Smart PET based organic scaffold exhibiting bright “Turn–On” green fluorescence to detect Fe3+ ion: Live cell imaging and logic implication

Article abstract of DOI:10.1016/j.jphotochem.2018.03.011

An efficient and simple photoinduced electron transfer (PET) based fluorescence turn–On probe 7 has been designed and synthesized by bridging naphthalimide and thiophene moieties through a piperazine unit. The photophysical behavior of probe and its affinity toward different metal ions have been investigated in partial aqueous medium, protein medium and live cells. The probe exhibited fluorescence “turn-On” response for Fe³⁺ ion with high sensitivity (limit of detection 0.373 μM) and selectively along with naked-eye sensitive visible green color in the medium. The significant change in photophysical behavior of the probe, upon a complexation between probe and Fe³⁺ is attributed to restriction in PET process. The probe 7 showed excellent biocompatibility and has been utilized in cellular imaging experiment to detect Fe³⁺ in MCF-7 live cells. Also, at molecular level probe 7 mimics the functions of a sequential logic circuit, corresponding to a memory device in which the two inputs (Fe³⁺ and AcO^?) based sequential logic operations mimic the function of a memory element.

Full text of DOI:10.1016/j.jphotochem.2018.03.011

Accepted Manuscript
Title: Smart PET based organic scaffold exhibiting bright
3
+
“
Turn–On” green fluorescence to detect Fe ion: Live cell
imaging and logic implication
Authors: Sushil K. Dwivedi, Ramesh C. Gupta, Rashid Ali,
Syed S. Razi, Sumit K. Hira, Partha P. Manna, Arvind Misra
PII:
S1010-6030(17)31853-1
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.03.011
JPC 11180
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
23-12-2017
7-3-2018
8-3-2018
Please cite this article as: Sushil K.Dwivedi, Ramesh C.Gupta, Rashid Ali, Syed
S.Razi, Sumit K.Hira, Partha P.Manna, Arvind Misra, Smart PET based organic
scaffold exhibiting bright “Turn–On” green fluorescence to detect Fe3+ ion: Live
cell imaging and logic implication, Journal of Photochemistry and Photobiology A:
Chemistry https://doi.org/10.1016/j.jphotochem.2018.03.011
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Smart PET based organic scaffold exhibiting bright “Turn–On” green
3
+
fluorescence to detect Fe ion: Live cell imaging and logic implication
§
Sushil K. Dwivedi, Ramesh C. Gupta, Rashid Ali, Syed S. Razi, Sumit K. Hira , Partha P.
§
Manna and Arvind Misra*
Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi – 221 005
UP INDIA
§Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi –221005
UP INDIA
Corresponding author: arvindmisra2003@yahoo.com; amisra@bhu.ac.in
Tel: +91-542-6702503; Fax: +91-0542-2368127, 2368175.
1

GA
Highlights
1
2
. Photoinduced electron transfer (PET) based fluorescence turn - On probe
3
+
. The probe exhibited fluorescence “turn-On” response for Fe ion with high sensitivity and
selectively along with naked-eye sensitive visible green color.
3
+
3
4
. The excellent biocompatibility and cellular imaging for selective detection of Fe in live cells.
. The molecular level probe mimics the functions of a sequential logic circuit, corresponding to
a memory device.
Abstract
An efficient and simple photoinduced electron transfer (PET) based fluorescence turn -On probe
7
has been designed and synthesized by bridging naphthalimide and thiophene moieties through
a piperazine unit. The photophysical behavior of probe and its affinity toward different metal
2

ions has been investigated in partial aqueous medium, protein medium and live cells. The probe
3
+
exhibited fluorescence “turn-On” response for Fe ion with high sensitivity (limit of detection
0
.373 µM) and selectively along with naked-eye sensitive visible green color in the medium. The
significant change in photophysical behavior of the probe, upon a complexation between probe
and Fe3+ is attributed to restriction in PET process. The probe 7 showed excellent
3
+
biocompatibility and has been utilized in cellular imaging experiment to detect Fe in MCF-7
live cells. Also, at molecular level probe 7 mimics the functions of a sequential logic circuit,
3
+
−
corresponding to a memory device in which the two inputs (Fe and AcO ) based sequential
logic operations mimic the function of a memory element.
3
+
Key word: Turn–On Probe, PET, Fe , Memory Element, Cell Imaging.
Introduction
Contemporary scientific investigations are paying more attention toward the design and
development of smart photoactive organic molecular scaffolds and/or fluorescent probes for the
recognition of biologically and environmentally important cations [1, 2]. The synthesis of small
3

molecules that enable recognition event selective and quantitative for a specific analyte are
important because it may provide a reliable fluorescent response under the specific
microenvironment. In this direction potential organic scaffolds exhibiting metal ion induced
enhanced, “turn-on” emission is more attractive and advantageous in terms of sensitivity,
selectivity, and response time [3, 4].
3
+
Further, among the various important metal ions iron, (Fe ) is the most abundant essential trace
element in the human body. Due to frequent conversion of Fe3+ to Fe2+ and vice versa iron
become vital in many biological processes [5] and metabolic activities [6, 7]. For instance, iron
in the form of hemoglobin [8, 9] work as oxygen carrier in all tissues and assists to transport
electrons as cytochrome [10-12]. It is also an important cofactor for many enzymatic reactions
[
13, 14]. The deficiency of iron in primary stages may cause anemia, and if proper attention is
not taken it may become fatal for organisms due to deficiency in optimum oxygen level [15, 16].
On the other hand, the high concentration of iron in human body may lead to diseases like,
Parkinson, Alzheimer, hepatitis, hemochromatosis, dysfunction of organs, and even cancer [17-
1
9]. In a healthy adult person the total iron content is 4 g (70% in Hgb, 25% in storage) [20].
3
+
Thus, the development of a suitable method to detect Fe in solution and intracellularly in a live
cell is highly demanded.
3
+
Recently, great efforts have been made to develop efficient chemosensors for Fe [21-23]. The
most of the reported fluorescent probes for the recognition of paramagnetic Fe3+ ions exhibit
fluorescence quenching, “turn-Off” emission [24-28]. Contrary to this, the PET mechanism based
fluorescence–Off–On molecular probes which can display distinct colorimetric naked-eye
sensitivity are relatively more reliable in specific affinity assays. Moreover, PET based smart
probes are much efficient to provide qualitative and quantitative information with ease and
4

inexpensive manner through fluorescence “turn-On” response in solution as well as in biological
medium [29-32]. There are three best possible ways to attain enhanced “turn-On” emission. First,
by chelation enhanced fluorescence (CHEF) process in which the designed small molecular
probe can interact with a specific metal ion through a suitable receptor or coordinating sites.
Second one, is by a chemical reaction based chemodosimeter strategy [33] in which the probe
dissociate or generate a new entity in the medium and the third one is by the popular photo-
induced electron transfer (PET) approach wherein enhanced emission can be achieved by the
restriction in the charge transfer upon an interaction with guest species [34, 35]. Nevertheless,
the diversity of CHEF and PET based molecular probes that may detect paramagnetic iron
selectively, through “turn–On” emission are quite rare [5, 36, 38]. However, some rhodamine
and fluorescein dyes based molecular probes [39-42] have been developed for Fe3+ which
showed enhanced emission as a result of opening and/or closing of spirolactam ring.
Therefore, keeping in mind the challenges associated we herein report a photoinduced electron
transfer mechanism (PET) based simple small molecular probe to detect Fe3+ through
fluorescence “turn–On” response in solution and live cells [43-48]. Recently, our research group
has developed some attractive molecular scaffolds for selective detection of biomolecules, metal
ions and anions under different conditions [49-54]. The naphthalimide based molecular probes
previously developed from our group [52-54] have been designed elegantly to get enhanced,
“turn-On” emission through fluorescence resonance energy transfer (FRET) process and were
2
+
2+
explored to detect metal ions (like, Hg and Zn ions) and anions through fluorescence On-
OFF or OFF-On responses. In continuation to that in the present structural motif we have
utilized naphthalimide moiety, as a signaling unit, in conjugation with thiophene and piperazine
3
+
rings to detect iron (Fe ions) selectively and sensitively in solution and live cells. As expected,
5

probe 7 upon interaction with different metal ions displayed fluorescence “turn-On” response
selectively with iron though PET mechanism as a result of selective interaction with coordination
sites available in the form of N and S hetero atoms. The probe 7 has shown good
biocompatibility to detect iron in live cells. Moreover, the observed photophysical properties
have been explored to mimics the functions of a sequential logic circuit, corresponding to a
memory device.
Results and Discussion:
Design and Synthesis of Probe 7: Naphthalimide (NPI) systems are known for good optical
properties and fluorescence quantum yield. The typical fluorescence property of NPI can be
easily manipulated with simple chemical transformations. For instance, the incorporation of
electron donor species such as, amino function, tertiary amines, pyridine or piperazine moieties
at 4-position of NPI unit, promote fluorescence quenching due to efficient photoinduced electron
transfer (PET) mechanism [55]. On the other side, if the flow of electron toward the NPI unit get
restricted either due to some chemical reaction or by the interaction with guest species like metal
ion the quenched emission of NPI unit renovate easily and one can observe enhance, “turn-on”
emission in the medium. Thus, by reducing the electron donating capacity of PET donors with a
metal ion like Fe3+ the quenched NPI fluorescence recovered easily and we can observe
significant “turn-on” emission [56, 57]. Based on this concept the probe 7 has been synthesized
as shown in Scheme 1. In the sequence of reactions, naphthalic anhydride was first subjected to
bromination and then refluxed with methylamine in methanol to get compound 2. Compound 2
and piperazine were refluxed in 2-methoxyethanol under inert atmosphere to get compound 3 in
good yield. Similarly, thiophene-2-carboxaldehyde was first reduced with NaBH in methanol
4
and then treated with thionyl chloride to get compound 6. Finally, the compound 3 and 6 were
6

refluxed in DMF to get desired probe 7 in good yield (58%). The compounds were purified by
column chromatography and well characterized by H NMR, 13C NMR, FTIR and HRMS
spectroscopic data (Supporting information Figure S1-S8).
1
Scheme 1: (i) CH
3
NH
2
/MeOH (ii) Piperazine/2-methoxyethanol/reflux (iii) NaBH
4
/MeOH (iv)
DCM/SOCl (v) K
2
2
CO
3
/DMF.
Photophysical behavior and response of probe 7 for different metal ions:
The observed photophysical behavior of 7 (10 µM) in the solvents of different polarity and water
gradient systems (Figure-S14) have been summarized in table S-1. The molar absorbtivity of the
probe in different solvents system were fairly good. It is worth to mention that the photophysical
behavior of the probe 7 varied in the solvents of different polarity and the emission spectra
showed shift toward longer wavelength region along with increase in Stokes shift. The observed
changes may be attributed to modulation in charge transfer process and energy perturbation
associated with solvents of different polarity. It is interesting to mention that in 40% aqueous-
THF and in 40% aqueous DMSO gradient system the probe 7 showed a weak emission band
almost, “switched-Off” state around at 528 nm. However, when the metal ion interaction studies
were performed in pure THF and 40% aqueous DMSO gradient system metal ion selectively was
3
+
3+
3+
2+
obscure and probe displayed affinities for different metal ions like, Fe , Al , Cr , Cu and
Hg2+ (Figure S15). Moreover, in THF-H
O gradient system probe 7 showed noticeable high
2
7

selectivity for iron. Thus, keeping in mind the compatibility of probe for aqueous and biological
medium optimum 40% aqueous-THF (40% H
2
O-THF; v/v) gradient system has been chosen to
carry out further studies.
Further, the electronic transition spectrum of probe 7 showed a low energy absorption band at
4
-1 -1
4
08 nm (ɛ = 1.1x10 cm M ) and a weak emission band at 528 nm (Ф = 0.01; λex. = 408 nm).
This could be attributed to photo-induced electron (PET) transfer process in which charge
transfer occurred from piperazine unit to naphthalimide moiety. Upon addition of Fe3+ to the
solution of 7 the absorption band centered at 408 nm disappeared and a new band appeared at
4
-1 -1
3+
3
80 nm (ɛ = 1.6x10 cm M ) (Figure 1a). Similarly, upon interaction with Fe the weak
emission intensity of probe exhibited enhanced “turn-On” emission (switched–On) with a blue
shift of 12-14 nm and increase in quantum yield (~ 40 fold, Ф = 0.40). Moreover, the color of
solution changed from a non-fluorescent color to a fluorescent green color, “switched-on”.
+
+
2+
2+
2+
2+
2+
2+
However, the other tested metal ions such as, Na , K , Ca , Cu , Co , Ni , Zn , Fe and
2
+
3+
3+
Hg showed insignificant change in the optical behavior of probe except for Al and Cr ions
Figure 1b). To ascertain the high selectivity of 7 for Fe3+ interference studies have been
(
performed by the addition of competitive metal ions in excess to a solution of probable complex,
3
+
3+
n+
7
.Fe and reversibly, by the addition of Fe to the solution of 7.M . The insignificant change
3
+
3+
in absorption and emission intensity of 7.Fe indicated high selectivity of probe for Fe (Figure
S16, S17). Moreover, to discriminate Fe3+ and Fe ions we also examined the photophysical
behavior of probe 7 in the presence of both the ions in solution, separately and found that probe 7
2+
3
+
has high selectivity for Fe only (Figure S18).
8

Figure 1: (a) Absorption and (b) emission spectra of probe 7 upon interaction with different
metal ions in 40% aqueous THF solution. Image: Naked-eye sensitive fluorogenic response of
probe 7 in the presence of tested metal ions (under UV light; at 365 nm).
The absorption and emission titration studies were carried out to understand the binding affinity
3
+
3+
of probe with Fe (Figure 2a, b). Upon a gradual increase in the concentration of Fe to a
solution of probe the absorption maxima centered at 408 nm exhibited blue-shift and a new
3
+
absorption band appeared at 380 nm. Similarly, upon addition of Fe to a solution of probe the
emission intensity centered at 528 nm (λex = 408 nm) enhanced, “turn-on” significantly. Job’s
plot analysis revealed maxima at mole fraction of 0.5 consistently (Figure 2c). Thus, suggested
3
+
about a 1:1 stoichiometry between probe 7 and Fe . As reported previously [50] the limit of
detection (LOD) for Fe3+ has been estimated through the fluorescence spectroscopic method
-6
(
please see supplementary information) and was found to be 0.373x10 M (Figure S22a, b).
Similarly, the binding constant was estimated through a nonlinear fitting of emission titration
9

4
spectra, using Benesi-Hildebrand (B-H) method and was found to be Kass = 2.2x10 (Figure 2d)
which is comparable with other reported methods (Table – 1).
Figure 2: (a) Absorption and (b) emission titration spectra of probe 7 with increasing
3
+
concentration of Fe in 40% H
2
O-THF solution. (c) Job’s plot and (d) Benesi Hildebrand plot
based on emission data.
3
+
Table-1: Comparable data of probe 7 with previous reported sensor for Fe .
1
0

Detection
Binding
Sensing
S.No. References
Medium of detection
-
1
Limit (M) constant (M ) Response
1
2
3
[58]
[59]
[29]
Water
2.3x10-5
33.0x10-6
0.388x10-6
9.6 × 103
--
2.5x103
Turn-off
Turn-on
Turn-on
CH
3
CN:H
2
O (1:1)
2
THF:H O (1:1)
Tris–HCl buffer
medium(50% MeOH)
4
[34]
0.58x10-6
--
Turn-on
Tris–HCl buffer
5
6
[60]
10.5x10-6
4.0x105
2.2x104
Turn-on
Turn-on
medium
Probe 7
2
THF:H O (6:4)
0.373x10-6
3
+
pH Dependence of probe 7 and 7.Fe :
3
+
The fluorescence behavior of probe 7 as a function of pH in the absence and presence of Fe has
been examined in phosphate buffer (Figure 3a, b). It is interesting to note that relative
fluorescence intensity of probe 7 remained insensitive to alkaline conditions (pH 7 to 14)
whereas, under acidic condition there was significant increase in the relative fluorescence
intensity of probe 7. However, the rise in relative fluorescence intensity was around ~ 40%
between pH 7 to 5. Thus, suggesting that in acidic medium protonation occurred at the nitrogen
atoms of piperazine unit of probe 7 which restrict the photo induced electron transfer process
(PET-Off). Further, we performed the reversibility effect on probe 7 with respect to pH. Under
the strong acid (pH, 1) and alkaline (pH, ≥10) conditions the relative intensity of probe 7 get
enhanced (switched – On) and diminished (switched – Off) respectively, due to the protonation
and deprotonation at piperazine unit. Moreover, upon repeating the cycle for almost 10 cycles the
extent of relative fluorescence intensity remained stable without any considerable loss (Figure
S19). Moreover, we also acquired the emission spectra of probe 7 in the presence of iron at
3
+
different pH. Notably, probe 7 could able to detect Fe in the range of pH 6 to 14. However,
below pH 5.5, probe 7 could not interact with Fe3+ due to the formation of quaternary ions.
3
+
3+
Reversibly, when the probe 7 was first treated with Fe the probable complex, 7.Fe generated
1
1

in the medium, which upon further variation of pHs, (acid and alkaline conditions) remained
insensitive (Figure S20). Thus, it is worth to mention that the distribution of 7 in a broad pH
range is susceptible to protonation below pH ≤ 6.0 while remain unaffected at neutral to alkaline
pHs (pH ≥ 6.0). Therefore, probe 7 can be utilized as an appropriate probe to work under
physiological conditions, (pH, ~7.0).
Figure 3: Relative change in (a) absorption and (b) emission spectra of probe 7 at different pH in
phosphate buffer (10 µM; pH 7.0; 40% H
emission intensity of 7 and 7.Fe at different pH.
2
O-THF). Inset: A pH-emission plot shows change in
3
+
3
+
Behavior of 7.Fe with anions:
1
2

3
+
Figure 4: (a) Emission spectra of 7.Fe upon interaction with different anions (b) Emission
3
+
-
spectra of 7.Fe upon titration with AcO in 40% H
2
O-THF solution, (c) Stern-Volmer plot
based on emission spectra. Images: (a) Naked-eye sensitive fluorogenic response of complex
3
+
3+
3+
7
.Fe in the presence of tested anions, (b) Change in color of probe 7 with Fe and 7.Fe with
-
AcO (under UV light; at 365 nm).
3
+
In order to understand the high selectivity of probe 7 for Fe the photophysical behavior of
-
-
-
-
-, N -
2-
2-
PO -
2-
probe 7 in presence of different class of anion (F , Cl , Br , I , NO
3
3
, SO
4
, S , H
2
4
, CO
3
-
,
AcO , and SCN (as their inorganic salt) were examined under the similar experimental
condition. Notably, the probe 7 displayed insensitive behavior toward tested anions (Figure S21).
3
+
Next, we examined the emission behavior of the probable complex, 7.Fe toward tested anions.
It is interesting to mention that upon interaction with different class of anions the probable
complex, 7.Fe3+ showed significant fluorescence quenching (switched–Off) selectively with
-
acetate anion (AcO ; 20 equiv) and the relative fluorescence intensity centered at, 528 nm
diminished considerably. Moreover, the naked-eye sensitive distinct green fluorescence of the
3
+
probable complex, 7.Fe also diminished. The other anions failed to exhibit any considerable
1
3

change in the photophysical behavior of the probable complex, 7.Fe3+ (Figure 4). Thus, the
−
interesting change observed with AcO may be attributed to the formation of iron acetate
complex in the medium as reported previously [61]. In order to estimate the extent of
3
+
-
fluorescence quenching Stern-Volmer plot was obtained between the complex, 7.Fe and AcO .
2
From the linear Stern-Volmer plot (R = 0.9991) quenching constant was estimated [62] and was
4
-1
found to be Ksv= 1.13 x 10 M (Figure 4c). Moreover, the estimated limit of detection (LOD) for
-5
acetate anion was found to be 1.4 ppm (1.7 x 10 M) (Figure S22a, c) which is relatively low but
very much comparable to the reported method [61].
Nature of Interaction: To get a deep insight about the nature of interaction between probe 7 and
3
+
1
1
Fe ion we followed H-NMR spectra (Figure 5). The H-NMR spectrum of probe 7 in CDCl
showed typical resonance corresponding to piperazine unit protons at δ 3.32 (Ha; -CH ) and δ
.832 (Hb; -CH ) ppm while methyl proton (Hc; -CH ) linking piperazine and thiophene unit
appeared at δ 3.89 ppm. The methine proton (Hd; -CH) of thiophene ring was observed at δ 6.98
3
2
2
2
2
1
3+
ppm. In contrast, the H-NMR spectra of the probable complex, 7+Fe showed downfield shifts
wherein the piperazine unit proton shifted downfield (∆δ, 0.020 and 0.01 ppm) to appear at δ
2
.851ppm and δ 3.329 ppm, respectively. Similarly, the Hc (∆δ, 0.023 ppm) and Hd protons
showed downfield shift and appeared at δ 3.913 and 6.994 ppm, respectively. Moreover, the FT-
IR spectrum of probe 7 showed typical stretching vibration bands corresponding to C=O and C-
-1
3+
N functions at 1692, 1654, 1285, 1232,1080, 1044 cm which, upon interaction with Fe
exhibited respective shifts in the C-N stretching vibrations (Figure S7, S13). Moreover, in
3
+
HRMS spectra, upon complexation with Fe the molecular ion peak of probe 7 (Figure S8) m/z
at 393.1444 (calc. 391.1354 +2H) changed to appear at 635.0496 (calc. 633.0338+2H) (Figure
S12). Thus, the experimental observation clearly suggesting about the interaction of probe 7 with
1
4

3
+
Fe in a 1:1 stoichiometry in which S atom of thiophene unit and the N atoms of most favorable
boat conformation of piperazine units are involved.
1
-2
3+
Figure 5: Stacked H NMR spectra of 7 (2.1 x 10 M) upon addition of Fe (0-2.0 equiv.) in
CDCl
3
.
Quantum chemical calculations:
The geometry optimization and quantum chemical calculations of probe 7 and for the complex,
3
+
7
0
7
.Fe were performed by density functional theory (DFT) method as implemented in Gaussian
3 suits of program employing basis set B3LYP/6-31G* for 7 and B3LYP/LANL2DZ* for
.Fe3+ to make sure that the optimized structure is a real minimum (Figure 6). The DFT
3
+
optimized structure revealed that probe has interacted with Fe ions through the coordination
sites, N and S atoms available on the piperazine and thiophene units. The two bond lengths
3
+
between Fe and N atoms of piperazine unit were found to be around 2.01 Å and 1.93 Å while
3
+
the bond length between Fe and S atom of thiophene unit was around 2.33Å, respectively.
1
5

Figure 6: DFT optimized minimum energy structure for a plausible mode of binding between
3
+
3+
probe 7 and Fe and DFT optimized HOMO-LUMO energy distribution for 7 and 7.Fe .
3
+
Moreover, the minimum energy linear structure of the probe upon interaction with Fe revealed
a slight bending of the thiophene unit and come close to the piperazine ring along with a twisting
in the naphthalimide ring. Additionally, the optimized structure of the probe 7 clearly suggested
that upon complexation with iron, 7.Fe3+ the stable conformation of piperazine unit, changes
1
6

from chair form to boat form via the stable 5-membered cyclic ring (Figure 6). Furthermore, the
energy distribution parameters HOMO and LUMO for 7 suggested about maximum electron
localization and delocalization around the piperazine and NPI unit, respectively. Similarly, the
LUMO shows more electron density around NPI unit along with energy difference, ∆E = 2.2582
3
+
eV. Similarly, upon complexation with Fe the electron density corresponding to HOMO and
LUMO remained toward thiophene and NPI units respectively, with a decrease in energy level,
∆
E = 0.2166 eV. Thus, the corresponding changes observed in minimum energy structure and
decrease in the energy level between HUMO and LUMO clearly supported about the
involvements of PET process in the current sensing event in which the charge transfer from
piperazine to NPI unit get restricted upon interaction with Fe3+ ions. Consequently, enhanced
emission was observed in the medium.
Analytical application:
3
+
Detection of Fe on cellulose paper strip:
To examine the naked-eye sensitive colorimetric response of probe 7 on solid surface, cellulose
2
TM
paper test strips (1.5 × 0.5 cm ) (Whatman ) were prepared by dipping the pieces of strip in the
solution of probe 7 (10 µM) in THF for 5 min (remained stable, overnight). Similarly, the stock
-2
-3
-4
solutions of ferric nitrate of three different concentrations (2 × 10 , 2 × 10 and 2 × 10 M)
were prepared in water. Now, the air dried test paper strips were dipped in different
concentration solutions of ferric ion for 10 min and after drying in air, strips were visualized
under UV light (at 365 nm). The considerable change in the color of paper strips from a weak
blue fluorescence to green fluorescence clearly suggested about the potential analytical
3
+
application of probe 7 for Fe (Figure 7).
1
7

Figure 7: Fluorogenic response of probe 7 (10 µM) on test paper strips containing different
3
+
-4
-3
-2
concentration of Fe (i) 2.0 x 10 , (ii) 2.0 x 10 and (iii) 2.0 x 10 M.
Detection of Fe3+ in real contaminated water sample: To validate the practical analytical
3
+
utility of probe 7 we made an attempt to determine concentration of Fe in real contaminated
water samples by standard addition method [63]. First, we quantified the fluorescence of probe 7
3
+
(
10 µM) in the presence of various concentration of Fe (0-0.4 M) and the corresponding
calibration plot was used as a standard curve (Figure 8). Prior to real sample detection when
probe 7 was added directly to the water samples, no significant fluorescence enhancement
occurred. However, when the emission spectra of treated contaminated water samples were
3
+
acquired, the recovery of Fe with respect to the standard calibration curve was good. We could
3
+
able to quantify recovered Fe in real water samples within the range of 110 to 92%. Moreover,
the intensity of the weak fluorescent color of the probe changed in to a naked-eye visual strong
green color under UV light at 365 nm (Figure 8).
1
8

3
+
Figure 8: (a) Calibration sensitivity plot of probe 7 for Fe and (b) change in color of 7 and 7+
3
+
Fe solution (A = 0.1, B = 0.2, C = 0.4 µM) in 40% aqueous THF solution. Table: Detection of
3
+
Fe in real water sample.
Off-On switching behavior of 7 and Logic implication:
The fluorescent organic scaffold demonstrating sensitive optical behavior (as output signals)
upon providing specific chemical/photonic inputs, according to logic operations may be utilized
to construct miniaturized electronic devices, logic gates, logic circuits, molecular switches and
computing devices. Recently, our group [47] and others [64, 65] showed the importance of
sequential logic circuits in creating memory devices to store information and perform operation
through feedback loops. In memory devices one of the outputs of the device function as an input
and is memorize as ‘memory element’. To achieve such goals, we intended to analyze the optical
behavior of probe 7 to construct logic circuits. The relative fluorescence intensity higher than the
threshold value (70% at 516 nm R
f
~ 0.7) has been assigned as “1” whereas, lower one as “0” and
has been correlated as “On” and “Off” states for the read out signals, respectively. The observed
1
9

3
+
photophysical behaviors of probe 7 upon providing two chemical inputs viz. ln1 (Fe ) and ln2
-
3+
3+
-
(
AcO ), as 7+Fe and 7+Fe +AcO correlate the “Off-On-Off” switching mechanism.
According to truth table (Figure 9) sequential logic operations 7+Fe3+ displayed high output
emission (at 660 a.u.) whereas, low output emission was obtained when logic operations
3
+
-
7
+Fe +AcO was performed, respectively. Thus, the equipped logic circuit corresponds to a
memory device in which the two inputs: In1 and In2, based sequential logic operations mimic the
3
+
function of a memory element. Figure 9 shows that addition of Fe (2.0 equiv.) to a solution of 7
3
+
revealed enhanced emission (switched-On; at 660a.u.) due to the formation of 7.Fe complex.
−
3+
However, addition of AcO (20 equiv) to a solution of complex, 7.Fe led to fluorescence
quenching (switched-Off) due to formation of iron acetate salt [61] in the medium. Further
3
+
addition of Fe regained the emission which was almost close to the intensity of a complex,
3
+
7
.Fe . As we repeated these sequential logic operations for six cycles, the probe 7 showed good
rewritable switched–On and switched–Off characteristics as well as corresponding visual color
change, with retention of ∼70–80% of relative emission intensity (Figure S23).
2
0

Figure 9: Emission spectra and bar diagram of relative intensities of probe 7 upon applying
3
+
-
inputs (ln1 = Fe ) and (ln2 = AcO ). (a) Memory unit with two inputs (In1 and In2) and one
output and (b) Truth table (1 = On; 0 = Off) for the sequential logic circuit.
Studies of Probe 7 in biological medium:
Further we designed the experiments to investigate the behavior of probe in a biological medium
3
+
and its potential application to detect Fe in live cell. First, we examine the emission behavior of
in a protein medium such as, bovine serum albumin (BSA). This is because the possibility of
7
any sort of nonbonding interaction, H-bonding and electrostatic interactions with macromolecule
as well as interaction with different natural electrolytes present in blood serum cannot be ruled
3
+
out. To ensure, the emission spectra of probe 7 and probable complex, 7.Fe in the presence of
BSA (3μM) were acquired. It has been observed that the emission of probe 7 and probable
3
+
complex, 7.Fe remained independent to even high concentration of BSA (Figure S24). The
3
+
molecular probe 7 in the presence of BSA (10.0 equiv.) showed high affinity for Fe with
fluorescence “turn–On” response. Notably, the increase in relative fluorescence intensity was
2
1

3
+
almost similar to the emission behavior observed for 7.Fe without BSA (Figure S24). Thus, it
3
+
is worth to mention that probe 7 may be utilized to detect Fe in biological medium.
Studies in Living cells and fluorescence imaging with probe 7:
In order to understand the susceptibility of probe 7 for live cell imaging in vitro studies have
been performed in different live cells through fluorescence microscopy. We have used MCF-7
cells, normal human peripheral blood mononuclear cells (PBMC) and red blood cells (RBC) to
understand the biocompatibility, cytotoxicity and cellular uptake ability of our probe and their
3
+
ability to detect Fe ion. The MCF-7cell is a widely studied epithelial cancer cell line derived
from human breast adenocarcinoma. Also, the RBC was used to demonstrate the percent
hemolysis in the presence of probe molecule. In animal body the RBC is the prime candidate to
encounter the effect of any administered agent. Our results suggested that cells were tolerant for
1
8hr in a broad concentration spectrum of probe 7 without any significant loss in cell viability.
Even upon incubation of high concentration of probe 7 around ~92% MCF-7 cells remained
viable (Figure 10A).
Figure 10: Assessment of viability of live cells with different molar concentration of probe 7 (A)
MCF-7 cells (B) Normal human peripheral blood mononuclear cells and (C) Hemolysis of RBC
3
+
in presence of Fe and probe 7.
2
2

A colorimetric assay for the nonradioactive quantification of cellular proliferation, viability, and
cytotoxicity was performed using cell proliferation Kit II (XTT; the second generation
tetrazolium dye, similar to MTT). XTT biological assays suggested that about the good
cytocompatibility of the probe 7 in PBMC cells (Figure 10B). Moreover, hemolysis assays also
3
+
indicated that RBC is tolerant to probe 7 in the absence and presence of Fe ions and very low
or negligible hemolysis (<1%) in RBC population occurred (Figure 10C).
Figure 11: Fluorescence imaging in MCF-7 cells (under bright field, blue and green channel,
3
+
respectively) in the presence different molar concentration of probe 7 and Fe .
Furthermore, the cell imaging studies were performed in MCF-7 cells with probe 7 in the
3
+
-3
-4
-5
-6
absence and presence Fe ions. First, the different concentration (10 , 10 , 10 , and 10 M) of
2
3

probe 7 was incubated in MCF-7 cells and images were acquired. The incubated cells showed
insignificant fluorescence color (Figure 11, bright field). However, when the probe incubated
cells were treated extracellularly with iron a clear fluorescence pattern was observed which was
more prominent in green channel in comparison, to blue channel (Figure 11). Importantly, the
observed fluorescence pattern remained prominent when the concentration of probe 7 and Fe3+
-4
-5
-6
-7
ions were around 10 and 10 M and 10 and 10 M respectively (Figure S25). Thus, the
observed fluorescence pattern suggested about the potential applicability of probe 7 to recognize
3
+
Fe in live cells and also about the spontaneous localization of probe around the nucleus and in
cytosol but not inside the nucleus.
Conclusion:
In summary, we have developed a new efficient PET based molecular probe for a selective
detection of Fe3+ solution and biological medium. The probe 7 displayed enhanced emission,
fluorescence “turn–On” response, upon interaction with iron ion selectively. The bright
fluorescent green color appeared in the medium is sensitive to naked-eyes. The molecular probe
has shown good stability in wide pH range with detection limit in the micro molar range. The
probe has shown excellent biocompatibility and it holds great promise for selective detection of
3
+
Fe in live cells and in further biomedical applications. Moreover, the fluorescence “turn Off –
On” switching response displayed by the probe 7 has been utilized to mimic the function of a
sequential logic circuit, a memory device in which, sequential logic operations of the two
3
+
−
chemical inputs, Fe and AcO ions correspond to a memory element. Overall, the experimental
3
+
observation further strengthens the designing of good sensor motif for the detection of Fe in the
solution, on paper strip and as well as in live cells.
2
4

Acknowledgments:
The authors are thankful to the Council of Scientific and Industrial Research
(CSIR02(0199)/14/EMR-II), and University Grant Commission (UGC) New Delhi for financial
support and fellowships (to SKD, RCG, R.A.) to carry out research works. We are also thankful
to the Head, Department of Chemistry for their valuable support (through DST-FIST, DST-
PURSE, CAS, UGC-UPE programs) and encouragements.
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