Insights into the phenomenon of acquisition and accumulation of Fe3+ in Hygrophila spinosa through fluorimetry and fluorescence images

Article abstract of DOI:10.1016/j.tetlet.2019.151520

Rhodamine functionalized fluorescent probe IP has been synthesized to investigate the phenomenon of Fe³⁺ acquisition and accumulation in Hygrophila spinosa. H. spinosa is a tropical medicinal plant which is iron rich and consumed for the treatment of the patients suffering from anaemia. IP is capable of selectively binding Fe³⁺ by enhancing fluorescent intensity via “turn on” mechanism due to complex formation. Spectroscopic studies and microscopic tools helped in better understanding about the acquisition as well as the quantitative accumulation of Fe³⁺ in different parts of the plant.

Full text of DOI:10.1016/j.tetlet.2019.151520

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Insights into the phenomenon of acquisition and accumulation of Fe3+ in Hy-
grophila spinosa through fluorimetry and fluorescence images
Ayndrila Ghosh, Saurodeep Mandal, Sujoy Das, Pallab Shaw, Ansuman
Chattopadhyay, Prithidipa Sahoo
PII:
S0040-4039(19)31319-X
https://doi.org/10.1016/j.tetlet.2019.151520
TETL 151520
DOI:
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
6 November 2019
9 December 2019
12 December 2019
Please cite this article as: Ghosh, A., Mandal, S., Das, S., Shaw, P., Chattopadhyay, A., Sahoo, P., Insights into the
phenomenon of acquisition and accumulation of Fe3+ in Hygrophila spinosa through fluorimetry and fluorescence
images, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.151520
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Insights into the phenomenon of
acquisition and accumulation of
Fe3+ in Hygrophila spinosa through
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fluorimetry and fluorescence images
Ayndrila Ghosh†[a], Saurodeep Mandal†[a],
Sujoy Das[a], Pallab Shaw[b],
Ansuman Chattopadhyay[b]
and Prithidipa Sahoo*[a]
†These authors contributed
equally to this work.

1
Tetrahedron Letters
Insights into the phenomenon of acquisition and accumulation of Fe3+ in Hygrophila
spinosa through fluorimetry and fluorescence images
Ayndrila Ghosh†a, Saurodeep Mandal†a, Sujoy Dasa, Pallab Shawb, Ansuman Chattopadhyayb and
Prithidipa Sahoo*a
aDepartment of Chemistry, Visva-Bharati, Santiniketan-731235, West Bengal, India.
bDepartment of Zoology, Visva-Bharati, Santiniketan-731235, West Bengal, India.
† These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Rhodamine functionalized fluorescent probe IP has been synthesized to investigate the
phenomenon of Fe3+ acquisition and accumulation in Hygrophila spinosa. H. spinosa is a
tropical medicinal plant which is iron rich and consumed for the treatment of the patients
suffering from anaemia. IP is capable of selectively binding Fe3+ by enhancing fluorescent
intensity via "turn on" mechanism due to complex formation. Spectroscopic studies and
microscopic tools helped in better understanding about the acquisition as well as the quantitative
accumulation of Fe3+ in different parts of the plant.
Available online
Keywords:
Hygrophila
Iron accumulation
Chemosensor
2009 Elsevier Ltd. All rights reserved.
Fluorescent Sensing
Estimation of Fe(III)
1. Introduction
absorbed in the duodenum cells and rapidly oxidized to Fe3+.
Then, the Ferric ions bind with intracellular carriers and
Hygrophila spinosa- a medicinal plant mostly found in
tropical climate and the leaves of the plant are often used as a
part of the daily cuisine especially in eastern India. Traditionally,
the stem and leaves are being used for centuries to improve in
haemoglobin level in blood to treat patients suffering from
anaemia as it is Iron rich in nature. The plant also shows anti-
transported to different cells and organs of the human body.[17] 1
Ferric ions are incorporated in the catalytic site of many
proteins and enzymes.[18-21] Formation of met-haemoglobin,
which is incapable of binding oxygen, induced by the oxidation
of haeme iron to ferric ion.[12] Increased accumulation of ferric
ions in a living cell can generate reactive oxygen species (ROS)
via the Fenton reaction[19], which can damage lipids, nucleic
acids, and proteins. The cellular toxicity of ferric ions are
connected to severe diseases like Alzheimer’s, Huntington’s, and
Parkinson’s disease, colorectal cancer.[22-25]
inflammatory [1-3], antipyretic [4,5], hematopoietic[6], antioxidant[7-
9], antibacterial[10,11] and anthelmintic
effects. Previous studies
[10]
on different other plant systems indicate, acquisition of iron
occurs via two major pathways:(i) by releasing H+- ATPase from
the root of the rhizosphere which extrudes H+ ions to lower the
pH and increasing the solubility of Fe3+. Consequently an
inducible ferric chelate reductase FRO2 reduces Fe3+ to Fe2+.[12,13]
The Fe2+ being transported by a major transporter IRT1 from root
Our interest is to find out the quantitative accumulation of
Fe3+ within H. spinosa. Several analytical methods such as
colorimetry, UV-Vis spectrometry, atomic absorption and
inductively coupled plasma mass spectrometry, flow injection
analysis, column preconcentration, catalytic cathodic stripping
voltammetry are commonly used for iron determination.[26-29] But
fluorescent chemosensors become predominantly popular for
their lion's share in the intrinsic features of a practical sensing
system.[30-36] Hence, the present work relates to the designing of a
to other parts of the plants[14,15]
;
(ii) by secreting
Phytosiderophores and the chelated Fe3+ then transported to
different organisms of the plant.[16] Literature suggests that Iron
mostly exists in the form of Fe2+ and Fe3+ in the soil. However,
the intake process and method of accumulation of iron within the
physiological system of Hygrophila spinosa has not been
definitively established yet, whether this accumulation occurs in
the form of Fe3+ or by transforming it into Fe2+. The current
knowledge on human metabolic processes indicates iron is easily
absorbed in the form of Fe2+ and therefore, all the external iron
supplements are found to exist in Fe2+ state. Fe2+ ions get
———
1  Corresponding author. Prithidipa Sahoo, e-mail: prithidipa@hotmail.com

2
Tetrahedron
new fluorescent chemosensor with the ability of selective
Rhodamine B (2.40 g, 5 mmol) was dissolved in methanol (20
mL). Ethylene diamine (5 mL, excess) was added drop-wise
to the solution and refluxed overnight (15 h) until the solution
loses its red color. The solvent was removed by evaporation.
Water (20 mL) was added to the resultant and extracted with
CH2Cl2 (20 mL × 2). The combined organic phase was
washed twice with water and dried over Na2SO4. The solvent
was removed by evaporation and dried in vacuum, affording a
pale-pink solid of 1(3.80 g, yield, 79%).
detection and estimation of Fe3+ in H. spinosa. Here we are
introducing a rhodamine-pyrene conjugate fluorescence probe
(IP) which has been applied to selectively determine and
estimate the Fe3+ ions present in various parts of the H. spinosa.
Though a large number of fluorescent probes have already been
developed to detect Fe3+, but most of them are not exclusively
selective for Fe3+. Probe IP is capable of detecting Fe3+ at very
low concentration compared to others. Moreover, IP doesn't show
any toxicity inside plant cells as well as tissues and hence it could
be a potential dye to be used in living system. As the literature
suggests, no previous work has been done in which qualitative
accumulation of Fe3+ has been measured in living plant tissues to
obtain the idea of acquisition and accumulation of inside plant
cells (Comparison table SI).
2.2.2. Synthesis of 2
Compound 1 (1.5 g, 3 mmol) mixed with K2CO3 (4.27 g,10
mmol) is suspended into a mixture of ethyl acetate (25 mL)
and water (25 mL) and stirred for 30 minutes. Then,
bromoacetyl chloride (1.22g, 2.5 mmol) in ethyl acetate (5
mL) is added dropwise into the solution. After 4 h stirring at
room temperature, the organic layer is isolated and dried by
MgSO4. The ethyl acetate solvent is removed by rotary
evaporation to give the crude product that is purified by
Probe IP was synthesized through three simple steps starting
from a very well-known fluorophore rhodamine and the structure
of IP was deduced by 1H and 13C NMR spectral studies(Fig. S1&
Fig.S2).
2. Experimental Section
column
chromatography
(silica,
220–400
mesh,
EtOAc/MeOH = 10:1 v/v). The product is isolated as a pale-
pink powder 2 (1.26 g, 84%). Synthesis of intermediate 1 and
2 has been performed by following previously published
protocols.[37]
2.1. Materials and Methods
Rhodamine B, ethylene diamine, bromoacetyl chloride, 1-
pyrrenemethylamine, potassium carbonate, all the metal salts
used for selectivity test were purchased from Sigma-Aldrich Pvt.
Ltd. (India). All the materials were bought from commercial
suppliers and were used without further purification. Standard
procedures were obtained for solvent drying. Double distilled
2.2.1. Synthesis of IP
water was used throughout all experiments. H and 13C NMR
1
To a solution of anhydrous K2CO3 (2.5g, 18 mmol) in dry
acetone was added 1-pyrrenemethylamine (0.55 g, 2.0 mmol).
The mixture was stirred for 45 minutes. Then compound 2 (1.26
g, 2.0 mmol) was added to the solution and refluxed for 24 h
(Scheme 1). Acetone was dried and the mixture was treated with
brine (3×20mL). The organic layer was extracted with CH2Cl2
(3×50 mL), and the combined organic layer was washed with 5%
aqueous HCl (50 mL), 10% aqueous Na2CO3 (50 mL) and finally
with water and then was dried over anhydrous MgSO4. After
removal of solvents, the residue was chromatographed on silica
gel (220–400 mesh) with chloroform/ethyl acetate=5:2 v/v as
eluent to give 0.98 g (60%) of IP as a pink solid. 1H NMR
(DMSO-d6, 400 MHz): δ (ppm) 8.39-8.37 (d, J = 9.2Hz, 1H),
8.31 (s, 1H), 8.26-8.22 (t, J = 16.4 Hz, 2H), 8.18-8.16 (d, J = 7.6
Hz, 1H), 8.12-8.09 (t, J = 9.2 Hz, 2H), 8.07-8.02 (m, J = 19.2 Hz,
2H), 7.80-7.78 (m, J = 8.4 Hz, 1H), 7.68-7.65 (t, J = 11.2 Hz,
1H), 7.48-7.46 (m, J = 8.8 Hz, 2H), 6.96-6.94 (m, J = 8.4 Hz,
1H), 6.35-6.34 (d, J = 2.4 Hz, 2H), 6.29 (s, 1H), 6.27 (s, 1H),
6.21-6.20 (d, J = 2.4 Hz, 1H), 6.19-6.18 (d, J = 2.4 Hz, 1H), 4.33
(s, 2H), 3.29-3.21 (m, J = 29.6 Hz, 8H), 3.14-3.12 (d, J = 9.6 Hz,
4H), 2.87-2.82 (m, J = 19.2 Hz, 2H), 1.23-1.20 (d, J = 8.0 Hz,
1H), 1.03-0.99 (t, J = 13.6 Hz, 12H). 13C-NMR (DMSO-d6, 400
MHz): δ(ppm) 170.86, 167.51, 153.63, 152.51, 148.31, 134.05,
132.69, 130.76, 130.29, 130.02, 129.87, 128.44, 128.15, 127.36,
127.15, 126.90, 126.78, 126.08, 125.01, 124.94, 124.59, 124.00,
123.96, 123.52, 123.48, 122.33, 108.01, 104.78, 97.24, 79.15,
64.02, 51.66, 50.33, 43.58, 40.14, 39.93, 39.72, 39.51, 39.30,
39.10, 38.89, 36.93, 20.72, 12.33. Anal.Calcd.for C49H49N5O3: C,
spectra were recorded on a Bruker 400 MHz instrument. For
NMR spectra and titration DMSO-d6, D2O were used as solvent
using TMS as an internal standard. Chemical shifts are expressed
1
1
in δ ppm units and H–1H and H–C coupling constants in Hz.
Fluorescence spectra were recorded on a PerkinElmer Model LS
55 spectrophotometer. UV spectra were recorded on
a
SHIMADZU UV-3101PC spectrophotometer. The following
1
abbreviations are used to describe spin multiplicities in H NMR
spectra: s = singlet; d = doublet; t = triplet; m = multiplet.
2.2. Synthesis procedure.
Scheme 1: Synthesis procedure of probe IP
2.2.1. Synthesis of 1

3
77.65; H, 6.78; N, 9.24; O, 6.33; Found: C, 77.61; H, 6.70; N,
9.19; O, 6.30.
analytes viz. various metal cations including Fe2+, anions,
phosphates, amino acids and vitamin (Fig. S7) . The non-linear
fitting analysis determined the association constant in the order
of 25.5×104 M-1 for IP-Fe3+complex (Fig. S3).The detection limit
of probe IP was found to be 82 nM for Fe3+ (Fig. S4). The
binding stoichiometry of probe IP and Fe3+ (Fig. S5) was
evaluated 1:1 y Jo ’s plot. Plot of fluorescence intensity as a
function of time confirms that maximum of 20 minutes are
required to complete this host-guest complexation (Fig. S6). pH
titration of probe IP with Fe3+ revealed that the probe IP started
to open the closed spirolactum ring at pH 6 and pink coloration
is observed below pH 6. But surprisingly after complexation with
Fe3+ the deep pink color persists even upto pH 8 (Fig. S8).
2.3. Computational calculations
Ground state optimizations of the structures were performed
using Density functinal theory (DFT) for probe and the probe
analyte complex i.e. IP and IP-Fe3+ complex have been studied
using hybridexchange-correlation functionalB3LYP (Becke,
three-parameter, Lee-Yang-Parr) and 6-31G**/LANL2DZ basis
set implemented at Gaussian 09 program[26]. From the optimized
structures of the IP-Fe3+ complex gives insight into the mode of
binding of iron ion with the probe. CPCM (conductor-like
Polarizable continuum model) solvent model has been used for
H2O medium to incorporate solvent effect in the computational
calculations.
2.4. Fluorescence imaging
Two sets (A and B) of H. Spinosa saplings have been taken for
the experiment. Where A has been submerged in H2O designated
as control (C). B has been treated with 1 mM FeCl3 solution
designated as treated (T). Two sets were kept for 25days.
Dissections of all the parts (Root, stem and leaf) of both the
matured plants were taken for fluorescence imaging. The samples
after treating with IP solution for 1 hr, were observed with 10x
magnification using FITC filter (519 nm) with excitation
wavelength 490 nm under a fluorescence microscope (Dewinter,
Italy) and the photographs were acquired through Biowizard
Figure 1.(a) UV-vis absorption spectra of IP (1 µM) in DMSO-
water (1:8, v/v), 10 mM phosphate buffer (pH 7.0), upon addition
of 1.5 equiv. Fe3+ solution. (Inset) Absorption spectra in the range
of wavelength 310-350nm (b) Fluorescence emission spectra of
IP (1 µM) in after addition of 1.5 equiv. of Fe3+ in DMSO-water
(1:8, v/v), pH 7.0 (10 mM phosphate u er) (λex = 490 nm).
image
analysis
software,
Dewinter
Optical
Inc.
3.2. Selectivity of IP
2.5.Quantitative estimation of Fe3+
The colorimetric observation illustrates that the addition of
Fe3+ to the IP turned the colorless clear solution into dark pink
while the addition of the other metal cations- Al3+, As3+, Fe2+,
Cu2+, Cu+, Zn2+, Cd2+, Pb2+, Hg2+ and Ag+ to IP did not show any
color change. When the similar experiment has been performed
under UV lamp, it was observed that the complex showed a
strong orange-red fluorescence but the presence of other metal
cations in IP could not make any change to its inherent non-
fluorescence (Fig. S9).
From the treated plant used for imaging, 2 g of root, stem and
leaves have been taken and aqueous extracts were prepared by
grinding. Double distilled H2O has been used for extract
preparation. The extracts were filtered through Whatman 41 filter
paper, and then centrifuged at 6800 rpm at 25oC for 8 minutes to
eliminate unwanted particles. Using fluorescence spectroscopic
data the concentration of Fe3+ present in plant cells has been
estimated.
3.3. NMR experiments
3. Results and discussion
NMR titration studies have been performed to elucidate the
interactions of IP with Fe3+. In 1H NMR titration, upon sequential
addition of 1 equiv. Fe3+ solution, the pyrene protons of IP
shifted downfield gradually and the amide proton peak vanished.
All the peaks became broader at the end of the titration (Fig.S10).
This phenomenon is due to change in electron distribution
between two fluorophores of probe IP. Also, the observation of
the downfield shifting of spiro cycle carbon peak from 69 ppm to
150 ppm and disappearance of one of the carbonyl carbon peak at
170 ppm in 13C NMR titration spectra just specifies a strong
interaction between probe IP and its analyte Fe3+ ion (Fig. S11).
3.1. UV-vis and fluorescence spectral behavior of IP with Fe3+
Absorbance and fluorescence titrations were carried out in
DMSO/H2O (1:8, v/v), pH 7.0 (10 mM phosphate buffer) to
investigate the interaction between probe IP and Fe3+ ion. In
presence of Fe3+ , IP gives rapid responses with an absorption at
558 nm (Fig. 1a) along with the formation of two sharp isosbestic
points at 330 nm and 344 nm. The molar extinction coefficient
has been calculated from UV-absorption experiment.
Considering λmax at 342 nm ,Ԑ was found to be 292600 M-1 cm-1
(see SI, UV-vis spectral studies). n luorescence titration spectra
a out18 old enhancement in intensity at 580 nm (λex = 490 nm)
has been observed after the incremental addition of Fe3+ solution
(Fig. 1b) whereas no change in intensity was found for other
3.4. DFT calculations
The energy optimized structures of IP and IP-Fe3+ complex
depict the mode of binding of FeCl3 with probe IP (Fig. S12).

4
Tetrahedron
Oxygen atom of the carbonyl group close to rhodamine moiety,
several lone pair antibonding orbitals of iron centre has been
found to be participating as acceptor orbitals where as the bond
pair orbitals of both the corbonyl groups, N-Hand C-N act as
donor orbitals (Table S5). Thus these theoretical calculations
evident the stabilization of IP-Fe3+complex through E(2) values
deduced for various molecular interactions.
another carbonyl oxygen from the linker chain and nitrogen atom
adjacent to pyrene moiety have satisfied three coordination sites
of Fe3+and other three sites of Fe3+ have been coordinated with
chlorine atoms- these rise an octahedral geometry for IP-Fe3+
complex (Fig. 2).
3.6. Plausible mechanism and explanation
All the above experimental and theoretical findings firmly
correlate the electronic properties of IP-Fe3+ complex. In UV-vis
titration spectra, hike in absorption at the wavelength 558 nm
indicates spirolactum ring opening of rhodamine moiety after the
interaction between probe IP and Fe3+. This ring opening is
responsible for the strong coloration and fluorescence of IP-Fe3+
complex. Moreover, the presence of two distinct isosbestic points
at 330 nm and 344 nm evident the formation of IP-Fe3+ complex
and electron flow from pyrene moiety to electron deficit
rhodamine part. The above interactions have also been
Figure 2. DFT optimized structure of IP-Fe3+ complex.
1
established from the downfield shift of pyrene proton peaks in H
NMR titration.
The total electron density has been calculated and mapped
with ESP for IP and IP-Fe3+ complex. Figure 3(a) indicates that
the electron density is much higher in oxygen atoms of the linker
chain and partially higher in the amine nitrogen compare to that
of pyrene moiety. Possibly electronic transition occurs from the
electron donating centres (O,N) to Fe3+ to form a stable IP-Fe3+
complex (Fig. 3(b)).
Scheme 2: Proposed binding mechanism of IP on addition of Fe3+.
Furthermore, the disappearance of amide proton peak from
1H-NMR spectra and spirolactum carbonyl carbon peak from 13C-
NMR spectra approve the non-covalent interaction of Fe3+ ion
with them. Hence, the plausible binding approach of IP with Fe3+
has been shown in Scheme 2.
3.7. Fluorescence microscopic imaging of different parts of H.
spinosa
Figure 3.The total electron density (iso value=0.0004, -0.131e0-
0.131e0) mapped with ESP for (a) IP and (b) IP-Fe3+complex
Two healthy H. spinosa saplings were chosen and treated
differently for imaging (Fig. S14 & Fig. 4).Root, stem and
leaves from both the plants were dissected and observed under
fluorescence microscope.
Further NBO (Natural Bond Orbitals) analysis has been
carried out to obtain a vivid picture about the binding
phenomenon. Estimation of donor-acceptor (bond-antibond)
interactions in the NBO basis has been done through second-
order perturbative calculations. This analysis has been executed
by examining all possible interactions between donor Lewis-type
NBOs and acceptor non-Lewis NBOs. The hyperconjugative
interaction energy was deduced from the second-order
perturbation approach.[35]
The most important interactions between Lewis and non-
Lewis orbitals with lone pairs are the second order perturbation
energy values E(2). Parenthesized label numbers in Fig S13 ,
Table S3 and S4 show the number of bond pair and lone pair
orbitals at each center. From the hyperconjugative interaction
energy major interactions has been identified. In all the cases
Figure 4. Matured Hygrophila spinosa a) in H2O, b)
treated with 1 mM Fe3+ solution.

5
From the fluorescence imaging studies several interesting
phenomena of accumulation of Iron(III) can be observed and it
can be compared with the control plant (Fig. 5).
Scheme 3: Preparation of plant extracts
Figure 6. Estimation of Fe3+ ion in treated sapling of
Hygrophila spinosa plant extracts. Standard deviations are
given by error bars where, n=3.
Figure 5. Fluorescence microscopic images of dissections of
root, stem and leaf of Hygrophila spinosa plant ( C= control ,
T= Treated with 1 mmolar Fe3+ solution) (A-F) are the
fluorescent images, (G-L) are the corresponding rightfield
images, and (M-R)are the corresponding overlay images.
To validate this experiment, Fe3+ has been added externally to
the extract and for each sample almost 90% of fluorescence
recovery has been found (Table S6).
In root, accumulation has been observed in endodermis
region, cortex and in pith. Where as in the dissection of the
treated stem reveals that the accumulation of Fe3+ occurred
mainly in secondary xylem, cortex, vascular bundle regions. In
case of leaf, the accumulation was in xylem, phloem and cortex
region. The above images describe that Fe3+ accumulation in leaf
and stem is substantially high rather than root.
4. Conclusion
Probe IP has been successfully synthesized, characterized and
applied as a potential chemosensor for selective detection of Fe3+
ion in aqueous medium. Fe3+ interacts with the probe IP through
three binding sites i.e. two carbonyl oxygen and one amine
nitrogen present in IP. During stronger interaction the
spirolactam ring of the rhodamine gets opened up and the
electrons start flowing from pyrene to electron deficient
rhodamine moiety. Spirolactum ring opening is responsible for
the fluorescence “turn on” response of IP in presence of Fe3+ and
the electron flow helps in the formation of stable IP-Fe3+
complex. All these phenomena has been well established by
different spectrometric experiments along with theoretical
calculations. The efficacy of IP has been employed to investigate
the visualization as well as accumulation of Fe3+ ions in various
parts of Hygrophila spinosa through fluorimetry even at
nanomolar concentration. Fluorescence microscopic imaging
studies suggests that accumulation of Fe3+ is more in leaf and
stem compared to root. In future we are in progress to investigate
3.8.Quantitative estimation of Fe3+ in plant extracts
Estimation of Fe3+ in the treated plant extracts (root, stem and
leaf) using fluorescence titration plot has been measured (Scheme
3).While plotting the data in the standard fluorescence titration
curve, concentration of Fe3+ in 2ml of extract has been found out
to be 0.35 µM and 0.74 µM for stem and leaf, respectively (Fig.
6).

6
Tetrahedron
the phenomenon of inter conversion between Fe3+ - Fe2+ in
[20] Bonda, D. J.; Lee, H.; Blair, J. A.; Zhu, X.; Perry, G.; Smith, M.
A. Metallomics. 2011, 3, 267-270.
plant system and storage of iron in such iron containing plants.
[21] Pithadia, A. S.; Lim, M. H. Curr. Opin. Chem. Biol. 2012, 16,
67-73.
[22] Zheng, H.; Weiner, L. M.; Bar-Am, O.; Epsztejn, S.;
Cabantchik, Z. I.; Warshawsky, A.; Youdim, M. B. H.; Fridkin,
M. Design, Bioorganic Med. Chem. 2005, 13 (3), 773–783.
https://doi.org/10.1016/j.bmc.2004.10.037.
5. Acknowledgements
[23] Glendening, E. D.; Badenhoop, J.K.; Reed, A. E.; Carpenter,
J.E.; Bohmann, J. A.; Morales, C. M.; Weinhold, F. NBO 5.0,
Theoretical Chemistry Institute, University of Wisconsin,
PS acknowledges CSIR, India for awarding her the start-up
grant [Project file no. 02(0384)/19/EMR-II dated 20/05/2019].
AG and SD are sincerely thankful to UGC and CSIR, India
respectively for the research fellowship. AG and SM also thank
Kazi Tawsif Ahmed, Dept. of Botany, Visva-Bharati for his
sincere help.
Madison, 2001
[24] Kshirsagar, A. D.; Ingale, K. G.; Vyawahare, N. S.; Thorve,
V. S. Pharmacogn Rev. 2010 (8), 167–171.
.
,
4
https://doi.org/10.4103/0973-7847.70912.
[25] Micronutrient Information Center, Linus Pauling Institute,
Oregon State University, Corvallis, Oregon. April 2016.
Retrieved 6 March (2018).
6. Notes
[26] Chen, Y.T.; Jiang, J. Org. Biomol. Chem. 2012, 10, 4782.
[27] Pandey, R.; Gupta, R. K.; Shahid, M.; Maiti, B.; Misra, A.;
Pandey, D. S. Inorg. Chem. 2012, 51, 298.
AUTHOR INFORMATION
[28] Goswami, S.; Chakrabarty, R. Eur. J. Org. Chem. 2010, 20,
3791.
[29] Ratha, J.; Majumdar, K. A.; Mandal, S. K.; Bera, R.; Sarkar, C.;
Saha, B.; Mandal, C.; Saha, K. D.; Bhadra, R. Mol. Cell.
Biochem. 2006, 290, 113.
Corresponding Author* E-mail: prithidipa@hotmail.com (P.S.).
ORCID Prithidipa Sahoo: 0000-0001-8493-7068
Ayndrila Ghosh: 0000-0003-1779-3863
Saurodeep Mandal : 0000-0003-0515-2209
Sujoy Das: 0000-0003-3921-0999
[30] Das, S.; Mukherjee, U.; Pal, S.; Maitra, S.; Sahoo, P. Org.
Biomol. Chem. 2019, 17, 5230-5233.
[31] Ghosh, A.; Das, S.; Kundu, S.; Maiti, P. K.; Sahoo, P. Sensors
and Actuators B: Chemical. 2018, 266, 80–85.
[32] Das, S.; Rissanen, K.; Sahoo, P. ACS Omega. 2019, 4,
5270−5274.
7. References
[33] Sarkar, H. S.; Das, S.; Uddin, M. R.; Mandal, S.; Sahoo, P.
Asian J. Org. Chem. 2017, 6, 71.
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7
Research Highlights

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

Rhodamine pyrene conjugate (IP) has been
employed for selective estimation of Fe3+.
Investigation of accumulation of Fe3+ in Hygrophila
spinosa by chemosensing method.
Visualization of Fe3+ in different parts of H. spinosa
through fluorescence microscopy.
Spectroscopic and theoretical analysis were done to
establish IP-Fe3+ interaction.