A Novel Cr3+ Fluorescence Turn-On Probe Based on Rhodamine and Isatin Framework

Article abstract of DOI:10.1007/s10895-015-1684-0

A novel turn-on fluorescent dye (E)-3′,6′-bis(diethylamino)-2-((1-(naphthalen-2-ylmethyl)-2-oxoindolin-3-ylidene)amino)spiro[isoindoline-1,9′-xanthen]-3-one (RBNI) based on a rhodamine-isatin hybrid molecular architecture was synthesized by condensation of isatin derivative with rhodamine hydrazide. The dye RBNI is selective and sensitive for recognition of Cr³⁺ ion in aqueous CH₃CN media over other tested metal ions. The sensor shows large fluorescence enhancement upon complexation with Cr³⁺ and simultaneous color change occurs from colorless to pink-red. Spectroscopic study predicted 1:1 binding stoichiometry between RBNI and Cr³⁺ ion and this was again verified through ESI-MS (Electrospray Ionisation Mass Spectrometry). Detection limit of Cr³⁺ ion by this dye was calculated to be 2.4 μM. Furthermore, the potential application of this dye for the monitoring of Cr³⁺ ions in pond water and tap water samples was demonstrated.

Full text of DOI:10.1007/s10895-015-1684-0

J Fluoresc
DOI 10.1007/s10895-015-1684-0
ORIGINAL ARTICLE
3+
A Novel Cr Fluorescence Turn-On Probe Based on Rhodamine
and Isatin Framework
1
1
1
Anamika Dhara & Nikhil Guchhait & Susanta K. Kar
Received: 23 July 2015 /Accepted: 28 September 2015
#
Springer Science+Business Media New York 2015
Abstract A novel turn-on fluorescent dye (E)-3′,6′-
bis(diethylamino)-2-((1-(naphthalen-2-ylmethyl)-2-
oxoindolin-3-ylidene)amino)spiro[isoindoline-1,9′-xanthen]-
Introduction
Fluorescent sensing via suitable sensors is an attractive and
efficient chemical tool because of its high sensitivity, selectiv-
ity, and simple instrumentation [1–5]. Fluorescent sensors
could be considered as molecular receptors that convert their
fluorescent messages into analytically useful signals upon
binding to specific guests [6]. Chromium (III) is an essential
micronutrient required in amounts of 50–200 μg per day for
humans and animals [7]. It affects human metabolism by mod-
ulation of the action of insulin through glucose tolerance fac-
tors (GTF), thereby activating certain enzymes and stabilizing
3
-one (RBNI) based on a rhodamine-isatin hybrid molecular
architecture was synthesized by condensation of isatin deriv-
ative with rhodamine hydrazide. The dye RBNI is selective
3
+
and sensitive for recognition of Cr ion in aqueous CH CN
3
media over other tested metal ions. The sensor shows large
3
+
fluorescence enhancement upon complexation with Cr and
simultaneous color change occurs from colorless to pink-red.
Spectroscopic study predicted 1:1 binding stoichiometry be-
3
+
tween RBNI and Cr ion and this was again verified through
3
+
ESI-MS (Electrospray Ionisation Mass Spectrometry). Detec-
proteins and nucleic acids [8–11]. Insufficient intake of Cr
3
+
tion limit of Cr ion by this dye was calculated to be 2.4 μM.
increases the risk for diabetes and cardiovascular diseases in-
cluding elevated levels of circulating insulin, glucose, triglyc-
erides and total cholesterol, and impaired immune function
[12]. However, higher concentration of Cr is detrimental to
cellular functions and structures [13]. Serious environmental
pollution can be caused due to the discharge of chromium by
industrial and other activities. Due to its biological impact, the
United States Environmental Protection Agency (USEPA) has
Furthermore, the potential application of this dye for the mon-
3
+
itoring of Cr ions in pond water and tap water samples was
demonstrated.
3
+
3
+
.
.
.
Keywords Chemosensor Cr ion Fluorescence Job’s
.
plot Rhodamine derivative
3
+
set strict standards on the permissible concentration of Cr in
−1
natural water (0.1 mg mL ) in an attempt to control build-up
due to industrial and agricultural activities [14, 15]. At the
same time, the higher oxidation state of Cr (VI) is an extreme-
ly toxic and potentially carcinogenic. It can penetrate through
cell membranes and causes toxic effects including cancer by
oxidizing DNA and some proteins [16–18]. Thus, there is
need for the development of sensitive and selective way for
Cr ions detection of environmental and biological samples.
Current approaches for environmental and clinical samples
rely on costly, time-consuming methods like atomic
absorption/emission spectroscopy [19] or inductively coupled
plasma mass spectrometry [20] which are not very convenient
and handy for “in-field” detection. These limitations have
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-015-1684-0) contains supplementary material,
which is available to authorized users.
*
*
Nikhil Guchhait
nguchhait@yahoo.com
3
+
Susanta K. Kar
skkar_cu@yahoo.co.in
1
Department of Chemistry, University College of Science, University
of Calcutta, 92, A.P.C. Road, Kolkata 700 009, India

J Fluoresc
actually set off an interest among chemists for the develop-
ment of efficient, cost-effective, and reversible chemosensors
for Cr ion. Generally a ‘turn-off’ response, i.e., fluorescence
dissipation causing enhancement of fluorescence upon bind-
ing. The data obtained from fluorimetric titration and compe-
tition experiments indicate that RBNI is pH-stable and highly
3
+
3
+
quenching is observed in sensing events due to paramagnetic
selective towards Cr over other metal ions. As far as our
knowledge is concerned, this is one of the rare examples of
a rhodamine derivative employed for the selective detection of
3
+
tendency of Cr ion [21]. There are very limited reports
where a ‘turn-on’ response has been reported for sensing of
3
+
3+
Cr ion and the fluorescence enhancement were reported to
be about 15 to 30 folds [22–25]. Whereas, fluorescence
quenching is not conductive to a high signal output upon
recognition and interferes with temporal separation of similar
complexes with time-resolved fluorometry [26]. Hence, the
sensors with “turn-on” fluorescence response are more attrac-
tive than the “turn-off” ones as they offer the potential for high
sensitivity [27–29]. During the last decade, a few numbers of
Cr .
Experimental
Synthesis
Synthesis of Compound 3
3
+
fluorescent probes have been reported for the purpose of Cr
sensing. However, there are several shortcomings such as
cross-sensitivity toward other metal cations, lack of selective
multi-chelating ligand, low water solubility, slow response,
low fluorescence enhancement and cytotoxicity. It is apparent
that there is a need to design new fluorescent probes for chro-
mium ions which can overcome these limitations. For these
reasons, we have taken care to design an isatin-appended rho-
damine based scaffold as a chemosensor. Rhodamine dyes are
extensively employed in the study of complex biological sys-
tems as molecular probes because of their high absorption
coefficients, high fluorescence quantum yields, and long
wavelength absorption and emission [30]. These unique prop-
erties make rhodamine dyes a good candidate in constructing
Indoline-2,3-dione (1 g, 6.79 mmol) was dissolved in DMF
(15 mL) and treated with cesium carbonate (3.54 g,
10.86 mmol) and 1-(bromomethyl)naphthalene (1.99 g,
9.03 mmol). The mixture was stirred at ambient temperature
for 2 days. The reaction mixture was quenched with ice and
then extracted with ethyl acetate and finally washed with brine
(100 mL×3), then dried over sodium sulphate and concentrat-
ed under reduced pressure to give an orange solid product.
−
1
Yield: 0.8 g, 40 %. IR (KBr, cm ) 400–4000: υ=3447,
2924, 2364, 1729, 1607, 1465, 1338, 1171, 1089, 1022,
1
862, 812, 753, 466; H-NMR (500 MHz, CDCl ): δ (ppm),
3
5.10 (s, 2H, −CH -), 6.82 (d, 1H, naph-H, J=7.9 Hz), 7.09 (d,
2
1H, Ar-H, J=5 Hz), 7.50–7.42 (m, 4H, naph-H & Ar-H), 7.63
(d, 1H, naph-H, J=7.3 Hz), 7.85–7.79 (m, 4H, naph-H & Ar-
H); Anal. Calc. for C H NO : C, 79.53; H, 4.58; N, 4.89;
3
+
a “turn-on” fluorescent probe for Cr ion [31]. Incorporation
of isatin moiety into a rhodamine-based system makes our
probe more promising because of their strong binding affinity
towards metals. Isatin and its derivatives are potent inhibitors
of monoamine oxidase, caspase-3 and so on [32, 33].
1
9
13
2
Found: C, 79.43; H, 4.56; N, 4.88.
Synthesis of Compound RBNI
In the present work, we have synthesized new dyad RBNI,
where in the fluorophore rhodamine core is linked to an isatin
derivative which acts as a soft metal chelating site, to achieve
the “off-on” type fluorescence enhanced sensing protocol
based upon coordination method. The hallmark of its superi-
ority over the reported methods is, the ease of synthesis as well
as very low detection limit (2.4 μM) observed during the
Rhodamine B hydrazide (5) was prepared according to the
literature procedure as described previously and was charac-
1
terized by H NMR, mass data and FT-IR [39, 40]. Rhoda-
mine B hydrazide (0.5 g, 1.095 mmol) was dissolved in 10 mL
ethanol and 1-(naphthalen-1-ylmethyl)indoline-2,3-dione
(0.31 g, 1.095 mmol) was added. The mixture was then heated
under reflux condition for 6 h. After completion of the reac-
tion, a yellow precipitate was formed within the reaction flask
(Scheme 1). Then, it was cooled and the resulting precipitate
was filtered off, washed with methanol/ether (1:1) by three
times and dried under vacuum. Yield: 0.25 g, 31 %.
3
+
selective detection of Cr . We anticipated that RBNI binds
3
+
to Cr ion result in the fluorescence enhancement [34–38].
Chelation-enhanced fluorescence (CHEF) [34–37] can be
possible in presence of metal ion as CHEF is an attractive
design principle for developing luminescent chemical devices,
which combine the ability to recognize and respond to an
external input mostly with photoinduced electron transfer
−
1
M.P.−231 °C (decomp.). IR (KBr, cm ) 400–4000: υ=
3062, 2926, 1701, 1613, 1516, 1468, 1542, 1263, 1219,
1
(
PET) process. Upon complexation of ligand with a certain
1117, 817, 782; H-NMR (500 MHz, DMSO-d ): δ (ppm),
6
metal ion, a large fluorescence enhancement can be observed
because the stable chelation abrogates the PET process from
the electron donating group to the fluorophore (“turn-on
state”). In some cases binding of metal to a flexible ligand
causes decrease of non-radiative decay channels of energy
1.10 (t, 12H, NCH CH , J=6.7 Hz), 3.33 (q, 8H, NCH CH ,
2
3
2
3
J=5 Hz), 5.10 (s, 2H,-CH -), 6.41–6.36 (m, 4H, xanthene-H),
2
6.98 (s, 2H, xanthene-H), 7.06 (d, 1H, isatin Ar-H, J=7.4 Hz),
7.12 (d, 1H, naph-H, J=7.4 Hz), 7.31 (d, 1H, naph-H, J=
7.2 Hz), 7.36 (s, 1H, isatin Ar-H), 7.45 (d, 1H, naph-H, J=

J Fluoresc
Single-Photon Counting (TCSPC) using a HORIBA
JobinYvon Fluorocube-01-NL fluorescence lifetime spec-
trometer. The sample was excited using a nanosecond laser
diode at 340 nm and the signals were collected at the magic
angle of 54.7° to eliminate any considerable contribution from
fluorescence anisotropy decay [41]. The typical time resolu-
tion of the experimental set-up is~800 ps. The decays were
deconvoluted using DAS-6 decay analysis software. The ac-
2
ceptability of the fits was judged by χ criteria and visual
inspection of the residuals of the fitted function to the data.
Mean (average) fluorescence lifetimes were calculated using
the following Eq. (1):
X
αiτ2i
τav ¼ X
ð1Þ
Scheme 1 Synthesis of chemosensor RBNI
αiτi
in which α is the pre-exponential factor corresponding to the
ith decay time constant, τi.
8
2
.2 Hz), 7.52–7.50 (m, 1H, Ar-H), 7.57 (s, 2H, Ar-H), 7.61 (s,
H, naph-H), 7.95–7.88 (m, 5H, Ar-H, isatin Ar-H & naph-
i
+
H); ESI-MS: m/z calculated for C H N O [M+H] 726.34,
4
7 43 5 3
found 727; Anal. Calc. for C H N O : C, 77.79; H, 5.99; N,
4
7 43 5 3
General Method of UV-vis and Fluorescence Titration
9
.64; Found: C, 77.77; H, 5.97; N, 9.65.
Stock solutions of the probe RBNI were prepared in 50 %
3
+
Synthesis of RBNI-Cr Complex
(
v/v) H O/CH CN buffered by 10 mM HEPES pH 7.2, in
2 3
−5
the concentration range 10 M. 2 mL of the receptor solution
was taken in a cuvette. Stock solutions of guests in the con-
centration range 2×10 M were prepared in the same sol-
A 5 mL methanolic solution of CrCl (0.010 g, 0.0688 mmol)
3
was added drop wise to a magnetically stirred solution of
RBNI (0.05 g, 0.0688 mmol) in methanol (5 mL). The colour
of the ligand solution was changed from almost colorless to
pink-red upon addition CrCl . After 2 h of stirring at room
temperature, the solution was dried using rotary evaporator
−5
vents and were individually added in different amounts to
the receptor solution. The same stock solutions for the recep-
tor and guests were used to perform the UV-vis and fluores-
cence titration experiments. Both fluorescence and UV-vis
titration experiments were carried out at 25 °C. Fluorescence
measurements were carried out with excitation and emission
3
3
+
which yielded a pink-red RBNI-Cr complex. The complex
1
was characterized by H NMR, MS and FT-IR studies.
slit of 2.5 and 2.5 nm (λ =530 nm).
ex
Materials and Instruments
All other chemicals used were of analytical grade. The solu-
tions of metal ions were prepared from their perchlorate salts.
All solvents used in spectroscopic tests were of spectrostropic
grade. Distilled-deionized water was used throughout the ex-
periment. A Perkin Elmer (Model LS-50B) fluorimeter was
used for fluorescence measurements. The absorption spectra
were recorded with a Hitachi model U-3501 spectrophotom-
Quantum Yield Measurements
The fluorescence quantum yields were determined using rho-
damine 6G as a reference [42] with a known Φ value of 0.95
in EtOH. The area of the emission spectrum was integrated
using the software available in the instrument and quantum
yield was calculated according to the following equation [43]:
R
eter. NMR spectra were recorded on a Bruker spectrometer at
h
i
Â
Ã
1
2
2
5
00 ( H NMR) MHz in DMSO-d . Chemical shifts (δ values)
ΦS=ΦR ¼ ½AS=ARŠ  ðAbsÞ =ðAbsÞ Â η =η
ð2Þ
6
R
S
S
R
were reported in ppm down field from internal Me Si. Ele-
4
mental analyses were carried out with a Perkin–Elmer CHN
analyzer 2400 instrument. Mass spectra were recorded in
methanol solvent in Qtof Micro YA263. IR spectra (KBr pel-
where Φ and Φ are the fluorescence quantum yield of the
S
R
sample and reference, respectively; A and A are the area
under the fluorescence spectra of the sample and the reference,
respectively; (Abs) and (Abs) are the corresponding optical
densities of the sample and the reference solution at the wave-
S
R
−1
let, 400–4000 cm ) were recorded on a Perkin–Elmer model
83 infrared spectrophotometer. Melting points were deter-
S
R
8
mined using a Buchi 530 melting apparatus. Fluorescence
lifetimes were measured by the method of Time Correlated
length of excitation; η and η are the refractive index of the
sample and the reference, respectively.
S
R

J Fluoresc
Calculation of the Detection Limit
enhancement of absorbance (about 40 fold) in the visible
range of 500–600 nm and a new peak at 560 nm was ob-
served. With increasing Cr ion concentration, a shoulder
3
+
3+
The detection limit (DL) of RBNI for Cr was determined
using the following equation:
peak at ~520 nm is also observed. The absorbance of RBNI
at 560 nm was proportional to Cr ion concentration over a
3
+
.
DL ¼ K Â Sb S
ð3Þ
range of 0-45 μM. The colour of the solution changed from
3
+
colorless to pink-red upon titration with Cr ions which may
be due to opening of spirolactum ring of the rhodamine moi-
Where K=2 or 3 (we take 3 in this case), Sb is the standard
deviation of the blank solution and S is the slope of the cali-
bration curve.
3
+
ety. The absorbance at 560 nm as a function of Cr concen-
tration is depicted in inset Fig. 1. Addition of separately
+
+
2+
2+
4
5 μM each of other metal ions viz., Na , K , Mg , Mn ,
2
+
2+
3+
3+
2+
2+
2+
Co , Ni , Ga , In , Zn , Cd and Hg , did not perturb
the UV-visible spectral pattern of RBNI to a significant extent
Results and Discussion
3
+
2+
3+
(
Fig. S7 in Supplementary data). However Fe , Cu , Al
2
+
and Pb did affect to an extent by changing of absorbance and
slight blue shifting of the absorption band with respect to the
Design and Synthesis of RBNI
3
+
band observed in presence of Cr ion. In these four cases the
colour of the RBNI categorically became light pink. Thus it
was not possible to discriminate any single metal ion through
RBNI only by UV-visible spectral changes. Moreover, when
being excited by 365 nm UV lamp it emits orange lights only
Rhodamine B hydrazide was synthesized according to a pre-
vious report [39, 40]. The synthesis of RBNI is depicted in
Scheme 1. Sensor RBNI was synthesized by simple Schiff-
base condensation between rhodamine B hydrazide (5) and
1
-(naphthalen-1-ylmethyl)indoline-2,3-dione (3) in methanol
+
3
1
13
in case of Cr ion, which acts as a typical “OFF-ON” based
optical response (Fig. 2).
and thoroughly characterized by H NMR, C NMR, ESI-
+
MS , FT-IR and CHN elemental analysis (Figs. S1–S6, in
The fluorescence spectrum of chemosensor RBNI (10 μM)
exhibited very weak fluorescence emission (quantum yield,
Supplementary data).
[
44, 45] Φ=0.03) when excited at 530 nm in 50 % (v/v)
UV–vis and Fluorescence Studies
H O/CH CN buffered by 10 mM HEPES, pH 7.2 at 25 °C.
2
3
3
+
As seen in Fig. 3, upon addition of only Cr ions to the
solution of RBNI a remarkable enhancement of emission in-
tensity was observed at 590 nm (Φ=0.36). Fluorescence en-
hancement observed for RBNI in the presence of Cr ions is
ascribed to be the formation of the RBNI-Cr complex. The
generated complex shows strong emission at 590 nm due to
ring opening of the spirolactam ring which causes fluores-
cence enhancement (~65 fold) by the generation of free rho-
damine unit in the complex (Scheme 2). In addition, binding
of metal ion insists rigid structure at the binding site of the
complex which expected to reduce non-radiative channels
causing fluorescence enhancement. Thus, ‘Turn-On’ of fluo-
Steady state absorption and fluorescence spectra were record-
ed to investigate spectroscopic changes of probe RBNI
(
10 μM) in presence of various metal ions in 50 % (v/v)
3
+
H O/CH CN buffered by 10 mM HEPES, pH 7.2 at 25 °C.
2
3
3+
As shown in Fig. 1, the absorption spectra of RBNI exhibited
only a very weak band over 500 nm region, indicating that the
rhodamine core is in the ring closed isomeric form. Addition
3
+
of Cr into this solution immediately resulted in a significant
3
+
rescence with addition of Cr ions indicates high selectivity
of RBNI towards Cr ions.
3
+
3
+
Under the same conditions as was used for Cr , we also
tested the fluorescence behaviour of RBNI (10 μM) toward
+
+
2+
2+
2+
2+
3+
other metal ions (Na , K , Mg , Mn , Co , Ni , Ga ,
3
+
2+
2+
In , Zn and Cd ). Very mild fluorescence enhancement
factors (FEF) were also detected for Al (~17 fold), Fe
~8 fold), Pb (~6 fold), and Hg (~4 fold), and other metal
3
+
3+
2
+
2+
(
ions showed nearly no response (Fig. 4). These findings indi-
cated that RBNI behaved as a highly sensitive and selective
fluorescent chemosensor towards Cr . Relative fluorescence
3
+
Fig. 1 UV-vis spectra of chemosensor RBNI (10 μM) in 50 % (v/v)
H
2
O/CH
presence of different amount Cr . [Cr ]: 0, 2.5, 5, 7.5, 10, 12.5, 15,
7.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45 μM. Insert:
3
CN buffered by 10 mM HEPES at pH 7.2 at 25 °C, in the
3+
3+
enhancement of RBNI in the absence and presence of various
3
+
other metal ions and thereby its selectivity for Cr was shown
in Fig. 5.
1
3+
absorbance at 560 nm as a function of Cr concentration

J Fluoresc
Fig. 2 Photographs of
chemosensor RNBI (10 μM) in
5
0 % (v/v) H
by 10 mM HEPES at pH 7.2 at
5 °C, in the presence of different
2 3
O/CH CN buffered
2
metal ions (100 μM) under a
visible light b UV light
The cross-sensitivity of any probe towards different metal
ions limits its utility in selective detection of a metal ion, and
that has also been tested with this probe. We also carried out
contaminant level (MCL) for chromium in the drinking water
(1.9×10 M, 0.1 mg/L) defined by the U.S. Environmental
−6
Protection Agency (EPA) [49], suggesting that RBNI has high
3
+
3+
competitive experiments in the presence of Cr at 100 μM
specificity toward Cr under current working conditions.
+
+
2+
2+
2+
2+
3+
3+
mixed with Na , K , Mg , Mn , Co , Ni , Ga , In ,
(Fig. S9, in Supplementary data).
2
+
2+
2+
2+
2+
2+
2+
2+
2+
Zn and Cd Fe , Zn , Hg , Cu , Pb , Ni , Co ,
Cd , Mg , Na , K at 100 μM. As shown in Fig. 6, no
significant variation in the fluorescence spectra of RNBI
For the determination of the stoichiometry the complex
2
+
2+
+
+
3+
between RBNI and Cr , Job’s plot [50] analysis was also
3
+
carried out. When molar fraction of Cr was 0.5, the absor-
bance at 560 nm band reaches to maximum (Fig. 7), indicating
2
+
3+
was observed with any other metal ion except Cu and Fe
3
+
which induce similar fluorescence emission but to a small
extent. Most likely, the paramagnetic nature of these ions
the formation of 1:1 complex between RBNI and Cr . It also
+
corroborated with the results obtained by TOF MS ESI mass
2
+
3+
(
Cu and Fe ) is responsible for quenching of fluorescence
spectroscopy in which the peak at m/z 902.72 in the mass
3
+
−
of the resulting RBNI complexes.
spectrum is assigned to the mass of [(RBNI + Cr + 2Cl +
3
+
The response of RNBI with Cr ion obtained from differ-
ent chromium salts such as chromium sulfate, chromium ni-
trate and chromic chloride were also investigated when addi-
3H O)] (Fig. S10, in Supplementary data). This is also con-
2
firmed by the Benesi-Hildebrand method [51]. When assum-
3
+
ing a 1:1 association between RBNI and Cr , the Benesi-
3
+
tion of Cr (10 mM, 50 mM, 100 mM, respectively) to
Hildebrand equation is given as follows:
chemosensor RBNI (10 μM), the response of RNBI with
3
+
1=ðA−A0Þ ¼ 1=fKðAmax−A0Þ½CŠg þ 1=ðAmax−A0Þ
the Cr from chromic chloride was in accorded with the
3
+
Cr from other chromium salts (Fig. S8 in Supplementary
data). For practical application, the detection limit [46] of
RBNI was also estimated. The fluorescence titration profile
A0 is the absorbance of RBNI at absorbance maxima (λ=
560 nm), A is the observed absorbance at that particular wave-
3
+
of RBNI (10 μM) with Cr demonstrated that the detection
length in the presence of a certain concentration of the metal
ion (C), Amax is the maximum absorbance value that was ob-
tained at λ=560 nm during titration with varying [C], K is the
3
+
limit of Cr is 2.4 μM [47, 48] . The obtained detection limit
was quite lower than the recommended maximum
−1
association constant (M ). As shown in Fig. S11 in Supple-
3
+
mentary data, the plot of 1/(A–A ) against 1/[Cr ] shows a
0
liner relationship, indicating that RBNI indeed associates with
3
+
Cr in a 1:1 stoichiometry. The association constant, K, be-
3
+
tween RBNI and Cr , is determined from the slope to be
3
−1
3
.4×10 M .
Fig. 3 Emission spectra of chemosensor RBNI (10 μM) in the presence
3
+
of increasing concentrations of Cr (0, 15, 25, 30, 35, 40, 45, 50, 55, 60,
5, 70, 75, 80, 100 μM) in 50 % (v/v) H O/CH CN buffered by 10 mM
HEPES at pH 7.2 at 25 °C. Excitation was performed at 530 nm
6
2
3
3+
Scheme 2 Proposed binding mode of RBNI with Cr

J Fluoresc
Fig. 6 Metal ions selectivity of RBNI (10 μM) in 50 % (v/v) H
2
O/
CH CN buffered by 10 mM HEPES pH 7.2 at 25 °C. Blue bars:
3
Fig. 4 Fluorescence spectra (excitation at 530 nm) of RBNI (10 μM) in
0 % (v/v) H O/CH CN buffered by 10 mM HEPES at pH 7.2 at 25 °C,
by addition of 100 μM different metal ions (Na , K , Mg , Pb , Al ,
fluorescence intensity of RBNI with the addition of the respective
cations (100 μM). Maroon bars: fluorescence intensity of RBNI with
5
2
3
+
+
2+
2+
2+
2+
3+
3+
3+
2+
3+
2+
2+
2+
3+
3+
2+
the addition of the respective competing cations (100 μM) and Cr
100 μM). The fluorescence intensities were detected at 590 nm
Cr , Mn , Fe , Co , Ni , Cu , Ga , In , Zn , Cd , Hg )
(
Since reversibility is a prerequisite in developing
chemosensors for practical applications, we also studied the
reversibility of the sensing protocol as proposed. The RBNI-
3
+
obtained in case of chemosensor RBNI, as well as Cr -bound
form can be fitted to a triexponential functions and the relevant
data were compiled in Table S1 (in Supplementary data). The
minor and short-lived component was assigned to the decay
time for the excited state related to the spirolactam moiety, while
the long-lived major component was attributed to the xanthene
form of the chemosensor RBNI. The relative increase in the
percentage of the open-ring xanthene form upon addition of
3
+
Cr complex was titrated with EDTA to determine the nature
of binding. As seen in the Fig. S12, in Supplementary data,
with the increase of the concentration of EDTA, the fluores-
cence intensity at 590 nm gradually decreased. Excess EDTA
completely quenched the fluorescence at 590 nm. This obser-
3
+
vation predicts the removal of Cr ion from RBNI, leading to
the reconstitution of the spirolactam ring in the rhodamine
moiety and hence fluorescence at 590 nm vanishes.
3+
3+
Cr confirms the interaction of Cr with the receptor.
The spirolactam ring of the rhodamine moiety in RBNI is
susceptible to change in pH. Experimental results show that,
for free RBNI, at acidic conditions (pH<5), an obvious off–
on fluorescence appeared due to the formation of the open-
ring state because of the strong protonation (Fig. 9). There-
fore, we evaluated the fluorescence properties of RBNI in
solutions with different pH values (2–14). It was found that
the fluorescence intensity of the probe remains stable in the
TCSPC Study
3+
The Cr recognition has been further supported by measuring
the fluorescence life-time of the species by time-correlated sin-
gle photon counting (TCSPC) technique during the titration.
Fig. 8 represents the time resolved fluorescence decay profile
3+
pH range 5–14. Upon the addition of Cr ions, there was an
of RBNI and its metal complex in aqueous CH CN media using
3
obvious fluorescence off–on change of RBNI under different
pH values. Thus, the sensor RBNI has the maximal sensing
response at physiological pH, indicating that the sensor RBNI
is promising for biological applications.
a 340 nm nano-LED as the excitation source. The TCSPC data
Fig. 5 Bar graph shows the relative emission intensity of RBNI at
3
+
3+
5
90 nm upon treatment with various metal ions in 50 % (v/v) H
2
O/
Fig. 7 Job plot of RBNI and Cr ([RBNI] + [Cr ] = 45 μM) in 50 %
(v/v) H O/CH CN buffered by 10 mM HEPES at pH 7.2 at 25 °C
CH CN buffered by 10 mM HEPES at pH 7.2 at 25 °C
3
2
3

J Fluoresc
1
3+
Fig. 10 H NMR titration of RBNI and with Cr +ion (DMSO-d
6
,
3
3
00 MHz); a RBNI only b RBNI with 1 equiv. of Cr
Fig. 8 Time-resolved fluorescence decay of RBNI (blue), Prompt
3+
(
black) and RBNI-Cr (green)
employed to detect its concentration. The results were sum-
marized in Table S2 (Supplementary data), which showed
satisfactory recovery values for all of the samples. Thus, the
present probe seems useful for the determination of Cr in
natural water samples.
1
H NMR Titration
3
+
3
+
For further insight into the binding event of RBNI with Cr ,
we performed H NMR titration experiment by the addition of
1
1
3
+
3+
equiv. of Cr to RBNI (Fig. 10). Upon addition of Cr , the
peak shape of RBNI broadened and almost all aromatic pro-
tons shifted downfield. This indicates that the opening of the
spirolactum ring in RBNI was occurred in the presence of
Conclusion
3
+
3+
In summary, we have designed and synthesized a novel
rhodamine-based chromo-/fluorogenic dual signalling probe
for selective recognition of Cr in aqueous CH CN media.
3
It has been well characterized by various spectroscopic tech-
niques. The colorimetric and fluorescent response to Cr can
be conveniently detected even by the naked eye, which pro-
vides a facile method for visual detection of Cr . Upon the
Cr . Further, IR spectrum of chemosensor RBNI with Cr
ion also confirms the proposed mechanism (Fig. S13, in Sup-
3
+
3+
plementary data). Upon addition of 1 equiv. of Cr , the char-
acteristic carbonyl amide stretching frequency shifts from
701 cm− in RBNI to 1587 cm in the complex, indicating
1
−1
3+
1
3
+
coordination of carbonyl oxygen with Cr ion. These find-
ings clearly support the ring-opening mechanism.
3
+
3
+
In order to examine the reliability of our sensing system,
practical application was evaluated by determining the recov-
addition of Cr , the spirolactam ring of RBNI was opened
and a 1:1 metal-ligand complex was formed. All biologically
relevant metal ions and toxic heavy metals did not interfere
3
+
eries of spiked Cr in pond water and tap water samples,
respectively. The samples collected were simply pretreated
3
+
with the Cr ion detection with this sensor. The excellent
3
+
3+
with filtration before further determination. No Cr was
detection limit (2.4 μM) of RBNI chemosensor toward Cr
3
+
3
+
ion can be used for the detection of trace quantities of Cr ion
in biological and environmental sample. We believe that the
sensor can be used for practical applications in chemical, en-
vironmental and biological systems.
found in these water samples. Then Cr stock solution was
separately spiked in these samples and RBNI probe was
Acknowledgments A.D. thanks to CSIR, New Delhi, India for finan-
cial support by awarding senior research fellowship (Sanc. No. 01(2401)/
1
0/EMR-II, dated 05.01. 2011).
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