead
to environment pollution and deleterious effects on human health
[1]. Therefore, the continuing concern over mercury pollution has
boosted interest in the design of new probes/new methods for mer-
cury detection that are cost effective, rapid, facile and applicable
to monitor Hg2+ in biological and environmental samples. Toward
this goal, a wide variety of Hg2+ sensors based on electrochemi-
cal [2] response, metal nanoparticles [3], thin film sensors [4] as
well as fluorescence [1,5] changes have been developed. Among
all the reported methodologies, fluorescence based detection is
highly attractive owing to its high sensitivity, selectivity, simplic-
ity and versatility [1]. Considerable efforts have also been devoted
to the development of Hg2+ ion selective reversible [6] as well as
irreversible [7] fluorescence chemosensors. However, the favorable
∗ Corresponding authors at: Department of Chemistry, Indian Institute of Tech-
nology Guwahati, Guwahati 781039, Assam, India.
E-mail addresses: ssbag75@iitg.eret.in, ssbag75@yahoo.co.in (S.S. Bag),
cmukherjee@iitg.ernet.in (C. Mukherjee).
ties for design of new fluorescent probes for the detection of Hg2+
ion in water. Most of the reported small organic molecules based
sensors, suffers from several shortcomings such as use of organic
solvent, quenched emissions and poor selectivity [5a]. Only a few
such sensors can detect Hg2+ ions in water with high sensitivity and
selectivity [5b]. Therefore, there is a real need for new probe design
to address fluorescence based selective detection of Hg2+ in water.
As a part of our continuous research efforts for the design of new
fluorescence sensor for biologically and environmentally impor-
tant metal ions and biomolecules [8], herein, we report a highly
selective fluorescent probe for Hg2+ in water based on meta-di-4-
methylpyridyl benzene (m-DPB) core. We envisaged that the probe
would be effective in binding Hg2+ through the coordination with
two pyridyl nitrogens and ultimately would result in a remarkable
change in the fluorescence emission intensity of the probe in water.
Therefore, in this report we present the newly designed simple but
smart fluorescence sensor meta-di-4-methylpyridyl benzene (1, m-
DPB, Fig. 1) for fluorescence switch-on sensing of Hg2+ ion. The
sensor is very easy to synthesize and it displays a high selectivity
and sensitivity (1 ␮M) for Hg2+ among the metal ions examined
with a switch-on fluorescence response.

http://dx.doi.org/10.1016/j.snb.2016.07.139
0925-4005/© 2016 Published by Elsevier B.V.
904 U. Ghosh et al. / Sensors and Actuators B 238 (2017) 903–907
Fig. 1. (a) The energy optimized-structure of the fluorescence probe m- di-4-methylpyridyl benzene (m-DPB) and (b) its fluorescence emission spectra in various solvents.
2. Materials and methods
2.1. General experimental
Organic extracts were dried over anhydrous sodium sulphate.
Solvents were removed in a rotary evaporator under reduced
pressure. Silica gel (60–120 mesh size) was used for the column
chromatography. Reaction was monitored by TLC on silica gel 60
F254 (0.25 mm). 1 H NMR spectrum was measured with Varian
400 (400 MHz) and 13 C NMR spectrum was measured with Var-
ian 400 (100 MHz) spectrometer. Coupling constants (J value) were
reported in hertz. The chemical shifts were shown in ppm down-
field from tetramethylsilane, using residual methanol (␦ = 3.35 and
4.78 in 1 H NMR, ␦ = 49.3 in 13 C NMR), as an internal standard. Mass
spectrum was recorded using WATERS MS system, Q-tof premier
and data analyzed using Mass Lynx 4.1. IR spectrum was recorded
in KBr on a Perkin Elmer Spectrum one FT-IR spectrometer.
2.2. Synthetic procedure
A 100 mL round-bottom flask was warmed with NiCl2 ·6H2 O
(0.06 g, 0.25 mmol) and DMF (5 mL). The resulting solution was
stirred and heated to 50 ◦ C, and then 2-bromo-4-methylpyridine
(0.173 g, 1 mmol), and 2-(3-bromophenyl)-4-methylpyridine
(0.411 g, 1.66 mmol), anhydrous LiCl (0.147 g, 3.5 mmol), and zinc
dust (0.273 g, 4.2 mmol) were added. At this temperature a grain
of iodine crystal and two drops of acetic acid were added to the
mixture. An immediate rise in temperature and color change to
black was caused. The mixture was stirred at 70–80 ◦ C for 24 h. To
the cooled reaction mixture was added 2 N HCl aqueous solution
(15 mL) to consume the remaining zinc dust. The resulting mixture
was made alkaline with aqueous ammonia (30%) to make the pH
of the solution 10–12 and taken up with CH2 Cl2 . The organic layers
were collected, dried over anhydrous Na2 SO4 , and concentrated.
The crude material was purified by column chromatography.
3. Photophysical study
All the UV–visible spectra of the compounds (10 ␮M) in pres-
ence of various metal ions with different concentration were
measured using a UV–vis spectrophotometer with a cell of 1 cm
path length. All the sample solutions were prepared as described
in UV measurement experiments. Fluorescence spectra were
obtained using a fluorescence spectrophotometer at 25 ◦ C using
1 cm path length cell. The fluorescence quantum yields (f ) were
determined using quinine sulphate as a reference with the known
f (0.54) in 0.1 molar solution in sulphuric acid.
4. Results and discussion
Anticipating that meta-di-4-methylpyridyl benzene (m-DPB)
core could form a strong complex with mercury via the coor-
dination through two pyridyl nitrogens, we decided to prepare
the m-DPB fluorophore and to study its complexing and photo-
physical properties. The synthesis of the probe was proceeded
by employing Zn-catalysed aromatic C C bond forming reaction.
Thus, 2-bromo-4-methylpyridine (2) was reacted with 2-(3-
bromophenyl)-4-methylpyridine (3) in presence of anhydrous LiCl,
NiCl2 ·6H2 O and zinc dust in DMF as solvent at 70–80 ◦ C to form
the product 1, m-DPB (Scheme 1). After purification via silica-gel
column chromatography, and characterisation by NMR and mass
spectrometry [9], we turned our attention to study the photophys-
ical properties in various organic solvents as well as in water.
Thus, from the UV–vis absorption spectra it was observed that
the fluorophore exhibited absorptions at around 277–285 nm in
various organic solvents as well as in water. As the polarity of
the solvents increased, the absorption-intensity decreased with a
red shift of about 7–10 nm. Upon excitation at 280 nm the probe
showed fluorescence emission at 350 nm in hexane and cyclohex-
ane, while, in other organic solvents two bands appeared at around
350 and 400 nm. Interestingly, in polar protic solvents like MeOH
and H2 O along with a weak band at 350 nm, it exhibited emission at
420 nm, which was characterised as intramolecular charge transfer
(ICT) band (Fig. 1b).
Inspired by the interesting dual emission behaviour in water, we
next examined the optical sensory property of our probe for metal
ions. Thus, the divalent metals Mg2+ , Ca2+ , Fe2+ , Co2+ , Ni2+ , Cu2+ ,
Zn2+ , Cd2+ , Hg2+ , Pb2+ ions were added individually to the aque-
ous solution of 1 and photophysical properties were studied. From
the UV–vis absorption spectra it was observed that an increasing
amount of Hg2+ ion induced a gradual decrease in absorption, while,
other metals have negligible/inconsistent effect on the absorption
property of the probe (See SI, Section 4). This fact implies that only
the Hg2+ ion interact significantly in a well-defined manner with
the probe under the experimental condition. Titration experiments
were carried out to investigate the binding event and the stoichiom-
etry of the probe-Hg2+ complexation. Thus, a Job’s plot [variation
of the absorbance (A0 –A1 ) at 280 nm vs. molar ratio of Hg2+ ion]
suggested a clear formation of 1:1 probe-Hg2+ complex (Fig. 2a).
To get insight into the metal binding properties, we then studied
the fluorescence changes upon addition of various concentrations
of different metal cations to the aqueous solution of the probe upon
excitation at 280 nm. Thus it was observed that the addition of vari-
ous concentrations of metal ions, other than Hg2+ , showed irregular
and negligible change of the emission spectra at 420 nm (See SI,
Section 4). Interestingly, upon addition of Hg2+ ion about 150-fold
enhancement of fluorescence intensity was observed compared to
 U. Ghosh et al. / Sensors and Actuators B 238 (2017) 903–907 905

Scheme 1. Synthesis of the m-di-4-pyridyl benzene (m-DPB) as fluorescence sensor of Hg2+ ion.

Fig. 2. (a) Absorbance Job’s plot showing 1:1 complexation and (b) bar diagram of fluorescence responses of 1 (10 ␮M) in water upon addition of 2.5 equiv. of various metal
ions.

Fig. 3. (a) Fluorescence titration of 1 (10 ␮M) with various concentration of HgCl2 in water (␭ex = 280 nm]) and (b) Benesi-Hildebrand Plot of 1 with Hg2+ metal ion showing
association constant of 1-Hg2+ complex. (c) Plot of (Imin − I)/(Imin − Imax ) vs. log[Hg2+ ] for calculating the detection limit.

other metal cations (Fig. 2b). A titration experiment on the probe’s
solution with Hg2+ revealed a gradual increase in emission inten-
sity at 420 nm and a saturation point was attained at 10 equivalents
of Hg2+ ion concentration (Fig. 3a). All these phenomena along with
a presence of an isosbestic point at 330 nm in the emission spectra
indicated a well-defined binding of Hg2+ ion to the probe and the
formation of a probe-Hg2+ (m-DPB −Hg2+ ) complex.
The association constant determined from a Benesi-Hildebrand
plot was found to be 8.48 × 104 M−1 indicating a strong and stable
complexation between Hg2+ ion and m-DPB (Fig. 3b). Furthermore,
906 U. Ghosh et al. / Sensors and Actuators B 238 (2017) 903–907
Fig. 4. (a) 1 HNMR of 1 with different concentration of HgCl2 . (b) LUMO-of 1-Hg2+ complex shows a good amount of interaction between CH-␲ and metal.
the detection limit was calculated as three times the standard
deviation of the background noise [5a]. The linear fit line of plot
(Imin − I)/(Imin − Imax ) vs. log[Hg2+ ] was extended till it crossed the
ordinate axis. A good linear relationship (R2 = 0.977) in the range of
Hg2+ concentrations from 1 ␮M to 30 ␮M indicated that our probe
could be utilized for the detection of the submilimole concentration
range of Hg2+ ions found in many chemical and biological systems
[8a,10a]. The crossing point is the detection limit which came in
the region of 1.0 ␮M (Fig. 3c). It means the lowest concentration of
1.0 ␮M of Hg2+ ion in the sample can be determined with acceptable
precision and accuracy level under the test conditions. The preci-
sion test under the same test conditions indicated the proximity of
repeated experiments and sensitivity/selectivity of Hg2+ detection
[10c]. The competition experiments in presence of various back-
ground metal ions (50 equiv.) showed little to no interference for
the detection of Hg2+ ion (Hg2+ ion 10 ␮M, probe 1 ␮M) (See SI, Fig.
S6).
The advantage of our probe over the reported small organic
molecules based Hg2+ sensors lies on the fact that the present probe
is very simple, easy to synthesise, low cost, less time consuming
which is an issue for the success of a sensor [5]. There is also no
precedent on such a fluorescent family. Furthermore, the probe is
reversible, does not suffer from a quenched incidence of fluores-
cence and above all the probe works well in aqueous media similar
to very few existing competitive fluorescence switch-on sensor [5].
The detection limit, though, does not lie below the level defined by
the World Health Organization, but is promising for further design
of such efficient sensor based on simple m-DPB core for Hg2+ ions
[10b].
Finally, we have titrated a solution of the probe 1 with Hg2 +
ion and recorded the NMR spectra to support the binding inter-
action between the probe and the metal. Thus, the metal-probe
binding was evident from a change in chemical shift of the proton
on benzene which is ortho- to two pyridine units. We observed a
gradual shifting of the signal from ␦ 8.4–8.9 ppm when an increas-
ing concentration of Hg2+ was gradually added to a solution of the
probe (Fig. 4a). Moreover, we carried out a density functional cal-
culation to support the binding event using Gaussian 09 program
package [11]. Thus, based on time dependent density functional
(TD-DFT) calculation at B3LYP/6-31G* level of theory, we observed
that the nitrogen atoms of both the pyridine units and carbon atom
ortho- to both the pyridine units had a significant amount of LUMO
coefficient with maximum coefficient remaining with Hg2+ . The
significant overlap of the Hg2+ and the probe orbitals supported
the strong binding events between Hg2+ and the probe as well as
chemical shift of the phenyl-proton.
The switch-on fluorescence response of the probe might be
attributed to the formation of a stable complex between the probe’s
pyridyl nitrogens and Hg2+ ion. This complexation prohibited
the nitrogens’ lone pairs to participate in photo electron trans-
fer process, which is the cause of fluorescence quenching [8a,12].
Therefore, the Hg2+ binding event has ultimately led to an enhance-
ment of fluorescence emission. The low fluorescence intensity of
the probe in water in absence of Hg2+ ion may be attributed to radi-
ationless channel from n-␲* state of emission by the nitrogeneous
donor pyridine of the probe. When Hg2+ ion coordinates with the
pyridins’ nitrogens, the energy of the n-␲* state would be raised so
that the ␲-␲* state of the emission of m-DPB becomes the lowest
excited state, leading to a substantial increase in the fluorescence
intensity.
5. Conclusion
In conclusion, we have developed a new and smart fluorescence
switch-on chemosensor for Hg2+ ion. The probe is highly sensitive
for sensing Hg2+ ion in the presence of other interfering cations with
a high binding constant for 1:1 probe-Hg2+ complexation as evident
from the absorption and fluorescence spectroscopic studies. The
use of our present probe for selective sensing of Hg2+ -ion with low
and promising detection limit, may open the opportunity for the
design of new, efficient, and simple fluorescence light-up probe for
metal ion detection. All the results indicate that the simple probe
might find applications in chemical and biological systems as an
efficient Hg2+ selective fluorescence light-up probe.
Acknowledgments
C.M. and S.S.B. are thankful to BRNS [2012/37C/28/BRNS/1375]
and DBT [BT/PR5169/BRB/10/1065/2012], respectively, Govt. of
India. Authors are thankful to CIF, IIT Guwahati for NMR facility.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2016.07.139.
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Biographies
Ujjal Ghosh has received his B.Sc major in Chemistry from The University of Calcutta,
India, in 2011. In 2013 he obtained his M.Sc from the Indian Institute of Technology
Guwahati, India, and continuing his doctoral study in the same institute under the
supervision of Dr. Chandan Mukherjee. His research work is based on the synthesis
of new dyes for dye-sensitized solar cell.
Subhendu Sekhar Bag, born at Tikarpara, Bankura, West Bengal, India, received
his M.Sc. (1st class 1st) and Ph.D. degrees in Chemistry from IIT Kharagpur, in
1998 and 2005, respectively. After a post-doctoral exposure (JST and JSPS) he
started his independent career in the Department of Chemistry, IIT Guwahati,
India, in 2008 and became an Associate Professor in 2013. His research group is
actively involved in designing Unnatural-Fluorescent Nucleobase Pair(s) which
could drive the synthesis of Unnatural Protein(s) containing their Designer Amino
Acid(s). “Click-chemistry” is being utilized as a key step for synthesis in both the
research. His research interests also include the (a) Developement of Chemical and
Biological Sensors, (b) Development of Potent Fluorescent ␤-Lactam Antibiotics
to assay of Enzymatic Cleavage, (c) Monitoring the DNA Cleavage by Enediyne-
Nucleoside/Amino Acid Hybrid Molecules with Fluorescence Spectroscopy, (d)
Organic Synthesis, (e) Development of New Synthetic Methods and (f) Theoretically
Interesting Molecules. His research in the interface of chemistry and biology takes
help of “Click-Chemistry” and Fluorescence Spectroscopy as two major tools. He
served as a Lead Guest Editor of Journal of Nucleic Acids and is serving as an editorial
board member of Biological and Biomedical Reports, STM Journals, and World Research
Journal of Chemistry. He is a recipient of The Venus International Faculty Awards
– VIFA 2016 (Outstanding Faculty) for his contribution in the field of Bioorganic
Chemistry.
Chandan Mukherjee received his B.Sc major in Chemistry at The University of
Calcutta in 2001 and M.Sc in Chemistry in 2003 at the Indian Institute of Technol-
ogy Kharagpur, India. He then pursued his doctoral studies with Prof. Dr. Phalguni
Chaudhuri at the Max-Planck Institute for Bioinorganic Chemistry, Muelheim,
Germany. Later on he moved to Bielefeld University, Germany, and The University
of Tokyo, Japan for his post-doctoral studies with Prof. Dr. Thorsten Glaser and Prof.
Shū Kobayashi, respectively. Dr. Mukherjee embarked on his independent career as
an Assistant Professor, The Department of Chemistry, Indian Institute of Technology
Guwahati, India, in 2011. In 2015, he has been promoted to the rank of Associate
Professor in Chemistry. His current research focuses on the Oxidation Catalysis,
Molecular Magnetism, Synthesis of Single-Molecule Magnets (SMMs), MRI Contrast
agents and Water Oxidation Chemistry.