A highly sensitive “Off-On” optical and fluorescent chemodosimeter for detecting iron (III) and its application in practical samples: An investigation of Fe3+ induced oxidation by mass spectrometry

Article abstract of DOI:10.1016/j.jphotochem.2017.07.048

We describe a highly sensitive turn on colorimetric and fluorescent probe for trivalent iron ions (Fe³⁺). The newly synthesized compound (RCD) was characterized by FTIR, NMR and mass spectral studies. Competitive experiments reveal that probe R

Full text of DOI:10.1016/j.jphotochem.2017.07.048

Accepted Manuscript
Title: A highly sensitive “Off-On” optical and fluorescent
chemodosimeter for detecting Iron (III) and its application in
3
+
practical samples: An investigation of Fe induced oxidation
by mass spectrometry
Authors: Pattan Sirajuddin Nayab, Mohd. Shkir, Parveez Gull,
S. AlFaify
PII:
S1010-6030(17)31139-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.048
JPC 10779
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
3-6-2017
15-7-2017
27-7-2017
Pleasecitethisarticleas:PattanSirajuddinNayab, Mohd.Shkir, ParveezGull, S.AlFaify,
A highly sensitive “Off-On” optical and fluorescent chemodosimeter for detecting
Iron (III) and its application in practical samples: An investigation of Fe3+ induced
oxidation by mass spectrometry, Journal of Photochemistry and Photobiology A:
Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.048
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A highly sensitive “Off-On” optical and fluorescent chemodosimeter for detecting Iron
3
+
(
III) and its application in practical samples: An investigation of Fe induced oxidation
by mass spectrometry
a
b
c
b
Pattan Sirajuddin Nayab *, Mohd. Shkir , Parveez Gull , S. AlFaify ,
a
Synteny Life Sciences Private Limited, Hyderabad-500044, INDIA
bAdvanced Functional Materials & Optoelectronic Laboratory (AFMOL), Department of
Physics, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi
Arabia
cDepartment of Chemistry, Government Degree College Pulwama kashmir, J&K-192301
India
*
Author for Correspondence. Mob: +918106450727
Email address: psiraj2016@gmail.com (Pattan Sirajuddin Nayab)
Graphical abstract:
CH3
CH
CH3
CH3
CH
CH3
Fe3+
O
N
O
N
Fe3+
N
N
EDTA
N
O
N
N
O
N
RCD
RCD + Fe (III)
RCD+Metal
RCD +Metal +Fe(III)
0
.3
.25
.2
.15
.1
.05
0
0
0
0
0
0
1

Highlights:

New
Rhodamine-based
colorimetric
and
fluorescent
reversible
chemodosimeter was developed for detecting Fe3+



The probe could be serve as a ‘naked eye’ chemosensor for Fe3+ and exhibits
greater emission response with 48-fold fluorescence enhancement
The probe is insensitive to pH under normal conditions(5-8) therefore can be
applicable for biological and environmental samples
The probe exhibits greater sensitivity and specificity for Fe3+ over other
biologically important metals tested with low LOD and high association
constant values.

The high recoveries and low RSD % indicates that RCD can acts as a potential
3
+
probe for the accurate recognition of Fe in water samples.
Abstract
We describe a highly sensitive turn on colorimetric and fluorescent probe for trivalent iron
3
+
ions (Fe ). The newly synthesized compound (RCD) was characterized by FTIR, NMR and
mass spectral studies. Competitive experiments reveal that probe RCD exhibits greater
3
+
sensitivity and specificity towards Fe among other biologically important metals. Binding
of Fe3+ to RCD triggers spirolactum ring opening of rhodamine and evokes a 48-fold
fluorescence enhancement accompanied by color variation from colorless to pink. The
3
+
formation of 1:1 complex between RCD and Fe was supported by Job’s plot, DFT and
Mass spectral studies. The detection limit for RCD is found to be 5.05µM even lowers than
3
+
the permissible [Fe ] in drinking water as per WHO standard. The high recoveries and low
RSD % indicates that RCD can acts as a potential probe for the accurate recognition of Fe3+
in water samples. Moreover, the probe is insensitive to pH 5-8 therefore can be applied for
biological and environmental samples.
2

Key words: Naked eye sensor, Fluorescent, Reversible, DFT study, Turn on, Rhodamine
1
. Introduction
Fluorometric and colorimetric chemodosimeters are powerful tools for the recognition of
biometals because of their simplicity, high specificity and great sensitivity [1]. In past
decades, various analytical techniques like anodic stripping vol-tammetry, atomic emission
spectrometry (AAS), atomic absorption spectrometry (AAS) and inductively coupled plasma-
atomic emission spectrometry (ICP-AES) have been explored for the detection of
environmentally and biologically important ions [2]. Despite their numerous applications, the
limitations such as huge maintenance cost, long analysis time and operational difficulties
associated with these techniques hinder their widespread utility [3]. Hence, there is a
considerable need for the emergence of new techniques to determine the analytes present in
the chemical, biological and environmental systems. Recently, photo physical methods have
been attracted significant interest to researchers due to their high selectivity and low-cost
instrumentation.
Iron is the most versatile transition metal performing different biological functions in living
systems [4]. Among them, physiological processes such as electron transfer, oxygen
metabolism and transportation could not occur without Iron [5]. Furthermore, Iron
participates in transcriptional regulation and structural enhancement of red blood proteins.
Even though, Iron is an essential dietary mineral, under overloading conditions this metal can
cause Alzheimer’s disease, cancer and fatal damage to the liver and brain [6].Whilst Iron
deficiency lead to anemia. As a consequence, detection and quantification of Iron has
become a major concern in biomedical analysis. Literature is enriched with significant
number fluorescent probes for iron ion sensing such as calcein derivatives, anthracene
analogues, coumarin and fluorescein derivatives [7-9].The success accomplished by previous
rhodamine turn-on sensors in intracellular Zn (II) and Cu (II) detection has led to the
investigation of novel candidates for other biologically important metals. Schiffbase
3

derivatives have been extensively studied in the fields of inorganic and analytical chemistry
as chemosensors because of their chelating ability [10-11]
Currently, rhodamine fluorophores have gained tremendous interest in the field of biosensors
because of their advantages such as greater photo stability, longer emission wavelength, high
fluorescence coefficient and quantum yields [12]. In general, rhodamine derivatives with
spirocyclic form are non fluorescent and colorless, interestingly upon binding with protons
and metals they become fluorescent and produce intense color because of ring-opened
structure [13]. Owing to their structural property, rhodamine derivatives would serve as ideal
candidates for constructing off-on fluorometric and colorimetric sensors. Construction of
probes on the basis of rhodamine spirolactum facilitates both absorbance and emission
enhancement and “naked eye” detection [14]. Therefore, rhodamine derivatives have been
intensively studied as colorimetric, fluorometric and ratiometric sensors.
Recently, we explored a novel rhodamine appended conjugate as a selective and sensitive
probe for Fe3+ in which, rhodamine spirolactum ring-opening process led to a “turn-on”
emission signal and colorimetric change toward Fe3+ [15]. As we anticipated in the
development of efficient probes for Fe3+ ions, herein we report a new conjugate base on
3
+
rhodamine as a dual responsive molecular probe for Fe .
2
. Experimental
2
.1. Reagents and Equipments
The reagents used for synthesis were procured from commercial suppliers and used without
further treatment. Deionized water was used throughout the experiment. The metal salts used
2
+
2+
2+
2+
2+
2+
3+
3+
3+
+
2+
were chloride salts of Hg , Ni , Cd , Zn , Cu , Co , Fe , Cr , Al , K , and Cd . The
progress of the reaction was checked by means of TLC (Thin layer chromatography) with
detection by UV. Absorbance experiments were accomplished on Perkin Elmer Lamda 40
UV-vis. spectrophotometer. Fluorescence titrations were done on Jobin-Yvon Fluorolog 3-22
spectrofluorometer. FTIR spectra were obtained on Perkin Elmer Spectrum RXI-IR
Instrument. Mass spectral analysis performed using MALDI MS-ABI Sciex 5800 TOF/TOF
System with LC-MALDI.
4

2
.2. Preparation of Rhodamine B hydrazide (RBH):
Rhodamine B hydrazide (RBH) was prepared using earlier procedure [16].The analytical data
for structural characterization of RBH is presented in supporting information.
2
.3. Synthesis of chemosensor RCD
The presented probe RCD was facilely synthesized in single step condensation between RBH
and cuminaldehyde in methanol solvent. Methanolic solution of Rhodamine B hydrazide (20
mL) was added to ethanolic solution of Cuminaldehyde. The resulting mixture was stirred
with reflux for 12 h. On cooling the reaction mixture, a white precipitate was generated
which filtered and washed with cold ethanol and dried under vacuum.
+
-1
Yield: 66 %; white solid; TOF MS ES : m/z 587.29; IR ʋmax(cm ): 1710 ʋ(C=O), 1571
1
ʋ(CH=N), 693 ʋ(Ar-CH); H-NMR (300 MHz, CDCl
3
), δ
)), 2.777-2.865 (m, 1H, CH(CH
,), 6.203-6.241 (dd, J = 8.4 Hz, J = 2.1 Hz, 2H,
H
: 1.124-1.147 (t, J =6.9 Hz, 12H,
NCH
2
CH
3
), 1.192-1.214 (d, J = .6.6 Hz, 6H, CH(CH
3
)
2
)
3 2
),
2
.889-3.346 (q, J = 6.6 Hz, 8H, NCH
2
CH
3
1
2
Xanthene-H,), 6.426-6.433 (d, J = 2.1 Hz, 2H, Xanthene-H), 6.506-6.535 (d, J = 8.7 Hz, 2H,
Xanthene-H), 7.088-7.097(d, J = 2.7 Hz,1H, Phen-H), 7.116-7.143 (d, J = 8.1 Hz, 2H, Ar-H),
7
7
.412-7.424 (d, J = 3.6 Hz, 1H, Phen-H), 7.452-7.463 (d, J
1
= 3.3 Hz, 2H, Phen-H,), 7.471-
.498(d, J = 8.1 Hz, 2H, Ar-H), 7.972-7.994(d, J = 6.6 Hz,1H, Phen-H), 8.620 (s, 1H,
1
3
CH=N); C-NMR (75 MHz ,CDCl
3
) δ
08.43, 123.38, 124.25, 127.19, 127.28, 128.06, 129.20, 129.51, 132.78, 134.17, 148.46,
48.91, 151.36, 151.47, 153.19, 164.06;
C
: 12.86, 24.02, 33.78, 44.10, 65.93, 97.77, 106.09,
1
1
2
.4. Absorbance and fluorescence spectral experiments
The absorbance and fluorescence measurements were performed at room temperature in
CH CN- aq. HEPES buffer. (5:5, v/v; pH = 7.2) The selectivity and sensitivity of probe RCD
against various metal ions like Ni , Cd , Hg , Fe , Cu , Zn , Co , K , Na ,Cr and
3
2
+
2+
2+
3+
2+
2+
2+
+
+
3+
3
+
Al were investigated. The metal salts were dissolved in the mixture of distilled water. The
5

absorbance and fluorescence spectra of RCD were recorded with and without metal ions. All
the solutions were mixed and equilibrated for 10 min before the spectra were taken.
Based on spectro photometric and spectro fluorometric data detection limit and binding
constant values were calculated. The interaction capacity of RCD toward Fe3+ was
determined by Benesi-Hildebrand equation [17].
+
1
/ (I − I
0
) = 1/K (I max − I
0
) [Mn ] + 1/ (I max − I
0
)
(1)
Where I
concentration and Imax, corresponding to the maximum fluorescence in the presence of Fe at
85 nm respectively. The equation which used to calculate detection limit is as follows [18].
0
is the emission intensity of probe RCD, I refers to the emission intensity at a given
3
+
5
Detection limit=3SD/S
(2)
Where SD and S refers to standard deviation and slope of the calibration line.
2
.5. DFT Study
Density functional theory (DFT) is found to be an excellent tool to predict the various key
properties like: geometrical and opto-electronic of enormous class of organic, inorganic and
their metal complexes [19, 20]. Moreover, DFT has been developed as a mutual contrivance
in first-principles calculations intended to predict and describe the characteristic properties of
molecular and condensed matter schemes. Quantum chemical calculations for probe RCD
were performed using Gaussian 09program at the B3LYP/6-31G* level. The optimized
structures of the ligand and its metal complex were visualized by Gauss view 5 programs.
3
+
The ground state molecular geometry of RCD and its Fe complex are shown in fig and data
of HOMO, LUMO bond energies were compiled in Table 1.
3
. Results and Discussion
The synthetic procedure for title compound is given in scheme 1. The condensation of RBH
with cumminaldehyde in the presence of methanol produces RCD. The solution of RCD in
buffered solution is colorless suggesting the formation of spirocyclic form. Structural
6

elucidation of synthesized compound was carried out by means of UV, FTIR, Mass and
NMR spectroscopy.
3
.1 spectral characteristics
−
1
The FTIR spectrum of compound RBH exhibited a sharp signal at 3362 cm ascribed to NH
vibrations (Fig S1). The disappearance of this band and emergence of strong band at 1571
−
1
cm in the FTIR spectrum of RCD indicate the formation of azomethine moiety. The bands
−
1
−1
corresponding to C=O vibrations were appeared at 1710 cm . At 1121cm , a medium
intense band was appeared due to C-O-C stretching vibrations. However, the bending
−
1
vibrations corresponding to aromatic CH bending bond were found at 693 cm respectively.
The NMR spectra of RCD are given in Fig. 1 and Fig. 2. The broad peak observed at 3.61
ppm in the NMR spectra of RBH attributed to amine group suggesting the formation of
1
rhodamine B hydrazide (Fig. S2). However this peak was disappeared in the H NMR spectra
of RCD indicating the condensation of amine group with cuminaldehyde. Moreover, the
appearance of a sharp signal at 8.62 ppm due to azomethine proton further confirms the
schiff base formation. The proton signals related to xanthenes were observed between 6.42
and 6.53 ppm whereas aromatic protons were appeared between 7.08 and 7.99 ppm
13
respectively. A sharp signal at 65 ppm in the C NMR spectra of RCD further provides
evidence for the formation of spirolactum ring form [21]. The peak appeared at 148 ppm was
attributed to azomethine moiety.
3
.2. Absorbance response of probe RCD to metal ions
2
+
The binding capacity of compound RCD was investigated towards diverse metal ions (Ni ,
2
+
2+
3+
2+
2+
2+
+
+
3+
3+
Cd , Hg , Fe , Cu , Zn , Co , K , Na ,Cr and Al ) by absorption and fluorescence
spectroscopy. The absorption spectrum of RCD exhibited absorption bands at 350 nm, but no
bands were observed over 500 nm which might be due to the closed spirolactam form of
3
+
RCD [22]. The metal ions except Fe induce minute or no changes in the absorption spectra
3
+
of RCD. On the addition of 5 equivalents of Fe , RCD exhibited a band at 550 nm implying
3
+
that the test compound exhibiting selectivity towards Fe among other metal ions (Fig. 3a).
Besides the color change of the solution from colorless to pink indicating that RCD could
7

3
+
serve as “naked-eye” chemo sensor for Fe (Fig. 4) [23]. The emergence of new band at 550
nm is because of the existence of ring-opened amide form of title compound (Fig. 3b).
Job’s plot experiments were also carried out in order to find out the binding stoichiometry
3
+
between probe RCD and Fe . As illustrated in the Fig. 3b maximum absorbance was
3
+
appeared at the ratio of 0.5 suggesting the 1:1 complex formation between RCD and Fe .
24]
[
3
.3. Fluorescence response of probe RCD to metal ions
The fluorescence responses of probe RCD towards different metal ions are illustrated in Fig.
3
+
5
a. As depicted in the figure only Fe cause a 48-fold fluorescence emission enhancement
where as other metal ions induce mild or no apparent changes in the fluorescence intensity of
3
+
probe RCD. As the concentration of Fe increased, the fluorescence intensity at 585 nm was
3
+
significantly enhanced due to the increased concentration of Fe -RCD complex (Fig. 5b).
Furthermore, competitive binding experiments were also performed to investigate the
sensitivity and practical applicability of probe RCD as a Fe3+ selective fluorescent
3
+
chemosensor [25]. The fluorescence intensity of probe RCD to Fe complex is unaffected by
other metal ions indicate that proposed probe could applicable for practical applications (Fig.
9
5
). The detection limit and association constant of probe RCD to the receptor is found to be
−
6
4
−1
.05 X 10 M and 3.42 X10 M (Table 2).
3
.4. pH responses of RCD
The absorbance and fluorescence response of probe RCD was also investigated under various
pH conditions. As the pH decreases the absorbance and fluorescence intensity of RCD
increases which could be ascribed to the spirolactum ring opening of test compounds due to
the strong protonation [26]. While at pH 4, the enhanced absorbance and fluorescence
intensity was gradually reduced. The disappearance of absorbance (at 550 nm) and
fluorescence (at 585 nm) was observed between pH 5-8. The experimental results reveal that
the newly synthesized probe is insensitive to pH 5-8 therefore practically applicable under
8

physiological conditions. These results indicated that, the probe RCD can be applied for
environmental and biological samples.
3
.5. Proposed sensing mechanism by FTIR and Mass spectral analysis
In order to intend the sensing mechanism FTIR and mass spectral analysis were done for
3
+
RCD with and without Fe ions. The IR spectrum of RCD showed a sharp signal at 1710
-1
-1
Cm corresponding to carbonyl vibrations. However this band shifted to 1681Cm (lower
3
+
frequency) suggesting the involvement of C=O group in complexation with Fe ions (Fig.
).
6
3
+
The reaction products of probe RCD with Fe were subjected to mass spectral analysis in
order to scrutinize sensing mechanism. A plausible mechanism is proposed on the basis of
intermediates formed in the reaction (scheme 2). As shown in the Fig. 7a, the apparent peak
observed at m/z value 587 is corresponding to newly synthesized probe RCD. However,
3
+
upon addition of five equivalents of Fe to the solution, intense peaks were obtained at m/z
values of 471 and 443(Fig. 7b). The fragment peak detected at 471 is attributed to an
intermediate 1 which subsequently oxidized to rhodamine B characterized by an intense pink
color [27]. The results indicate that interaction of Fe3+ to RCD lead to Fe (III) induced
oxidation reaction where 1 formed as an initial product which is unstable and further
hydrolyzed to Rhodamine B(2) as a final product.
3
.6. Theoretical investigations
Quantum chemical calculations were also performed to scrutinize the binding pattern of RCD
3
+
3+
with Fe and to resolve the geometry of RCD- Fe complex. The spatial distribution and
energies of HOMO and LUMO were determined by means of DFT calculations. As
illustrated in the Fig. 8, the oxygen atom form the amide group of RCD and nitrogen atom of
3
+
azomethine moiety is involved in the complexation with Fe . In case of compound RCD the
electron density in HOMO is distributed over the phenylene and cumunaldehyde moieties
while LUMO it is localized on the xanthene moiety. On the other hand the HOMO and
3
+
LUMO of RDFB-Fe complex were spread over the half of xanthane and spirolactum ring
and central metal ion. The energy gap between HOMO and LUMO for probe RCD is
9

calculated to be 3.76 eV. Whereas for RCD-Fe3+ complex it is found to be 0.841 eV
3
+
respectively demonstrating that binding of RCD to Fe decreases the HOMO-LUMO energy
gap of RCD-Fe3+ complex and stabilizes the system. As results indicated the cleavage of
3
+
spirocyclic C-N bond occur in order to facilitate the interaction of Fe to oxygen atom of
amide moiety of RCD [28].
3
.7. Determination of Fe 3+ in practical samples
Furthermore to check the practical applicability, RCD was employed for the determination of
3
+
Fe in drinking, distilled and tap water samples. Initially, the analyzed samples contain no
3
+
Fe , indicating the absence of ferric ions in these samples. Then the water samples were
3
+
spiked with known concentrations of Fe and subjected to recovery studies. For each spike
sample three replicate determinations were done and the results were reported in Table 3.An
excellent harmonization was observed between added and determined values which validate
our proposed sensing method. In addition, the high recoveries and low RSD % indicate that
RCD could acts as a potential probe for the accurate detection of Fe 3+ in practical samples.
4
. Conclusions
We synthesized a novel dual responsive colorimetric and fluorescent chemodosimeter which
showed greater sensitivity towards Fe3+ ions over other tested metal ions. The presented
probe displayed remarkable absorbance enhancement accompanied with a color change from
3
+
colorless to pink upon interaction to Fe . In addition to that probe RCD exhibited 48-fold
fluorescence enhancements suggesting the high potential of this compound to serve as an
3
+
effective “Off-On” switch for Fe . A job plot and mass spectral analysis confirmed the 1:1
3
+
binding mode between synthesized probe and Fe . DFT studies and FTIR spectral analysis
provided the evidence of involvement of oxygen and nitrogen atoms of RCD in complexation
3
+
with Fe . The detection limit for RCD (5.05µM) is found to be lowers than the permissible
3
+
[
Fe ] in drinking water as per WHO standard. The low RSD % high and recoveries
demonstrates that RCD can serve as a potential sensor for the detection of Fe3+ in water
samples. Moreover, this probe is insensitive to pH between 5-8 which property makes it
practically applicable for biological samples under physiological conditions.
1
0

Acknowledgements
The author P.S. Nayab is thankful to. Mr. K. Ashok Reddy, for his kind permission to do
research work at Synteny Lifesciences Pvt Ltd, Hyderabad.
1
1

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5

Figures
1
Fig. 1. H NMR spectrum of RCD
1
6

1
3
Fig. 2. C NMR spectrum of RCD
1
7

Fig. 3a. UV-visible spectra of RCD (10µM) in the presence of various metal ions in buffered
-5
solution. (10 M)
1
8

Fig. 3b. Absorption spectra of RCD in buffered solution upon addition of different amounts
3
+
3+
3+
−5
of Fe (1-25µM). Job's plot for Fe -RCD system [Fe ]+ [RCD] =1.0×10 M.
1
9

Fig. 4. Color changes of RCD upon addition of various metal ions.
Fig. 5a. Fluorescence spectroscopic response of RCD to different metal ions in CH
3
CN- aq.
HEPES buffer medium.
2
0

3
+
Fig. 5b. Fluorescence spectra of RCD upon addition of increasing concentrations of Fe (1-
25µM) in CH CN- aq. HEPES buffer medium.
3
2
1

3
+
Fig. 6. FTIR spectra of RCD alone and RCD-Fe complex.
2
2

Fig. 7a Mass spectrum of RCD.
2
3

CH3
CH
O
CH3
N N
O
N
N
O
RCD
H N N H
O
+
N
N
1
O
OH
O
N+
N
2
3
+
Fig. 7b Mass spectrum of RCD-Fe adduct
2
4

3
+
Fig. 8 HOMO and LUMOdistributions of RCD and RCD–Fe complex.
2
5

RCD+Metal
RCD +Metal +Fe(III)
0
.3
.25
.2
.15
.1
.05
0
0
0
0
0
0
RCD+Metal
RCD +Metal +Fe(III)
2
500
000
500
000
2
1
1
5
00
0
Fig. 9. Histograms representing A) Absorbance B) Fluorescence changes in the presence of
different metal ions.
2
6

CH3
CH
O
O
O
O
HCl
CH3
Methanol
H2N NH2
Reflux
N NH2
N N
Cuminaldehyde, Reflux
O
N
N
O
N
O
N
N
N
RBH
RCD
Rhodamine B
Scheme 1: Synthesis of RBH and sensor, RCD
2
7

CH3
CH
O
O
CH3
Fe(III)
HN NH
N N
O
N+
N
O
N
N
1
RCD
Colorless, non-fluorescent
m/z= 471
O
OH
O
N+
N
2
m/z= 443
Pink, Strong fluorescent
Scheme 2: Moleculat structures of RCD, 1 and 2
2
8

Tables:
Table 1
The HOMO energy (EHOMO), LUMO energy (ELUMO) and the HOMO-LUMO energy gap of
3
+
RCD and its Fe complex.
Entry
HOMO
LUMO
E
HOMO LUMO
-
RCD
-5.012 eV.
-1.252 eV
-0.786 eV
∆E = EHOMO
-
-
LUMO = 3.76 eV
LUMO = 0.841 eV
RCD+Fe3+
-1.627 eV
∆E = EHOMO
2
9