Ferrocene appended fluorescein-based ratiomeric fluorescence and electrochemical chemosensor for Fe3+ and Hg2+ ions in aqueous media: Application in real samples analysis

Article abstract of DOI:10.1016/j.ica.2019.119097

A novel visually detectable fluorescein-based chemosensor (4) containing a ferrocene backbone has been designed and synthesized successfully by simultaneously introducing ferrocene with an azide appendage and fluorescein with an alkyne functional group vi

Full text of DOI:10.1016/j.ica.2019.119097

InorganicaChimicaActa498(2019)119097
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Research paper
Ferrocene appended fluorescein-based ratiomeric fluorescence and
electrochemical chemosensor for Fe3+ and Hg2+ ions in aqueous media:
Application in real samples analysis
Sushil Ranjan Bhattaa, Adwitiya Pala, Ujwal K. Sarangib, Arunabha Thakura,⁎
a Department of Chemistry, Jadavpur University, Kolkata 700032, India
b Department of Chemistry, Vikram Deb (auto) College, Jeypore, Odisha 764001, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ferrocene
Ratiometric fluorescence
Electrochemistry
Fluorescein
A novel visually detectable fluorescein-based chemosensor (4) containing a ferrocene backbone has been de-
signed and synthesized successfully by simultaneously introducing ferrocene with an azide appendage and
fluorescein with an alkyne functional group via a [2 + 3] cycloaddition reaction. Further, its sensing response
towards various metal cations was examined via multiple channels. The probe shows a remarkable specificity
towards Fe3+ and Hg2+ ions through “naked-eye” detection as well as via ratiometric fluorescence emission with
limits of detection of 9.75 × 10–8 M and 39 × 10–8 M for Fe3+ and Hg2+ ions, respectively. Further, shifts in the
voltammogram towards anodic potential of ΔE1/2 = 31 and 33 mV are observed upon binding Fe3+ and Hg2+
ions, respectively. Characterization of compound 4 has been done by 1H NMR, 13C NMR, high-resolution mass
spectrometry (HRMS) and CHN analysis studies. The stoichiometry of the complex of the present probe with
either Fe3+ or Hg2+ is indicative of a 1:1 ratio, which was determined by Job’s plot as well as 1H NMR titration
data. The incorporation of compound 4 in solid support has made the work more practicable. In addition, the
practical application of receptor 4 was successfully explored towards the detection of Fe3+ and Hg2+ ions in real
water samples by using the different water sources.
Click chemistry
1. Introduction
properties [10], high absorption coefficient, excitation and emission
wavelengths in the visible region with a high fluorescence quantum
Molecular fluorescence detection, among the various detection
techniques possible, is a well-grounded method for the analysis of a
variety of biological events [1–3]. However, fluorescence “turn-off” or
“turn-on” responses where the measurement is based on the single
signal (the change of fluorescence intensity) encountered many draw-
backs such as limitations with respect to excitation intensity, probe
concentrations, and instrumental efficiency etc. [4,5]. Further ad-
vancement in this field has led to ratiometric fluorescence studies,
which utilize the ratio of the emission intensity at two different wa-
velengths, and are always advantageous as they can overcome the
above-mentioned problems to make the results precise and accurate
[6,7]. Ratiometric fluorescence sensing, among all other optical sensing
methods currently being explored, has received particular attention as a
technique with the potential to provide precise and quantitative ana-
lyses with high sensitivity and inherent reliability [8,9]. In our work,
we have chosen fluorescein as our basic fluorophore unit. This interest
stems from its manifold advantages, such as its favorable photophysical
yield and high photostability [11–14], easy synthesis and functionali-
zation [15], excellent biocompatibility and cellular membrane-pene-
trating capacity [16]. It is a widely used fluorescent probe in bios-
ciences [17–20] for the lipophilic properties of its alkyl, ester and ether
derivatives [21]. On the other hand, the wide chemical functionality of
ferrocene and its derivatives has resulted in their use as extra-ordinarily
robust building blocks for the preparation of multi-responsive chemo-
sensors. They have been considered as prototype chemosensors owing
to their interesting electrochemical and optical sensing properties [22].
Therefore, the combination of these two moieties, in principle, should
develop a potential molecular system for the detection of toxic metal
ions.
Detection of a toxic metal ion that influences the physiological and
pathological processes in living systems [23] has attracted great interest
around the globe and is considered one of the forefront research areas
in supramolecular chemistry. Fluorescence chemosensors, especially
ratiometric fluorescence chemosensors, prone to give precise and
⁎ Corresponding author.
E-mail address: arunabha.thakur@jadavpuruniversity.in (A. Thakur).
https://doi.org/10.1016/j.ica.2019.119097
Received 19 June 2019; Received in revised form 26 August 2019; Accepted 26 August 2019
Availableonline27August2019
0020-1693/©2019ElsevierB.V.Allrightsreserved.

S.R. Bhatta, et al.
InorganicaChimicaActa498(2019)119097
quantitative optical output by addition of proper analyte (e.g. metal
ions, anions or biologically relevant small molecules), have come to
prior action [24]. The extension to a real time procedure owing to its
sensitive responses, instrumentation viability, economically affordable
nature and profuse biocompatibility has added up to the importance of
optical methods compared to other contemporary methods [25–31].
Fe3+, being one of the important trace elements, helps in carrying
oxygen through the heme group, but its deficiency and overdose can
lead to anemia and hemochromatosis, respectively [32]. Since iron is a
bioactive species [33], it becomes necessary to distinguish one oxida-
tion state from the other to assess their course of action, as there are
different properties attributed to the different oxidation states. Many
probes for the simultaneous detection of Fe2+ and Fe3+ are reported in
the literature [34–42] whereas a smaller number is known with the
capability of discriminating the two oxidation states [43–45]. Apart
from the biologically important ions, there are many toxic metal ions,
which can cause severe damage even at very low concentration. In this
context, Hg2+ is considered as one of the most important toxic metal
ions, as it can lead to damage of kidney, liver, and neurological systems
leading to pyrexia [46,47]. Therefore, building up a potential practical
sensor system, which efficiently determines toxic metal ions, is the focal
point of supramolecular chemistry.
2.2. Instrumentation
1H and 13C NMR spectra were obtained with BRUKER 400 MHz FT-
NMR spectrometer, and the chemical shifts are reported in ppm, using
tetramethylsilane or the following residual solvent peaks as an internal
reference: CDCl3 = 7.26 (1H), 76.16 (13C). For 1H NMR, coupling
constants J are given in Hz and the resonance multiplicity is described
as s (singlet), d (doublet), t (triplet), m (multiplet). The absorption
spectra were recorded with
a SHIMADZU-2450 UV–vis spectro-
photometer at room temperature. Fluorescence was recorded with a
HORIBA Scientific Fluoromax-4 Spectrophotometer. CH Instruments
Electrochemical Analyzer was used to perform the cyclic voltammetry
(CV) and differential pulse voltammetry (DPV) studies. HRMS were
taken using a Quadruple-TOF (Q-TOF) micro MS system using the
electrospray ionization (ESI) technique. CHN analysis was performed
on a Vario EL elementar CHNS analyser. Melting points were measured
in a Labotech melting point apparatus as well as manually by thermo-
meter in silicon oil.
Caution! Metal perchlorate salts are potentially explosive in certain
conditions. All due precautions should be taken while handling per-
chlorate salts!
Niu and his co-workers recently developed several fluorescent
chemosensors for the detection of various metal cations including Hg2+
and Fe3+ ions [48–55]. Some fluorescent chemosensors have also been
reported for the simultaneous detection of Hg2+ and Fe3+ species in the
literature [56–60]. However, to the best of our knowledge, our probe is
the 1st example of a ferrocene-appended fluorescein moiety connected
through a triazole linker, which is found to detect Hg2+ and Fe3+ ions
simultaneously. Our continuous research interest in the field of triazole-
appended ferrocene-based electrochemical and fluorescence chemo-
sensors led us to rationally design a functionalized fluorescein deriva-
tive attached to a ferrocene backbone to develop an easy and reliable
chemosensor, with good sensitivity, selectivity and ease of applicability
in real samples.
2.3. Synthesis of compound 4
A solution of ethyl 2-(3-oxo-6-(prop-2-yn-1-yloxy)–3H-xanthen-9-
yl)benzoate, 2 (0.268 g, 0.674 mmol) and 0.5 equiv of 1,1′-bis(azido-
methyl)ferrocene, 3 (0.100 g, 0.337 mmol) in dry DMF were taken in a
Schlenk flask equipped with a stirrer bar. It was degassed for 30 min.
After that CuI (0.4 equiv) and DBU (0.5 equiv) were added to it and the
resulting solution was heated at 65 °C for 5 h, where upon it was diluted
with methanol/water (1:1, v/v) and extracted with dichloromethane.
The combined organic extracts were washed with brine (50 mL) dried
with Na2SO4 and the solvent was removed in vacuo to afford a yellow
residue. Purification by column chromatography on silica gel with
EtOAc/hexane (8:2, v/v) as the eluent afforded a yellow solid com-
pound 4 (0.316 g, 85.86%).
4: 1H NMR (CDCl3, 400 MHz): δ = 8.25–8.22 (m, 2H, Hfluorescein),
7.73–7.69 (m, 2H, Hfluorescein), 7.68–7.64 (m, 2H, Hfluorescein), 7.61 (s,
2. Experimental section
2H, Htriazole), 7.28 (d, 2H, Hfluorescein
fluorescein, J = 1.2 Hz), 6.90–6.82 (m, 4H, Hfluorescein), 6.79–6.76 (m,
2H, Hfluorescein), 6.54–6.52 (m, 2H, Hfluorescein), 6.43 (d, 2H, Hfluorescein
, J = 7.6 Hz), 7.06 (d, 2H,
2.1. Materials and reagents
H
,
The perchlorate salts of Li+, Na+, K+, Fe2+, Fe3+, Cu2+, Hg2+, as
well as 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) and CuI were pur-
chased from Sigma Aldrich and used directly without further purifica-
J = 1.6 Hz), 5.25 (d, 8H, CH2, J = 1.6 Hz), 4.26 (s, 4H, HCp), 4.22 (s,
4H, HCp), 4.01 (q, 4H, OCH2ester, J = 7.2 Hz), 0.95 (t, 6H, OCH2CH3,
J = 7.2 Hz); 13C NMR (CDCl3, 100 MHz): δ = 185.7, 165.3, 162.4,
158.9, 154.0, 150.3, 142.6, 134.1, 132.7, 132.6, 131.2, 131.1, 130.6,
130.5, 130.4, 129.9, 129.7, 129.1, 129.0, 122.8, 117.8, 115.3, 113.6,
105.7, 101.4, 101.3, 82.0, 70.0, 69.6, 62.3, 61.4, 52.4, 49.7, 13.6. Mp:
195–197 °C. HRMS (ESI) m/z calcd for C62H48FeN6O10 [M + 23]+
1115.2681; found 1115.2626; Anal. Calcd for C62H48FeN6O10 C, 68.12;
H, 4.43; N, 7.69. Found: C, 68.02; H, 4.28; N, 7.34.
tion. The perchlorate salts of Ag+, Ca2+, Mg2+, Mn2+, Zn2+, Pb2+
,
Co2+, Cr3+, Al3+, and n-butyllithium (1.6 M in hexane) were pur-
chased from Alfa Aesar. Ferrocene, N,N,N′,N′-tetra-
methylethylenediamine (TMEDA), propargyl bromide, fluorescein,
NaN3, NaBH4, K2CO3 were purchased from local chemical providers.
DMF and acetonitrile (HPLC) were purchased from Thermo Fisher
Scientific and freshly distilled prior to use. Chromatography was carried
out using 60–120 mesh silica gel in a column of 2.5 cm diameter. All the
necessary solvents were dried by conventional methods and distilled
under N2 atmosphere before use. 1,1′-Bis(azidomethyl)ferrocene was
synthesized as per the literature procedure [61,62]. The cyclic vol-
tammetry (CV) was performed with a conventional three electrode
configuration consisting of glassy carbon as working electrode, pla-
tinum as an auxiliary electrode and Ag/Ag+ as a reference electrode.
The experiment was carried out with 10–5 M solution of sample in
CH3CN/H2O (3/7) containing 0.1 M (TBAP) [(n-C4H9)4NClO4] as sup-
porting electrolyte. The working electrode was cleaned after each run.
3. Results and discussion
3.1. Synthesis of receptor 4
Our long-term interest in the synthesis and study of the reactivity of
the triazole moiety has led us to the exploration of a novel and simple
triazole-appended ferrocene derivative (4) starting from the corre-
sponding alkyne and azide. Receptor 4, having a ferrocene skeleton, has
been synthesized by following the synthetic route as depicted in
Scheme 1. A click reaction between 2 equiv. of ethyl 2-(3-oxo-6-(prop-
2-yn-1-yloxy)–3H-xanthen-9-yl)benzoate (2) and 1,1′-bis (azidomethyl)
ferrocene (3) yielded receptor 4, which was well-characterized using
various spectroscopic techniques such as 1H NMR, 13C NMR, HRMS and
elemental analysis. The HRMS (ESI, Fig. S4) spectrum shows the major
peak at m/z 1115.2626 [M + Na]+, which perfectly matches the
The cyclic voltammograms were recorded at a scan rate of 0.05 Vs−1
The UV–vis spectra were recorded in CH3CN/H2O (3/7, v/v) solutions
at c = 10–5 M and the fluorescence spectra were recorded at c = 10–7
M, as it is stated in the corresponding Figure captions.
.
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S.R. Bhatta, et al.
InorganicaChimicaActa498(2019)119097
complexed species. Moreover, the absorbance at 430 nm remained
constant in the presence of more than 1 equiv. of Fe3+ or Hg2+
,
strongly suggesting the formation of 1:1 ligand-to-metal complexes.
Furthermore, the formation of a 1:1 complex of 4 with Hg2+ and Fe3+
ion was confirmed by elemental analysis of the isolated [4·Hg2+] and
[4·Fe+3] species. A linear regression plot has been obtained from the
absorption intensity of the ligand versus metal ion concentrations and
indeed, a linear increment of absorption intensity up to 1 equiv. for
both metal ions was observed (ESI, Fig. S6).
3.3. Fluorescence study
To further carry forward our investigation of interaction of receptor
4 with Hg2+ and Fe3+ ions, the fluorescence emission spectra were
measured in CH3CN/H2O (3/7, v/v) at 25 °C, (Fig. 2). The unaltered
fluorescence spectra of 4 upon addition of any of the aforementioned
cations, except Hg2+ and Fe3+ ions, infer us the selectivity of our probe
(ESI, Fig. S7). On excitation at 430 nm, in the absence of any analyte,
the probe displayed emission peaks at 521 and 545 nm which are ty-
pical for the fluorescein moiety [66,67]. Addition of Hg2+ and Fe3+
generates a new emission peak at 473 nm corresponding to the binding
of metal ions to the receptor 4. With the increasing concentration of
Hg2+ ion up to 1 equiv., the fluorescence intensity at 473 nm gradually
increases, while that at 521 and 545 nm decreases. However, the
change in fluorescence intensity at 545 nm outweigh change in fluor-
escence intensity at 521 nm. Therefore, a change in ratio of the fluor-
escence intensities at the two wavelengths (545 nm and 473 nm) is
considered to explain the ratiometric phenomenon. As the ratio change
at 545 nm and 473 nm is the key factor in the detection of Hg2+ and
Fe3+ ions, the probe 4 is considered a ratiometric probe. The fluores-
cence intensity ratio of 4 (I473/I545) and its metal derivatives shows a
linear relationship (R2 greater than 0.99) with increasing concentra-
tions of Hg2+ and Fe3+ ions (Fig. 3). Addition of an excess amount of
analyte beyond 1 equiv. did not exhibit any further changes in the
fluorescence intensities. This 1:1 ligand-to-metal binding ratio was
further verified via Job’s plot for both [4·Hg2+] and [4·Fe3+] species
(ESI, Figs. S8 and S9). The association constant (Ka) was calculated to
be 3.35 × 105 M−1 and 2.67 × 105 M−1 for Hg2+ and Fe3+ respec-
tively according to a fit plot [68] (ESI, Figs. S10 and S11).
Scheme 1. Synthesis of compound 4.
calculated molecular weight of [L + Na]+ (1115.2681). After having
obtained compound 4 in good yield, its cation sensing ability has been
thoroughly studied by UV–vis, fluorescence spectroscopy, electro-
chemical studies and colorimetric detection method along with 1H NMR
titration.
3.2. UV–vis absorption study
The changes in the UV–vis absorption spectra of receptor 4 in so-
lution of CH3CN/H2O (10–5 M, 3/7) in the presence of perchlorate salts
of K+, Na+, Ag+, Fe2+, Fe3+, Al3+, Cr3+, Co2+, Cu2+, Zn2+, Ca2+
,
Mg2+, Mn2+, Cd2+, Ni2+, Hg2+ and Pb2+ ions were measured and
recorded (ESI, Fig. S5). As shown in Fig. 1, a very weak band above
350 nm is ascribed to a trace amount of the ring-open form of 4 [63]
and the bands at 430, 454 and 485 nm are the characteristic peaks of
the fluorescein moiety itself [64]. The peak at 454 nm is attributed to
the π-π* transition of the fluorescein chromophore [65]. Addition of 1
equiv. each of Hg2+ and Fe3+ to the receptor 4 separately has notice-
ably reduced the intensity of the band at 485 nm with a concomitant
appearance of a new peak at 430 nm. Two well-defined isosbestic points
were observed at 404 and 451 nm for Hg2+ and at 422 and 447 nm for
Fe3+, indicating a net inter-conversion between un-complexed and
The sensitivity of a probe is determined by its quantitative response
to a particular species. To investigate the sensitivity of probe 4, fluor-
escence titration experiments were carried out with Fe3+ and Hg2+
ions (4.87 × 10–7 M). A good linear relationship between the fluores-
cence intensity ratio (data extracted from Fig. 2) and the concentration
of Fe3+ and Hg2+ was presented with R2 = 0.992 and 0.972 for Fe3+
and Hg2+ respectively (ESI, Fig. S12). The limits of detection (LOD)
Fig. 1. Changes in the absorption spectra of 4 in CH3CN/H2O (3/7, v/v) (10−5 M) at room temperature and pH = 7.2, upon addition of increasing amounts of (a)
Hg2+ and (b) Fe3+ ions up to 1 equiv.
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Fig. 2. (a) Fluorescence emission titration of compound 4 (10–7 M) upon addition of Hg2+ ion and (b) Fe3+ ion up to 1 equiv. in CH3CN/H2O (3/7; v/v) solution at
room temperature and pH = 7.2, Inset of Fig. 2; shows the plot of emission intensity variation at 473 and 545 nm (c) as a function of Hg2+ concentration, (d) as a
function of Fe3+ concentration respectively.
were calculated by the widely used 3σ/S method [69] where ‘σ’ is the
standard deviation of the blank and ‘S’ is the slope of the calibration
curve. The LOD was found to be 9.75 × 10–8 M and 39 × 10–8 M for
Fe3+ and Hg2+ ions respectively.
very stable in the temperature range of 25˚ C to 80° C. For [4·Hg2+] and
[4·Fe3+], the fluorescence intensity remains the same up to 60˚C and
decreases abruptly beyond 60˚C (ESI, Fig. S13). These results demon-
strate that Hg2+ and Fe3+ complexes of probe 4 are stable over a wide
temperature range of 25˚ C to 60° C.
To determine the sensitivity of a colorimetric or fluorescence probe,
response time plays a significant role. Thus, the effect of time on the
fluorescence intensity of probe 4 (10–7 M) has been determined in
CH3CN solvent (Fig. 4). Upon addition of 1 equiv. of Hg2+/Fe3+ ion
(10–7 M), the ratio of fluorescence intensities increases instantaneously
and reaches a maximum value within 40 s for Hg2+ and 30 s for Fe3+
ion. Fluorescence intensities remain constant over an extended period
of time (200 s). This suggests that the present probe 4 may provide a
proficient “zero-wait” detection method for the detection of Hg2+ and
Fe3+ ions.
Further, in order to compare our probe with the other reported
probes, we have thoroughly searched the literature and tabulated the
data in Table 1, demonstrating that our probe is comparable or even
better than other reported fluorescein-based Hg2+ and Fe3+ ion sensor
molecules. Further, from the literature survey, it is evident that the
present probe 4 is highly selective as it recognizes only two metal ions
among various other metal ions and is fairly sensitive on the basis of its
corresponding low limits of detection and high binding constant values.
Moreover, this is the 1st example of a ferrocene based-fluorescein de-
rivative for the simultaneous detection of Hg2+ and Fe3+ ions in the
literature.
Fluorescence intensities of ligand 4, [4·Hg2+] and [4·Fe3+] were
measured in the pH range of 2–12 (ESI, Fig. S14). The fluorescence
intensities remain constant over the neutral pH range i.e. within pH
6–7. However, changes in fluorescence intensity were observed in the
acidic or basic pH range as the fluorescein moiety is sensitive to pH
changes [83] due to the presence of sensitive functional groups.
Therefore, the sensing ability of a fluorescein-based sensor is expected
to be found precisely in the neutral pH range only. From the plot of
fluorescence intensity vs pH of metal-bound ligand, it is evident that the
present probe 4 can bind to Hg2+ and Fe3+ most effectively at neutral
3.4. Temperature, time and pH effect
To investigate the range of applicability of the ligand in metal
sensing, temperature, time and pH effects on the bare ligand as well as
of the metal-bonded probe have been studied [80–82] in CH3CN
medium. The temperature of CH3CN solutions of ligand 4, [4·Hg2+] and
[4·Fe3+] has been increased gradually from room temperature to 80° C.
In case of free ligand, the fluorescence intensity was almost unaffected
with rising temperature (ESI, Fig. S13). This suggests that the probe is
Fig. 3. Linear graph representation of fluorescence intensity ratio of receptor 4 for (a) Hg2+ and (b) Fe3+ ions.
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Fig. 4. Reaction time profile: changes of fluorescence intensity of receptor 4 (10–7 M) in the presence of 1 equiv. of (a) Hg2+ and (b) Fe3+ ions as a function of time
(0–200 s).
pH.
S15). The addition of Hg2+ and Fe3+ gave a clean response with re-
ceptor 4. The free receptor exhibited a reversible one-electron redox
wave, typical of a ferrocene derivative, with the half-wave potential
value E1/2 = 0.615 V for 4 due to the ferrocene/ferrocenium redox
couple. After addition of 1 equiv. of Hg2+ or Fe3+, respective anodic
shifts of ΔE1/2 = 33 mV and 31 mV were observed due to the formation
of new complex species (Figs. 6a and 7a). Also, a differential pulse
voltammetry (DPV) titration study was performed for a better under-
standing of the interaction of the Hg2+ and Fe3+ ions with receptor 4,
and the results are concurrent with that obtained from the cyclic vol-
tammetry study (Figs. 6b and 7b).
Cyclic voltammetry is one of the analytical tools that allows calcu-
lating the detection limit of a probe. Thus, to analyze the LOD we have
plotted the linear calibration graph between the change in potential
difference (ΔE1/2) vs conc. of increasing amount of metal ions in
CH3CN/H2O (3/7) as a solvent (ESI, Fig. S16). A linear response was
observed in the concentration range of 10–5 M from the calibration plot.
LODs were calculated by using the equation [88] ((3σ/S); σ = standard
deviation and S = slope of the calibration plot) and were found to be as
13.7 × 10–8 M and 30 × 10–8 M and for Fe3+ and Hg2+ ions, respec-
tively. These values are almost consistent with those of obtained from
the fluorescence plot.
3.5. Solvatochromism of receptor 4
Investigation of the solvatochromic effect of 4 in selected organic
solvents yielded some interesting results. Although the structured ab-
sorption bands at ca. 430, 454, 485 nm and emission bands at 521 and
545 nm of compound 4 correspond to characteristics of the quinoid
form of fluorescein [84], they are devoid of remarkable wavelength
shift in a range of different organic solvents. However, a negative sol-
vatochromism is observed for protic solvents like ethanol, which can be
explained by the larger charge separation and therefore larger dipole
moment of the excited state as compared to the ground state. Hence,
there occurs a decrease in the energy state difference on going from
polar to non-polar solvent [85–87]. It is worth noticing that hydrogen
bonding between the fluorescein moiety within our structured frame-
work and the polar solvent led to a hypsochromic shift of the absor-
bance as well as fluorescence emission band (Fig. 5).
3.6. Electrochemical study
The electrochemical properties of receptor 4 were investigated by
cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in
CH3CN/H2O (3/7, 10–5 M). Remarkably, the presence of a range of
other metal ions (as perchlorate salts) as mentioned above was ex-
amined for the electrochemical behavior in CH3CN/H2O (3/7, v/v)
containing 0.1 M [(n-Bu)4N]ClO4 as supporting electrolyte (ESI, Fig.
3.7. Naked eye detection
Visual detection by naked eye, one of the preliminary techniques for
the metal cation binding study, was performed via colorimetric
Fig. 5. (a) UV–vis absorption and (b) fluorescence spectra of 4 in various solvents at room temperature and pH = 7.2.
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Fig. 6. (a) Evolution of CV and (b) DPV of 4 (10−5 M) in CH3CN/H2O (3/7) at room temperature and pH = 7.2, using [(n-Bu)4N]ClO4 as supporting electrolyte on
addition of 1 equiv. of Hg2+ scanned at 0.05 V s−1
.
experiments with CH3CN/H2O (3/7) solutions of 4 (10–5 M) with the
different metal cations. Distinctive color changes from yellow to faint
green for both Hg2+ and Fe3+ ions with probe 4 were observed during
the investigation with 1 equiv. of perchlorate salts of different metal
convenient to use. Keeping this idea in mind, we have set out to in-
vestigate the action of receptor 4 on a solid support of silica, which
demonstrates its practical analytical applicability. Here, the silica gel
(60–120 mesh, 6.0 g, colorless), soaked with 4 (10–3 M) in 15 mL of
acetonitrile and dried, was added to aqueous solutions of Hg2+ and
Fe3+ ions (10–3 M) separately. The faint yellow color of the treated
silica gel instantly changed to green upon treatment with Hg2+ and
Fe3+ ions (Fig. 9). This experiment clearly demonstrates the practical
applicability of the present probe for the development of material-based
sensors.
A competitive experiment of compound 4 with other metal ions was
carried out using the fluorescence emission study, inferring that re-
ceptor 4 displays a high specificity towards only Hg2+ and Fe3+ ions.
Further, another competition experiment was performed between Hg2+
and Fe3+ ions with probe 4 to ascertain the particular selectivity to-
cations (Na+, Ag+, Ca2+, Mg2+, Mn2+, Hg2+, Fe2+, Fe3+, Al3+, Cr3+
,
Co2+, Cu2+, Zn2+, Cd2+, Ni2+ and Pb2+) (Fig. 8), which indicates the
selective and sensitive ‘naked eye’ identifying capacity of receptor 4 for
Hg2+ and Fe3+ ions.
3.8. Studies on the reversibility of the Hg2+ and Fe3+ binding to receptor 4
For a superior sensor molecule the important perspectives are to
recognize a particular analyte (selectivity), a low limit of detection
(sensitivity), and reversibility. For the reversibility point of view, a
fluorescence study for the Hg2+ and Fe3+ ions has been performed by
exposing the Hg2+ ion to the solution of 4. A greenish blue color
emanates with enhancing fluorescence intensity, which reverses back to
its nearly original form upon the introduction of iodide as a seques-
tering agent to the solution, owing to the formation of HgI2. Repeated
changes in color and fluorescence intensity on further addition of Hg2+
and masking it by the sequestering agent proves the reversible nature of
the interaction of receptor 4 with the Hg2+ ion (ESI, Fig. S17). Simi-
larly, the reversibility of the sensing of the Fe3+ ion was explored using
EDTA as the binding agent (ESI, Fig. S18). In both the cases, the above
experiments can be repeated at least 3 times without much loss in
sensitivity.
wards only one ion. The fluorescence enhancement of the [4·Hg2+
]
system (10–7 M) was found to be perturbed in the presence of 1 equiv.
of Fe3+ ions. However, the fluorescence emission of the [4·Fe3+
]
system did not display any significant change in presence of Hg2+ ions
(Fig. 10). Therefore, probe 4 could be used for the detection of Fe3+ in
the presence of other competing metal ions, including the Hg2+ ion. In
addition, 4 can be utilized for the detection of Hg2+ ions in the pre-
sence of other competing ions, except for the Fe3+ ion.
3.9. Real sample preparation and analysis
Ease of applicability of a sensor is a quality that makes it more
The effectiveness of receptor 4 can only be justified when it can
Fig. 7. (a) Evolution of CV of 4 (10−5 M) and (b) DPV of 4 (10−5 M) in CH3CN/H2O (3/7) at room temperature and pH = 7.2, using [(n-Bu)4N]ClO4 as supporting
electrolyte on addition of 1 equiv. of Fe3+ scanned at 0.05 V s−1
.
7

S.R. Bhatta, et al.
InorganicaChimicaActa498(2019)119097
Fig. 8. Visual colour changes observed for 4 in CH3CN/H2O (10–5 M) after addition of 1 equiv. of several metal cations.
Table 2
Determination of recovered conc. of Fe3+ and Hg2+ in pond water and tap
water samples.
Samples
Added metal
conc. (M)
Recovered metal
conc. (M)
% of
Recovery
% Error
Pond water Fe3+
0.4014 × 10–7
0.3973 × 10–7
0.4075 × 10–7
0.2031 × 10–7
0.1787 × 10–7
98.98
1.02
1.51
2.00
2.76
Hg2+ 0.4014 × 10–7
101.53
102.00
102.76
Tap water
Fe3+
0.1991 × 10–7
Hg2+ 0.1739 × 10–7
filter off the unwanted dust particles. The pH of the water samples was
maintained around 6.5–7. Known concentrations of Fe3+ and Hg2+
ions were added to the water samples separately and they were in-
troduced to the receptor 4 to determine recovered concentrations of
Fe3+ and Hg2+ ions, respectively. Fluorescence spectra were recorded
after each addition of metal ions to the ligand solution (10–7 M) and
enhancement of fluorescence intensities was observed for both real
samples. With each addition, the corresponding recovered metal ion
concentration was calculated from the equations y = (145.4x + 110.7)
and y = (123.5x + 160.7), with correlation coefficient R2 = 0.962 and
R2 = 0.968 for Fe3+ and Hg2+ ions respectively for pond water.
Equations y = (254.9x + 126.5) and y = (277.3x + 137.7) and cor-
relation coefficient R2 = 0.981 and R2 = 0.959 were found for Fe3+
and Hg2+ ions respectively for tap water sample (ESI, Figs. S19 and
S20). The experimental concentration and percentage recovery values
Fig. 9. Color changes of silica gel soaked with 4 upon addition to aqueous
solutions of Hg2+ ion and Fe3+ ion.
sense the selected metal ions in real water samples. For such an ex-
periment, pond water and tap water were collected from Jadavpur
University campus area and filtered on a Whatman 41 filter paper to
Fig. 10. Bar plot representation of the fluorescence emission intensity of 4 upon the addition of several potentially competing cations (1:5) in CH3CN/H2O.
8

S.R. Bhatta, et al.
InorganicaChimicaActa498(2019)119097
Fig. 11. Proposed binding mode between receptor 4 with (a) Hg2+ and (b) Fe3+ ions in CDCl3.
of the Fe3+ and Hg2+ ions are shown in Table 2.
shifted downfield by 0.05 ppm, resulting in a signal at 7.65 ppm.
Similarly, the Hb proton attached to the OCH2 unit experienced a
downfield shift of 0.07 ppm, giving rise to a signal at 5.32 ppm
(Fig. 11(a)). Interestingly, the other protons of receptor 4, corre-
sponding to the fluorescein moiety, do not show any shift in the 1H
NMR spectrum compared to those of the protons of the unbound re-
ceptor. This suggests the possible binding mode of the receptor with the
metal ion as given in the diagram below. Similarly, the host–guest
chemistry between receptor 4 and the Fe3+ ion was quickly identified
by a proton NMR study (Fig. 11(b)). Here also Ha and Hb protons were
downfield-shifted by 0.04 and 0.14 ppm, respectively, with no changes
in the chemical shifts of the other protons, even in the presence of more
than 1 equiv. of Fe3+ ion in the solution of receptor 4. Thus, the 1H
NMR titration study concludes the binding site for Hg2+ and Fe3+ to be
a nitrogen atom of the triazole ring and the oxygen atom of the OCH2
group linked to the fluorescein moiety.
3.10. IR and 1H NMR spectroscopic titration
Further, we have performed FT-IR spectral measurement of free li-
gand and its complexes with Hg2+ and Fe3+ ions. Upon addition of
metal ions, no significant change or shift in streching frequency of the
carbonyl group as well as any other peak in the finger print region was
observed. One new peak appeared at 1103 cm−1 which may be at-
–
tributed to the Cl-O streching frequency of the ClO4 unit (ESI, Fig.
S21). This result demonstrated that metals are certainly not binding
with to the carbonyl group, they rather bind to other heteroatoms
present in the molecule. As an IR spectral titration is not sufficent to
demonstrate the binding mode unambigously for the present probe 4,
we have further performed a 1H NMR titration which is more convin-
cing in order to ascertain a metal ion binding mode.
A preliminary 1H NMR cation binding analysis of receptor 4 with
Hg2+ and Fe3+ ions was investigated by 1H NMR spectroscopic titra-
tion experiment in CDCl3 as solvent. Upon addition of 1 equiv. of Hg2+
ion to the solution of receptor 4, the Ha proton with an initial chemical
shift of 7.60 ppm, corresponding to the triazole unit of the free receptor,
4. Conclusion
In conclusion, a novel fluorescein-based ratiometric fluorescent
chemosensor with a ferrocene backbone has been successfully designed
9

S.R. Bhatta, et al.
InorganicaChimicaActa498(2019)119097
and synthesized. It shows good selectivity towards Fe3+ and Hg2+ ions
via multiple channels with a very low limit of detection, a high binding
constant and “zero-wait” response time. The present probe exhibits a
negative solvatochromism in protic solvents, which may be due to a
hydrogen bonding interaction between the fluorescein moiety and the
corresponding polar solvent. Further, the present probe exhibits an
anodic shift of ΔE1/2 = 33 mV and 31 mV upon binding Hg2+ and Fe3+
ions, respectively. To the best of our knowledge, our probe is the 1st
example of ferrocene based-fluorescein derivative for the detection of
Hg2+ and Fe3+ ions simultaneously via ratiometric fluorescence and
electrochemical studies. Thus, in real-time detection based on the
promising ratiometric feature, the protocol described in this article
opens a new entry for the design of fluorescent and electrochemical
sensors for an easy and reliable detection of metal ions in real en-
vironmental samples.
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Generous support of the Department of Science and Technology,
DST, New Delhi for Inspire Faculty fellowship (IFA/14/CH-161) is
gratefully acknowledged.
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