Hg(II) ion specific dual mode signalling in a thiophene derivatized rhodamine based probe and their complexation cooperativity

Article abstract of DOI:10.1016/j.jphotochem.2012.05.010

The rhodamine based chemosensor 1 exhibited highly selective and sensitive detection of Hg(II) ion in MeCN-H₂O mixture observable through amplified absorption and emission signals with a colour change from colourless to pink. Its thiophenyl subunit plays a predominant role in observed allosteric cooperativity in binding of both the Hg(II) ions of its 1:2 (L:M) complex stoichiometry. The anion induced 'turn-off' signalling responses of the Hg(II)-complex of 1 and subsequent 'turn-on' responses with further addition of Hg(II) here demonstrated its repetitive usability as a probe for Hg(II) ion detection.

Full text of DOI:10.1016/j.jphotochem.2012.05.010

Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Hg(II) ion specific dual mode signalling in a thiophene derivatized rhodamine
based probe and their complexation cooperativity
Ajoy Pal, Bamaprasad Bag∗
Colloids and Material Chemistry Department, Institute of Minerals and Materials Technology (CSIR-IMMT), P.O. R.R.L., Bhubaneswar 751013, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The rhodamine based chemosensor 1 exhibited highly selective and sensitive detection of Hg(II) ion in
MeCN–H2O mixture observable through amplified absorption and emission signals with a colour change
from colourless to pink. Its thiophenyl subunit plays a predominant role in observed allosteric cooperativ-
ity in binding of both the Hg(II) ions of its 1:2 (L:M) complex stoichiometry. The anion induced ‘turn-off’
signalling responses of the Hg(II)-complex of 1 and subsequent ‘turn-on’ responses with further addition
of Hg(II) here demonstrated its repetitive usability as a probe for Hg(II) ion detection.
© 2012 Elsevier B.V. All rights reserved.
Received 20 January 2012
Received in revised form 12 April 2012
Accepted 11 May 2012
Available online 19 May 2012
Keywords:
Signalling probe
Chromogenic
Fluorogenic
Mercury specificity
Rhodamine delactonization
1. Introduction
known to be a potential quencher [22,23] of excited fluorophores
through the spin–orbit coupling effect, a modular design of probe
The existence of transition and heavy metal ions in living organ-
isms is highly essential for various biological functionalities [1,2],
however, their presence or absence deviating requisite concentra-
tion threshold imparts lethal toxic effect to pose serious human
health hazards and environmental impacts [3]. In this regard,
the toxic Hg(II) ion contamination [4–10] in environment which
occurs through various natural and human activities is a matter
of concern. Mercury rapidly bioaccumulates [11,12] in the phys-
iological functionalities due to microbial conversion of its both
elemental and ionic form into methyl mercury, which subsequently
passes through skin, respiratory and gastrointestinal tissues into
the human body. Further, its exposure lead to dysfunction of cells
and consequently cause many health problems [11–15] in the brain,
kidney, central nervous, mitosis and endocrine systems because of
its higher affinity for thiol-group in proteins and enzymes. Owing
to its highly carcinogenic and mutagenic nature, its on-site, real
time, selective analysis for biological, environmental and clini-
cal monitoring purposes is highly desirable [13–16]. Considering
the selectivity, sensitivity and operational advantages over other
analytical methods, development of fluoro- and chromogenic sig-
nalling probes through various methodological designs as well
as manifestation of signal transduction pathways have received
considerable attention recently [17–21]. Although Hg(II) ion is
is expected to nullify such effect and enable to exhibit a turn-on
fluorescence signalling response of the probe upon complexa-
tion, which may subsequently lead to a practical implementation
in terms of detection limit for environmental and in situ intra-
cellular Hg(II) ion analysis [24]. Further, chromogenic signalling
module offers a visual detection while fluorogenic module mostly
addresses the sensitivity issue, hence, it should be advantageous
to design chemosensors that facilitate both chromogenic and flu-
orogenic modes of signalling. Rhodamine based probes [17,25–27]
are ideal in this regard as the dye (a) exhibits excellent spectro-
scopic characteristics such as large molar extinction coefficient,
high emission quantum yield, absorption and emission at longer
wavelengths, (b) modulates the optical signal in aqueous media
that facilitates in vitro and/or in vivo monitoring of biologically
relevant species and (c) renders a contrasting structure–function
correlation through its lactonization–delactonization process, i.e.
its commonly existing spirolactam form which is colourless and
non-fluorescent in protic solvents structurally equilibrates to the
coloured and highly fluorescent ring-opened amide form upon
complexation. Following this protocol, many rhodamine based
probes for Hg(II) ion detection have been reported [25–32] where
the signal transduction pathway involves delactonization of its
spiro-ring through either metal ion coordination or ion-induced
chemical reactions. The signalling action of many of these probes
is limited in terms of practical utility as they have either exhib-
ited irreversible signalling with Hg(II) ion that defy the reusability
or pose a restricted detection limit and often are cross sensitivity
∗
Corresponding author. Tel.: +91 674 237 9254; fax: +91 674 258 1637.
E-mail address: bpbag@immt.res.in (B. Bag).
1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2012.05.010

A. Pal, B. Bag / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
43
towards other metal ions. Hence, the quest for higher selectiv-
ity, sensitivity, reversibility in signalling with Hg(II) ion demands
development of new rhodamine based probes with altered struc-
tural and functional modifications. In this direction, we have
recently shown [28] that few ‘amino-ethyl-amino’ derivatized rho-
damine based probes exhibit dual channel signalling with Hg(II) ion
for their highly selective and sensitive detection in organic-aqueous
medium.
where ꢀS and ꢀR are the radiative quantum yields, FS and FR are the
area under the fluorescence spectra, AS and AR are the absorbance
(at the excited wavelength) of the respective sample and the refer-
ence; ꢁS and ꢁR are the refractive indices of the solvent used for the
sample and the reference. The quantum yield of Rhodamine G was
measured using quinine sulphate in 1 N H2SO4 as reference [35]
excited at (ꢂex) 350 nm. The standard quantum yield value thus
obtained was used for the calculation of the quantum yield of the
samples.
The association constants were determined [36] from the
change in absorbance or fluorescence resulted from the titration
of dilute solutions (∼10−4–10−6 M) of the probes against metal ion
through non-linear regression curve fitting 1:2 (L:M) stoichiometry
of complexation following the equation (Eq. (2)) as depicted below:
With a prejudice idea of enhancing the selectivity, sensitiv-
ity and faster regeneration of Hg(II)-selective rhodamine based
probes by exploiting thiophilic nature of Hg(II) ion, we report
herein the dual channel ‘turn-on’ signalling responses of a bis-
rhodamine derivatized thiophenyl probe 1 selectively with Hg(II)
ion and the subsequent reversible ‘turn-off’ signalling responses of
1 ⊂ (Hg(II))2 complex with AcO−, CO3 and HCO3 anions. A pos-
itive allosteric cooperativity in binding of both Hg(II) ions to the
probe was observed during complexation. In this context, the ear-
lier reports on Hg(II)-selective bis-rhodamine appended systems
either have shown [29] a 1:1 (L:M) stoichiometry of Hg(II) coordina-
tion or did not exhibit [30,31] any positive cooperativity in binding
of both Hg(II) ions in case of 1:2 (L:M) complex stoichiometry. In
order to investigate the role of thiophenyl S-atom on the probe’s
Hg(II)-ion selectivity and sensitivity of detection, the signalling pat-
tern of a structurally modified probe 2 with central phenyl C-atom
in its architecture was evaluated and compared with that of 1 under
similar experimental conditions.
2−
−
2
A ꢃHG
H
k1 G + B
H
k1k2 G
[ ]
[
]
[
]
[
]
2
ꢃHG1
(
)
(
)
0
0
ꢃXobs
=
(2)
1 + k1 G + k k
G
]
[
]
[
1
2
where ꢃXobs is the change in absorption (ꢃA) or fluorescence (ꢃF)
of the probe (H) solution after addition of metal ions (G), k1 and
k2 are the first and second stepwise association constants, A(ꢃHG)
is either the molar absorptivity (absorption) or proportionality
constant (fluorescence) of the complex (HG), B(ꢃHG1) is either the
molar absorptivity (absorption) or proportionality constant (fluo-
rescence) of the complex HG1.
2.3. Synthesis
2. Experimental
The probes were synthesized as per the schematic representa-
tion (Scheme 1).
2.1. Materials
2.3.1. Synthesis of 1a, aminoethyl rhodamine
This compound was synthesized following the procedure
reported [28,29,37,38] earlier.
All the reagent grade chemicals were used without purifica-
tion unless otherwise specified. 2,5-Thiophene-dicarboxaldehyde,
rhodamine B hydrochloride, ethylenediamine, 1,3-benzene dicar-
boxaldehyde and the metal-perchlorate salts were obtained from
Sigma–Aldrich (India) and used as received. Anhydrous sodium
sulphate, sodium borohydride, neutral alumina for column chro-
matography, acids and the solvents were received from S.D. Fine
Chemicals (India). All the solvents were freshly distilled prior to
use for absorption and fluorescence measurements.
2.3.2. Synthesis of 1, thiophene-2,5-di-(methyl-amino-ethyl
rhodamine)
To a stirring solution of 1a (1.02 g, 2.1 mmol) in EtOH (30 mL);
2,5-thiophene-dicarboxaldehyde (0.145 g, 1.03 mmol) was added
and allowed to react at RT for 36 h with constant stirring. The
Schiff-base thus formed was reduced with NaBH4 (0.12 g, 3.2 mmol)
stirring at RT for overnight followed by reflux for 2 h to ensure
complete reduction. The solvent was then removed under reduced
pressure and the solid mass thus obtained was added with 50 mL of
water and extracted with CHCl3 (3× 30 mL). The combined organic
layers, after drying over anhydrous Na2SO4 was filtered and dried
under reduced pressure to obtain a faint-pink coloured solid as the
desired product 1, which was purified and isolated by column chro-
matography (silica gel, 100–200 mesh) using chloroform in hexane
(3%, v/v) as eluent.
2.2. Analysis and measurements
The compounds were characterized by elemental analyses, 1H
NMR, 13C NMR and mass (ESI) spectroscopy. 1H NMR and 13C
NMR spectra were recorded on a JEOL JNM-AL400 FT V4.0 AL 400
(400 MHz and 100 MHz, respectively) instrument in CDCl3 with
Me4Si as the internal standard. Electrospray mass spectral data
were recorded on a MICROMASS QUATTRO II triple quadruple mass
spectrometer. The dissolved samples of the compounds in suitable
solvents were introduced into the ESI source through a syringe
pump at the rate of 5 ␮L/min, ESI capillary was set at 3.5 kV with
40 V cone voltage and the spectra were recorded at 6 s scans. Melt-
ing points were determined with a melting point apparatus by
PERFIT, India and were uncorrected. Elemental analyses were done
in an Elementar Vario EL III Carlo Erba 1108 elemental analyzer.
UV–visible spectra were recorded on a Perkin Elmer Lambda 650
UV/VIS spectrophotometer at 298 K in 10−4–10−6 M concentration.
Steady-state fluorescence spectra were obtained with a Fluoromax
4P spectrofluorometer at 298 K. Fluorescence quantum yield was
determined in each case by comparing the corrected spectrum with
that of Rhodamine G (ꢀF = 0.95) in EtOH [33] by taking the area
under total emission using the following equation [34]:
Yield: 0.88 g (79%); mixed mp: 98–100 ◦C; ESI-MS [Mw
(C66H76N8O4S) = 1077.43)], m/z+ (%): 1077.4 [1]+, (100%); 1H NMR
(400 MHz,CDCl3, 25 ◦C, TMS): 7.882 (d, J = 1.8 Hz, 2H), 7.424 (t,
J = 2.7 Hz, 4H), 7.062 (br s, 2H), 6.544 (s, 2H), 6.401 (d, J = 9.0 Hz,
4H), 6.364 (s, 4H), 6.234 (d, J = 8.7 Hz, 4H), 3.629 (s, 4H), 3.07 (t,
J = 7.2 Hz, 20H), 2.402 (t, J = 6.3 Hz, 4H), 1.148 (t, J = 6.9 Hz, 24H); 13
C
NMR (100 MHz, CDCl3, 25 ◦C, TMS): 168.63, 153.77, 153.38, 148.87,
142.63, 132.46, 131.25, 128.86, 128.09, 124.44, 123.89, 122.87,
108.24, 105.68, 97.89, 65.04, 48, 47.28, 44.48, 39.98, 29.81, 12.74.
Anal. Calcd. for C66H76N8O4S: C, 73.57, H, 7.11, N, 10.40; found: C,
73.15; H, 7.49; N, 10.28.
2.3.3. Synthesis of 2, benzene 1,3-di-(methyl-amino-ethyl
rhodamine)
This compound was synthesized following the procedure
reported earlier [37] from our laboratory.
ꢀ
ꢁ ꢀ
ꢁ
2
FSAR
FRAS
ꢁS
ꢁR
ꢀS = ꢀR
·
(1)

44
A. Pal, B. Bag / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
O
. HCl
OOC
H2N
NH2
N
NH2
N
(i)
N
O
1a
N
O
N
RB.HCl
OHC
CHO
OHC
CHO
(ii)
(ii)
S
O
O
N
O
S
O
N
HN
NH
N
N
NH
N
N
HN
N
O
N
N
O
N
O
N
O
N
1
2
Reaction conditions: (i) EtOH, Reflux, 16h. (ii) (a)EtOH, 36h, RT, (b)NaBH4
Scheme 1. Synthetic route to the probes 1 and 2.
2.3.4. Synthesis of 1-[Hg(II)]2 complex
few other metal ions also displayed smaller enhancements in fluo-
rescence (FEF), although were much lower in comparison to that
with Hg(II) ion. Apart from the enhanced fluorescence intensity
To a stirring solution of the probe 1 (0.1 mmol) in MeCN,
an acetonitrile solution of Hg(II)(ClO4)2·xH2O (0.1 mmol) was
added, allowed to react for 2 h at RT and then mixture was fil-
tered off. The filtrate was allowed to evaporate at RT to afford
a dark brown solid as the desired product. The solid were col-
lected by filtration, washed, dried under vacuum and characterized
through ESI-MS spectra and CHN analyses prior to use. ESI-MS [Mw
(C66H76N8O4S.2Hg) = 1478.51)] m/z+ (%): 1477.3 [(1.2Hg)−1]+ (8%),
1277.2[1.Hg]+ (11%), 1077.3 [1]+ (100%).
of 1 in presence of Hg(II) ion, a 30 nm red-shift in the fluores-
max
cence maxima (ꢂ
= 550 → 581 nm) was observed in MeCN. Like
em
absorption spectral behaviour, fluorescence of 1 also enhanced
(ꢀFT = 0.697) selectively in presence of Hg(II) ion (Fig. 1b) and other
metal ions failed to render any appreciable change (ꢀFT ≤ 0.001)
when measured in MeCN–H2O (9:1/7:3/1:1, v/v) mixture. The
change in absorption and fluorescence intensities of 1 due to metal
ion coordination in varying proportion of acetonitrile–aqueous
binary mixture revealed that the selectivity of 1 towards Hg(II) is
more apparent in MeCN–H2O mixed medium than in pure MeCN.
This infers that presence of water molecules in the medium pro-
motes only Hg(II) coordination to delactonized rhodamine of 1 and
restricts coordination of other metal ions to the probe through their
preferential hydration. This typical behaviour of aqueous promoted
Hg(II)-selectivity was also consistently observed in other substi-
tuted ‘amino-ethyl-amido’-rhodamine based Hg(II) ion selective
probes [28]. Similarly, probe 2 has also exhibited absorption and
fluorescence enhancement selectively in presence of Hg(II) ion,
although the extent of enhancement is less in comparison to that of
1. The ‘turn-on’ signalling responses of these probes in MeCN–H2O
mixed medium, which can be visually perceived as a function of
colour change as well as instrumentally monitored through absorp-
tion and fluorescence spectral amplification, have rendered a high
selectivity towards Hg(II) ion for their detection.
3. Results and discussion
The absorption and emission spectral pattern of 1 in various sol-
vents revealed that it exhibits absorption transition at ∼260–270
and ∼315 nm which attributed to the higher energy ligand local-
ized ꢄ−ꢄ* transition and does not absorb in 500–600 nm region
irrespective of the solvent medium. This characteristic behaviour
attributed to the spirolactam conformation of rhodamine, which
has also been indicated by the characteristics peak at ∼65 ppm
(9-Cq) in 13C NMR spectrum in CDCl3. 1 has exhibited a weak fluo-
rescence (ꢀFT < 0.001) due to lactonized conformation of rhodamine
that facilitates various non-radiative pathways to quench its fluo-
rescence, apart from an operative photo-induced electron transfer
processes from distal Amino-donor groups present in its receptor
moiety. The absorption and fluorescence spectral responses of 2 in
various solvents have shown similar features as that of 1.
A similar trend of Hg(II)-induced absorption and fluorescence
signal amplification in 1 was observed when HgCl2 was used
instead of Hg(ClO4)2 as ionic input under similar experimental con-
ditions. On the contrary, its optical spectral pattern did not exhibit
such signal amplification and remained almost unchanged with
Hg(OAc)2 as input. This infers that Hg(II)-selective chemo-sensing
behaviour of 1 also depends upon the counter anions present in the
medium. Further, no appreciable change in absorption (A557) or flu-
orescence (F581) spectral pattern of 1 was observed when measured
in varied pH in the range of 4.0–10.0. The spectral responses of 1
in presence of Hg(II) in varying pH revealed that 1 yields a stable
Hg(II) induced amplified signals upon addition of Hg(II) in the same
region without any interference by protons. However, the intensity
of the metal free 1 has increased with decrease in pH values under
more acidic conditions (pH < 4.0). Nevertheless, the stability of the
probe and its Hg(II) complex over a wide pH range indicates that 1
can be potentially utilized as a probe for Hg(II) ion in complicated
molecular and biological systems.
In order to investigate the metal ion induced signalling pattern
of 1 and 2, perchlorate salts of various metal ions such as Na(I), K(I),
Mn(II), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Ag(I), Cd(II)
and Hg(II) were added to the probes in either MeCN or MeCN–H2O
(v/v, varied proportions) mixture. Addition of various metal ions
to the colourless solution of 1 in MeCN resulted in appearance of
an absorption peak at 557 nm with subsequent change in colour
to pink due to metal ion induced delactonization of rhodamine’s
spiro-ring. The absorption enhancement (A557) of 1 in MeCN was
observed to be maximum in presence of Hg(II) (ε/ε0 = 1223 fold).
Presence of other metal ions has also induced appreciable absorp-
tion spectral change (ε/ε0 = 67 and 62 fold for Cu(II) and Fe(II) ions,
respectively). However, when measured in MeCN–H2O mixture
(variable compositions, 9:1/7:3/1:1, v/v) medium, 1 exhibited an
absorption spectral amplification and rendered a colourless → pink
colour change selectively in presence of Hg(II) while other metal
ions imparted either no or negligible change (Fig. 1a). Expectedly,
the metal ion induced spiro-ring opening of rhodamine also lead to
significant fluorescence enhancement of 1 in MeCN. The maximum
fluorescence recovery was observed with Hg(II) ion in MeCN while
In order to validate Hg(II) selectivity of 1 and quantify the
extent of interference of other competitive metal ions, few

A. Pal, B. Bag / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
45
3
2
1
0
(b)
(a)
1
II
Hg
I
II
blank, Na, KI, Mn ,
II
II
II
Fe ,Co , Ni ,
Cu , Zn , Ag,
blank, Na, KI,
II
II
I
I
II
Hg
II
II
II II II
Cd , Pb
Mn , Fe ,Co , NiII,
II II I
Cu , Zn , Ag,
II
II
Cd , Pb
300
400
500
600
700
550 600 650 700 750 800
Wavelength (nm)
Wavelength (nm)
Fig. 1. (a) Absorption and (b) emission spectra of 1 alone and in presence of various metal ions in MeCN–H2O (1:1, v/v). Absorption: [1] = 1 × 10−4 M, [M(II/I)] = 1 × 10−3 M.
Emission: [1] = 1 × 10−6 M, [M(II/I)] = 2 × 10−5 M, RT, em./ex. b.p. = 5 nm, error < 5%.
estimated to be 2.2 × 10−8 M. The observable Hg(II) detection limit
alkali/heavy/transition metal ions were added to the solution con-
taining 1 with Hg(II) in MeCN. Addition of these competitive metal
ions did not change the absorption and emission intensity of the
solution of 1 with Hg(II) ion. On the contrary, when Hg(II) ion was
added to the solution containing 1 and one of these competitive
metal ions in MeCN (where ꢃε557 or ꢃF581 of 1 ⊂ M(I/II) either
negligible or much lower), the absorption in visible region and flu-
orescencewas observedto increasesignificantlyupto a comparable
extent of that of 1 in presence of Hg(II) ion alone, inferring to the
remarkably high selectivity of 1 for Hg(II) ion (Fig. 2). Similar Hg(II)-
selective spectral responses with non-interference of competitive
metal ions were also observed with probe 2.
of 1 as a function of colour change observable through naked
eye was found to be 4.0 × 10−6 M. Similarly, the detection limit
(LOD) of Hg(II) binding to 2 was estimated to be 2 × 10−7 M (flu-
orescence experiment) and the observable detection was found
to be 1.5 × 10−5 M through naked eye due to change in colour.
The corresponding fluorescence intensity change [(F/F0)580] of time
course study of 1 (1 ␮M) with Hg(II) (5 ␮M) in MeCN–H2O (1:1, v/v,
pH = 7.0) revealed that the probe–metal complexation occurred in
almost 1 min and remained constant for at least 12 h. Hence, the
probe 1 promises its potentiality in real time monitoring of Hg(II)
concentration, which is an essential factor for a probe if to be used
in practical chemo-sensing application.
The absorption spectra (Fig. 3a) of 1 (20 ␮M) in presence of
varying concentration of Hg(II) in MeCN–H2O (1:1, v/v, pH = 7.0)
revealed that the absorption intensity at 557 nm has enhanced
non-linearly with increase in Hg(II) concentration, although addi-
tion of Hg(II) ion in large excess (>100 equiv.) resulted in decrease
in the extent of such enhancement. Two clear isobestic points at
334 nm and 288 nm observed in the absorption titration, which
may be correlated to the two components of complexation. The
non-linear nature of the plot of change in absorbance of 1 (A − A0,
ꢂmax = 557 nm) as a function of added Hg(II) ions has been shown
in Fig. 3b. The stoichiometry of complexation of 1 with Hg(II) was
determined to be 1:2 (L:M) from the plot of absorbance of 1 as a
function of molar fraction of added Hg(II) in MeCN (continuous vari-
ation method, Fig. 3c). The ESI mass spectra of 1 ⊂ (Hg(II))2 complex,
synthesized by addition of 2.5 equivalents of Hg(II) to 1, also have
supported to a 1:2 (L:M) complex formation. The spectral pattern
exhibited peaks at m/z = 1477.3 and 1277.2 and 1077.3 correspond-
ing to [1+ 2Hg(II)]+, [1+ Hg(II)]+ and [1]+ fragments, respectively,
in comparison to the molecular ion peak of metal free probe 1 at
1077.4 (m/z+).
Based on the spectral signal perturbation, the detection limit
(LOD) of Hg(II) binding to 1 was calculated as a function of stan-
dard deviation (ꢅbl) of blank measurements over slope (m) between
intensity versus sample concentration as 3ꢅbl/m, which was
The fluorescence spectral pattern of the titration of 1 (0.6 ␮M)
with Hg(II) in MeCN–H2O (1:1, v/v, pH = 7.0) also revealed that
the emission at 581 nm has increased with increase in Hg(II) con-
centration (Fig. 4). It gradually enhanced up to 8 equiv. of Hg(II)
and remained constant thereafter even up to addition of 45 equiv.
of Hg(II) ion. The association constants (Ka) for Hg(II) binding to
1 were determined [36] through the non-linear curve fitting for
Fig. 2. Extent of fluorescence enhancement in 1 with various metal ions (M) in
absence and presence of Hg(II) ion in MeCN.

46
A. Pal, B. Bag / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
Fig. 3. (a) Absorption spectra of 1 (2 × 10−5 M) with different equivalents of Hg(II) added in MeCN:H2O (1:1, v/v). (b) Change in absorption (A − A0, ꢂmax = 557 nm) of 1 as a
function of added Hg(II) ions ([1] = 2 × 10−5 M, MeCN:H2O (1:1, v/v)). (c) Absorbance of 1 as a function of mole fraction of Hg(II) ion added (Job’s plot) in MeCN.
1:2 (L:M) stoichiometry of the absorption or fluorescence titration
data. In these cases, the linear regression fitting of the titra-
tion did not converge appropriately (r2 < 0.75) with conventional
Benesi–Hildebrand plot [32,39] for either 1:1 or 1:2 (L:M) complex-
ation. Assuming a 1:2 (L:M) stoichiometry (as obtained from Job’s
plot of its complexation, Fig. 3c) and from the non-linear regression
analysis of the fitting of fluorescence intensity of 1 as a func-
tion of Hg(II) concentration, two association constants (log k1 = 4.72
and log k2 = 12.32) with good linear coefficient (r2 = 0.99) were
obtained. The fluorescence enhancement (FEF) and subsequent
may also be described by the interaction parameter [42] (˛ = k2/k1
or k1k2/k2) where ˛ > 1 correlates to a positive cooperativity and
˛ < 1 correlates to a negative cooperativity of binding. Both the
association constants due to complexation in a two-binding site
receptor if found to be almost same to the corresponding refer-
ence receptor with one binding site (k1 = k2 = k), then it indicates
the absence of such cooperativity of binding. Here, the estimated
higher interaction parameter (˛ > 1) of Hg(II)-complexation to 1 as
obtained through absorption/fluorescence titration data suggests
a positive cooperativity in step-wise binding of both Hg(II) ions.
The higher interaction parameter, in turn, the higher k2 in com-
parison to k1 also suggests that formation of a 1:2 (L:M) complex
is more favourable over formation of 1:1 (L:M) complex in this
case, which was also complimented well by the Job’s plot, ESI-
MS analysis and the observed isobestic points in the absorption
spectral pattern as a function of increase in Hg(II) concentration.
Further, it suggests that 1 binds selectively to the first Hg(II) ion
through one of its ‘amino-ethyl-amido’ unit along with its central
Sthiophenyl-atom, which further facilitates a spatial reorientation of
the ligand and leads to a faster binding of second Hg(II) ion. No
such positive cooperativity was observed between binding of both
the Hg(II) ion to 2 despite it has also exhibited a 1:2 (L:M) complex
stoichiometry. The association constants for binding of both the
Hg(II) ion with 2 [k1 = 2.06 × 106 M−1 and k2 = 2.13 × 106 M−1 (flu-
orescence titration) and log k1 = 5.48 and log k2 = 4.85 (absorption
higher ka value (>1010
M
−1) for 1 with Hg(II) corresponding to
its higher binding ability infer to its potentiality in detecting
Hg(II) ion at a very low concentration level. The higher value of
k2 (2.1 × 1012
M
−1) in comparison to that of k1 (5.4 × 104 M−1
)
obtained through fluorescence titration here argues for a step-wise
binding of both Hg(II) ions to 1, apart from contributing towards
sensitivity level of detection with respect to its concentration. Asso-
ciation constants obtained from the absorption titration of 1 with
Hg(II) ion were (log k1 = 3.61 and log k2 = 10.11) also found to be
consistent (within the experimental error) with those from fluo-
rescent titration. Further, the higher k2 values in comparison to
k1 obtained from non-linear regression plots of both absorption
and fluorescence titrations suggest the existence of cooperativity
[40,41] between coordination of both Hg(II) ions to 1. The coop-
erativity of complexation in a two-binding site receptor as in 1

A. Pal, B. Bag / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
47
N
II
N
Eq. Hg added
9.9
8.8
7.7
6.6
5.5
4.4
3.3
2.8
2.2
2
1.8
1.5
1.3
1.1
0.9
0.7
0.4
0.2
0
O
HgII
O
O
O
N
N
N
N
N
H
HN
HgII
HgII
S
1
S
HgII
NH
O
0.0
0.5
1.0
1.5
2.0
-5
2.5
3.0
HgII
NH
O
[Hg(II)] (x 10 )
N
N
N
O
N
N
O
N
II
II
[1.(Hg ) ]
1
[1.Hg ]
2
550
600
650
700
750
800
Fig. 5. Pictorial representation of sequential complexation of Hg(II) ions to 1.
Wavelength (nm)
Fig. 4. Fluorescence spectral pattern of 1 (0.6 ␮M) in MeCN–H2O (1:1, v/v) upon
addition of different equivalents of Hg(II) ion (RT, ꢂex = 500 nm, excitation and emis-
sion b.p. 5 nm). Each spectrum was taken after 1 min of Hg(II) addition. Inset:
Fluorescence enhancement change (normalized with the emission of free probe 1)
as a function of concentration of Hg(II) added and subsequent fitting through non
linear regression.
Further addition of Hg(II) to these colourless solutions of
anion-mediated Hg(II)-decomplexed 1 (ꢀFT ≤ 0.002) resulted in
appearance of pink colour and exhibited the A558 and F580 spec-
tral enhancement almost to the extent of those upon initial Hg(II)
addition to 1. A comparison of time course studies between the
A557 or F580 spectral responses of (a) Hg(II) addition to 1 in
MeCN:H2O (1:1, v/v) and (b) subsequent Hg(II) addition to the solu-
tion containing anion-induced-Hg(II)-decomplexed 1 has shown
that the Hg(II) induced enhancement in absorption and emis-
sion, in turn, spiro-ring opening of rhodamine occurs faster in
former case than the later condition. The reversible signalling pat-
titration), respectively] were found to be much lower in magni-
tude in comparison to those with 1, which also establishes the
significance of thiophenyl S-atom of 1 towards its faster Hg(II)-
complexation. In order to verify the role of thiophenyl S-atom in
1 towards its sequential Hg(II) coordination and in cooperativity of
both the metal ion binding, a controlled experiment was carried out
by addition of urea and thiourea to the solutions (MeCN:H2O::9:1,
v/v) containing either Hg(II) complex of 1 or that of 2. Upon addi-
tion of thiourea, the intensity of absorption and emission spectral
tern of Hg−(II)-complexation to 1 in presence of only AcO−, CO3
2−
and HCO3 has revealed that these counter anions promote com-
plete decomplexation of amido-rhodamine binding unit of 1 with
Hg(II) due to higher binding affinity of these anions towards Hg(II)
and subsequently lead to a structural transformation of the ring-
opened amide form of rhodamine in 1 to its original spirolactam
conformation. The anion-induced ‘turn-off’ signalling responses
were more pronounced when measured with solution of solid
1 ⊂ (Hg(II))2 complex dissolved in MeCN–H2O (1:1, v/v) in com-
parison to the solution of 1 added with Hg(II) ion. In this context,
responses (A
and F∼580) decreased slightly for 1 ⊂ (Hg(II))2
∼550
complex, however, quenched substantially for 2 ⊂ (Hg(II))2 com-
plex almost to the extent of that of free probe. Addition of urea to
the solutions containing either of the complexes resulted in no or
negligible change in their spectral pattern. The quenched spectral
responses of 2 ⊂ (Hg(II))2 complex in presence of thiourea may be
attributed to the effective coordination of Hg(II) ion to thiourea due
to thiophilic nature of Hg(II) ion, which subsequently promoted
decomplexation of 2. In case of 1 ⊂ (Hg(II))2 complex, one of the
Hg(II) ion remained bound to 1 through thiophenyl S-atom even in
presence of thiourea due to higher Hg(II) affinity of Sthiophenyl than
Sthiourea-atom. However, the thiourea might have decomplexed the
second Hg(II) ion in 1 ⊂ (Hg(II))2 which has not coordinated to the
Sthiophenyl atom of 1, as a result, the Hg(II)-induced enhancement
in absorption and emission of 1 has reduced slightly. The pictorial
representation of stepwise Hg(II) binding to 1 is given in Fig. 5.
The reversibility being the pre-requisite criteria for repetitive
usability of a chemosensor for selective detection of metal ions,
the absorption and emission signalling responses of 1 with Hg(II)
ion were evaluated upon addition of ammonium salts of var−i-
it is worth mentioning that the reversible signall−ing of Hg(II) com-
2−
plexation to 1 in presence of CO3
and HCO3 anions was not
observed within the time frame in the case of mono-substituted
‘amino-ethyl-amido’ rhodamine based probes [28] reported ear-
lier. Further, the precursor to the probes (1a) had exhibited [28]
an non-selective ‘turn-on’ signalling pattern with Hg(II) and a sub-
sequent irreversible ‘turn-off’ responses with acetate and iodide
anions. Apart from these anions, 1 also regenerates its initial
spirolactam state upon subsequent addition of chelating agents
such as EDTA and ethylenediamine to the solution of 1 ⊂ (Hg(II))2
complex as their addition leads to immediate quenching of the
enhanced absorption and emission spectral responses of 1 that
resulted earlier due to Hg(II)-complexation, to the extent of that
in metal free probe. Here, this interesting aspect of reversible
signalling pattern thus may be attributed to the corresponding
stereo-electronic situation rendered by 2,5-thiophenyl unit of 1
during Hg(II) complexation–decomplexation process; however, a
further investigation is required for a conclusive opinion on this
complicated process. Nevertheless, this establishes a predomi-
nant fact that the stereo-electronic nature of probe as well as the
metal–probe and metal–counter anion interactions plays a crucial
role in inducing mutual signal reversibility in probes for selective
metal ion and corresponding counter anions detection as demon-
strated.
ous counter anions, such as Cl−, F−, Br−, I−, AcO−, CO32−, HCO3
,
Cr2O72−, NO3−, N3−, SO42−, PO4
and SCN−, in MeCN:H2O (1:1,
3−
v/v) medium. The Hg(II)-induced enhancement in absorption (A558
)
and emission (F581) of 1 significantly decreased almost up to that of
the metal free probe 1 and the pink coloured solution turned colour-
less within 1 min only in presence of AcO−, CO3 and HCO3 ions
(Fig. 6). Addition of other counter anions has shown a negligible
decrease in spectral responses and colour of the solution remained
pink up to at least 12 h.
2−
−

48
A. Pal, B. Bag / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 42–49
II
1 + HgII (blank)
(b)
(a)
1 + Hg (blank)
1.0
0.8
0.6
0.4
0.2
0.0
N3 , SO42 , NO3
Cr2O72 , SCN , Cl
Br , I , F , PO43
N3 , SO42
NO3 , Cr2O72
SCN , Cl
Br , I , F
PO43
HCO
3
CO32
CO32
AcO
HCO3
AcO
1 only
500
600
700
550 600 650 700 750 800
Wavelength (nm)
Wavelength (nm)
Fig. 6. Change in (a) absorption and (b) fluorescence intensity upon addition of various anions (10 equiv.) to the solution containing 1 (1 equiv.) and HgII (5 equiv.). Conc. of
1 = 1.0 × 10−4 M (abs) and 1.0 × 10−7 M (em), ꢂex = 500 nm, MeCN–H2O (1:1, v/v).
4. Conclusion
Acknowledgements
The
thiophenyl
derivatized
bis-(amino-ethyl-amido)-
BPB wishes to thank the DST, New Delhi for the financial sup-
port (SR/FTP/CS-138/2006). The authors sincerely wish to thank to
CSIR, New Delhi and the Director, IMMT for a CSIR-diamond jubilee
research internship to AP.
rhodamine based probe
1
has exhibited both chromogenic
and fluorogenic signalling selectively with Hg(II) ion at very low
concentration level by exploiting the metal ion induced structural
transformation of signalling subunit. The thiophenyl subunit in
its architecture contributes significantly towards the enhanced
selectivity and sensitivity of Hg(II) ion detection, apart from
contributing towards a positive allosteric cooperativity between
binding of both the Hg(II) ions. The counter anions, such as car-
bonate/bicarbonate and acetate, have induced the reversibility in
its Hg(II)-complexation and the structural transformation thus
rendered a reversible dual channel signalling pattern, which infers
to the crucial role of metal–anion interaction in its sensing mech-
anism apart from the metal ion–probe interaction and suggests
further possibility of their reuse as chemosensors. Upon suitable
modification, 1 can potentially be used as a chemosensor for
environmental or in vitro analysis of Hg(II) ion considering the
advantages of its high sensitivity, selectivity, reversibility and real
time ‘turn-on’ signalling responses exploiting the thiophilic nature
of Hg(II) ion. Further, anion-induced optical signal modulation
exploiting their coordinating tendencies towards a particular
metal ion in signalling probes is advantageous in designing
strategic protocol for selective detection of anions itself. In this
strategy, the anions need not to follow the protocol for modulation
of photo-physical processes directly, but the metal ion provides
the requisite binding site. Rhodamine based probes exhibiting
both chromo- and fluorogenic signalling selectively with a metal
ion followed by a selective anion are still rare, baring few recent
reports [43,44]. In this context, although the anion induced
reversible ‘turn-off’ signalling responses of 1 ⊂ (Hg(II))2 complex
with carbonate/bicarbonate/acetate anions are not selective, the
protocol can be exploited in designing chemosensors selective for
these anions by tuning the probe’s stereo-electronic situation with
suitable structural and function modifications. We are presently
working along this direction.
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.jphotochem.
2012.05.010.
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