Aroyl hydrazone with large Stokes shift as a fluorescent probe for detection of Cu2+ in pure aqueous medium and in vivo studies

Article abstract of DOI:10.1016/j.jphotochem.2020.112501

An aroyl hydrazone based fluorescent probe, hpsh, has been developed for the selective detection of Cu²⁺ ions in pure aqueous medium by static fluorescence quenching. The fluorescence quenching of hpsh in the presence of Cu²⁺ takes place as a result of ground state complex formation through intramolecular charge transfer (ICT). Addition of Cu²⁺ ions changes the color of the solution from colorless to yellow-green which is clearly visible by naked eye. Large Stokes shift of hpsh prevents the self-quenching of the probe in absence of metal ions. The observed stoichiometry between Cu²⁺ and probe has been found as 1:2 (M: L). MTT assay of hpsh on fruit flies confirms that the probe is non-toxic and biocompatible. The plausible in vivo bioimaging application of the probe to detect Cu²⁺ in Drosophila gut tissues as well as in adult fruit fly has been investigated with excellent results.

Full text of DOI:10.1016/j.jphotochem.2020.112501

JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
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Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Aroyl hydrazone with large Stokes shift as a fluorescent probe for detection
of Cu2+ in pure aqueous medium and in vivo studies
Romi Dwivedia, Saumya Singha, Brijesh Singh Chauhanb, S. Srikrishnab, Anoop Kumar Pandayc,
Lokman H. Choudhuryc, Vinod Prasad Singha,*
a Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi, 221005, India
b Department of Bio Chemistry, Institute of Science, Banaras Hindu University, Varanasi, 221005, India
c Department of Chemistry, Indian Institute of Technology Patna, Bihta, Patna, 801106, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
An aroyl hydrazone based fluorescent probe, hpsh, has been developed for the selective detection of Cu2+ ions in
pure aqueous medium by static fluorescence quenching. The fluorescence quenching of hpsh in the presence of
Cu2+ takes place as a result of ground state complex formation through intramolecular charge transfer (ICT).
Addition of Cu2+ ions changes the color of the solution from colorless to yellow-green which is clearly visible by
naked eye. Large Stokes shift of hpsh prevents the self-quenching of the probe in absence of metal ions. The
observed stoichiometry between Cu2+ and probe has been found as 1:2 (M: L). MTT assay of hpsh on fruit flies
confirms that the probe is non-toxic and biocompatible. The plausible in vivo bioimaging application of the probe
to detect Cu2+ in Drosophila gut tissues as well as in adult fruit fly has been investigated with excellent results.
Fluorescent probe
Large stokes shift
Static quenching
Bio imaging
1. Introduction
pursuit [15,16].
For bio application, a practical imaging agent must be stable, non-
Significant possibilities in the field of fluorescent imaging tools
capable of monitoring specific targets are continuously being explored
in recent years [1,2]. In this regard, many attempts have been made to
design small size molecular fluorophores with oriented chemical,
photophysical and biological properties that monitor toxic transi-
tion metal ions [3,4]. Cu2+ sensors have been the subject of intense
study owing to its environmental significance, pivotal role in various
physiological processes and in a number of neurodegenerative diseases
[5–8]. Excess copper is considered as a biohazard as it is prone to
produce reactive oxygen species (ROS), activating oxidative damage of
protein, nucleic acid and lipids [9]. Although amount of copper can be
calculated precisely by modern analytical techniques viz. Atomic ab-
sorption spectroscopy, Ion selective electrode and inductively coupled
plasma mass spectroscopy [10,11], but these techniques bear some
underlying drawbacks as these are relatively complicated, labor-in-
tensive and costly. These techniques hamper the practical applications
of the probe in biological field. Owing to high sensitivity, selectivity,
easy detection by visual or instrumental methods and fast response,
fluorescence is the most promising tool to meet the demand of in-field
monitoring [12–14]. Therefore, the development of easily accessible
and environment-friendly molecular probes for Cu2+ detection is still in
toxic, fairly soluble in water and produce complete quenching of
fluorophore upon binding the analyte under study. However, in most of
the cases self-quenching takes place in fluorophores showing small
Stokes shifts viz. BODIPY, coumarin, rhodamine and fluorescein [17].
Though they are known to show strong fluorescence in dilute solutions
but turn weakly fluorescent in concentrated solutions or in the solid
state due to severe self-absorption among closely located molecules.
Thus, a large Stokes shift is a great and significant property of a
fluorophore [18]. Sie et al. (2017) [16] have reported a Schiff base
sensor for the detection of Cu2+, Co2+ and Hg2+ ions in water/DMSO
matrix with real sample analysis, Aderinto et al. (2017) [19] have re-
ported an ICT mechanism based Cu2+ sensor in tris-HCl buffer/DMF
matrix with its application in real water samples, Bu et al. (2014) [20]
have reported an indole base based Schiff base for the detection of Cu2+
in DMF, More and Shankarling (2017) [13] have reported a reversible
and turn off fluorescence sensor for Cu2+ ion in ACN-HEPES buffer
media. In all the above reported works there are some drawbacks viz.
absence of biological application, mixed matrix solution, reversibility of
the probe. Keeping these precedencies in view, in this paper, a highly
selective quenching fluorescent probe with large Stokes shift for Cu2+
in aqueous media has been reported, which acts by way of static
⁎ Corresponding author.
E-mail address: singvp@yahoo.co.in (V.P. Singh).
https://doi.org/10.1016/j.jphotochem.2020.112501
Received 24 June 2019; Received in revised form 12 March 2020; Accepted 14 March 2020
Availableonline20March2020
1010-6030/©2020ElsevierB.V.Allrightsreserved.

R. Dwivedi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
quenching mechanism. Herein, the current study attempts to gain a
clear and deeper understanding of this phenomenon by means of in vivo
trapping of Cu2+ in gut tissue of Drosophila.
added Cu2+. To find out standard deviation (σ), the emission intensity
of hpsh without Cu2+ was measured 10 times. The limit of detection
(LOD) was then calculated from the definition given by IUPAC and the
slope of the Stern-Volmer plot according to the equation (Eq. 2) [23]:
2. Experimental
LOD = 3σ/K
(2)
2.1. Reagents
Where, σ is the standard deviation of 10 independent blank measure-
ments and K is the slope of Stern-Volmer plot (Eq. 3) i.e. Fo/F versus
[Cu2+].
Analytical reagent grade 2-hydroxypropiophenone (Sigma–Aldrich,
USA), salicylic acid hydrazide (TCI Chemicals, India), solvents (Merck
Fo/F = 1 + KSV[Q] = 1 + kqτo [Q]
(3)
Chemicals, India), Chloride salts of Na+, K+, Mg2+, Ca2+, Ba2+, Al3+
,
Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ (Merck
Chemicals, India) were purchased and used without any further pur-
ification. Metal salt solutions were prepared in milli Q water.
KSV is the Stern–Volmer quenching constant, τo is average fluores-
cence lifetime of fluorophore i.e. 10−8 s; kq i.e. KSV/τo, is quenching rate
constant [24,25].
2.2. Synthesis of probe
2.5. Median lethal dose (LD50) determination
Synthesis of the probe hpsh was done by the reported method [21]
by refluxing a mixture of salicylic acid hydrazide (10 mmol, 1.52 g) and
2-hydroxypropiophenone (10 mmol, 1.50 ml) solutions in dry ethanol
in a round bottom flask for 3 h. After cooling the above solution to room
temperature, a pale-yellow solid product was obtained. The product
was filtered and washed thoroughly with ethanol. The compound was
purified by recrystallization from hot ethanol and dried over anhydrous
CaCl2 in a desiccator.
In toxicology, to examine the median lethal dose of hpsh, age
matched virgin female and male oregonR+ flies were collected. The 10
virgin females were genetically crossed with 10 male flies in separate
vials having different hpsh concentrations of 10 μM, 50 μM, 100 μM,
250 μM, 500 μM, 1000 μM and 2000 μM. The flies were grown in un-
treated food considered as control. After 15 days of treatment, their F1
adult progeny were scored in reference to control to assess lethality of
hpsh. All experiments were performed in triplicate.
2.3. Synthesis of hpsh-Cu2+ complex
Synthesis of hpsh-Cu2+ complex was done by reacting ethanolic
solution of copper (II) chloride (5 mmol) with hpsh solution (10 mmol)
in 1:2 (M: L) molar ratio in a round bottom flask. The reaction mixture
was stirred on a magnetic stirrer at room temperature for half an hour
to precipitate a green colored product. The product was filtered in a
glass crucible, washed several times with ethanol and finally with
diethyl ether, and dried in a desiccator at room temperature (Scheme
1).
2.6. Fly culture
The wild type Oregon-R+ fruit flies obtained from Bloomington
Drosophila stock center, Indiana, USA, were cultured on standard corn
meal agar media at 24
1
oC in BOD incubator. The wild type Oregon
R+ adult Drosophila (fruit fly) and the gut tissues of Drosophila third
instar larvae were used for the bioimaging studies. The untreated flies
considered as control. Flies were incubated with of 10 μM hpsh, 10 μM
Cu2+ alone and 10 μM hpsh+10 μM Cu2+ respectively in separate food
vials. All experiments were performed in triplicate. Their F1 progenies
had grown to adult. The 15 days old flies were selected for bioimaging
studies through fluorescence microscopy.
2.4. UV–vis and fluorescence measurements
For UV–vis titration and fluorescence titration experiments, 50 μM
and 5 μM aqueous solution of hpsh were used at room temperature.
Milli Q water was used to prepare solution of the cations. Host-guest
binding ratio was obtained from Job’s plot. The binding constant (Kb)
has been obtained from the fluorescence quenching data using modified
Benesi-Hildebrand equation (Eq. 1) [22].
The gut tissues were dissected in PBS (pH 7.4) with the aid of fresh
fine needles under the Stereomicroscope (Magnus Zoom Star-V). The
dissected tissues were incubated separately with 10 μM hpsh and 10 μM
Cu2+ in Maximo cavity slides for 60 min. at room temperature.
Simultaneously, gut tissues were incubated with hpsh for 40 min. and
were subsequently incubated with Cu2+ for 20 min. in a separate cavity
slide. The untreated gut tissues were considered as control. Tissues were
washed twice with PBS after each consecutive step of treatment. The
processed tissues were mounted with PBS on plane glass slide and ob-
served under the Nikon NiU-Upright fluorescence microscope to eval-
uate the intracellular fluorescence intensity.
log (Fo -F)/F = log Kb + n log[Q]
(1)
i.e. log [(Fo − F)/F] vs function of log[Q], a straight line appears in
the plot, whose slope corresponds to n (binding number), Kb is the
binding constant, Fo is the initial emission intensity of hpsh when there
was no metal ion, F is the emission intensity of hpsh after addition of
metal ion and [Q] is the concentration of Cu2+
.
The limit of detection of the receptor hpsh for Cu2+ was calculated
from the plot of fluorescence emission intensity vs concentration of
Scheme 1. Synthesis of hpsh and hpsh-Cu2+ complex.
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R. Dwivedi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
3. Results and discussion
3.1. Spectroscopic characterization of hpsh and hpsh-Cu2+
3.1.1. hpsh
Yield (77 %). M.P. 239 oC. Anal. Calc. for C16H16N2O3 (284.31): C,
67.50; H, 5.56; N, 9.76. Found: C, 67.35; H, 5.46; N, 9.87 %. HR-MS: [M
+ Na]+, m/z, 307.10. IR (cm−1, KBr): ν(O–H) 3415, ν (N–H) 3326, ν (C
= O) 1644, ν (C]N) 1614, ν (N–N) 926. 180. 1H NMR data: 1.23 (3H, J
=7.2 Hz, CH3); 2.90 (2H, CH2), 6.903–8.005 (Ar–H), 11.68 (s, 1H, NH),
13.19 (s, 1H, OH). 13C NMR (DMSO-d6): 11.05 (CH3); 21.22 (CH2);
113.47–132.84 (aromatic carbons);83 163.01 (C]N); 166.76 (C–OH);
185.27 (C = O).
3.1.2. hpsh-Cu2+
Green, Yield (73 %). M.P. 260 oC. Anal. Calc. for C32H30CuN4O6
(630.15): C, 60.89; H, 4.79; N, 8.75; Cu, 10.04. Found: C, 60.69; H,
4.78; N, 8.78; Cu, 10.01 %. HR-MS: [M+H]+, m/z, 631.16. IR (cm −1
,
KBr): ν(OHe), 3380; ν(N–H) 3323; ν(C]O), 1622; ν(CN), 1597; ν(CO]
e
−), 1361; ν(NNe), 957; ν(Cu–O), 536; ν(CueN). 436.
In the IR spectra of hpsh (Fig. S1), the bands for eCN and > CO]
e] groups observed at 1614 cm−1 and 1644 cm−1, respectively, are
shifted to lower frequency (17–22 cm−1) in its Cu2+ complex (Fig. S2)
suggesting coordination of the nitrogen atom of > CN and oxygen atom
of > CO]] groups with metal. The band corresponding to amine
(> NeH) group observed at 3326 cm−1 in hpsh shows a negligible shift
in hpsh-Cu2+ complex indicating non-involvement of > NeH group in
bonding [26]. The presence of a new (CeO−) band in hpsh-Cu2+
complex at 1361 cm−1, suggests bonding of hpsh to Cu2+ via pheno-
late-O [27]. The ν(NNe) band in hpsh is shifted to higher wave number
(31 cm–1) in the complex suggesting the involvement of one of the ni-
trogen atoms of > NN < egroup in bonding [28]. The bands at 3415
cm−1 in hpsh and 3380 cm−1 in Cu2+ complex, are assigned to phe-
nolic–OH [29].
The 1H NMR signals for eCOH and > NHee protons in hpsh (Fig.
S3 ESI) appear at 13.632 and 11.158 ppm, respectively [21]. The sig-
nals observed at 8.05–6.89, 2.49 and 1.23 ppm are assigned for aro-
matic, CH2 and CH3 protons, respectively. 13C NMR (Fig. S4 ESI)
spectrum of hpsh exhibits signals at 158.96, 156.27, 133.64,
131.18–116.86, 19.39 and 10.25 ppm for (C]O), (COH), (CNe]),
aromatic, CH2, and CH3 carbons, respectively. The molecular ion peak
observed at m/z = 307.10 in HR-MS spectrum of hpsh (Fig. S5, ESI)
correspond well to [M + Na]+ validating the molecular formula
Fig. 1. UV–vis spectra of 50 μM aqueous solution of hpsh (a) upon addition of 1
equivalent of aqueous Cu2+ ions. (b) Upon addition of 1 equivalent of aqueous
Na+, K+, Ba2+, Al3+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+ and
Cu2+ ions.
C
16H16N2O3. The HR-MS spectrum of the hpsh-Cu2+ complex shows a
molecular ion peak at m/z, 631.16 [M + Na]+ confirms the molecular
formula, C32H30CuN4O6 (Fig. S6, ESI).
3.2. Absorption studies
The strong peak observed at 340 nm in the absorption spectrum of
hpsh was mainly due to n to π* transitions of imine group. Another
strong peak present in the high-energy region of 294 nm was due to π to
π* transition of aromatic group of hpsh (Fig. 1a) [30]. The absorption
spectra of hpsh was investigated by the presence of several mono/bi/
trivalent metal salts viz. Na+, K+, Ba2+, Ca2+, Al3+, Cr3+, Mn2+
,
Fe3+, Co2+, Ni2+, Zn2+ and Hg2+ in 50 μM aqueous solution of hpsh
showed no significant change (Fig. 1b) while a distinct change was
observed upon the addition of Cu2+ ions. In the presence of Cu2+, the
absorption spectra of hpsh showed a major band at 374 nm with a red-
shift (34 nm) and 298 nm.
Fig. 2. UV–vis titration spectra of 50 μM aqueous solution of hpsh showing
increased absorbance up to 0.7 eq. of aqueous Cu2+ ion while further addition
up to 1 equivalent resulted in decreased absorbance.
UV–vis titration (Fig. 2) was performed to study the binding beha-
vior of hpsh. The successive addition of Cu2+ up to 0.7 eq. resulted an
increase in absorbance while further addition of Cu2+ up to 1 equiva-
lent caused a decrease in absorbance at λmax =374 nm indicating
complex formation by C]N coordination in ground state in 1:2 (M: L)
stoichiometric ratio [19].
To quantify the reaction ratio between hpsh and Cu2+ ions, a Job’s
plot from UV–vis spectra was constructed by changing the concentra-
tion of Cu2+ ions, which clearly indicated 1:2 (M: L) stoichiometry
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R. Dwivedi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
Fig. 5. Fluorescence titration of 5 μM aqueous solution of hpsh at λem: 517 nm
(λex: 298 nm) upon addition of 0-2 equivalents (10 μM) Cu2+ ion.
Fig. 3. Job’s plot for hpsh-Cu2+ complex showing 1:2 (M: L) stoichiometry.
fluorescence spectra of hpsh was not noticeably affected by cations
under test except Cu2+. On adding 2 equivalents aqueous Cu2+ ions to
5 μM aqueous solution of hpsh, there was drastic decrease in fluorescent
intensity resulted in complete quenching of fluorescence of hpsh (Fig. 4
b). Fluorescence turn-off of hpsh by Cu2+ was further explained by
emission titration of 5 μM aqueous solution of hpsh (Fig. 5).
(Fig. 3) for hpsh-Cu2+ complex.
3.3. Emission studies
Upon addition of successive amount of Cu2+ up to 1 equivalent, the
intensity of the maximum emission at 517 nm quenched completely.
Further addition of Cu2+ up to 2 equivalents reflected no change in the
emission intensity of hpsh. To evaluate the selectivity of hpsh towards
Cu2+, an interference experiment between Cu2+ and rest of the metals
with hpsh was performed. It was observed from Fig. 6 that no inter-
The fluorescence emission spectra of pure hpsh showed strong
fluorescence at 517 nm (λex: 298 nm) with 219 nm Stokes shift. The
peak at 590 nm is due to Rayleigh scattering. Emission intensity of hpsh
was examined against aforementioned ions. As shown in Fig. 4a,
ference was there for the detection of Cu2+ in the presence of Na+, K+
Ba2+, Ca2+, Al3+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+ and
Hg2+
,
.
3.4. pH effect
The influence of pH on the probe has been studied by recording the
absorption spectra of hpsh in the acidic, basic and neutral solution. The
UV–vis. spectrum of hpsh at acidic pH showed slight shift in wavelength
with a decrease in absorbance compared to neutral pH, while at basic
pH, a new peak generated at higher wavelength due to deprotonation of
hpsh followed by extension in conjugation. (Fig. S7) Like absorption
spectra, the influence of pH on the probe hpsh has also been explained
Fig. 4. a) Fluorescence bar diagram of aqueous solution of hpsh (5 μM) + Mn+
Fig. 6. Interference of other metal ions in binary mixture solution of aqueous
hpsh (5 μM) + Cu2+ (5 μM) + Mn+ (10 μM) at λem: 517 nm (λex: 298 nm); Mn
+ = Na+, K+, Ba2+, Ca2+, Al3+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+
(10 μM); Mn+ = Na+, K+, Ba2+, Ca2+, Al3+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+
,
Zn2+, Cd2+ and Hg2+. (b) Fluorescence spectra of hpsh on addition of 2
equivalents of aqueous Cu2+ ions at λem: 517 nm (λex: 298 nm).
and Hg2+
.
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R. Dwivedi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
expression [22]. From Fig. 7(b), very good linear relationship was ob-
served (R ≈ 0.998), and the value of n is close to 2. This means that
binding ratio of hpsh-Cu2+ is in the ratio of 1:2 (M: L). This information
was also supported by UV–vis Job’s plot analysis.
3.6. Binding mode of hpsh-Cu2+ complex and Mechanism of quenching
It is clear from the earlier discussion about the structural char-
acterization of hpsh-Cu2+ complex by FT-IR spectra and HR-MS data
that Cu2+ coordinates with nitrogen atom of > CN, oxygen atom
of > COe] and phenolate-O of the probe hpsh.
Intramolecular charge transfer takes place in system when there is
direct attachment of the receptor to the electron donating/withdrawing
groups in conjugation to fluorophore [34,35]. The hpsh is formulated
such in a way as all the functional groups present in probe come in the
proximity of the fluorophore moiety of the probe. Fig. 8(a). When the
system is irradiated, the fluorophore undergoes intramolecular charge
transfer between donor and acceptor. The donors are the eOH groups
in hpsh and the receptor is the Cu2+ chelating moiety. An effective
fluorescence quenching process happens when receptor Cu2+ chelating
moiety incorporates into fluorescence motifs and an apparent red shift
is observed in the absorption spectrum due to the ICT becoming more
developed. The chelation between hpsh and Cu2+ is facilitated by the
coordination from: (i) the imine group, C]N (ii) the carbonyl group
and (iii) the phenolate oxygen group (Fig. 8(b)).
UV–vis spectral changes of hpsh before and after the addition of
Cu2+ suggested the quenching mechanism to be static in nature due to
the complex formation in the ground state. (Fig. S9). Stern–Volmer
quenching constant calculated from the equation (Eqn. 3) was also
employed to differentiate static quenching from dynamic quenching.
For dynamic quenching kq i.e. KSV/τo (quenching rate constant) of
various quenchers is limited to 2.0 × 1010 L mol−1 s−1 [36,37]. While
in our case, calculated kq (KSV/τo) is 3.12 × 1015 L mol−1 −1, that is
s
Fig. 7. a) Emission intensities of hpsh (5 μM) vs. [Cu2+] at λem:517 nm (λex
:
50,000 times larger than 2.0 × 1010 L mol−1 s−1 supporting static
nature of the quenching, KSV is obtained from the detection limit plot of
hpsh-Cu2+ complex i.e. 3.12 × 107 L mol−1 (Fig. 7(a)).
298 nm) (b) Benesi–Hildebrand plot for hpsh with Cu2+ for 1:2 (M: L) com-
plexation.
by fluorescence study (Fig. S8). At λex: 298 nm, the emission spectra of
hpsh in acidic and neutral medium followed similar pattern while in
basic medium hpsh showed no emission. Since at this excitation wa-
velength there was no absorbance in the UV–vis spectrum.
3.7. Significance of large Stokes shift
Self-quenching takes place mainly in fluorophores having small
Stokes shift. Thus, large Stokes shift is an important property of fluor-
ophore to prevent self-quenching in solid state (Fig. S10) [17]. The
Stokes shift (υA − υF) explains the difference in the properties and
structure of the fluorophore between the ground state (So) and the first
exited state (S1) according to the equation (Eq. 4) [38].
3.5. Detection limit and binding constant
The detection limit (LOD) of hpsh (5 μM) at λem =517 nm (λex
=298 nm) was calculated according to the definition of IUPAC [31]
and the value from the linear fitting of Stern-Volmer plot [23] is 3.03 ×
(νA−νF) = (1/λA−1/λF) × 107 cm−1
(4)
10−7 M in the linearity range of 4.0 × 10–6–1.05 × 10–5 M (R2
=
Intramolecular charge-transfer between HOMO and LUMO of the
chromophore, inter and intra molecular hydrogen bonding of hpsh with
water due to the presence of the polar groups are the responsible causes
for the observed Stokes shift. Polar solvents yield larger Stokes shift
values than non-polar solvents owing to their more favored hydrogen
0.9951) (Fig. 7(a)). It is under the regulatory limits given by WHO and
USEPA [32,33]. Furthermore, the binding constant for hpsh-Cu2+
complex was calculated to be 8.21 × 1012 M-1 by the linear fitting of
the fluorescence titration plot in modified Benesi–Hildebrand
Fig. 8. a) Diagram showing inter and intramolecular H-bonding in hpsh (b) Proposed binding structure of hpsh-Cu2+ complex.
5

R. Dwivedi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
treated with hpsh show strong fluorescence in abdominal region in
green channel (Fig. 10C, D). Flies treated with Cu2+ alone exhibit re-
sults like control flies (Fig. 10E, F). It is clear from Fig. 10 that Cu2+
quenches the fluorescence of hpsh treated flies. (Fig. 10G, H) indicating
the bioimaging property of hpsh in living system.
3.8.3. Bioimaging of gut tissue of fruit fly
The bioimaging of dissected gut tissue of fruit fly exhibits similar
fluorescence properties like adult fruit fly (Fig. 11). Images (A–D) are
the images of untreated gut tissue considered as control. It is clear from
Fig. 11 (G) that tissue treated with hpsh exhibits strong green fluores-
cence while there is no fluorescence in tissue treated with Cu2+ alone
(Fig. 11 K). The last panel of Fig. 11 represents complete fluorescence
quenching of hpsh treated gut tissue after addition of Cu2+ in green
channel (Fig. 11O).
Fig. 9. The histogram represents percentage eclosed flies cultured in normal
and hpsh treated food. The hpsh concentration 2000 μM is median lethal dose
for Drosophila in which 50 % flies eclosion occurs.
4. Conclusions
In this study, we have reported a fluorescent probe, hpsh having
large Stokes shift for selective and distinct quenching of Cu2+ ion in
pure aqueous medium. The ground state complex formation of hpsh
with Cu2+ reveals the quenching to be static in nature. The functioning
of hpsh is regulated by intramolecular charge transfer (ICT) me-
chanism. Thus, hpsh could be applied as a highly selective fluorescence
probe for Cu2+ ion without any interference. Moreover, according to
the Jobs plot, a 1: 2 (M: L) stoichiometry between Cu2+ and hpsh is
formed. The selectivity and cell-permeability of the probe state its
plausible application in the biological signaling and tracking of copper.
bond formation or dipole-dipole interactions due to the presence of ICT
[19].
3.8. LD50 analysis and bioimaging
3.8.1. LD50 analysis
To assess the 50 % lethal effects of hpsh on Drosophila, wild type
flies were cultured in different concentrations of hpsh along with con-
trol in separate vials. Their F1 flies eclosion numbers were scored
shown in graph (Fig. 9). The graph shows that flies grown in normal
food represents total number of eclosed flies considered as control. Flies
treated with 10 μM hpsh also exhibits normal eclosion number near to
control. For evaluation of median lethal dose, result clearly shown in
the following graph exhibited gradual decrease in eclosion number of
flies with increase in hpsh concentrations. The administration of hpsh
concentration 2000 μM exhibited nearly 50 % eclosion and 50 % death
of fly population in contrast to control. Thus, present study confirmed
that lower dose of hpsh is non-toxic and biocompatible for flies.
CRediT authorship contribution statement
Romi Dwivedi: Conceptualization, Methodology, Visualization,
Investigation, Data curation, Writing - original draft, Software. Saumya
Singh: Resources. Brijesh Singh Chauhan: Investigation. S.
Srikrishna: Formal analysis. Anoop Kumar Panday: Software.
Lokman H. Choudhury: Software. Vinod Prasad Singh: Supervision,
Validation.
3.8.2. Bioimaging of adult fruit fly
Declaration of Competing Interest
In vivo studies of adult flies show that there is no fluorescence in
blue channel and basal level of fluorescence (auto-fluorescence) in
green channel in flies considered as control in (Fig. 10 A, B) while flies
There are no conflicts to declare.
Fig. 10. In vivo images of adult fruit fly in blue and green channel fluorescence at 4X objective. Images (A) and (B) are considered as control flies, (C) and (D) are the
hpsh treated flies showing fluorescence in green channel, (E) and (F) are the Cu2+ treated flies, (G) and (H) are the hpsh treated fly upon addition of Cu2+
.
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R. Dwivedi, et al.
JournalofPhotochemistry&PhotobiologyA:Chemistry395(2020)112501
Fig. 11. In vivo images of adult fruit fly gut tissues at 20X objective. The control images (A-D) exhibit no fluorescence in blue and green channel. Images. Panel (E-H)
represents fluorescence in green channel in hpsh (10 μM) treated tissue. Third panel (I–L) represents tissue incubated with Cu2+ only. Fourth panel (M–P) represents
fluorescence quenching of hpsh treated tissue after addition of Cu2+
.
Acknowledgements
[14] Z.C. Liu, Z.Y. Yang, T.R. Li, B.D. Wang, Y. Li, D.D. Qin, M.F. Wang, M.H. Yan,
Dalton Trans. 40 (2011) 9370–9373.
[15] W.L. Chang, P.Y. Yang, J. Lumin. 41 (2013) 38–43.
The authors thank the Head, S.A.I.F., Indian Institute of Technology,
Patna, India for recording mass spectra and fluorescence data.
[16] Y.W. Sie, C.F. Wan, A.T. Wu, RSC Adv. 7 (2017) 2460–2465.
[17] P. Horváth, P. Sebej, T. Solomek, P. Kian, J. Org. Chem. 80 (2015) 1299–1311.
[18] B. He, H. Nie, L. Chen, X. Lou, R. Hu, A. Qin, Z. Zhao, B.Z. Tang, Org. Lett. 17
(2015) 6174–6177.
[19] S.O. Aderinto, Y. Xu, H. Peng, F. Wang, H. Wu, X. Fan, J. Fluoresc. 27 (2017) 79–87.
[20] J. Bu, H. Duan, X. Wang, Res. Chem. Intermed. 40 (2014) 3119–3126.
[21] M. Mishra, K. Tiwari, A.K. Singh, V.P. Singh, Polyhedron 77 (2014) 57–65.
[22] H.A. Benesi, J.H. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703–2707.
[23] B.P. Joshi, J. Park, W.I. Lee, K.H. Lee, Talanta 78 (2009) 903–909.
[24] M.R. Eftink, Fluorescence Quenching Reactions, Plenum Press, New York, 1991, pp.
1–44.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2020.
112501.
[25] J.R. Lakowiz, G. Weber, Biochemistry 12 (1973) 4161–4170.
[26] D.P. Singh, B.K. Allam, K.N. Singh, V.P. Singh, RSC Adv. 4 (2014) 1155–1158.
[27] P. Krishnamoorthy, P. Sathyadevi, A.H. Cowley, R.R. Butorac, N. Dharmaraj, Eur. J.
Med. Chem. 46 (2011) 3376–3387.
[28] R. Dwivedi, D.P. Singh, B.S. Chauhan, S. Srikrishna, A.K. Panday, L.H. Choudhury,
V.P. Singh, Sens. Actuators B Chem. 258 (2018) 881–894.
References
[1] D.T. Hogan, T.C. Sutherland, New J. Chem. 42 (2018) 16469–16473.
[2] D.P. Singh, R. Dwivedi, A.K. Singh, B. Koch, P. Singh, V.P. Singh, Sens. Actuators B
238 (2017) 128–137.
[29] P. Sathyadevi, P. Krishnamoorthy, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvanesh,
N. Dharmaraj, Dalton Trans. 40 (2011) 9690–9702.
[3] S. Nagl, O.S. Wolfbeis, Analyst 132 (2007) 507–511.
[4] A.J. Sanchez, B. Ortiz, V.O. Navarrete, N. Farfan, R. Santillan, Analyst 140 (2015)
6031–6039.
[5] L. Yu, Y. Qu, F. Chai, L. Chen, New J. Chem. 42 (2018) 17478–17485.
[6] E.D. Harris, Nutr. Rev. 59 (2001) 281–285.
[7] J.L. Groff, S.S. Gropper, S.M. Hunt, Advanced Nutrition and Human Metabolism,
West Publishing Company, New York, 1995.
[8] S.G. Liu, N. Li, Y.Z. Fan, N.B. Li, H.Q. Luo, Sens. Actuators B Chem. 243 (2017)
634–641.
[30] V.L. Sii, M.R. Sudarsanakumar, S. Suma, Polyhedron 29 (2010) 2035–2040.
[31] G.L. Long, J.D. Winefordner, Anal. Chem. 55 (1983) 712A–724A.
[32] G.R. You, G.J. Park, J.J. Lee, C. Kim, Dalton Trans. 44 (2015) 9120–9129.
[33] H.S. Jung, P.S. Kwon, J.W. Lee, J.I.I. Kim, C.S. Hong, J.W. Kim, S. Yan, J.Y. Lee,
J.H. Lee, T. Joo, J.S. Kim, J. Am. Chem. Soc. 131 (2009) 2008–2012.
[34] A.P. de Silva, B. McCaughan, B. McKinney, M. Querol, Dalton Trans. 10 (2003)
1902–1913.
[35] J.F. Callan, A.P. de Silva, D.C. Magri, Tetrahedron 61 (2005) 8551–8588.
[36] C. Yang, Y. Liu, D. Zheng, J. Zhu, J. Dai, J. Photochem. Photobiol. A 188 (2007)
51–55.
[9] Y. Christen, Am. J. Clin. Nutr. 71 (2000) 621–629.
[10] T. Saha, A. Sengupta, P. Hazra, P. Talukdar, Photochem. Photobiol. Sci. 13 (2014)
1427.
[37] Y. Hu, Y. Liu, X. Xiao, Biomacromolecules 10 (2009) 517–521.
[38] N.I. Georgiev, V.B. Bojinov, J. Lumin. 132 (2012) 2235–2241.
[11] V. Dujols, F. Ford, A.W. Czarnik, J. Am. Chem. Soc. 119 (1997) 7386–7387.
[12] L.P. Singh, J.M. Bhatnagar, Talanta 64 (2004) 313–319.
[13] P.A. More, G.S. Shankarling, Sens. Actuators B Chem. 241 (2017) 552–559.
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