Colorimetric indicators for specific recognition of Cu2+ and Hg2+ in physiological media: Effect of variations of signaling unit on optical response

Article abstract of DOI:10.1016/j.ica.2018.09.074

Easy to synthesize probes are designed using bispicolyl moiety as the receptor unit with two different signaling moieties, anthraquinone and bisindolyl. Both the compounds show ‘naked-eye’ sensing of Cu²⁺ and Hg²⁺ in ～100% aqueous me

Full text of DOI:10.1016/j.ica.2018.09.074

Accepted Manuscript
Research paper
Colorimetric Indicators for SpecificRecognition of Cu2+and Hg2+in Physiolog-
ical Media: Effect of Variations of Signaling Unit on Optical Response
Nilanjan Dey, Namita Kumari, Dipen Biswakarma, Satadru Jha, Santanu
Bhattacharya
PII:
S0020-1693(18)31152-6
https://doi.org/10.1016/j.ica.2018.09.074
ICA 18532
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 July 2018
25 September 2018
26 September 2018
Please cite this article as: N. Dey, N. Kumari, D. Biswakarma, S. Jha, S. Bhattacharya, Colorimetric Indicators for
SpecificRecognition of Cu2+and Hg2+in Physiological Media: Effect of Variations of Signaling Unit on Optical
Response, Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/j.ica.2018.09.074
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Colorimetric Indicators for Specific Recognition of Cu2+ and Hg2+ in Physiological Media:
Effect of Variations of Signaling Unit on Optical Response
Nilanjan Dey,a† Namita Kumari,a† Dipen Biswakarma,a Satadru Jha,a,b Santanu Bhattacharya*a, c
aIndian Institute of Science, Bangalore-560012, Karnataka, India,
Contact No: (+91) 080-2293-2664, Fax: (+91) 080-2360-0529
bDepartment of Chemistry, Sikkim Manipal Institute of Technology, Sikkim 737132, India
cDirector’s Research Unit, Indian Association of Cultivation of Science, Kolkata 700032, India
ABSTRACT
Easy to synthesize probes are designed using bispicolyl moiety as the receptor unit with two
different signaling moieties, anthraquinone and bisindolyl. Both the compounds show ‘naked-eye’
sensing of Cu2+ and Hg2+ in ~100% aqueous medium. Not only selectivity, the compounds also
show high sensitivity towards the target metal ions, as evident by their sufficiently low detection
limits. The present system can detect Cu2+ as low as 0.06 ppm and Hg2+ up to 0.03 ppm from visible
color change. The sensors are found equally effective in the detection of metal ions even in the
protein-bound state. The impact of the signaling unit on the sensing properties of probes having
same binding units has been observed and investigated. Mechanistic investigations revealed that
variations in signaling unit influence the pKa of the bispicolyl nitrogens, which subsequently
controls the stoichiometry of the probe-metal ion complexes as well as their luminescence property.
Additionally, high specificity and good accuracy with recovery values ranging from 98.0 to
104.0 % were obtained during Cu2+/Hg2+ estimation in spiked water samples. Low cost, reusable
paper strips were developed for on-site detection of the metal ions which does not require the
assistance of an optical instrument.
KEYWORDS: Naked-eye sensing, Signaling unit, toxic metal ions, Albumin protein, Paper strips
Highlights:



Naked-eye sensing of Cu2+ and Hg2+ in physiological media
Role of signaling unit in determining the stoichiometry of probe-metal ion complexes
Estimation of toxic metal ions in natural water samples and also in presence of serum albumin


Low-cost reusable paper strips for on-location detection purpose
*Corresponding author. e-mail: sb@orgchem.iisc.ac.in
†Both the authors contributed equally.
1. INTRODUCTION
Transition metals, up to a certain level, are essential in human body due to their relevance in
biological systems. Among these copper is the third most abundant transition metal ion found
in the human body. It takes part in a number of physiologically relevant redox processes.1 The
serum albumins (human, bovine) have specific Cu2+ binding site and act as a Cu2+ transport
protein in blood plasma.2 But at the same time, short term exposure of high level of Cu2+
produces gastrointestinal disturbance, while a longer exposure triggers a liver or kidney
damage.3 ‘Free’ copper is an indicator of Wilson’s disease and Menkes’ kinky hair syndrome.4
Moreover, copper ions in free state catalyses the formation of reactive oxygen species and
causes oxidative damage of cellular components.5 Therefore regulation for allowed levels of
copper in drinking water has been imposed. The maximum permitted limit of the Cu2+ ion in
drinking water is <1.3 ppm (EPA).6 On the other hand, mercury is one of most toxic metal ions
heavily engaged in different chemical industries, as it irreversibly inhibits the activities of
different selenium or sulfur-dependent enzymes. Thus the exposure to excess Hg2+ ion results
in severe kidney problems as well as neurological disorders.7 Mercury enters in the food chain
through organomercury species.8 Because of the high biomagnification of Hg2+ ion in aquatic
food chain, its permitted limit in drinking water is only 2 ppb.6 There are many sensors which
report the detection of Cu2+ and Hg2+.9 But many of these work only in organic media and often
fail to work in water. Therefore there is an urgent need of developing sensors that can detect
these ions in water as well as in biological media. The selective as well as sensitivity has always
been the primary concern while designing new sensing probes. However, the efficiency of the
probes get affected by the electronic characteristic of the signaling as well as receptor moiety,
overall structure and the surrounding environment.10 Though plenty of work has appeared in
the literature addressing the effects of surrounding enviroment (polarity, pH, viscosity of the
medium) , till date, no systematic effort has been made to explore the role of signaling moiety

on the metal ion sensing behavior of small molecular probes. Considering this, herein we have
investigated how the structural and electronic changes in the signaling unit affect the metal ion-
chelating behavior of the receptor moiety.
For this, we chose tridentate 2-substituted pyridine based (picolyl) ligands, which are known
for binding various metal ions.11 Metal ion coordination leads to changes in the electronic state
of the bispicolyl moeity, which subsequently alter the optical properties of the covalently-
linked signaling units. We have coupled this bispicolyl amine (BPA) moiety with two different
signaling units based on i) anthraquinone (1) and ii) bisindolylmethane (BIM) moiety (2)
(Scheme 1). Both the probes experience metal ion-triggered modulation in the intramolecular
charge transfer interaction and allow ratiometric ‘naked-eye’ sensing of Cu2+ and Hg2+ at pH
6.5. On the contrary, specific binding is observed solely with Cu2+ at pH 7.4. However, the
mode of metal ion binding is found to be dependent on the nature of the signaling moiety.
Compound 1, with anthraquinone as the signaling unit, shows 2 : 1 binding with metal ions,
while compound 2 having bis-indolyl unit forms a 1 : 1 complexes with metal ions. Another
striking observation was made during the fluorimetric studies, where compound 1 and 2
respectively showed ‘turn-on’ and ‘turn-off’ response with both Cu2+ and Hg2+. Additionally,
color change with Cu2+ was also observed in presence of the excess of serum albumin at
physiological pH. We further checked the applicability of these probes for the detection of the
Cu2+ and Hg2+ ions in the real-life water samples. Furthermore, low-cost paper strips were
prepared for rapid, on-site detection of these toxic metal ions even at distant locations.
2. EXPERIMENTAL SECTION
2.1.Material and methods: All reagents, including starting materials, and silica gel (for TLC and
column chromatography) were obtained from the best known commercial sources and used
without purification. FT-IR spectra were recorded on a Perkin-Elmer FT-IR Spectrum BX
system and were reported in cm−1. 1HNMR and 13C-NMR spectra were recorded with a Bruker
1
Advance DRX 400 spectrometer operating at 400/300 and 100/75 MHz for H and 13C NMR
spectroscopy respectively. Mass spectra were recorded on Micro mass Q-TOF Micro TM
spectrometer.

2.2.Spectroscopic studies: UV−vis and fluorescence spectra were recorded on a Shimadzu model
2100 UV-vis spectrometer and a Cary Eclipse spectrofluorimeter respectively. In the emission
experiments, the slit widths were kept 10 nm and 10 nm for the excitation and emission channel
respectively. The excitation wavelength was chosen as 480 and 465 nm for 1 and 2 respectively.
For sensing in different buffered media, solutions of different pH (HCO2Na/HCl buffer for pH
2-4.0, CH3CO2Na/HCl buffer for pH 4.5-6.5, Tris/HCl for pH 7-9 and Na2B4O7·10H2O/NaOH
for pH 9.5-10.5) were prepared. Sensing of metal ions in aqueous medium was carried out by
adding 10 μL DMSO solution of either 1 or 2 from a stock (1 × 10−3 M) in buffered medium to
make the final volume of 1 mL ([1]=[2]=1 × 10−5 M) followed by addition of aqueous solution
of metal ions. The final concentration of DMSO in all the studies were less than 1%.
2.3.Preparation of paper strips for sensing: To prepare dye-coated filter paper, a methanolic
solution of compound 1 (1 mM) was drop cast onto the filter paper to form a rectangular
luminescent spot with a size of about 1 x 1.5 cm. The solution was completely absorbed on the
filter paper within 20 min and then the filter papers were left overnight for air-dry. To check
metal ion sensing, the dye-coated paper strips were exposed to aqueous solutions of different
metal ions. For recovery studies, the metal ion treated paper strips were dipped into EDTA (1
mM) solution for ~5 min between each reading. Similarly, iodide solution (1 mM) was utilized
to discriminate between Cu2+ and Hg2+.
3. RESULTS AND DISCUSSION
3.1.Synthesis and photophysical properties of receptors: The two sensors were synthesized
using a simple synthetic strategy as shown in Scheme 1. The key starting material p-BPA
benzaldehyde was being synthesized via Vilsmeier-Haack reaction, and then coupled with
either 1, 2-diaminoanthraquinone to afford 1 or with 2 equiv. of indole followed by oxidation
using DDQ to get 2 (See ESI†). All of the compounds were adequately characterized using FT-
1
IR, mass, H and 13C NMR spectral methods (See ESI†). To evalute the metal ion binding
ability of the probes, Uv-visible spectra of compounds were monitored at different pH in
buffered medium. The two pKa of the bispicolyl (BPA) unit were determined as 3.6 and 8.6 for
the protonation of pyridine and the tertiary nitrogen respectively for 1 (Fig. S1a, ESI†). For

compound 2, the pKa values were estimated as 3.9 and 6.5 respectively (Fig. S1b, ESI†). These
pKa values indicate that at pH 6.5, the tertiary nitrogen of compound 1 will be more protonated
compared to the compound 2. On the other hand, at pH 6.5 bisindolyl site of compound 2 will
be in the partially protonated state.12 Both these factors module the internal charge transfer
states of the compounds in different ways. Protonation at the bispicolyl end restricts the charge
transfer process in 1, while protonated bisindolyl group of 2 makes the process energetically
more favorable. Thus the position of ICT band of compound 2 (λmax 510 nm) was found to be
more redshifted compared to 1 (λmax 487 nm). This was also supported by the HOMO-LUMO
energy gap of 1 (2.5 eV) and 1-H+ (3.7 eV) (Fig. S2, ESI†).13 Moreover, microscopic images
(AFM) indicated that compound 2 also forms self-assembled random aggregates at pH 6.5 (Fig.
S3, ESI†). Variable temperature and concentration-dependent emission studies further
substantiate this fact (Fig. S4, ESI†). Both these factors together markedly affect the selectivity
as well as sensitivity of thr probe compounds towards metal ions.
3.2.Metal ion sensing behavior in the aqueous medium: The metal ion sensing properties of each
probe was explored both at pH 6.5 and 7.4 in 0.05 M HEPES buffer medium. Compound 1
showed interaction with Cu2+ and Hg2+ at pH 6.5 with a prominent color change from red to
yellow (Fig. 1a and S5a, ESI†). However, at pH 7.4, a selective binding with Cu2+ was observed
(Fig. 1b and S5b, ESI†). Most importantly, no such color change was witnessed when other
metal ions, such as Ag+, Cd2+, Co2+, Ni2+, Zn2+, Pb2+, Ca2+, Mg2+, Mn2+, Al2+, Cr3+, and Fe3+
were added as controls to the aqueous solutions of either 1. The changes in the color of 1 upon
addition of Cu2+ and Hg2+ are shown in the Fig. 1. The UV-Vis titration of 1 with increasing
Cu2+ at pH 7.4 led to decrease in absorbance at 497 nm with a concomitant increase at 433 nm
(Fig. S6, ESI†). The presence of an isosbestic point at 453 nm indicates the existence of a one-
to-one equilibrium between 1 and the Cu2+ ion. The plot of absorbance ratios at 435 and 505
nm vs. [Cu2+] gave a straight line indicating a visible, ratiometric detection of Cu2+ at
physiological pH. Job plot analysis under similar condition showed a 2:1 complexation between
1 and Cu2+ ion (Fig. S7, ESI†).14 On the other hand, titration studies at pH 6.5 showed that there
is a blue shift of ~80 nm with both the Cu2+ and Hg2+ ions (Fig. 1c & 1d). The Job plots indicated

the formation of 2:1 complexes with either ion in this condition (Fig. S8, ESI†). Thus, we
realized that both the selectivity as well as sensitivity towards metal ions largely depends on
the pH of the medium. At pH 7.4, the selectivity gets improved and exclusive interaction was
observed with Cu2+. However, the extent of metal ion-induced blue shifts of CT band (∆ ~ 80
nm at pH 6.5 and ∆ ~ 65 nm at pH 7.4) were reduced when the pH of medium was changed
from 6.5 to 7.4. Further, association constant of 1 was calculated (for the 2:1 complex) with
each of Cu2+ and Hg2+ at both pH 6.5 and 7.4 in HEPES buffer medium (Fig. S9, ESI†).15 The
detection limit for Cu2+ was estimated to be ~0.06 ppm from UV-Vis spectral titration of the
compound 1 (Table S1, ESI†). Thus with 1, one can detect Cu2+ even ~21 times lower than the
permitted limit of Cu2+ in water and ~0.15 ppm level of Hg2+.16
On the contrary, compound 2 possessing the BPA and a BIM moiety showed an altered
complexation behavior with the above ions. At pH 6.5, it showed similar interaction with both
Hg2+ and Cu2+ ion (Fig. 2a & S10a, ESI†), but did not show any interaction with metal ions at
pH 7.4 (Fig. S10b, ESI†). The complexation of 2 resulted in a decrease in the UV-Vis spectral
intensity of the 497 nm band and the emergence of a new band at 403 and 438 nm with Cu2+
and Hg2+ ions respectively (Fig. S11-S12, ESI†). The titration studies showed ratiometric
sensing for both Cu2+ and Hg2+. Job plot analysis at this condition indicates that compound 2
formed complexes at 1:1 stoichiometry with both Cu2+ and Hg2+ ion (Fig. 2b & S13, ESI†). The
association constants were calculated using Benesi-Hildebrand equation (Fig. S14, ESI†). The
detection limit of 2 was estimated to be ~0.07 ppm and ~0.03 ppm respectively for Cu2+ and
Hg2+ based on UV-vis titrations (Table S1, ESI†). However, both probes showed reversible
binding with the metal ions when EDTA was used. Thus using EDTA, the sensors could be
used multiple times for detection (Fig. 2c-d, S15, ESI†). A comparison study with some of the
structurally-related optical probes has been made in terms of their interaction medium,
selectivity, optical response and detection limits to evaluate the potential of the present system
(Table S2, ESI†).17
The fluorescence responses of both the probes towards various metal ions were checked.

Compound 1 was faintly fluorescent at pH 6.5, but the addition of both Cu2+ and Hg2+ ions
resulted in the ‘turn on’ emission (Fig. 3a & S16a, ESI†). Compound 2 showed fluorescence
emission at 567 nm upon excitation at 465 nm. The fluorescence was however quenched by
both Cu2+ and Hg2+ ions (Fig. 3b & S16b, ESI†). Most importantly, the other metal ions had
negligible effect on the emission intensity of both the ligands (Fig. S17, ESI†).
1
3.3.Mechanistic insight into the metal ion binding profile: H NMR spectral titrations were
1
employed to understand the binding mode. H NMR titration of 1 with Cu2+ resulted in a
complete quenching of all the peaks. This may be due to a paramagnetic behavior of Cu2+ ion
when complexes with 1. The NMR titration of 1 with Hg2+ ion, however, resulted in a downfield
shift of all the protons with maximum changes to the protons of the BPA moiety (Fig. 3c). It
1
indicates that the metal ion are bound to the picolyl moiety. The H NMR titration of 2 with
1
both metal ions showed similar downfield shifts in H-NMR (Fig. S18-S19, ESI†). The
complexations of both 1 and 2 with metal ions were further verified by mass spectrometry.
Each probe furnished mass spectral peaks due to the respective metal complexes of the
stoichiometry consistent with Job plots (Fig. S20, ESI†). Based on the stoichiometry data
derived from the Job plots, NMR titrations and mass spectra, it may be concluded that the 1
forms an octahedral complex with Cu2+, whereas 2 forms a tetrahedral complex (See DFT
optimized structures of 1 and 2 with Cu2+ and Hg2+ as well, Fig. 4a).18
3.4.Signaling unit-controlled alteration in sensing behavior: Thus both the probe showed
preferential interactions with both Cu2+ and Hg2+ ions at pH 6.5 and selective detection of Cu2+
at physiological pH 7.4. The mechanism of interaction indicates that both the probes interact
with metal ions through the bispicolyl unit. Inspite of having identical binding site, the
compounds showed significant differences in their metal ion-chelating properties: i) 1 showed
1:2 stoichiometry with metal ions whereas, 2 showed 1:1 stoichiometry; ii) 1 showed an
increase in emission intensity upon addition of metal ions, while quenching of emission
intensity was observed with 2. The changes in the medium of detection (either solvent system
or the pH of medium) are known to influence the binding properties of the probe towards the
metal ions.19 Here we observed even the change in signaling unit affects the binding properties

of the same binding site.
As described earlier, compound 2 formed self-assembled nanostructure in aqueous medium
owing to its highly ampiphatic nature. Due to aggregation, some of the binding sites of 2 will
be in the buried state and will be inaccessible to the incoming metal ions. Thus for compound
1, saturations in optical signal were achieved upon addition of ~15 µM of metal ions, while for
compound 2, the amount was as high as ~40 µM. Moreover, compound 1 exhibited ~80 nm
blue shift in ICT band, while with 2, the addition of metal ions induce a smaller shift of ~14
nm of the CT band. On the other hand, from pH variation studies, we assumbed that the tertiary
nitrogen center (of BPA unit) of 1 is in protonated form at pH 6.5 and therefore will not
participate in metal ion binding process. Thus, to provide proper coordination sites for metal
ions, two molecules of 1 is required (it showed 2:1 stoichiometry with both the metal ions). To
confirm this we have determined the stoichiometry of interaction between 1 and metal ion
(Cu2+) in acetonitrile medium where no protonation is possible. In CH3CN medium, 1 showed
1:1 interaction with metal ion (Fig. 4b). In a similar way, we have determined the stoichiometry
interaction of 2 with metal ion at pH 4.0, where the tertiary nitrogens of the BPA unit will
mostly be in the protonated state. At this pH, compound 2 showed 2:1 interaction with metal
ion (Fig. S21a, ESI†). Thus from the above shred of observations, it was clear that the
electronics as well as orientation of the signaling meoity markedly influence the protonation
equilibrium of the bispicolyl moeity, which inturns affect the sensitivity of the compounds
towards metal ions as well as the stoichiometry of probe-metal ion complexes (Fig. 5).
The change in turn-on and turn-off behaviour of fluorescence with the change in stoichiometry
is known.20 Here the results indicated the similar phenomenon; when the probes form 2:1
complex preferably with the metal ions, they show turn on emission while 1:1 interaction results
in turn off emission. Further, we checked their emission response towards the metal ions with
varying pH. It has been known that the emission property of the sensors with bispicolyl binding
site changes with change in pH.21 Interestingly, we found that both the compounds showed a
similar trend with a change in pH (Fig. 4c & S21b, ESI†). The addition of metal ions to both
the molecules showed an increase in emission intensity at the pH range where the nitrogens are

protonated. However, at higher pH, where both the nitrogens are deprotonated showed a
decrease in emission intensity upon addition of metal ions (Fig. S22, ESI†).
3.5.Sensing of metal ions in presence of serum albumin protein: Mammalian serum albumin has
a specific Cu2+ ion binding site and is proposed to act as a Cu2+ transport protein in blood
plasma.22 Thus the ability of probes to sense protein-bound Cu2+ can be extremely important to
probe the transportation pathways inside the cells through different membrane-bound carrier
proteins or mechanistic investigations. On the other hand, binding of metal ions like Hg2+, Ni2+
and Co2+ to HSA can occur during acute intoxication. Thus detection of such metal ions in the
bound state with HSA has high clinical importance. To detect the presence of endogenous Cu2+
ion in the human serum albumin (HSA), the sensing ability of 1 and 2 were checked in HSA.
Even in concentrated (0.1 mg/ml) BSA, 1 showed a ratiometric detection of Cu2+ at biological
pH of 7.4 (Fig. 6a). Both the sensors showed ratiometric sensing of either Cu2+ or Hg2+ in HSA
at pH 6.5 (Fig. S23-S24, ESI†).
3.6.Sensing of metal ions in natural water samples: Though metal ions play important roles in
maintaining a wide range of physiological processes, their excess intake may cause severe
health problems, even can lead to death. Considering this, here we have collected water from
institute supply system and determined presence of toxic metal ions, such as Cu2+ and Hg2+. In
both the cases, the water samples spiked with different amounts of toxic metal ions (0-2.5 ppm)
were subjected to spectral analysis without any purification (Fig. S25, ESI†). All the analytical
parameters, such as recovery values, standard deviations were calculated based on the changes
in absorbance values at 490 nm (Fig. S25, ESI†).23 A standard equation of Yꢀ= 0.980-0.0474 x
was used for the quantification of Cu2+ and Yꢀ= 1.0417-0.0344 x for estimation of Hg2+ in the
spiked samples (x = conc. of analyte in µM). In most of the cases, the recovery values varied
from 98% to 105% with relative standard deviations (RSDs) in the range of 2.3 – 8% (Fig. 6c,
Table S3, ESI†). Small standard deviation values indicate a high accuracy of the present
method.
3.7.Sensing of metal ions using dye-coated paper strips: Considering the severity of metal
toxicity in drinking water and the much needed tools for on site detection of ions, we have

developed an alternative strategy for rapid, on-location sensing of Cu2+ and Hg2+ using low-
cost paper strips.24 A gradual change in color from red to yellow was observed when the strips
were dipped into either Cu2+ or Hg2+ solutions (0–4ꢀμM) (Fig. 6b). Thus, using the current
method, toxic metal ions as low as ∼1ꢀμM can be identified from the visible color change. The
selectivity experiment was performed by applying other metal ion solutions as controls onto
these paper strips. The results of such study clearly indicate that the present system is fairly
selective towards Cu2+ and Hg2+ and will not face significant interference from other competing
metal ions. Additionally, the color strips can be reused by subsequently washing them with
dilued EDTA solution (at least for 5 cycles) (Fig. S26a, ESI†). Further, to differentiate between
Cu2+ and Hg2+, we have employed the aqueous solution of iodide as a ‘selective masking agent’.
When the metal ion-treated paper strips were exposed to the iodide solution, the original red
color could be recovered specifically for Hg2+ ion (Fig. S26b, ESI†). Since in this case, no
sophisticated instrument is required (naked-eye sensing), people with limited knowledge in
science will be able to use these strips without much difficulty. Further, the reusability will
significantly lower the cost of these strips.
4. CONCLUSION
In summary, we have synthesized bispicolyl based small molecular probes comprising with two
different chromophore (signalling) units. Both the compounds allow naked eye, ratiometric
sensing of Cu2+ and Hg2+ in buffered aqueous medium and in various biological conditions.
The attributes of pH of the medium of detection on the metal ion sensing behavior is
demonstrated, where a fairly selective response was observed for Cu2+ at physiological pH.
Overall, the present system can detect Cu2+ as low as 0.06 ppm from visible color change, while
the detection limit was even lower for Hg2+, only 0.03 ppm in aqueous medium. The study also
revealed that the modes of metal ion binding largely depend on the nature of the signaling unit.
Though for both the probes, it is evident that metal ion coordination occurs only through
bispicolyl unit, with compound 1 we observe 2:1 binding interaction, while for 2, it is 1:1
complexation. Moreover, probe 1 shows ‘turn-on’ detection of target metal ions, whereas with
2, ‘turn-off’ response was witnessed. The signaling unit controls the protonation equilibrium

of the bispicolyl unit to a significant extent, which likely influences the self-assembly of the
probe molecules in buffered medium as well as their metal ion binding property. Thus the
present study provides an insight on how the same binding moiety behaves differently just by
changing the signaling unit. Additionally, the probes were utilized for estimation of toxic metal
ions in natural water samples. Low-cost dye-coated paper strips were designed for on-location
detection purpose. Therefore new probes of such kind will not only offer significant practical
utility in terms of naked-eye sensing but also added a new understanding on the mechanism of
interaction.
5. ACKNOWLEDGEMENTS
S.B. thanks DST (J. C. Bose Fellowship) for the financial support of this work. ND, NK and DB
thank IISc for fellowship and IACS, Kolkata for the financial support for the work presented in this
manuscript.
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Scheme 1:
Scheme 1. Scheme shows
involved in the
Figure 1:
synthesis of compound 1 and 2
N
N
present study
N
N
N
Cl
N
N
HCl
NH2
N
K2HPO4/CH3CN
POCl3/DMF
90 oC/2 h
N
3
CHO
reflux/24 h
N
O
O
HN
N
N
O
O
NH2
N
Nitrobenzene
130oC, 8h
NH2
N
1
Compound
3
CHO
N
N
N
N
N
1.I2/CH3CN, rt, 1h
N
2. DDQ/CH3CN, rt, 1h
N
H
HN
N
3
CHO
2
Compound

0.45
0.36
0.27
0.18
0.09
0.00
(b)
0.30
0.24
0.18
0.12
0.06
0.00
(a)
+Cu2+
+Cu2+
+Hg2+
300
400
500
600
300
400
500
600
Wavelength (nm)
Wavelength (nm)
(d) 0.8
(c) 0.24
0.18
1 with Hg2+
(0-25 µM)
1 with Cu2+
(0-25 µM)
0.6
0.4
0.2
0.0
0.12
0.06
0.00
350
420
490
560
630
350
420
490
560
630
Wavelength (nm)
Wavelength (nm)
Figure 1. (a) UV-visible spectra of 1 (10 µM) with different metal ions (25 µM) at pH 6.5 in HEPES
buffered medium. (b) UV-visible spectra of 1 (10 µM) with different metal ions (25 µM) at pH 7.4 in
HEPES buffered medium. (c) UV-visible titration of 1 (10 µM) with Cu2+ (0- 25 µM) at pH 6.5 in
HEPES buffered medium. (d) UV-visible titration of 1 (10 µM) with Hg2+ (0- 25 µM) at pH 6.5 in
HEPES buffered medium.
Figure 2.
(a)0.30
3.0
Figure 2. (a) UV-
(b)
With 1
visible spectra
With 2
0.24
0.18
0.12
0.06
0.00
2.4
1.8
1.2
0.6
0.0
of 2 (10
µM)
with
+Cu2+
(1:1)
+Hg2+
(1:2)
0.2
0.4
0.6
0.8
280
350
420
490
560
Cu2+/ [1 + Cu2+]
Wavelength (nm)
(c)
100
80
60
40
20
0
(d)
60
40
20
0
+
+
+
+
2
+
+
+
+
2
g
2
g
2
2
2
2
2
1
2
A
A
A
A
A
A
T
T
T
T
T
T
g
g
g
g
g
g
D
D
D
D
D
D
H
H
H
H
H
H
H
H
E
E
E
E
E
E
+
+
+
+
+
+
+
+
+
+
+
+
+
+

different metal ions (40 µM) at pH 6.5 in HEPES buffered medium. (b) Job’s plot analysis of 1 and 2
with Cu2+ at pH 6.5 in HEPES buffered medium. (c) EDTA-mediated (25 µM) reversibility checking
during interaction of 1 (10 µM) with Hg2+ (25 µM) at pH 6.5 in HEPES buffered medium. (d) EDTA-
mediated (40 µM) reversibility checking during interaction of 2 (10 µM) with Hg2+ (40 µM) at pH 6.5
in HEPES buffered medium.
Figure 3.
Figure
3.
(a)
(b) 80
(a) 50
2 with Cu2+
1 with Cu2+
40
30
20
10
60
40
20
510
540
570
600
630
520
560
600
640
680
720
Wavelength (nm)
Wavelength (nm)
(c)
7
6
5
4
3
2
1
N
e
N
f
N
a
d
g
l
c
b
O
O
HN
N
h
k
j
i
0.5 eq.
0.25 eq.
1
8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8
f1 (ppm)
Fluorescence titration of 1 (10 µM, ex = 480 nm) with Cu2+ (0-25 µM) at pH 6.5 in HEPES buffered
medium. (b) Fluorescence titration of 2 (10 µM, ex = 465 nm) with Cu2+ (0-40 µM) at pH 6.5 in HEPES
1
buffered medium. (c) H-NMR titration of 1 (5 mM) with Hg2+ (0-2 equiv.) in DMSO-d6/D2O (5:1)
mixture medium (the relevant protons were assigned in the structure for convenience).
Figure 4.

High Luminescence
(a)
O
H
Poor ICT
N
Donor
H
N
O
H
N
N
N
N
X
M
N
N
Acceptor
N
X
N
N
H
O
N
N
H
N
N
O
H
O
Metal coordination (1:2)
Probe 1
O
Low Luminescence
X
Strong ICT
(b)
M
Donor
N
N
N
N
High Luminescence Low Luminescence
N
N
Acceptor
N
H
N
H
N
H
N
H
Self-assembly of 2
Metal coordination (1:1)
Probe 2
Figure 4. (a) Scheme shows formation of highly fluorescent 1:2 metal ion-probe complex of 1 (10 µM)
with Hg2+/Cu2+ at pH 6.5 in water. (b) Scheme shows formation of nonfluorescent 1:1 metal ion-probe
complex of 1 (10 µM) with Hg2+/Cu2+ at pH 6.5 in water.
Figure 5.
2.0
(b)
Figure
5.
(a)
(a)
In acetonitrile
Energy-
In water (pH 6.5)
1.5
1.0
0.5
0.0
1 + Cu2+
1:1
1:2
0.2
0.4
0.6
0.8
Mol fraction (1/1+Cu2+)
(c)
1 + Hg2+
60
40
20
0
Probe 1
z
With Cu2+
With Hg2+
z
z
z
2
4
6
8
10
2 + Hg2+
2 + Cu2+
pH of the medium
minimized structures of 1 + Hg2+, 1 + Cu2+, 2 + Cu2+ and 2 + Hg2+ using B3LYP/LANL2DZ level of
theory. (b) Job’s plot analysis of 1 with Cu2+ at pH 6.5 in HEPES buffer and acetonitrile medium. (c)
Change in fluorescence intensity of 1 at 472 nm upon addition of Hg2+ and Cu2+ in different buffered
medium.
Figure 6.

(a)0.60
Metal ion checking with 1
(b)
With Cu2+
With Hg2+
Cu2+ in HSA
0.45
0.30
0.15
0.00
280
350
420
490
560
630
0
2
4 6
8 10 µM
Wavelength (nm)
Hg2+
added
(µM)
Hg2+ Calcd (µM)
Y = 0.980-0.0474 x
Hg2+
found
(µM)
Recovery RSD
(%)
(%)
0
0
0
0
0
0
0
1.54
3.08
4.62
6.16
7.70
1.45
2.92
4.78
5.83
7.38
1.32
3.20
4.54
5.92
7.52
1.65
2.87
4.92
6.20
7.85
1.47
3.00
4.75
5.98
7.58
99.9
99.9
100.1
99.6
99.0
8.0
6.1
4.2
3.2
3.1
Figure 6. (a) UV-visible titration of 1 (10 µM) with Cu2+ (0-25 µM) at pH 7.4 in presence of 0.1 mg/mL
HSA. (b) Job’s plot analysis of 1 with Cu2+ at pH 6.5 in HEPES buffer and acetonitrile medium. (c)
Estimation of Hg2+ using compound 1 (10 µM) in natural water sample.
Colorimetric Indicators for Specific Recognition of Cu2+ and Hg2+ in Physiological Media:
Effect of Variations of Signaling Unit on Optical Response
Nilanjan Dey,a† Namita Kumari,a† Dipen Biswakarma,a Satadru Jha,a,b Santanu Bhattacharya*a, c
aIndian Institute of Science, Bangalore-560012, Karnataka, India,
Contact No: (+91) 080-2293-2664, Fax: (+91) 080-2360-0529
bDepartment of Chemistry, Sikkim Manipal Institute of Technology, Sikkim 737132, India
cDirector’s Research Unit, Indian Association of Cultivation of Science, Kolkata 700032, India
Highlights:




Naked-eye sensing of Cu2+ and Hg2+ in physiological media
Role of signaling unit in determining the stoichiometry of probe-metal ion complexes
Estimation of toxic metal ions in natural water samples and also in presence of serum albumin
Low-cost reusable paper strips for on-location detection purpose