Upper rim modified calix[4]arene towards selective turn-on fluorescence sensor for spectroscopically silent metal ions

Article abstract of DOI:10.1016/j.ica.2020.120133

This work describes the synthesis of a novel sensor calix[4]arene thiosemicarbazone (Lig), consisting of a calix[4]arene thiosemicarbazone moiety capable of detecting Cu²⁺, Hg²⁺, Zn²⁺ colorimetrically and for the selective detection of Zn²⁺ by “Turn On” fluorescence change in DMSO-H₂O (9:1, v/v, pH = 7.2) solution amongst various toxic heavy metal ions. The sensor shows about 240-fold fluorescence enhancements at 474 nm (Stokes shift-140 nm) when bound to a Zn²⁺ ion with a detection limit of about 15 μM for the Zn²⁺ ion. Such fluorescence could be visualized by normal unaided eyes offering a simple, visual detection process. DFT theoretical study has been used to further support the high efficiency of the sensor. The binding ability of the Lig with Zn²⁺ ions has been used to evaluate the biofunctional properties of the synthesized ligand. The synthesized Lig is found to possess the cytotoxic activity and a better penetrating ability towards the A549 human lung cancer cell line.

Full text of DOI:10.1016/j.ica.2020.120133

Inorganica Chimica Acta xxx (xxxx) xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Upper rim modified calix[4]arene towards selective turn-on fluorescence
sensor for spectroscopically silent metal ions
,
Ambigapathi Anandababua, Sambandam Anandana *, Asad Syedb, Najat Marraikib,
Muthupandian Ashokkumarc
a Department of Chemistry, National Institute of Technology, Tiruchirappalli 620 015, India
b Department of Botany & Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
c School of Chemistry, University of Melbourne, Vic 3010, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
This work describes the synthesis of a novel sensor calix[4]arene thiosemicarbazone (Lig), consisting of a calix
[4]arene thiosemicarbazone moiety capable of detecting Cu2+, Hg2+, Zn2+ colorimetrically and for the selective
detection of Zn2+ by “Turn On” fluorescence change in DMSO-H2O (9:1, v/v, pH = 7.2) solution amongst various
toxic heavy metal ions. The sensor shows about 240-fold fluorescence enhancements at 474 nm (Stokes shift-140
nm) when bound to a Zn2+ ion with a detection limit of about 15 µM for the Zn2+ ion. Such fluorescence could be
visualized by normal unaided eyes offering a simple, visual detection process. DFT theoretical study has been
used to further support the high efficiency of the sensor. The binding ability of the Lig with Zn2+ ions has been
used to evaluate the biofunctional properties of the synthesized ligand. The synthesized Lig is found to possess
the cytotoxic activity and a better penetrating ability towards the A549 human lung cancer cell line.
Calix[4]arene
Thiosemicarbazone
Fluorescence sensor
Colorimetry
Mercury
Zinc ion
1. Introduction
are well coordinated with zinc uptake by the human body. Hence,
monitoring the level of zinc in the human body is an indirect way of
Metal toxicity is caused by both essential and non-essential metals
when exceeding their limits in the human environment and the accu-
mulation of excessive amounts of these metals within the body [1,2].
Among the essential metals, zinc cation is the second most abundant that
plays crucial functional roles in the human body such as structural [3],
catalytic [4], Lewis acid [5] regulation of transcription and translation
via its DNA and RNA binding [6], brain function [7] and immune
function [8]. Compared to other toxic metals, zinc is considered to be
relatively harmless to humans [9]. Normally, the human body contains
about 2 g of zinc found in reproductive organs, muscle, liver, and brain.
Despite zinc is considered to be a harmless element, exposure to high
concentrations results in serious health disorders such as interference of
copper uptake for the metabolic process [10]. Alzheimer’s disease is a
chronic neurodegenerative disease that affects most of the human pop-
ulation worldwide, which may be related to an imbalance in copper,
iron, and zinc in human physiology [11]. Deficiency of zinc causes
multiple systemic effects such as growth retardation, weight loss,
infertility, mental disorders, impaired immune function, skin lesions,
and hair loss [12]. Cell growth and cell proliferation metabolic processes
monitoring the uncontrolled cell proliferation processes such as tumor
growth [13–15]. Copper is an essential redox-active metal, which reg-
ulates many biological functions by its bivalent oxidation states (Cu2+
,
Cu+) [16,17]. The usage of copper extensively in chemical industries,
particularly in electronics, leads to the contamination of the environ-
ment. Imbalance in copper levels in the human body disrupts normal
biological functions that lead to health disorders such as oxidative stress,
hepatocellular carcinoma, neurodegeneration, respiratory deficiency,
cardiomyopathy, and pigmentation defects [18]. United States Envi-
ronment Protection Agency (USEPA) quoted that the permissible con-
centration of Cu2+ in water is 1.30 mg/L [19]. Mercury is one of the
potentially harmful elements to living organisms when continuously
exposed. Mercury vapor easily accumulates in the lungs and it can
penetrate the blood–brain barrier (BBB) through vain and gets accu-
mulated in the brain [20]. Once mercury is accumulated in the brain, it
reduces the neurotransmitter production, disturbs the nerve cellular
process, and decreases the secretion of important hormones, thyroid,
and testosterone [21].
According to the United States Environment Protection Agency
* Corresponding author.
E-mail address: sanand@nitt.edu (S. Anandan).
https://doi.org/10.1016/j.ica.2020.120133
Received 12 September 2020; Received in revised form 8 November 2020; Accepted 8 November 2020
Available online 14 November 2020
0020-1693/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Ambigapathi Anandababu, Inorganica Chimica Acta, https://doi.org/10.1016/j.ica.2020.120133

A. Anandababu et al.
Inorganica Chimica Acta xxx (xxxx) xxx
(USEPA) and the World Health Organisation (WHO), the concentration
of mercury in drinking water should not exceed 30 nM Lꢀ 1 [22]. So
monitoring the concentration level of zinc, copper, and mercury in the
human body as well as in the environment is necessary. Spectroscopi-
cally, some heavy metals do not show a unique signature in UV–visible
and IR regions because they either have completely filled or empty d-
orbitals [23]. So the detection of such “spectroscopically silent” heavy
metals is quite difficult and need sophisticated instruments like syn-
chrotron [24], X-ray absorption spectroscopy (XANES) [25], flame
atomic absorption spectrometry (FAAS) [26], graphite furnace atomic
absorption spectrometry (GFAAS) [27], inductively coupled plasma-
optical emission spectrometry (ICP-OES) [28] and inductively coupled
plasma-mass spectrometry (ICP-MS) [29]. UV–visible and fluorescence
spectroscopies are quite common than the above mentioned sophisti-
cated techniques. There is an endless research effort to develop a new
colorimetric and fluorescence sensor for spectroscopically silent heavy
metals [30–37].
bromoethane, hydrazine monohydrate and trifluoroacetic acid were
purchased from Alfa Aesar. Dry solvents and spectroscopic grade sol-
vents were used for the entire synthesis and spectral studies, respec-
tively. Metal salts used were nitrate or chloride salts. Double distilled
(DD) water was used in all experiments. T90 + UV/Visible spectrometer
was used for UV–visible spectral studies and Shimadzu (RF-5301 PC)
spectrofluorometer was used for fluorescence experiments. Thermo
Scientific Nicolet iS5 FT-IR spectrometer was used for FT-IR spectral
measurements. 1H and 13C NMR analysis were recorded by Bruker
Advance DPX 500 MHz spectrometer. pH adjustment was carried out by
Eutech digital pH meter. Time-resolved fluorescence lifetime experi-
ments were carried out using Horiba Jobin Yvon nanosecond pulse diode
laser-based time-correlated single-photon counting (TCSPC) spectrom-
eter and data was processed by IBH DAS 6.2 data analyzer.
2.2. Synthesis of N(naphthalen-1-yl)hydrazinecarbothioamide (1)
Thiosemicarbazones are sulfur analogs of semicarbazones derived
from the condensation of aldehyde or ketone with thiosemicarbazide,
which have been the focus of chemists because of their therapeutic ap-
plications [38–41]. Recently, chemists use thiosemicarbazones exten-
sively in various fields that include catalysis [42], sensors [43–45], and
corrosion [46]. Thiosemicarbazones exhibit sensing property against
both cations and anions owing to their chemical skeletal formulae. The
lone pair electrons in azomethine nitrogen and thiocarbonyl moiety are
responsible for the cation recognition and the hydrogen present in the
nitrogen moiety is responsible for the anion recognition [47]. The
applicability of thiosemicarbazone and their metal complexes are
extended in the field of biology particularly in cytotoxicity [48–51].
Cytotoxicity means toxic for cells via irreversible lethal cellular damage
and alters normal metabolic functions of cells which cause cell death
called necrosis (induced) or apoptosis (programmed) [52]. Generally,
cytotoxic drugs impair the cellular reproductive integrity by inhibiting
the enzymes responsible for cell reproduction or by the interference of
the molecular assembly of a protein responsible for cellular reproduc-
tion via non-covalent interaction [53]. When thiosemicarbazone enters
the biological system, it can coordinate with bio-available metal ions.
These complexes are responsible for the release of reactive oxygen
species (ROS) which will impair various biomolecules and causes cell
death [54]. Thiosemicarbazone and its metal complexes are found to
cause cellular death via apoptosis mechanism for cancer cells [55].
The field of supramolecular chemistry starts from the discovery of
crown ethers by Charles Pedersen [56]. Supramolecular chemistry has
grown massively in various branches of chemistry especially in sensing
[57,58]. Calix[4]arenes are oligomers largely used to sense various
harmful elements [59–63]. Calix[4]arene mainly acts as a great platform
in the sensor field because of its rigid structure, modifications possible
with a lower and upper rim, host–guest properties, and self-assembly
behaviour [64,65]. With the aid of calix[4]arene platform, numerous
strategies are used to sense cations and anions. Familiar strategies such
as photoinduced electron transfer (PET), excimer formation, photoin-
duced charge transfer (PCT) and fluorescence resonance energy transfer
(FRET) are broadly studied with calix[4]arene for sensing [66]. So the
synthetic receptor having calix[4]arene and thiosemicarbazone moieties
may act as a great sensor towards cations and anions. In this work, we
The compound 1 was synthesized using a modified procedure
available in the literature [67]. In a 100 ml RB flask, 1-Napthylisothio-
cyanate (740.96 mg, 4 mmol) was weighed and 30 ml of dry ethanol was
ˆ
added. Hydrazine monohydrate (250 Aµl slightly excess than 4 mmol)
was added dropwise. The reaction mixture was stirred at room tem-
perature for 24 h and the resultant precipitate was filtered off from the
reaction mixture. The precipitate was washed with ethanol and then
recrystallized in methanol. The colorless product yield was 87%. FT-IR
(KBr, cmꢀ 1) (Fig. S1) data showed 3345, 3299, 3248, 3188, 3047,
1595, 1492, 1213, 1073, 892, 805, 768, 507. 1H NMR (500 MHz, CDCl3)
(Fig. S2) δ: 9.45 (s, 1H), 8.04 (s, 1H), 7.90 (m, 3H), 7.84 (t, 3H), 4.08 (s,
2H). 13C NMR (500 MHz, DMSO‑d6) (Fig. S3) δ: 181.7, 135.7, 134.1,
130.2, 128.5, 126.4, 125.8, 125.3, 123.1.
2.3. Synthesis of 1, 3, 5, 7-tetra-tert-butyl-3, 7-diethoxy-calix[4]arene
(2)
The compound (2) was synthesized as per a method available in the
literature [68]. In a 100 ml RB flask, p-tert-butylcalix[4]arene (1.2979 g,
2 mmol) and potassium carbonate (580.48 mg, 4.2 mmol) were
weighed. Then 70 ml of dry acetonitrile was added. This mixture was
refluxed for 2 h to dissolve p-tert-butylcalix[4]arene. After 2 h, this
mixture was cooled to room temperature and a slight excess of bromo-
ˆ
ethane (373 Aµl) was added to the reaction mixture. This reaction
mixture was further refluxed for 48 h. After the completion of the re-
action, acetonitrile was distilled out from the mixture and the precipi-
tate obtained was redissolved in dichloromethane. The dichloromethane
solution was washed with water and brine solution. The organic layer
was collected and dichloromethane was distilled off by a rotary evapo-
rator to obtain a colorless precipitate. This precipitate was purified by
column chromatography using hexane:ethylacetate (80:20) as eluent.
Yield: 83%. FT-IR (KBr, cmꢀ 1) (Fig. S4): 3455, 2961, 1635, 1485, 1194,
1032, 869. 1H NMR (500 MHz, CDCl3) (Fig. S5) δ: 7.83 (s, 2H), 7.03 (s,
4H), 6.85 (s, 4H), 4.32 (d, 4H), 4.10 (q, 4H), 3.33 (d, 4H), 1.64 (t, 6H),
1.27 (s, 18H), 1.01 (s, 18H). 13C NMR (500 MHz, CDCl3) (Fig. S6) δ:
150.6, 150.1, 146.7, 141.5, 133.1, 128.1, 125.5, 122.1, 71.9, 34.0, 33.8,
32.0, 31.7, 31.1, 15.4.
synthesized upper
rim
modified p-tert-butyl calix[4]arene
2.4. Synthesis of 1, 5-diformyl ꢀ 3, 7-di-tert-butyl-3, 7-diethoxy-calix[4]
thiosemicarbazone-based sensor (Lig) which was used to sense spec-
troscopically silent metal ions in aqueous solutions, and its cytotoxic
activity against A549 lung cancer cell line was evaluated.
arene (3)
The compound (3) was prepared following a procedure reported in
the literature [68]. In a 100 RB flask, 1, 3, 5, 7-tetra tert-butyl-3, 7-dieth-
oxy-calix[4]arene (705.03 mg, 1 mmol) and Hexamethylenetetramine
(HMTA) (5.7659 g, 41.13 mmol) were weighed. 80 ml of trifluoroacetic
acid was added. This mixture was refluxed for 48 h. After the completion
of the reaction, the mixture was poured into 500 ml of ice water. A
yellow color precipitate was formed. This precipitate was redissolved in
chloroform and washed with water and brine solution. The chloroform
2. Experimental details
2.1. Materials and methods
1-Napththyl isothiocyanate and hexamethylenetetramine (HMTA)
were purchased from Sigma-Aldrich. p-tert-butylcalix[4]arene,
2

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Fig. 1. Absorption change of Lig (10 µM) when addition (10 µL) of three pH
buffer solutions.
3. Results and discussion
The synthetic pathway of Lig is shown in scheme 1. The character-
ization details are provided in the supporting information (S1–S13). The
first step was the synthesis of naphthalene thiosemicarbazide in ethanol
at normal room temperature. The peak at 1213 cmꢀ 1 (Fig. S1) in FT-IR
spectrum confirms the formation of thiosemicarbazide. The product of
the first step was also confirmed by 13C NMR spectroscopy. The peak at
181.7 ppm (Fig. S3) indicates the formation of thiosemicarbazide. The
second step was the lower rim modification of p-tert-butyl calix[4]arene
with ethyl bromide at an elevated temperature in the presence of po-
tassium carbonate. This modification was confirmed by 1H NMR spec-
trum of compound 2. The two singlets appeared at 1.01 and 1.27 ppm
(Fig. S5) indicate that only two phenolic moieties are functionalized as
ether and the conformation is maintained as a cone structure. It is
possible to tune the upper rim selectivity of calix[4]arene by its lower
rim functionalization [68]. The lower rim bi-functionalization can help
for the selective bis-formylation of the upper rim of calix[4]arene. The
third step was the bis-formylation of calix[4]arene on its upper rim via
ipso substitution of a formyl group with hexamethylenetetramine and
trifluoroacetic acid at an elevated temperature. There is a strong ab-
sorption observed at 1685 cmꢀ 1 and weak doublet absorption observed
at 2798 and 2719 cmꢀ 1 (Fig. S7) due to the stretching vibration of the
carbonyl group and Fermi resonance of aldehyde protons, respectively.
The disappearance of strong singlet around 1.27 ppm, the appearance of
a strong singlet at 9.77 ppm in 1H NMR spectrum (Fig. S8), and the
appearance of a strong peak at 191.07 ppm in 13C NMR spectrum
(Fig. S9) confirm the ipso substitution of formyl group in the upper rim
of calix[4]arene moiety. After ipso formylation, the conformation of the
molecule is maintained in cone structure and is confirmed by the strong
singlet that appeared at 1.05 ppm in the proton spectrum of compound 3
(Fig. S8). The final step is the synthesis of thiosemicarbazone from bis-
formylated calix[4]arene and naphthalene thiosemicarbazide by stir-
ring at room temperature in an ethanol medium. The peak at 1196 cmꢀ 1
(Fig. S10) corresponds to the thiocarbonyl stretching frequency con-
firming the formation of Lig. The presence of a singlet at 1.18 ppm is
responsible for the cone conformation of the entire molecule (Fig. S11).
Scheme 1. Synthesis of calix[4]arene thiosemicarbazone (Lig).
was removed by a rotary evaporator to obtain a yellow precipitate. This
precipitate is purified by column chromatography using hexane:ethyl-
acetate (90:10) as eluent. Yield: 80%. FT-IR (KBr, cmꢀ 1) (Fig. S7): 3349,
2963, 2798, 2719, 1685, 1597, 1479, 1133, 1027. 1H NMR (500 MHz,
CDCl3) (Fig. S8): δ: 9.77 (s, 2H), 9.17 (s, 2H), 7.62 (s, 4H), 6.94 (s, 4H),
4.32 (d, 4H), 4.13 (d, 4H), 3.50 (q, 4H), 1.70 (t, 6H), 1.05 (s, 18H). 13
C
NMR (500 MHz, CDCl3) (Fig. S9) δ: 191.0, 159.5, 149.8, 148.1, 132.0,
129.1, 128.7, 126.0, 72.3, 54.5, 34.2, 31.6, 31.2, 15.3.
2.5. Synthesis of ligand Calix[4]arene thiosemicarbazone (Lig)
In a 100 ml RB flask, compound 1 (304.2 mg, 1.4 mmol) was weighed
and 30 ml of dry ethanol was added. This solution was stirred at room
temperature for 2 min. Compound 3 (390 mg, 0.6 mmol) was dissolved
in a minimum amount of dry ethanol and added dropwise to the above
solution. This mixture stirred at room temperature for 24 h. A yellow
color precipitate was formed. This precipitate was filtered off and
washed with ethanol. Yield: 84%. m.pt: 188 ◦C, FT-IR (KBr, cmꢀ 1
)
(Fig. S10): 3326, 3183, 2962, 1599, 1517, 1475, 1274, 1196, 1102,
1
1028, 771, 515. H NMR (500 MHz, DMSO‑d6) (Fig. S11) δ: 11.75 (s,
2H), 10.15 (s, 2H), 9.29 (s, 2H), 8.02 (d, 2H), 8.00 (s, 1H), 7.94 (d, 2H),
7.88 (d, 2H), 7.77 (s, 4H), 7.60 (m, 4H), 7.55 (m, 4H), 7.26 (s, 4H), 4.23
(d, 4H), 4.09 (q, 4H) 3.51 (d, 4H), 1.65 (t, 6H), 1.10 (s, 18H). 13C NMR
(500 MHz, DMSO‑d6) (Fig. S12) δ: 177.9, 154.8, 150.0, 148.0, 143.3,
136.2, 134.2, 133.6, 131.2, 129.9, 128.4, 127.4, 127.1, 126.5, 126.5,
126.3, 126.22, 125.9, 123.7, 72.2, 34.6, 31.5, 31.2, 15.4. ESI Mass
(Fig. S13) Calculated: 1047.39, Found: 1047.61.
–
The peaks at 11.75 and 10.15 ppm (Fig. S11) correspond to N H pro-
tons of thiourea moiety which show singlet confirming the cone
conformation. The presence of a singlet peak at 8.02 in 1H NMR spec-
trum (Fig. S11) corresponds to imine protons and peaks at 177.9 &143.3
ppm in 13C NMR spectrum (Fig.S12) correspond to thiocarbonyl carbon
and imine carbon, respectively, also confirming the formation of desired
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Fig. 4. Incremental addition of Cu2+(upto 1 eq.) with Lig (10 mg/L). Inset fig.
absorption variation in 380 nm with respect to [Cu2+] (1 eq.).
Fig. 2. Absorption spectral changes of Lig (10 µM) with various metal ions
(1 eq.)
Fig. 5. Incremental addition of Hg2+ (upto 1 eq) with Lig (10 mg/L). Inset fig.
absorption variation in 380 nm with respect to [Hg2+] (1 eq.).
Fig. 3. Incremental addition of Zn2+(up to 1 eq.) with Lig (10 µM). Inset fig.
absorption variation in 380 nm for [Zn2+] (1 eq.).
DMSO:H2O (9:1, v/v, pH = 7.2) solvent. The Lig shows a sharp ab-
sorption band in 255 nm (ε255 = 4,870 L molꢀ 1cmꢀ 1) and broad ab-
sorption band at 334 nm (ε334 = 5,840 L molꢀ 1cmꢀ 1). The absorption
p-tert-butylcalix[4]arene thiosemicarbazone Lig.
bands at 255 nm, 334 nm correspond to π-π* and n-π*, respectively. The
3.1. pH studies
sensing ability of the synthesized Lig was investigated with various
metal ions that included Ag+, Al3+, Cd2+, Cu2+, Fe3+, Hg2+, Mg2+
,
The synthesized Lig is enriched with acidic hydrogen in thio-
semicarbazone moiety. So the pH of the medium plays a major role
while sensing an analyte. To investigate the photophysical properties of
synthesized Lig with respect to pH, UV–visible spectra were recorded at
three different pH conditions. The absorbance difference at 400 nm of
the Lig with and without pH buffer was measured and plotted (Fig. 1).
The absorption difference is large at pH 9.2 (basic medium), and ligand
shows a considerable color change from colorless to a yellow color as a
consequence of tautomerization from thione to thiol [69]. These results
indicate that the synthesized Lig is best suitable for sensing at acidic and
neutral pH conditions and not suitable for metal ions in a basic medium.
In this work, all sensing studies were carried out in aqueous neutral pH
conditions.
Mn2+, Na+, Ni2+, Pb2+, and Zn2+. Among the metal ions, the Lig showed
selectively towards Zn2+, Cu2+, and Hg2+ (Fig. 2) (Fig. S14). To study
the UV–Visible spectral changes and sensing capability of Lig with these
three metal ions, absorption titration was carried out individually. While
titrating with Zn2+, the absorption intensity at 255 nm, 334 nm
decreased gradually and absorption intensity increased at 380 nm
(Fig. 3). The isosbestic point noticed at 360 nm indicates that there is
coordination between Lig and Zn2+ metal ion. While titrating with Zn2+
ion the absorption intensity at 380 nm gradually increases (6 fold) with
increasing concentration of zinc ion (Fig. S15). The linearity curve is not
straight: shows the first bend at first half equivalence and a second bend
at the second half equivalence of zinc metal ion concentration (inset
Fig. 3). The stoichiometry ratio between Lig and Zn2+ is 1:1 found by the
job’s plot method (Fig. S16). The binding constant and limit of detection
for the Zn2+ metal ion is 1.99X104 M, 6.05 (±3) mg/L, respectively
(Fig. S17) [67]. When Lig was titrated against Cu2+ ions peaks at 280
3.2. UV–Visible absorption studies
The absorption studies of the synthesized Lig were investigated in
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3.3. Fluorescence studies
The fluorescence property of synthesized Lig was investigated by
titrating with various metal ions such as Ag+, Al3+, Cd2+, Cu2+, Fe3+
,
Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, and Zn2+. When the synthesized Lig
was excited at 334 nm, it did not show any emission at 474 nm in free
form. The reason behind the non-fluorescent nature of Lig at 474 nm is
–
due to the absence of isomerization of the imine C N double bond in the
–
excited state [67]. After the addition of 1 eq. metal ions, only Zn2+ show
considerable fluorescence change at 474 nm indicating the isomeriza-
–
tion of imine C N double bond in the excited state (Fig. 6). In the case of
–
Cu2+ and Hg2+ ions, Lig did not show any considerable fluorescence
enhancement, due to the quenching behavior of these ions. The
quenching ability of Cu2+ and Hg2+ is a consequence of the para-
magnetic nature of copper and spin–orbit coupling of mercury ions.
Upon the addition of Zn2+ ions, the emission peak at 474 nm shows a
242 fold hyperchromic shift along with a 24 nm bathochromic shift
(Fig. 7). The selectivity of Lig towards Zn2+ ions is shown in the fluo-
rescence bar diagram (Fig. S22). The stoichiometric ratio of Lig with
Zn2+ calculated by fluorescence 15.25 (±3) mg/L, 140 nm respectively
(Figs. S24 & S25) [69]. The detection limit of synthesized Lig is
compared with earlier reported journals (Table S1). Competitive titra-
tion was carried out to study the fluorescence interference ability of
other cations towards Zn2+ ion-induced fluorescence of Lig. By the
addition of 2 equivalents of other cations, we did not observe any
fluorescence quenching of the Lig-Zn2+ complex. The fluorescence in-
tensity at 474 nm was increased during the addition of other interfering
cations which is the consequence of combining effect other of metal ions
with Zn2+ ion (Fig. S26). From the above results, the synthesized Lig
selectively senses Zn2+ ion over various toxic heavy metals without any
interference by fluorescence spectroscopy in aqueous solutions is
confirmed.
Fig. 6. Fluorescence spectrum of Lig (10 mg/L) with various metal ions (1 eq.)
(λEx = 334 nm, λEm = 474).
3.4. Naked-eye detection
A complete and efficient sensor should detect heavy metals via
visible color change; this will enhance the practical applicability of the
sensors. If the color change is detectable to normal unaided eyes, then
the sensing process will be simple and efficient. The naked eye detection
was carried out. For this analysis, a higher concentration of Lig (1 mM)
was used. Upon addition of one equivalent heavy metal ions to Lig so-
lution, only Cu2+, Hg2+ and Zn2+ showed a considerable visible color
change from colorless to a yellow color (Fig. S27) due to the metal ions
induced conjugation extension of imine double bond. When the same
solution was kept under UV light, only Zn2+ showed chartreuse color
fluorescence. Cu2+, Hg2+ ions quench the blue fluorescence of Lig. The
observed quenching may be due to the paramagnetic nature of Cu2+ and
spin–orbit coupling of Hg2+ ions. In order to find the lowest detection
limit of Zn2+ ion by normal unaided eye analysis, we titrated the syn-
thesized Lig with Zn2+ ion having various concentrations. We observed
the considerable color change from colorless while the addition of 100
µL of 0.05X10ꢀ 2 mol/L Zn2+ ion to the synthesized Lig solution
(Fig. S28). When exceeding more than 1 eq., the turbid solution was
observed. This turbidity is due to the metal-Lig complex reaches its
maximum level. If we add a suitable solvent to this complex dissolution
will occur. This may improve the detection limit of the synthesized Lig.
From this result, we confirmed that the synthesized Lig could sense
Cu2+, Hg2+, and Zn2+ ions via color change having the lowest detection
limit of Zn2+ by normal eye is 500 µM concentration range. The syn-
thesized Lig shows better selectivity towards Zn2+ ion over various
metal ions via fluorescence change also confirmed.
Fig. 7. Incremental addition of Zn2+ with Lig (up to 1 eq.) Inset fig. Intensity
variation in 474 nm with respect to concentration of Zn2+ (λEx = 334 nm, λEm
= 474).
nm, 380 nm gradually increased and the peaks at 255 nm and 334 nm
gradually decreased (Figs. 4 & S18). The presence of isosbestic point at
360 nm clearly shows the coordination between Lig and Cu2+ ion. The
absorbance at 380 nm increases gradually with increasing concentration
of Cu2+ and stable after 0.6 eq of Cu2+ ion concentration (Inset Fig. 4).
The calculated binding constant and limit of detection for the Cu2+
metal ion is 8.79X103 M and 15.03 (±3) mg/L, respectively (Fig. S19)
[67]. When the Lig was titrated against Hg2+ ions, the peaks at 255 nm
and 334 nm gradually decreased with increasing concentration of Hg2+
ions (Figs. 5 & S20). The 334 nm peak shows a bathochromic shift to-
wards higher wavelength along with hypochromic shift and the presence
of an isosbestic point at 360 nm infer the coordination between Lig and
Hg2+ ion. The absorption intensity at 380 nm with respect to the con-
centration of Hg2+ ion shows the first bend at 0.5 eq and increase further
addition (Inset Fig. 5). The calculated binding constant and limit of
detection for Hg2+ ion are 5.69X104 M and 8.21 (±3) mg/L, respectively
(Fig. S21) [70].
3.5. Lifetime study
Excited-state lifetime was investigated by the TCSPC fluorescence
technique. The synthesized Lig was excited at 455 nm laser and the
5

A. Anandababu et al.
Inorganica Chimica Acta xxx (xxxx) xxx
electron cloud found concentrated on the naphthalene moiety. Such an
electron cloud movement may be due to due to isomerization of imine
–
C
N double bond in the excited state. This will facilitate some sort of
–
charge-transfer complex which results in the electron injection between
the Lig moiety and the metal ions. The HOMO-LUMO energy difference
of metal-free Lig frontier molecular orbitals is 1.07 eV and metal bound
Lig frontier molecular orbital is 0.15 eV. Such decrement of the energy
gap from metal-free form to metal bounded form is the consequence of
stabilization by Zn2+ both HOMO and LUMO of Lig orbitals (Fig. 9). The
binding mode of synthesized Lig was confirmed by FT-IR, 1H NMR, and
Mass spectrometry. After the addition of one equivalent of Zn2+ to Lig
the disappearance of the peak at 3326 cmꢀ 1 and shift as well as intensity
decrement of 1517 cmꢀ 1, 1196 cmꢀ 1 peaks noticed which confirms the
binding of the Zn2+ ion to Lig (Fig. S31). The disappearance of NH
protons at 11.75, 10.15 ppm, and downfield shift of imine proton also
confirm the binding of synthesized Lig with Zn2+ (Fig. S32). The singlet
appeared around 1 ppm is remain singlet after the addition of metal ions.
This observation confirms that there is no conformation changes
occurred. Besides, found Lig + Zn2+ mass adduct peak at 1168.8 M/Z in
the HR-LCMS spectrum also confirms the binding ability of the synthe-
sized Lig towards Zn2+ ion (Fig. S33). The singlet appeared around 1
ppm is remain singlet after the addition of metal ions. This observation
confirms that there is no conformation changes occurred. Based on the
above results, how a binding mode occurs between Lig with Zn2+ ion is
described in Fig. 10. Further, the formation of azine may be responsible
for the color change of Lig after metal binding.
Fig. 8. TCSPC lifetime spectrum of Lig (10 mg/L) and Lig + Zn2+ (1 eq. of
Zn2+ ion).
emission was recorded at 474 nm. Similar experiments were carried out
with the addition of Zn2+ to the Lig solution. The data was processed by
IBH DAS 6.2 data analyzer using biexponential fit. From this analysis,
the excited state lifetime of synthesized Lig was determined to be 0.40
ns. After the addition of one equivalent of Zn2+ ion, there is a slight
change in a lifetime and the value is 0.086 ns (Fig. 8 prompt curve
omitted). This slight change in a lifetime is due to the polarity change in
the solvent medium. So the Zn2+ ion is not acting as a fluorescence
quencher to the synthesized Lig and does not affect the fluorescence
property of the molecule (Figs. S29 & S30). From the above results, it is
concluded that this Lig is very much useful to detect Zn2+ ion in the
aqueous medium.
3.7. Cytotoxicity study
Normally apoptosis occurs during aging to maintain the cell popu-
lation in tissues. Apoptosis leads to cell death through cell shrinkage
whereas necrosis leads to cell death through cell swelling. Drugs having
cytotoxic activity are used to trigger the apoptosis mechanism in cancer
chemotherapy treatment. The cytotoxicity activity of the synthesized Lig
was investigated against A549 human lung cancer cell lines by MTT
assay. This study revealed that the half-maximal inhibitory concentra-
tion (IC50) value of the synthesized Lig is 58 ± 0.5 µg/ml against cancer
cell lines after 24 h in vitro treatment (Fig. S34). This study shows that
the synthesized Lig possesses cytotoxic activity against cancer cells. To
investigate the mechanism for killing cancer cells, AO/EB (Acridine
3.6. Computational study and mode of binding
Based on DFT theoretical study, it is inferred that the electron clouds
are concentrated on the thiosemicarbazone moiety of the Lig in the
ground state (HOMO). Whereas in the excited state (LUMO), the
Fig. 9. DFT optimized structure of Lig and Lig + Zn2+ complex.
6

A. Anandababu et al.
Inorganica Chimica Acta xxx (xxxx) xxx
Fig. 10. Proposed binding mode of Lig with Zn2+ ion.
Fig. 11. AO/EB staining images of A549 lung cancer cell lines control sample (a), cancer cell lines + Lig treated cell (up to 24 h) (b), Hoechst staining images of A549
lung cancer cell lines control sample (c), cancer cell lines + Lig treated cell (up to 24 h) (d).
Orange/ Ethidium Bromide) and Hoechst staining studies were carried
out. The AO/EB study can show which type of morphology (apoptosis,
necrosis) is observed during the cell death process. These features are
not observed in Lig untreated control (normal) cancer cell lines (Fig. 11
(a)). During 24 h in vitro treatment, the Lig treated cells show early
apoptosis features such as cell shrinkage, condensation, and fragmen-
tation (Fig. 11 (b)). A very few necrosis types of cell morphology were
also observed with Lig treated cancer cells. In the Hoechst study control
sample (Fig. 11(c)) the apoptosis type of morphology is observed during
the cell death process after 24 h in vitro treatment of cancer cells with the
synthesized Lig (Fig. 11 (d)). This again confirms the observations of
AO/EB Staining study. From these observations, it may be concluded
that the synthesized Lig shows anticancer activity against A549 human
lung cancer cell lines and the mode of cell death majorly followed in
apoptosis (programmed cell death) pathway compared to necrosis
(Induced cell death) (Figs. S35 & S36).
7

A. Anandababu et al.
Inorganica Chimica Acta xxx (xxxx) xxx
Fig. 12. .
3.8. Cellular uptake study
the Rajiv Gandhi National fellowship for disability students’ financial
support. The author (N. Marraiki) extends her appreciation to The Re-
searchers Supporting Project number (RSP-2020/201) King Saud Uni-
versity, Riyadh, Saudi Arabia. The author SA & MA thank MHRD, New
Delhi for sanctioning them a joint Scheme for Promotion of Academic
and Research Collaboration project (SPARC/2018-2019/P236/ SL).
The relative extent of cellular uptake was observed in terms of
intracellular fluorescence intensity exhibited by the treatment of syn-
thesized calix[4]arene thiosemicarbazone Lig in the presence of Zn2+
ions. A549 cells were incubated with the Lig for 6 and 12 h followed by
exposure to Zn2+ ions to identify their localization. (Control sample
Fig. 12. (a)). The Lig mainly be present in the cytoplasm as well as in the
nucleus of the cells. Green fluorescence was observed in the treated cells.
It can be seen that only a small amount of the Lig penetrates through the
cell wall after 6 h (Fig. 12(b)) incubation, whereas after 12 h (Fig. 12(c)),
the Lig penetrates deeply into the cell line. Further studies are required
to confirm the interaction of cellular organelles with the synthesized Lig.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ica.2020.120133.
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CRediT authorship contribution statement
Ambigapathi Anandababu: Conceptualization, Formal analysis,
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