A styrylpyridinium dye as chromogenic and fluorogenic dual mode chemosensor for selective detection of mercuric ion: Application in bacterial cell imaging and molecular logic gate

Article abstract of DOI:10.1016/j.dyepig.2019.108054

The present work demonstrates design, synthesis and chemosensing application of a styrylpyridinium based donor-π-acceptor (D-π-A) chromophore (L1) containing NS₂O₂ binding site. The chemosensor L1 selectively and successfully detects

Full text of DOI:10.1016/j.dyepig.2019.108054

Journal Pre-proof
A styrylpyridinium dye as chromogenic and fluorogenic dual mode chemosensor for
selective detection of mercuric ion: Application in bacterial cell imaging and molecular
logic gate
Madhusmita Tripathy, Usharani Subuddhi, Sabita Patel
PII:
S0143-7208(19)32377-0
DOI:
https://doi.org/10.1016/j.dyepig.2019.108054
DYPI 108054
Reference:
To appear in: Dyes and Pigments
Received Date: 10 October 2019
Revised Date: 12 November 2019
Accepted Date: 16 November 2019
Please cite this article as: Tripathy M, Subuddhi U, Patel S, A styrylpyridinium dye as chromogenic and
fluorogenic dual mode chemosensor for selective detection of mercuric ion: Application in bacterial
cell imaging and molecular logic gate, Dyes and Pigments (2019), doi: https://doi.org/10.1016/
j.dyepig.2019.108054.
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Table of contents entry
The styrylpyridinium D-π-A chromophore L1 exhibits selective and reversible binding to
2
+
Hg with visual and turn-on fluorescence response along with possibilities of intracellular
imaging applications.
2
Hg
+
S
S
OH
OH
S
S
O
N
N
Hg2+
H C N+
H C N+
3
3
I-
I-
O
λem 566 nm
2
+
L1 + Hg Bound form
Highly fluorescent
λem 590 nm
L1 Free form
Weakly fluorescent
2
S
-
1

A styrylpyridinium dye as chromogenic and fluorogenic dual mode
chemosensor for selective detection of mercuric ion: Application in
bacterial cell imaging and molecular logic gate
*
Madhusmita Tripathy, Usharani Subuddhi and Sabita Patel
Department of Chemistry
National Institute of Technology Rourkela, Odisha, India-769008
Email: sabitap@nitrkl.ac.in; Ph: +91-6612462660
Abstract
The present work demonstrates design, synthesis and chemosensing application of a
styrylpyridinium based donor-π-acceptor (D-π-A) chromophore (L1) containing NS O2
2
2
+
binding site. The chemosensor L1 selectively and successfully detects Hg ions both
visually and spectro-photometrically in perfect aqueous medium as well as in an aqueous-
alcoholic mixture. Contrary to the reported literature on mercury sensing by cyanine dye
2
+
which were reported to be “turn-off” Hg sensor, probe L1 shows fluorescence “turn-on”
response. D-π-A chromophore L1 exhibits orange colour in aqueous and aqueous-alcoholic
solution due to intramolecular charge transfer (ICT) from the N, N-disubstitutedaminostyryl
2
+
donor to pyridinium acceptor moiety. It shows Hg -specific colour change with concomitant
generation of a new absorption band and large emission enhancement (∼ 10 fold) along with
2
+
a blue shift of emission peak, owing to the formation of a selective 1: 1 L1-Hg complex.
1
2+
2+
H-NMR analysis reveals the binding mode of L1 with Hg . Hg probing process was
found to be reversible in presence of sulphide anion. The reversible off-on-off fluorescence
2
+
2-
response of the sensor with Hg and S has been used to generate an INHIBIT molecular
logic gate. The probe not only provides a highly efficient, low cost, portable sensor for
2
+
recognition and naked-eye detection of Hg with a low detection limit in aqueous solution
but its turn-on fluorescence response and cell permeability offer the possibility of using it for
fluorescence imaging in living cells.
Keywords: Styrylpyridinium dye, Chemosensor, Mercuric ion, ICT, Fluorescence, D-π-A
chromophore
1

1
. Introduction
The recognition and sensing of metal ions has become increasingly important and
emerged as a significant goal in the field of chemical sensors due to their biological,
1
-4
medicinal, and environmental hazardous impact. Traditional analytical techniques such as
high-performance liquid chromatography, mass spectrometry, atomic absorption
spectroscopy, inductively coupled plasma atomic emission spectrometry, electrochemical
sensing, etc. used to analyze the target provide accurate result, however associated with
multiple shortcomings including extensive and time-consuming procedures, involve the use
of sophisticated instrumentation and requirement of large size sample. In this regard,
chromogenic and fluorogenic techniques by the use of chromoionophoric sensors have
emerged as promising alternatives in recent years for the sensing of ions and small molecules
1
-4
of biological and environmental relevance. Owing to a range of advantages e.g. high
sensitivity, selectivity, operational simplicity, economical perspective, minimal and cheaper
instrumental assistance, naked-eye detection, quantitative estimation of analytes, variety of
5
-8
chromoionophoric chemosensors are being designed and developed. Chromoionophoric
sensors comprise of two essential features (i) an ionophore with a specific binding pocket for
selective recognition and binding of ions and (ii) at least one chromophoric or fluorophoric
moiety capable of transducing the chemical information produced by the ionophore-ion
interaction into an resolvable distinct optical signal either through change in colour or
5
-10
through change in fluorescence response.
Mercury is found to be one of the most common pollutants and highly toxic heavy
metal ion. Both elemental and ionic mercury present in soil and water are methylated by
microorganisms and the resultant methylmercury is subsequently bio-accumulated through
1
1
food chain. Organomercury compounds, easily permeated through the cell membrane and
able to reach high levels in the central nervous system (CNS), leading to nephrological and
neurological dysfunctions. The direct chemical interaction between methylmercury and thiol
groups from proteins and non-protein molecules, such as glutathione (GSH), is one of the
1
2
major pathways in mercury induced neurotoxicity. Hence, the sensitive and selective
detection of mercury in biological and environmental samples is very important and highly
desirable. Numerous reports on design and development of chromogenic and fluorogenic
2
+
chemosensors for selective binding and detection of Hg ion are found in literature.
2
+
However, presence of Hg ion significantly quenches the fluorescence signal through spin-
2

1
3
orbit coupling making them less useful for practical purpose. Therefore, sensors which
display “turn-on” response upon binding with metal ions are mostly preferred. In the
2
+
designing of turn-on sensors for Hg , an important strategy is to incorporate covalently
binding donor atoms (particularly S-Donor atoms) in the binding site of potential sensor.
2
+
These covalently bound donor atoms strongly interact with Hg weaken the interaction of
,
1
3
the heavy metal with the fluorophore and thus facilitate to resume fluorescence.
With a view to get selective “turn-on” chemosensor for mercury atom, the greater
binding affinity of mercury towards sulphur atom is considered. In this regard a thioether
based metal binding unit containing NS O donor atoms is appended with a styrylpyridinium
2
2
chromophore. Styrylpyridinium dyes are a class of polymethine hemicyanine dyes, well
known for their applications in diverse and broad area of science, technology, engineering,
1
4-19
pharmacology and medicine.
easy electronic delocalization, and found to exhibit electronic transition in the visible and
These dyes possess excellent electronic properties due to
2
0-22
NIR region of the electromagnetic spectrum.
Labelling of proteins, DNA, and other
biopolymers with polymethine dye molecules, has become a very powerful research tool in
2
3
molecular biology to unveil various biological and biochemical processes. Notable use of
these dyes in biology, medicine and drug development are as fluorescent probes and labels
for cells, micelles and organelles, flow cytometry, fluorescence microscopy, DNA
sequencing, detection on microarrays, quantification of nucleic acids in capillary and gel
electrophoresis and single molecule detection of DNA. Considering numerous advantages of
polymethine styrylpyridinium framework such as aqueous solubility, stability, long
absorption and emission wavelength, high extinction coefficient and fluorescence quantum
yield, uses in two-photon absorption, low detection limit and most importantly their cell
permeability, non toxicity at low micro-molar level and cell imaging, herein we have reported
the chromoionophoric application of a styrylpyridinium dye (L1) (Scheme 1) in aqueous and
aqueous-alcoholic mixture. Probe L1 is designed to have a Donor-π-Acceptor framework and
characterized with intramolecular charge transfer (ICT) from the N,N-
disubstitutedaminostyryl donor to N-alkylpyridinium acceptor. Presence of pyridinium unit
made it highly soluble in aqueous medium. The chemosensor was found to display high
2
+
selectivity and sensitivity for Hg in both water and water-ethanol mixture irrespective of
2
+
other interfering ions. This is attributed to the strong binding interaction of Hg with NS O
2
2
donor ligand as compared to other metal ions. The selective binding of the metal ion could be
easily detected by “naked-eye” colour change, UV-Vis spectral change and through
3

fluorescence “turn-on” response. The significant fluorescence enhancement (∼ 10-fold) was
attributed to chelation enhanced fluorescence (CHEF) due to the arrest of excited state photo
electron transfer (PET), decreased nonradiative relaxation and increased conformational
rigidity as a result of the binding of metal ion with the donor atoms. Contrary to the reported
literature on mercury sensing of styryl based dye chromophore which were reported to be
2
+
24-26
“
turn-off” Hg sensor, probe L1 showed fluorescence “turn-on” response.
Exploiting this
2
+
advantage of turn-on response for Hg along with the water solubility, low toxicity and cell
2
+
permeability of styrylpyridinium dyes, probe L1 was used for fluorescence imaging of Hg
in bacterial cell. Further, reversible fluorescence response in presence of sulphide anion was
applied to construct an INHIBIT molecular logic gate.
2
.
Experimental Section
General materials and methods
All the solvents and reagents (analytical grade and spectroscopic grade) were obtained
commercially and used after purification. Reactions were monitored by TLC, and the residue
was chromatographed on a silica gel (100–200 mesh), using ethyl acetate-petroleum ether
mixture as eluent. Melting point of solid samples was monitored on Yanaco Melting Point
Apparatus SP-500. All NMR spectra were recorded on a Bruker Spectrometer 400 MHz (for
1
13
H NMR) and 100 MHz (for C NMR) NMR spectrometer, and chemical shifts were
expressed in δ ppm units relative to the TMS signal (as an internal reference), to CDCl taken
3
1
13
1
as 7.28 ppm for H and 77.00 ppm for C or to DMSO-d taken as 2.5 ppm for H NMR.
6
UV-vis absorption spectra were recorded on a double beam Shimadzu (UV-1800)
spectrophotometer using a quartz cuvette (path length = 1 cm). IR spectra were recorded
using a Shimadzu IR Affinity-1 SWL using thin film deposit on KBr plates. High resolution
mass spectra (HRMS) were acquired on a Bruker ESI-MS micro TOF-QII mass spectrometer.
Fluorescence measurements were performed using FluoroMax-4P fluorescence spectrometer
(HORIBA Scientific, USA) and a quartz cell (1 cm x 1 cm). Fluorescence lifetime
measurements were carried out using IBH 5000F Nano LED equipment (Horiba Jobin Yvon,
Edison, NJ) with Data Station software in the time-correlated single photon counting
(TCSPC) mode. The method for determination of fluorescence quantum yield and lifetime is
presented in electronic supporting information (ESI).
4

Synthesis of 4-[N,N-Bis(2-chloroethyl)amino]benzaldehyde (2)
2
7
Compound 2 was synthesized using a method reported in literature. 20 ml of
dimethylformamide was placed in a 100 ml flask equipped with a mechanical stirrer and
cooled to 0-5° as 10 ml of phosphorus oxychloride was added slowly over approximately 0.5
hr. A solution containing 3.62 g (20 mmol) of N, N-bis(2-hydroxyethyl) aniline was then
added with stirring and cooling. After stirring at 90° C for 2.5 hr, the mixture was cooled and
poured into 200 m1 of ice cold water. Concentrated ammonium hydroxide was then added
with vigorous stirring until the solution was strongly basic as checked by pH paper. The solid
yellow product precipitated was then collected and recrystallized from methanol-water to
1
give 1.73 g (59% yield) of the cream colored needles (M.Pt. 85-88° C). H NMR (400 MHz,
CDCl ) δ (ppm): 9.80 (s, 1H), 7.81 (d, J = 9.2 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H), 3.88 (t, J =
3
1
3.6 Hz, 4H), 3.72 (t, J = 13.6 Hz, 4H) (Figure S1: Electronic supporting Information (ESI))
1
3
C NMR (100 MHz, CDCl ) δ (ppm): 190.2, 150.9, 132.3, 126.7, 111.2, 77.0, 53.3, 40.0
3
(Figure S2: ESI).
Synthesis of 4-(N,N-bis-(2-hydroxyethylthioethyl))benzaldehyde (3)
2
7
Compound 3 was prepared as per the following procedure. Na metal (0.2 g, 10 mmol) was
added into ethanol (5.6 ml) along with stirring. After Na reacted completely, 2-
mercaptoethanol (0.7 ml, 10 mmol) was added into the solution with stirring for 1 hr at room
temperature. Solution of 2 (1.23 g, 5 mmol) in 2.5 ml DMF was then added in portions. After
0
stirring for 1 hr, the mixture was then stirred at 60 C for another 1hr. The reaction mixture
was then diluted with water and was extracted with DCM (6 x 10 ml) and dried over Na SO .
2
4
DCM was then removed from the mixture in a rotary evaporator and residue was vacuum
1
dried. The desired product was found as brown colored viscous oil 1.32 g (78% yield). H
NMR (400 MHz, CDCl ), δ : 9.74 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 9.2 Hz, 2H),
3
5
.31(s, 2H), 3.82 (t, J = 12 Hz, 4H), 3.69 (t, J = 15.2 Hz, 4H), 2.83 (m, J = 12 Hz, 8H)
1
3
(
Figure S3: ESI).; C NMR (100 MHz, CDCl ) δ: 190.2, 151.4, 132.4, 125.9, 111.4, 77.0,
3
+
6
3
9
1.2, 51.4, 35.4, 29.1 (Figure S4: ESI).; HRMS: m/z [M+Na] 352.1034 (Calcd); Found:
52.1012 (Figure S5: ESI).; IR(KBr): 3373, 2920, 1654, 1595, 1523, 1402, 1288, 1168, 1045,
-1
45, 817cm (Figure S6: ESI).
5

Synthesis of (E)-4-(4-N,N-bis-(2-hydroxyethylthioethylamino)styryl)-1-methylpyridinium
iodide (L1)
The Compound 3 (0.658 g, 2 mmol) and 1,4-dimethylpyridinium iodide (0.220 g, 2mmol)
were mixed in 6 ml of absolute ethanol in presence of 1 drop of piperidine as base catalyst.
The resulting mixture was heated to reflux overnight. After cooling, the mixture was filtered
to collect the solid followed by washing with ethanol. The crude product was recrystallized
twice from ethanol and then dried in vacuum to obtain red colored chemosensor 0.27 g (56%
0
1
yield) m.pt. 139-141 C. H NMR (400 MHz, DMSO-d ) δ (ppm) : 6.76-8.68 (m, 8H), 7.18 (d,
6
J = 16 Hz, 1H), 7.91 (d, J = 16 Hz, 1H), 4.87 (s, 1H), 4.17 (s, 3H), 3.60 (m, J = 13.2 Hz, 8H),
1
3
2
.76 (t, J = 14 Hz, 4H), 2.68 (t, J = 13.2 Hz, 4H) (Figure S7: ESI).; C NMR (100 MHz,
DMSO-d ), δ (ppm): 153.7, 149.4, 144.8, 130.9, 123.1, 117.7, 112.1, 61.6, 51.0, 46.8, 40.1,
6
+
3
9.2, 34.5, 29.1 (Figure S8: ESI).; HRMS: m/z [M] 419.1841(Calcd); Found: 419.1821
(Figure S9: ESI). ; IR (KBr): 3296, 2918, 1645, 1585, 1523, 1404, 1340, 1172, 1045, 972,
-1
9
45, 827cm (Figure S10: ESI).
3
.
Results and Discussion
Synthesis
of
chemosensor
4-(4-N,N-bis-(2-hydroxyethylthioethylamino)styryl)-1-
methylpyridinium iodide (L1)
The chemosensor L1 was synthesized by a simple condensation reaction of 1,4-
dimethylpyridinium iodide (4) and 4-(N,N-bis-(2-hydroxyethylthioethyl))aminobenzaldehyde
(3) in presence of piperidine in ethanolic medium to form the styrylpyridium dye L1
1
13
(
Scheme 1). Formation of the product was characterized from IR, NMR ( H and C) and
mass spectral analysis (Figure S7 –S10; ESI).
6

H
O
H
O
H
O
H
O
S
S
N
Cl
Cl
HO
OH
N
S
S
N
1
DMF
SH
N
Piperidine, EtOH
H C N+
CH3
HO
Na, EtOH
POCl3
3
I-
CHO
2
4
CHO
3
N+ I-
CH3
L1
Scheme 1 The stepwise synthetic route to chemosensor L1.
Solvatochromism of the probe L1
Styrylpyridinium dyes, a class of polymethine hemicyanine dyes are very well known for
2
0, 28-30
their solvent-induced reversible colour change termed as solvatochromism.
Due to the
presence of Donor-π-Accepter (D-π-A) moiety, upon excitation these molecules exhibit a
large change in dipole moment attributed to the relative contributions of both benzenoid and
quinonoid forms. Extensive study on the solvatochromism/fluorosolvatochromism of
different classes of polymethine dyes proved their sensitivity to environmental changes and
thus used as good probes for monitoring micropolarity, hydrogen bonding interactions, and
for the investigation of micro-environmental characteristics of biochemical and biological
systems.
OH
2
1
1
0
0
.0
.5
.0
.5
.0
S
S
DCM
N
DMSO
H C
N
3
Acetonitrile
MeOH
OH
OH
Quinonoid form
Water
S
S
N
H C N+
3
OH
3
50
450
550
650
Benzenoid form
Wavelength (nm)
Figure 1 ICT absorption band of styrylpyridinium dye L1 in different solvents
7

The styrylpyridinium chemosensor L1, contains a D-π-A chromophore with
conjugated tertiary nitrogen groups as the donor and quaternary pyridinium nitrogen centre as
the acceptor, flanked with styryl π-spacer. The large solvatochromic shift (i.e 450 nm in
water to 503 nm in dichloromethane (DCM)) clearly demonstrates intra molecular charge
transfer (ICT) characteristic of the chromophore (Figure 1).
Chromoionophoric evaluation
The aqueous solubility of the chemosensor L1 made it possible to study its metal ion binding
efficacy in aqueous as well as in aqueous-alcoholic medium. The binding properties of the
2
+
probe L1 with Hg and with other metal ions were studied by naked eye visualisation test as
well as UV-Vis and fluorescence emission experiments in water and in MeOH-H O (4:1 v/v)
2
mixture. Figure 2 depicted the visual change on adding 4 equivalents of different metal ions
-5
-5
(
4.8 x 10 M) to the solution of L1 (1.2 x 10 M) in water medium. Orange solution of probe
L1 became colourless with mercury ion, however in presence of other tested metal ions it
remained unaffected when observed in daylight. Similarly, under the influence of UV light
2
+
strong yellow fluorescence was observed only with Hg ion. This clearly demonstrated the
chromoionophoric chemosensor nature of the compound L1 for selective detection of
Mercury.
Figure 2 Free L1 and L1 in presence of different metal ions in aqueous medium (A)
photograph in daylight and (B) photograph under UV light (365 nm).
UV–Vis spectral characteristics of L1 with metal ions
In order to investigate the selective recognition and binding of metal ions and to study the
binding mechanism, the UV-Vis absorption characteristics of the chemosensor L1 was
2
+
2+
2+
3+
2+
3+
2+
studied in presence of different metal ions such as Ca Zn Pb Al Mg Cr , Ba ,
,
,
,
,
,
8

2
+
,
+
2+
2+
,
+
+
+
Cd Na , Cu Ni K , Ag and Fe3 (as their nitrate salts) in aqueous as well as in MeOH-
,
H O (4:1 v/v) mixture. The aqueous solutions of different metal ions were added separately
2
to the prepared solution of compound L1 and UV-Vis absorption spectra were recorded.
2
+
Upon addition of Hg solution to the aqueous solution and also to MeOH-H O (4:1 v/v) of
2
L1, the ICT band suffered hypochromicity and a new absorption band appeared at ∼ 355 nm
(Figure 3) However, no significant change in the absorption spectrum was observed in
presence of other tested metal ions. The observed change in the UV-Vis spectral
2
+
characteristics on addition of Hg was found to be greater in methanol-water mixture, as
compared to pure aqueous medium. This may be attributed to greater and stronger binding of
metal ion with the host molecule in methanol-water medium. In contrary, in aqueous solution,
a smaller and weaker binding of metal ion is anticipated due to greater and stronger solvation
2
+
of Hg ion in pure water which may restrict the interaction with the host molecule.
Figure 3 UV-Vis absorption band of the probe L1 (12 µM) in presence of different metal
ions (48 µM) in (A) H O medium (B) methanol-H O (4:1 v/v) medium.
2
2
9

2
+
To gain insight into the specific interaction with Hg ion, UV-vis absorption titration
of the chemosensor L1 (12 µM) was performed with gradual increase in the concentration of
2
+
Hg (0-108 µM) ion. The UV-Vis absorption spectrum of free chemosensor L1 was found to
2
+
be perturbed drastically. With increase in Hg concentration (0-108 µM), absorbance of the
ICT band centered at 455 nm started decreasing with concomitant increase in the absorbance
of the newly developed absorption band at lower wavelength centered ∼ 355 nm (Figure 4).
2
+
The new absorption band was thus attributed to L1-Hg bound species. Appearance of three
well defined isosbestic points ca. 390, 300 and 258 nm clearly demonstrated the
interconversion from free un-complexed species (L1) to complexed bound species (L1-
2
+
Hg ). The decrease in the absorbance of ICT band clearly showed the involvement of the
2
+
tertiary N-donor atom for the binding of Hg ion. Upon binding with the metal, N-donor
atom may acquire partial positive charge resulting in inhibition of intramolecular charge
transfer in the D-π-A chromophore L1.
0
0
0
0
.4
.3
.2
.1
0
Hg2+
2
00
300
400
500
600
Wavelength (nm)
Figure 4 Changes in the UV-Vis absorption spectra of L1 (12 µM) upon addition of
2
+
increasing amount of Hg ion (0-108 µM) in H O medium.
2
Fluorescence spectral characteristics of L1 with metal ions
Similar to UV-Vis absorption studies, fluorescence emission experiments were also carried
out for free L1 and metal bound species in methanol-H O (4:1 v/v) medium. Fluorescence
2
spectra of the chemosensor L1 at an excitation wavelength of 360 nm in the absence and
2
+
2+
2+
3+
2+
3+
2+
2+
+
presence of different metal ions such as Ca Zn Pb Al Mg Cr , Ba , Cd Na ,
,
,
,
,
,
,
2
+
,
2+
+
3+
+
Cu Ni K Fe , Ag (as their nitrate salts) have been collected and represented in Figure
,
,
5
.
1
0

Figure 5 Fluorescence emission spectra of L1 (12 µM) in presence of different metal ions
48 µM) in methanol-H O (4:1 v/v) medium (λ = 360 nm)
(
2
ex
The chemosensor L1 in its free state was found to be weakly fluorescent (quantum
yield (φ ) = 0.009) giving a low intense broad emission band at ca. 590 nm. Upon treating
f
2
+
with 4 equivalents of different metal ions separately, except in the case of Hg ion no
2
+
significant change in the fluorescence spectrum was observed (Figure 5). On adding Hg
ion, remarkable enhancement was observed in the fluorescence intensity (φ = 0.044) with a
f
blue shift of the emission wavelength to 566 nm. This turn-on fluorescence response may be
explained through the following two factors. (i) Principle of chelation enhanced fluorescence
6
,13, 31-33
(CHEF) as a consequence of inhibition of photo induced electron transfer (PET)
and
3
4-35
(ii) increase in conformational rigidity.
Most of the turn-on chemical sensors are non-fluorescent or weakly fluorescent in
their free state possibly due to PET (photo induced electron transfer) effect. The metal
binding host unit of these sensors contain lone pair of electrons on the hetero-atoms (such as,
nitrogen, oxygen, sulphur atoms or with any other functional groups), which are of higher
energy than the highest occupied molecular orbital (HOMO) of the fluorophore. Upon
excitation of an electron from the HOMO to the lowest unoccupied molecular orbital
(
LUMO) of the fluorophore, these lone pair of electrons jump down immediately to fill the
partially empty HOMO of the fluorophore, which inhibits the excited electron to return back
to the fluorophore ground state HOMO and there by quenching fluorescence (Scheme 2).
Involvement of these lone pairs for metal chelation drops the energy of lone pair below that
1
1

of the fluorophore HOMO, consequently prevents the process of PET resulting in enhanced
3
1-33
2+
fluorescence (CHEF).
Similarly, in the present case binding of Hg to the NS O binding
2 2
site of the chemosensor L1 through the lone pairs on nitrogen, oxygen and sulphur make
them unavailable for any possible PET effect, which may result in strong fluorescence
enhancement.
PET On
PET Off
Metal ion
Fluorophore
Binding Unit
Non-fluorescent Or weakly fluorescent
Strongly Fluorescent
CHEF effect
LUMO
HOMO
PET effect
Emission
E
Lone pair
Free Binding unit
Metal bound
binding unit
Fluorophore
Fluorophore
Scheme 2 Schematic representation of fluorescence quenching by PET effect and
3
1-33
fluorescence enhancement by CHEF effect.
Fluorescence intensity is often related to the rigidity of the chromophore structure. In
general, the restricted rotational motion of the fluorophore makes the rotation-related
nonradiative decay pathway less efficient, hence enhancing the fluorescence quantum yield
3
4-35
and intensity.
The strong enhancement of fluorescence intensity of L1 in its mercury
bound state may also be attributed to the restriction of the torsional motion of disubstituted
2
+
amino group imposed by the formation of L1-Hg complex making it conformationally more
rigid than the unbound L1 which possibly resulting in increased fluorescence intensity
through a reduction of non-radiative decay. In order to confirm this proposition, the
2
+
fluorescence lifetime studies were carried out for free L1 and L1-Hg complex in methanol-
H O (4:1 v/v) medium. The unbound ligand exhibited tri-exponential fluorescence decay with
2
an average fluorescence lifetime of 0.25 ns, which increased to 0.56 ns in presence of
mercury ion. From the average fluorescence lifetime and quantum yield values the radiative
1
2

(
k ) and non-radiative (k ) decay constants were computed and the values are represented in
f nr
Table 1. As evident from the data, there is a significant reduction in the k value in presence
nr
2
+
of mercury thus supporting the above proposition i.e. formation of L1-Hg complex induces a
conformational rigidity in the fluorophore. As as a consequence there is a reduction in non-
radiative decay, which in turn results in an enhanced fluorescence quantum yield.
Table 1 Comparison of average fluorescence lifetime, quantum yield, radiative (k ) and non-
f
2
+
radiative (k ) decay constants for L1 and L1-Hg complex in MeOH:H O(4:1) medium
nr
2
System
τ 1
α 1
τ 2
ns)
α 2
τ 3
(ns) (%)
4.5
α3
τ avg
(ns)
Φꢀ
kf
knr
(
ns)
(%)
−1
−1
(
(%)
(ns )
(ns )
L1
0.14
90.33 0.71 8.31
46.34 0.74 53.66 --
1.35 0.25 0.009
0.037 4.025
0.079 1.709
L1+Hg
0.35
--
0.56 0.044
Remarkable increase in fluorescence intensity, quantum yield and lifetime along with
2
+
a blue shift of ca. 26 nm in presence of Hg is thus a result of the binding of donor group
with the metal ion restriction in bond rotation by giving a rigid environment and inhibition of
,
2
+
PET induced quenching in the L1-Hg adduct. The selective fluorescence enhancement
2
+
could be due to more effective and strong binding of Hg with the donor atoms of L1 in
comparison with other metal ions. However, a detailed analysis of the photo-physics of the
fluorophore with and without metal ion is essential for a definite conclusion.
Selectivity and non-interference of L1
2
+
In order to establish the high selective nature of chemosensor L1 towards Hg , the
effect of other metal ions on the fluorescence emission was investigated. The interference
experiments were conducted by adding excess of the interfering metal ion (240 µM) namely
2
+
2+
2+
3+
2+
3+
2+
2+
+
2+
2+
+
+
3+
Ca , Zn , Pb , Al , Mg , Cr , Ba , Cd , Na , Cu , Ni , K , Ag and Fe
to the
2
+
solution of Hg ( 48 µM) and the probe L1 (12 µM). The fluorescence intensities were
analysed and plotted as a bar graph in Figure 6. No considerable change was observed in the
2
+
fluorescence emission profiles of L1-Hg solution, indicating that the probe has a strong
2
+
selective binding ability for Hg even in presence of different interfering metal ions
encountered in environmental and biological samples.
1
3

Figure 6 Bar graph of fluorescence intensity (at 566 nm) for the solution of free L1 (12 µM),
2
+
2+
L1 with Hg (48 µM), L1 with Hg (48 µM) in the presence of other competitive metal ions
240µM) in MeOH:H O(4:1) medium. The red bars and black bars represent the fluorescence
(
2
intensity of solution of L1 in the presence of 48 µM of other tested metal ions in the absence
2
+
2+
of Hg and in presence of Hg ion respectively. (λ = 360 nm)
ex
Binding stoichiometry, association constant and limit of detection (LOD)
2
+
The binding stoichiometry of the probe L1 with Hg ion was quantified by Jobs’s
method. For this the emission spectral data were collected at a constant total molar
concentration of 12µM with continuous variation of mole fraction of the two binding species
(Figure 7a). Figure 7b represents the change in fluorescence intensity with change in mole
2
+
fraction of Hg . A maximum at a mole fraction of 0.5 clearly established the formation of a
2
+
1
:1 binding of the probe with the metal ion to form L1-Hg complex in methanol-H O (4:1
2
3
6
v/v) medium.
2
+
The association constant (K) for the binding of the probe with Hg was determined
from fluorescence spectral emission data in water medium as well as in MeOH-H O (4:1 v/v)
2
3
7
medium using Benesi-Hildebrand equation (equation 1).
ꢁ
ꢁ
ꢁ
= ꢂ
+ ꢈꢉꢂ
(1)
ꢃꢂ ꢊꢋꢌꢍꢎꢏꢐ
ꢂꢃꢂꢄ
ꢅꢆꢇꢃꢂꢄ
ꢅꢆꢇ
ꢄ
2
+
Where I I , and I refers to fluorescence intensity of free L1 in the absence of Hg ,
0
,
max
2
+
maximum fluoresce intensity in presence of Hg at a concentration of complete interaction
2
+
and intermediate fluoresce intensity at varying [Hg ].
1
4

2
+
Using slope and intercept of the plot of 1/(I-I ) versus 1/[Hg ] K was calculated to
0
,
5
-1
5
-1
be 1.13 x 10 M and 1.46 x 10 M in water and in MeOH-H O (4:1 v/v) medium
2
respectively ( Figure 8 A and B). The lower value of association constant in water medium
2
+
compared to that of methanol-water mixture is probably due to greater solvation of Hg ion
in water which may restrict the interaction of L1 and metal ion.
A
B
2
+
Figure 7 (A) Fluorescence spectra of solution containing L1 and Hg with varying molef
2
+
fraction at a total molar concentration of 12µM and (B) respective Job’s plot of Hg mole
fraction vs. fluorescence intensity at 566 nm (λ = 360 nm).
ex
A
B
2
+
Figure 8 Plots of 1/(I-I ) versus 1/[Hg ] in (A) water medium (B) in MeOH-H O (4:1 v/v)
0
2
medium.
In order to perform as a suitable chemosensor one of the major concern is the
sensitivity or limit of detection (LOD) of the probe. To determine the sensitivity of the probe
L1, a fluorescence titration experiment in water medium was performed with increasing
2
+
2+
[
Hg ] (0-30 µM) at constant [L1] (12 µM) (Figure 9A) On addition of Hg up to a
2
+
concentration of 3.6µM no significant change was observed. However, when the [Hg ] is ≥
4
.8µM, detectable enhancement in fluorescence intensity with gradual blue shift of the peak
2
+
was observed. This experiment demonstrated a low detection limit (DL) of L1 towards Hg
1
5

i.e. 4.8 µM. Further, LOD was also calculated using the extensively used method i.e. LOD =
/S, where is the standard deviation intercept and S is the slope of the linear fitting
3
σ
σ
2
+
equation of the plot I/I versus [Hg ] (Figure 9B). LOD calculated using this method was
0
found to be 7.5 µM. DL and LOD for the present work is comparable with that of previously
3
8-43
reported literature (Table 2).
A
B
2
+
Figure 9 (A) fluorescence titration experiment with increasing [Hg ] (0-48 µM) at constant
2
+
[
L1] (12 µM) in water medium, (B) linear plot of (I/I ) versus [Hg ].
0
2
+
Table 2 Comparison of LOD for Hg of the present chemosensor with previously reported
literatures.
References Fluorophore/Chromophore Type of detection Solvent medium
LOD
Ref 38
Pyrazidine derivative
Colorimetric and
fluorescence
Water
14.54µM
Ref 39
Ref 40
HOE DNA binding dye
fluorescence
Water
0.87µM
20µM
Thiophene-based Schiff base Colorimetric and
fluorescence
MeOH:H O
2
Ref 41
Rhodamine B/
Colorimetric and
fluorescence
Water
10µM
Spirolactam ring
Ref 42
Ref 43
Rhodamine derivative
BODIPY
fluorescence
fluorescence
MeOH:H O
10µM
4.2µM
7.5µM
2
Water
Water
This work
Styryl pyridinium dye
Colorimetric and
fluorescence
1
6

1
Nature of binding Interaction: H NMR spectroscopic analysis
2
+
1
In order to confirm the binding mode of the compound L1 with Hg ion, H-NMR
1
spectroscopic studies were carried out. The H-NMR spectra of the chemosensor L1 before
2
+
1
and after treatment of Hg in DMSO-d are presented in Figure 10. All the H-NMR peaks
6
of L1 are found to be downfielded to more or less extent after addition of mercury. However
significant change in the proton signal for H1, H2, H3, H4, H5 H6 and H9 are observed as
2+
follows. (i) Peak for the -OH proton (H1) at δ 4.83 ppm disappeared on addition of Hg
probably because of binding of oxygen atom to metal ion which may weaken the O-H bond
leading to deprotonation. (ii) Proton signal for –CH proton attached to O atom (H2) and N
2
atom (H5) appeared as multiplet at δ 3.53 ppm are found to be slightly downfielded,
2
+
separated progressively with increasing concentration of Hg and appeared as two distinct
triplets at δ 3.56 and δ 3.7 ppm respectively. (iii) Peak for –CH proton attached to S atom
2
(H3 and H4) appeared as two distinct triplets at δ 2.62 ppm and δ 2.69 ppm respectively in
2
+
free L1 are found to be downfielded and merged with gradual addition of Hg to give a
distorted broad triplet at δ 3.15 ppm. This may be because of decrease in electron density at S
atom due to strong covalent type binding interaction with mercury ion. (iv) Doublet for H6
i.e. ortho to the electron donating N-atom at δ 6.73 ppm is also downfielded to a great extent
and appeared at δ 7.16 ppm which may be due to decrease in electron density on N-atom
1
upon association with the metal ion. Thus the change in H NMR chemical shifts
demonstrated a clear picture of an adduct formation between the sensor L1 with metal cation
chelated through N, S and O donor atom.
1
7

1
2+
Figure 10 H-NMR spectra of probe L1 and L1+ Hg in DMSO-d and a possible mode of
6
2
+
2+
2+
binding interaction (a) Free L1 (b) L1 + 0.2 eq. Hg (c) L1 + 0.5 eq. Hg (d) L1 + 1 eq. Hg
e) L1 + 1.5 eq. Hg (f) L1 + 1.8 eq. Hg (g) L1 + 2 eq. Hg
2
+
2+
2+
(
FESEM analysis
Cyanine dyes can spontaneously self assemble to form aggregates via intermolecular van der
Waals forces. By varying solvent or adding salt, the aggregates can be transformed into
various forms, for example, H- or J-aggregates, which can be easily distinguished by different
4
4
analytical techniques. To observe any morphological alteration of chemosensor L1 after
2
+
chelation to Hg , FESEM analysis was attempted. Whereas the neat styryl dye L1 possessed
wire like microstructure which was found to be disrupted to a flower like morphology upon
2
+
treatment with Hg (Figure 11A & B). The reorganization of the aggregated probe L1 might
2
+
2+
be attributed to the formation of L1-Hg adduct. The chemical composition of the L1+Hg
complex was also measured by the EDAX analysis which clearly showed the presence of
1
8

2
+
carbon (C), oxygen (O), nitrogen (N), sulphur (S) and mercury (Hg) in the prepared L1+ Hg
complex (Figure 11D).
(A)
(B)
cps/eV
1
1
1
4
2
0
8
6
4
2
0
(
C)
C
N
O
S
S
1
2
3
4
5
keV
6
7
8
9
10
cps/eV
(
D)
9
8
7
6
5
4
3
2
1
0
S
Hg
N
S
Hg
C
O
Hg
1
2
3
4
5
keV
6
7
8
9
10
2
+
Figure 11 SEM images of (A) Free L1 (B) L1 + Hg (C) EDX pattern of L1 (D) EDX
2
+
pattern of L1 + Hg adduct.
1
9

Reversibility of the chemosensor L1
The reversible binding behaviour of chemosensors towards specific analytes is a vital aspect
for practical application. The reversibility of the chemosensor L1 was tested with sodium
2
+
sulphide (Na S). Addition of Na S (24 µM) to the solution containing L1(12µM) and Hg
2
2
(48µM) resulted in a dramatic quenching of the fluorescence emission intensity (Figure 12).
The colour of the solution as well as fluorescence intensity at 566 nm was found to revert
back to that of free L1 solution. It may be explained through strong affinity of sulphide anion
2
-
2+
2+
(
S ) for the Hg cation, causing demetallation of the L1-Hg complex with a simultaneous
2
+
decrease in the fluoresce intensity due to non-availability of Hg for chelation. After the
2
+
addition of 1 equiv. (12 µM) of Hg again to the same solution, the fluorescence of the
2
-
2+
solution was revived. Furthermore, upon the sequential alternate addition of S and Hg into
the solution, the fluorescence switching response could be repeated for several cycles. These
results suggested that L1 can be used as a reversible fluorescent chemosensor for the selective
2
+
detection of Hg ions in aqueous as well as in aqueous-alcoholic mixtures under physiological
conditions.
A
B
2
+
2-
Figure 12 The reversible binding behaviour of L1 with Hg tested using S anion.(A)
Emission spectra and (B) Emission intensity (at 566nm) of L1 with sequential addition of
2
+
2-
Hg and S .
Construction of Logic Gate
The clear “off–on” fluorescence response of the chemosensors in response to the addition of metal
45
shows the digital action of molecules and thus applied to combinational logic circuit. The
2+
reversible binding behaviour of chemosensor L1 to Hg and consequent changes in fluorescence
2-
46
intensity at 566 nm in presence and absence of S mimics the INHIBIT logic gate. Figure 13
2+
demonstrated the circuits for the INHIBIT logic gate. This was constructed with Hg (Input 1)
2
0

2-
and S (Input 2) as two chemical inputs and the fluorescence intensity at 566 nm as output. The
2+
2-
presence of Hg and S was defined as binary “1”, and the absence of these inputs was defined as
binary “0” (Table 2). For the output, the value of I/I at 566 nm was considered as “1” for strong
0
enhanced fluorescence (turn-on) and “0” for weak quenched fluorescence (turn-off). As shown
2+
in Figure 14, when input of Hg (Input 1) was present alone, output “1” was obtained because the
L1 solution showed a prominent turn-on fluorescence response at 566 nm. In other input
combinations, the output signal was “0”, implying turn-off fluorescence. The behaviour is properly
fitting with INHIBIT logic gate. This molecular switching behaviour might be applied to the
development of smarter fluorescent material for multitasking biosensing applications.
2+
Hg
Input-1
Input-2
INH
2
-
566nm
S
Output
Figure 13 The circuit diagram for the INHIBIT logic gate
Input 1
Input 2
(S )
Output
(λ=566nm)
2
+
2-
(Hg )
0
1
0
1
0
0
1
1
0
1
0
0
Figure 14 The fluorescence intensity of L1 at 566 nm in the presence of four different inputs and truth table
for the INHIBIT logic gate
Based on the above collective results from UV-Vis absorption, Fluorescence
emission, Job’s plot analysis, NMR interpretation and reversibility test, the selective 1:1
2
+
binding of probe L1 with Hg ion and the probable mechanism of turn-on fluorescence
enhancement is schematically represented below (Scheme 4). The chemosensor L1 in its free
form has a visible absorption due to ICT transition, gives an orange colour in water medium
and is weakly fluorescent for possible electron transfer upon photo excitation (PET). Owing
2
1

to the involvement of donor tertiary amino N atom in the metal bound form, while it becomes
colourless as a result of absence of ICT, absence of PET and increased rigidity due to
chelation produce a large enhancement in fluorescence intensity (CHEF).
2
Hg
+
Free state
Metal bound state
PET ON
PET OFF/CHEF ON
S
S
O
S
S
OH
OH
N
Hg2+
H C N+
N
3
H C N+
3
I-
O
I-
λem 566 nm
λem 590 nm
2+
L1
L1 + Hg
Highly fluorescent
2
S
-
Weakly fluorescent
2
+
Scheme 4 Proposed binding mode of the chemosensor L1 with Hg and probable
mechanism of turn-on sensing.
Application of chemosensor L1 in cell imaging
Among numerous potential applications of cyanine dyes, bioimaging as fluorescence probes
4
7
in living cells has attracted a great deal of attention recently. Zhou et al. reported that
cyanine chromophore could interact with DNA in the mode of intercalation and be effectively
used as fluorescent staining dye for cell imaging applications. They have also reported the
non-toxic nature of the chromophore by examining cell viability data for MCF-7 (Human
breast carcinoma) cells at micromolar concentrations and can applied in brighter twophoton
4
8
fluorescent (TPF) bioimaging. In the present work, for practical applicability of sensor L1,
2
+
permeability and detection of Hg within living bacterial cell in an image wise manner was
explored. For this, the E.coli DH5-α bacterial cells were grown overnight in LB (Luria
Bertani) medium at 37 ᴼC in a shaker incubator. These bacterial cells were further incubated
with 48 µM of Hg(NO ) in PBS (phosphate buffered saline) buffer at 37 °C for 2 h. To
3
2
remove the remaining Hg(NO ) the bacterial cells were washed with PBS buffer and
3
2,
incubated further with 12 µM of sensor L1 in Methanol:PBS for half an hour at room
temperature. After washing again with PBS, these bacterial cells were transferred to glass
slide for imaging. Fluorescence images of bacterial cells were gleaned using a Leica TCS
2
2

SP8 confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence
microscope. Figure 16 displayed the fluorescence images of bacteria cell in the absence and
2
+
presence of Hg and probe L1. This test indicated that the bacterial cell emits red
2
+
fluorescence with L1 in presence of Hg under fluorescence microscope. It revealed that the
probe L1 has cell membrane permeability for cell imaging and thus can be utilised for
2
+
intracellular detection of Hg in living cells.
Figure 16 Confocal fluorescence images: (a) bright field image (b) confocal image (c) a merged
of (a) and (b) of E.coli DH5-α cells; (d) bright field image (e) confocal image of (f) a merged of
2
+
(
d) and (e) of E.coli DH5-α cells stained to Hg (48 µM); (g) bright field image (h) confocal
image (i) a merged image of (g) and (h) of E.coli DH5-α cells stained to L1 (12 µM); (j) bright
field image (k) confocal image (l) a merged image of (j) and (k) of E.coli DH5-α cells stained to
2
+
L1 (12 µM) and Hg (48 µM).
2
3

Application of chemosensor L1 in test paper strips
Based on the differential colour of L1 in its free form and in Hg bound form, it can be used
2
+
2
+
as a useful means for naked eye detection of Hg in aqueous and aqueous-organic media.
Further for practical usage and for easy detection of mercury content, paper strip method was
applied. For this paper strip made up of whatman filter paper were prepared by treating the
paper with a solution of L1 (0.3 mM) in methanol and dried with air. The test paper strip was
then immersed in an aqueous solution of Mercuric nitrate (1.2 mM) for 2 minutes and dried
2
+
in air. Figure 17 shows the change in colour of paper strips with and without Hg ions in day
light and under UV light at 365 nm. The clear change in colour of the L1 loaded test strip
2
+
upon dipping in Hg solution demonstrated the practical applicability for successful
2
+
49-50
detection of Hg metal ion on a solid paper strip.
(
A)
(B)
2+
L1
L1+Hg
2+
L1
L1+Hg
Figure 17 Photographs showing the colour change of probe L1 before and after addition of
2
+
Hg (A) in day light and (B) under UV light at 365 nm.
4
.
Conclusion
In conclusion, we demonstrate a simple and selective chromogenic and fluorogenic turn-on
2
+
chemosensor based on a styrylpyridinium chromophore (L1) to detect Hg ion in aqueous,
aqueous-organic medium and in biological systems. The change in color of the solution from
2
+
orange to colourless upon addition of Hg ion is clearly visible to the naked eye. Significant
2
+
increase in emission intensity of L1 upon interaction with Hg is ascribed to CHEF due to
2
+
the formation of a conformationally rigid stable L1 + Hg adduct. Based on the collective
experimental results from UV-Vis, fluorescence emission, Job’s plot analysis, NMR
2
+
interpretation and reversibility test, a selective and strong 1:1 binding of probe L1 with Hg
ion through the NS O donor atoms is proposed. The molecular recognition with fluorescence
2
2
2
4

signal is used to construct an INHIBIT molecular logic gate based on the input/output
characteristics of this molecular device. Moreover, chemosensor L1 can be utilised to track
2
+
the presence of Hg ion in real-time in aqueous medium using naked-eye paper test strip
method and in living cell by fluorescence imaging.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
SP gratefully acknowledge DST, New Delhi (SR/FT/CS-023/2009) and BRNS, Mumbai
(37(2)/14/15/2018-BRNS) for financial support through research projects. Authors are
grateful to Sambalpur University, Odisha, India for fluorescence lifetime measurement and
NISER, Bhubaneswar, India for HRMS measurement.
5
.
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Declaration of interests
√☐ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: