An “Off-On-Off” type fluorescent chemosensor for the relay detection of Zn2+ and H2PO4? in aqueous environment

Article abstract of DOI:10.1016/j.ica.2019.119348

A new fluorescence receptor 2-(diethylamino)-N-(quinolin-8-yl) acetamide (L) was synthesized by simple reaction condition. The ligand HL is highly selective and sensitive towards Zn²⁺ ions over other tested metal ions. Upon interaction HL with Zn²⁺, the receptor HL showed a distinct fluorescence enhancement at 496 nm due to chelation enhanced fluorescence (CHEF) leading to visible fluorescence color change from blue to cyan. Subsequently, an in-situ generated L-Zn²⁺ when interacted with various anions, only with H₂PO₄^? showed fluorescence quenching while other ions did not respond. Accordingly, the receptor HL was developed for the highly selective determination of Zn²⁺ and H₂PO₄^? with the lowest detection limit (LOD) of 8 nM and 55 nM respectively. The binding mechanism of HL with Zn²⁺ and L-Zn²⁺ with H₂PO₄^? were studied by both experimental and theoretical studies. The off-on-off sequential of HL with Zn²⁺ and H₂PO₄^? were successfully employed for the INHIBIT logic gate response and cellulose assisted test strips proved, the practical application for qualitative analysis of Zn²⁺ and H₂PO₄^? ions. Finally, the receptor HL was applied for the detection of Zn²⁺ in different water and pharmaceutical samples.

Full text of DOI:10.1016/j.ica.2019.119348

Journal Pre-proofs
An “Off–On–Off” Type Fluorescent Chemosensor for the Relay Detection of
2
+
-
Zn and H PO in Aqueous Environment
2
4
S. Saravana Kumar, Selva Kumar R, S.K. Ashok Kumar
PII:
S0020-1693(19)31271-X
https://doi.org/10.1016/j.ica.2019.119348
ICA 119348
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
27 August 2019
23 November 2019
5 December 2019
Please cite this article as: S. Saravana Kumar, S. Kumar R, S.K. Ashok Kumar, An “Off–On–Off” Type
2
+
-
Fluorescent Chemosensor for the Relay Detection of Zn and H PO in Aqueous Environment, Inorganica
2
4
Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119348
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An “Off–On–Off” Type Fluorescent Chemosensor for the Relay
2+
-
Detection of Zn and H PO in Aqueous Environment
2
4
a
a
a,*,ashok312002@gmail.com
S. Saravana Kumar , Selva Kumar R , S.K. Ashok Kumar
aDepartment of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore-
32014, TamilNadu, India
6
*Corresponding author.
Graphical abstract
Highlights
Designed sensor ‘HL’ and HL-Zn2+ significantly senses Zn2+ and H PO by
-
2
4
fluorescence with high selectivity.
The binding mechanism of HL and HL-Zn towards Zn and H PO were studied by
2
+
2+
-
2
4
(1
H, P) NMR, mass analysis and DFT calculations.
31
2
+
2+
-
The ligand HL and HL-Zn could detect the Zn and H PO down to be 8.8 nM and
2
4
5
5 nM respectively
Abstract
A new fluorescence receptor 2-(diethylamino)-N-(quinolin-8-yl) acetamide (L) was synthesized
2
+
by simple reaction condition. The ligand HL is highly selective and sensitive towards Zn ions
2
+
over other tested metal ions. Upon interaction HL with Zn , the receptor HL showed a distinct
fluorescence enhancement at 496 nm due to chelation enhanced fluorescence (CHEF) leading to
visible fluorescence color change from blue to cyan. Subsequently, an in-situ generated L-Zn2+
-
when interacted with various anions, only with H PO showed fluorescence quenching while
2
4

other ions did not respond. Accordingly, the receptor HL was developed for the highly selective
2
+
-
determination of Zn and H PO with the lowest detection limit (LOD) of 8 nM and 55 nM
2
4
2
+
2+
-
respectively. The binding mechanism of HL with Zn and L-Zn with H PO were studied by
2
4
2
+
-
both experimental and theoretical studies. The off-on-off sequential of HL with Zn and H PO
2
4
were successfully employed for the INHIBIT logic gate response and cellulose assisted test strips
2
+
-
proved, the practical application for qualitative analysis of Zn and H PO ions. Finally, the
2
4
2
+
receptor HL was applied for the detection of Zn in different water and pharmaceutical samples.
Keywords: Fluorescence, Chemosensors, Zinc, Phosphate, CHEF, DFT
1
. Introduction
Molecular fluorescence chemosensors for the selective recognition of cations and anions have
much attention due to diverse ecological, biological and clinical applications [1,2]. The design
and development of fluorescence chemosensors for quantitative detection of bioactive analytes
have gained increasing interest in supramolecular and analytical chemistry due to several
advantages such as high selectivity, sensitivity, cost-effective, low detection limit and on-site
monitoring without any much need of sophisticated instrumentation [3,4]. Upon selective
interaction of the analyte to the recognition site, the ligand optical property is perturbed which
leads to the change (quenching/enhancement/shift) in the original fluorescence signal of the
sensor due to photo induced electron transfer (PET), chelation enhanced fluorescence (CHEF),
internal charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT) and C=N
isomerization [5–7].
Zinc is the second most abundant transition metal in the human body and zinc ions play a vital
role in may cellular process such as DNA synthesis, neurotransmission, gene expression, and
signal transduction [8–10]. It is also responsible for different diseases such as Alzheimer’s
disease, epilepsy, and human ischemic stroke [11,12]. Therefore, the development of fluorescent
chemosensors for the selective and sensitive detection of zinc ions in a biological environment is
1
0
of great importance but is challenging due to zinc’s close-shell d configuration [10,13–15].
Phosphate is an indispensable constituent of two important biopolymers DNA and RNA and
many chemotherapeutic and antiviral drugs [16]. However, an excess agricultural use of

inorganic phosphate causes excessive algal growth, leading to decomposition and decreasing the
-
dissolved oxygen levels [17]. Dihydrogen phosphate (H PO ) is the predominant equilibrium
2
4
-
species of inorganic phosphates at physiological pH. Therefore, H PO sensing and detection
2
4
methods have received much attention [18,19].
In recent years, sequential detection techniques are reported in the literature, in which the
synthesized ligand upon interaction with target analyte-1 produces an optical change. In turn in-
situ generated ligand-analyte-1 complex will be used as recognition for analyte-2. The recent
2
+
2+
3+
literature on sequential detection reports such as Cu -cystine, Hg -picric acid, Al -
2
+
3+
pyrophosphate, Zn -pyrophosphate, and Al -picric acid [20–24]. In the present work, we have
2
+
synthesized a pincer type of ligand HL as a fluorogenic sensor for Zn via CHEF and in-situ
2
+
-
generated L-Zn complex as a sensor for H PO via demetallation in semi-aqueous media.
2
4
2
. Experimental Section
2
.1. Materials and Methods
All metal salts were purchased as nitrate and anion salts as sodium form from SD Fine chemicals
and Sigma Aldrich. 8-aminoquinoline, chloroacetyl chloride and diethylamine were purchased
from AVRA synthesis, India. All solvents were purchased from Merck chemicals and used
without further purification.
The absorption spectra were recorded on a JASCO V-730 UV-Vis spectrophotometer using 1 cm
quartz cell and fluorescence spectra were recorded on a JASCO FP-8200 spectrofluorometer
equipped with Xenon lamp as an excitation source. All instrumental parameters were controlled
by JASCO spectral manager-2. Infrared spectra (IR) were recorded on a Shimadzu Affinity FT-
-1
IR spectrometer in the range of 4000-400 cm . Mass spectra were recorded on a Perkin-Elmer
Clarus 680 GC and Clarus 600 MS. NMR spectra were recorded using 400 MHz Advance
1
13
Bruker NMR spectrometer. H and C NMR chemical shifts were recorded in ppm downfield
from TMS. Fluorescence lifetime analysis was performed using a HORIBA Jobin-Yvon
instrument. The elemental analysis was carried out using Perkin Elmer Elemental Analyzers.
2
+
The energy minimised ground state geometry of HL and L-Zn complex were optimised by
density functional theory (DFT) using gaussian 09W [25]. The Backe three-parameter Lee-Yang-

Parr (B3LYP) method with the standard basis set 6-311G (d, p) for C, H, O, N and S atoms and
the LANL2DZ basis set with effective core potential for Zn atom [26]. Time-dependent density
functional theory (TD-DFT) method was employed to calculate the excited state parameters [27].
2
.2. Synthesis and Characterization of HL and L-Zn2+
The synthesis of ligand HL follows two step process as shown in Scheme 1. Initially,
chloroacetyl chloride (0.8 ml, 1.2 eq.) was dissolved in dry dichloromethane (DCM) was added
slowly to a stirred mixture of quinoline-8-amine (1.16 g, 1 eq.) and triethylamine (0.3 mL) in
º
DCM at 0 C under nitrogen atmosphere and reaction mixture was stirred at room temperature for
overnight. After completing the reaction, the reaction mixture was quenched in water and then
aqueous layer was extracted with DCM. The combined organic layer was washed with 10%
aqueous HCl solution and dried over anhydrous sodium sulphate followed by solvent evaporation
under reduced pressure which afforded the solid product of 2-chloro-N-(quinolin-8-yl)
acetamide. The product was purified by recrystallization from ethanol to give a pure compound
-1
with a yield of 92%. Characterisation (C H ClN O): FTIR (ATR, cm ) 3269 (-NH), 2968 (-CH,
1
1
9
2
1
Ar), 2899 (-CH, Ali), 1678 (C=O), 1240 (C-N). H NMR (400 MHz, DMSO-d ) δ 10.71 (s, 1H),
6
8
.97 (dd, J = 4.2, 1.4 Hz, 1H), 8.62 (d, J = 7.5 Hz, 1H), 8.46 (dd, J = 8.3, 1.3 Hz, 1H), 7.75 (d, J
1
3
=
7.7 Hz, 1H), 7.68 (dd, J = 8.3, 4.2 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 4.62 (s, 2H). C NMR
(
100 MHz, DMSO-d ) δ 165.50, 149.57, 138.44, 137.44, 134.04, 128.36, 127.48, 123.28, 122.81,
6
+
1
17.43, 44.13. HR-Ms (M ): Calculated: 220.0403 and Obtained: 220.0400.
In the second step, a mixture of 2-chloro-N-(quinolin-8-yl) acetamide and diethyl amine in
acetone was refluxed for overnight. The reaction mixture was cooled to ambient temperature and
volatiles was evaporated under reduced pressure. The solid mass was washed with 10% aqueous
HCl solution and extracted using ethyl acetate and dried over anhydrous sodium sulphate
followed by solvent evaporation under reduced pressure afforded the crude product. Further, the
product was purified by using silica gel column chromatography offer the desired product HL
-1
with an yield of 93% [28,29]. Characterisation (C H N O): FTIR (ATR, cm ), 3273 (-NH),
1
5
19
3
1
2
1
7
968 (-CH, Ar), 2819 (-CH, Ali), 1681 (C=O), 1514 (C=N). H NMR (400 MHz, DMSO) δ
1.38 (s, 1H), 8.94 (d, J = 4.1 Hz, 1H), 8.70 (d, J = 7.6 Hz, 1H), 8.41 (d, J = 8.2 Hz, 1H), 7.76 –
1
3
.45 (m, 3H), 3.26 (s, 2H), 2.66 (q, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H). C NMR (100MHz,

CDCl )δ 171.15, 148.47, 139.00, 135.97, 134.49, 127.99, 127.11, 121.60, 121.47, 116.34, 58.95,
3
+
4
8.69, 12.54. HR-MS (M+H) : Calculated: 258.1528 and Obtained: 258.1670. Elemental
analysis (%) for C H N O (257.1528): Calculated C 70.04, H 7.39, N 16.34; Found C 70.09 H
1
5
19
3
8
.01 N 16.36.
To prepare complex, a mixture of HL (1eq.) and Zn(NO ) .6H O (1eq.) in ethanol was refluxed
3
2
2
for 1h. The content of the mixture was allowed to stand at room temperature to get a solid
product which is separated by filtration. The filtered product was washed with ethanol and dried
under vacuum, providing L-Zn2+ complex with a yield of 86%.
Characterisation
C H N O Zn): FTIR (ATR, cm ),3088 (-CH, Ar), 2875 (-CH, Ali), 1620 (C=O), 1481 (C=N).
-1
(
1
5
18
4
4
1
H NMR (400MHz, DMSO-d ) δ 8.93-8.94 (d, 1H), 8.68-8.69 (d, 1H), 8.40-8.42 (d, 1H), 7.5-
6
2
+
7
.68 (m, 3H), 3.25 (s, 2H), 2.65-2.68 (t, 4H), 1.08-1.14 (q, 6H). ESI-TOF mass (L-Zn ):
Calculated: 383.0776 and Obtained: 383.0697. Elemental analysis (%) for C H N OZn
1
5
18
3
(382.0620): Calculated C 47.12, H 4.71, and N 14.66; Found C 46.89, H 4.08 and N 13.96.
2
.3 Preparation of Pharmaceutical Samples
Zinc containing pharmaceutical samples such as Ascazin Syrup manufactured by Apex
Laboratories Pvt. Ltd., has zinc in the form of Zn(CH COO) .2H O while Z&D suspension
3
2
2
manufactured by Dr. Reddy’s Laboratories Ltd., in the form of ZnSO .H O was purchased from
4
2
a local pharmacy shop. To prepare an initial stock solution of these samples, 1 g of drug sample
was dissolved in 1mL of HNO and 10 mL of deionized water. The solution was heated to near
3
boiling for 30 min then contents were cooled and filtered. The pH of the solution was adjusted to
6
.5 using Tris-HCl buffer solution before diluting the contents of the flask to 100 mL deionized
2
+
water. The total concentration of Zn present in the prepared solution was standardized by using
ICP-OES analysis.
3
. Results and Discussion
In the design strategy of ligand HL, begin with 8-aminoquinoline fluorophore was reacted with
chloroacetyl chloride to get an intermediate product chloroquinoline acetamide. This product
further reacted with diethylamine to get final product HL with a yield of 92%. An intermediate
and final product was characterized by FTIR, NMR, and Mass analysis (Fig. 1S to 5S).
Previously, the synthesized HL was used as a ligand to make an organometallic complex with

2
+
2+,
2+
various metal ions like Cu , Ni and Pb as a catalyst in various reactions [28–31]. The ligand
typically called pincer ligand in organometallic chemistry [32] and structurally similar to earlier
2
+
reported Zinquin used for first time intracellular Zn imaging in living cells [33,34]. Till date no
such report to apply this HL as an optical sensor for the selective detection of cations and anions.
2
+
In this work, demonstrated as fluorogenic sensor for the selective detection of Zn and in-situ L-
2
+
-
Zn complex can selectively detect H PO .
2
4
3
.1 Visual Test and UV-Vis Spectral Studies
In order to check the binding ability of sensor HL towards various metal ions. The visible
colorimetric and fluorescence sensing ability were studied under normal and UV light. The
+
+
2+
2+
2+
sensor HL was interacted with 10 eq. of various metal ions such as Na , K , Ca , Mg , Mn ,
3
+
2+
3+
2+
2+
2+
2+
2+
+
3+
3+
3+
2+
3+
3+
4+
Cr , Co , Fe , Ni , Cu , Zn , Cd , Hg , Ag , Ru , Al , In , Pb , Bi , La and Th in
CH CN:H O (8:2, v/v) media. As shown in Fig. 1a, sensor HL show colourless upon interaction
3
2
with selected metal ions while under UV-light, the HL show no fluorescence, upon interaction
2
+
with Zn show cyan color fluorescence visible to naked eye while other metal ions did not show
2
+
such changes (Fig. 1b). This observation reveals that HL selective to Zn ions.
The UV-Vis absorbance spectra of HL (50 μM) in acetonitrile displays two absorption bands at
-1
-1
-1
-1
2
40 nm (ε = 39638 L.M .cm ) and at 315 nm (ε = 8206 L.M .cm ) which may be due to the π-
+
+
2+
2+
π* and n-π* transitions. Upon interaction with10 eq. of metal ions such as Na , K , Ca , Mg ,
2
+
3+
2+
3+
2+
2+
2+
2+
2+
+
3+
3+
3+
2+
3+
3+
Mn , Cr , Co , Fe , Ni , Cu , Zn , Cd , Hg , Ag , Ru , Al , In , Pb , Bi , La and
4
+
Th with HL, absorbance spectra of HL was slightly varied absorbance at 315 nm due to veru
weak complexation occurring between HL and other tested metal ions (Fig. 6S). Particularly,
2
+
when HL interacted with Zn , both absorbance bands are experiencing red-shift to 246 nm (ε =
-1
-1
-1
-1
3
4074 L.M .cm ) and 358 nm (ε = 4336 L.M .cm ). Further, the UV-Vis spectral titration was
2
+
carried out by adding incremental addition of Zn to HL. The spectral response of redshift with
isosbestic points at 287 nm and 331 nm confirms the formation of the host-guest complex (Fig.
2
+
2
). But the selective detection of Zn by using spectrophotometry data will be quite difficult
because of serious interference from other metal ions.

3
.2 ON-OFF Fluorescence Mode for Detecting Zn2+
The metal ions selectivity of HL was investigated by recording the fluorescence spectral
response. A distinct turn-on fluorescence emission at 496 nm was observed in the presence of
2
+
Zn ions upon excitation at 310 nm (Fig. 3a, 7S). The ligand HL doesn’t exhibit fluorescence
this may be due to combined photo-induced electron transfer (PET) induced by the lone pair of
electrons present on nitrogen atom transfer to quinoline ring system and excited-state
intramolecular charge transfer (ESIPT) process due to keto-enol tautomerism of amide proton. In
2
+
2+
the case of L-Zn , all three nitrogen atoms may involve in the complexation with Zn ions lead
to inhibition of the PET and ESIPT process but there is an increase in the chelation-enhanced
2
+
2+
fluorescence (CHEF). Metal ions such as Cd and Hg exhibits small fluorescence change upon
binding with HL while all other tested metal ions did not show any fluorescence change. The
2
+
fluorescence spectral change was very selective to Zn over the other tested metal ions. These
emission changes resulting in a visible fluorescence change of cyan color in the presence of Zn2+
as detected to naked-eye.
2
+
Effect of water content on L-Zn fluorescence change was studied by increasing water content
in the ACN medium. As seen in Fig. 8S, it reveals that spectral response did not alter till 20%
water in ACN medium. Beyond this level, a study decreasing in fluorescence intensity was
noticed may be due to aggregation causes quenching. Hence, all further studies were carried out
in ACN:H O (8:2, v/v) media. Effect of pH was studied in the range of 1.0 to 11.0 on the
2
2
+
emission intensity spectral response of HL and L-Zn . As seen in Fig. 3b, sensor HL did not
2
+
change its emission at 496 nm by varying the pH. But in the case of L-Zn , emission intensity
remains unchanged in the pH range of 4.0 to 9.0 and below pH 4.0, the protonation of the
binding site may decrease the complexation. Beyond pH 9.0, emission intensity decrease may be
due to the formation of insoluble Zn(OH)2.
2
+
To elucidate the sensing ability of HL towards Zn , fluorescence spectral titration was carried
2
+
out. As shown in Fig. 4a, upon interaction of HL with Zn , emission maximum appeared at 496
-1
nm and intensity increased by ⁓ 100 fold compared to HL with large Stocks shift (12097 cm ).
2
+
The binding constant of HL with Zn was calculated using fluorescence titration data using BH
2
+
plot [35]. The linear fit of inverse change in fluorescence intensity with 1/[Zn ] gave the best fit

4
-1
line and estimated association constant was found to be 3.28 ×10 M (Fig. 4b). The binding
2
+
stoichiometry of HL with Zn was studied by Job’s plot method [36]. As shown in Fig. 4c, the
fluorescence derived Job’s plot gave maximum emission at 0.5 mole fraction which confirms the
formation 1:1 stoichiometric complex. Further, the limit of detection (LOD) and limit of
2
+
quantification (LOD) of Zn were calculated using standard IUPAC definition and found to be
-9
-8
8
.8 ×10 M and 2.9×10 M respectively (Fig. 4c) [37,38].
The most important condition for the selectivity is the ability to detect a specific metal ion in the
vicinity of other competing ions. To explore further, the specificity of HL as a fluorogenic
2
+
2+
chemosensor for Zn , the fluorescence intensity was measured in the presence of 1 eq. of Zn
with 10 eq. of interfering ions. The obtained results show that intensity at 496 nm was not much
2
+
affected in the presence of other metal ions which indicates excellent selectivity of Zn over the
other competing ions.The fluorescence emission response of HL with different metal ions
2
+
revealed the remarkable selectivity of Zn over the other tested metal ions.
-
3
.3 OFF-ON Fluorescence Mode for Detecting H PO
2 4
The sensing ability of HL-Zn2+ complex was examined by adding different anions. Upon
-
2+
interaction of H PO with HL-Zn , the fluorescence intensity at 496 nm completely
2
4
-
disappeared. The addition of H PO ions resulted in the fluorescence color change from cyan to
2
4
colorless this change can be easily visual under a UV light (Fig. 6a,b). The fluorescence change
-
2+
upon interaction of H PO ions with HL-Zn can be accounted for strong binding interaction of
2
4
-
2+
2+
H PO with Zn . The selectivity of HL-Zn complex was also checked with other anions such
2
4
-
-
-
-
-
-
-
-
-
as F , Cl , Br , I , CN , NO , HSO , OH and AcO ions, but no significant changes were
3
4
observed (Fig. 6c).
2
+
In order to study, the dynamic binding ability of HL-Zn complex, the fluorescence titration
-
was carried by successive addition of H PO ions. The emission band at 496 nm started to
2
4
-
decrease gradually with an increasing amount of H PO (Fig. 7a). The fluorescence quenching
2
4

2
+
-
of complex at 496 nm due to formation of HL-Zn -H PO ternary complex. The binding
2
4
stoichiometry of HL-Zn2+ with H PO was calculated by applying Job’s plot, the obtained
-
2
4
results show that maximum quenching achieved at mole fraction of 0.66 which supports the
formation of 1:2 stoichiometry complex during the interaction (Fig. 7b). Effect of pH on the
fluorescence quenching of HL-Zn2+ with H PO was studied. The fluorescence intensity
-
2
4
remains constant in the pH range of 4 to 7. All other studies were carried out at pH 6.5 buffer
solution (Fig. 10S). The binding constant of HL-Zn2+ with H PO was calculated from
-
2
4
fluorescence titration data using Bindfit v0.5 program. The non-linear curve fitting of 2:1
7
-2
stoichiometry gave the association constant of 3.84 ×10 M (Fig. 7c). The Stern-Volmer (SV)
-
plot behave linearly at low concentration while at higher concentration of H PO deviates from
2
4
linearity [39]. The non-linear nature of the SV plot can be ascribed to a combination of both
static and dynamic quenching [40]. A linear SV plot was obtained at a lower concentration of
-
4
-1
H PO with an estimated quenching constant of 4.58 ×10 M (Fig. 7b). The LOD and LOQ of
2
4
-
-8
-7
H PO were calculated as per IUPAC guidelines and it found to be5.5 ×10 and 1.87 ×10 M
2
4
2
+
-
respectively (Fig. 11S). The selective quenching of HL-Zn complex with H PO ions in the
2
4
2
+
vicinity of other anions were studied. The emission at 496 nm of HL-Zn complex changes with
-
H PO ions while in the presence of other anions, it did not change. It shows that the HL-
2
4
2
+
-
Zn exhibits highest selectivity towards H PO ions (Fig. 12S).
2
4
3
.4. Binding Mechanism
2
+
The binding mechanism of HL with Zn was instigated using three different techniques such as
1
FTIR, ESI mass and H NMR titration. As shown in Fig 8a, the FTIR spectra of free HL show
-1
vibrational bands at 3273, 2968, 2819, 1681 and 1514 cm corresponds to amide νN-H, aromatic
2
+
vC-H, aliphatic vC-H, carbonyl vC=O, and aromatic vC=N respectively. After complexing with Zn , a
-1
-1
-1
peak at 3273cm of amide v was disappeared and 1681 cm of C=O and 1514 cm o v
are
N-H
C=H
-1
-1
shifted to 1620 cm and 1481 cm respectively. The IR data reveals that the participation of –
2
+
NH and C=N function groups are involved in the complexation with Zn . In order to carry out
NMR titration, initially, HL was dissolved in DMSO-d followed with the sequential addition of
6
Zn(NO ) .6H O in DMSO-d . During titration, the -NH peak appeared at δ 11.56 starts to
3
2
2
6
decrease its intensity and other aromatic signals 8.80-8.84, 8.08-8.11, 7.46-7.53, 7.38-7.41and

bridging -CH signal appeared at 3.29 starts to shielding. As shown in Fig. 8b, after the addition
2
2
+
of 1eq of Zn the -NH signal completely disappeared and other aromatic signals are shifted to
8
.93-8.94, 8.68-8.69, 8.40-8.42, 7.5-7.68, bridging -CH signal at 3.15 and there is a minor
2
1
change in ethyl group signals. The overall change in the chemical shift of the various H NMR
signals clearly indicated the binding of quinoline nitrogen, amide group nitrogen and diethyl
amine nitrogen are coordination to Zn2+ in HL-Zn2+ complex formation. Also, upon
complexation the C=O peak of HL shifted from 171.15 ppm to 161.82 ppm, which indicated the
2
+
formation of HL-Zn complex (Fig. 4S). Further, ESI-time-of-flight (TOF) mass analysis of
2
+
HL-Zn exhibits two major mass peaks appear at m/z 320.0742 and 383.0697. The first peak
2
+
appeared m/z at 320.0742 corresponds to the formation of HL+Zn and the calculated mass is
2
+
-
+
3
20.0736. The second peak appeared m/z at 383.0697 corresponds to HL+Zn +NO +H and
3
calculated mass is 383.0620 (Fig. 8c). Hence, the ESI mass analysis also reveals the formation of
:1 host-guest with one nitro group as its counter ions.
1
31P NMR spectral response studied to confirm ternary complex formation. Accordingly, the 31P
2
+
NMR spectral analysis of neat NaH PO and HL-Zn -NaH PO in DMSO-d were taken (Fig.
2
4
2
4
6
-
31
9
S). The free H PO show P NMR signal at 0.05 ppm upon interaction it got shifted to 0.57
2 4
ppm. This shielding effect reveals the formation ternary complex.
3
.5. Theoretical Studies
In order to study insights into observed photophysical properties of HL and L-Zn2+ were
examined using density functional theory (DFT) calculation. The initial ground state geometries
of HL and L-Zn2+ were optimized by applying B3LYP exchange-correlation method using
standard 6-311G(d,p) basis set for lighter elements such as C, H, N, and O atoms, whereas
LanL2DZ effective core potential used for Zn atom. In order to avoid the computational time, the
geometry was pre-optimized using the semi-empirical method. The ground state optimized
structure of HL show highly pre-organised geometry by exhibiting an intramolecular hydrogen
bonding of amide hydrogen with both the quinoline nitrogen and diethylamine nitrogen and the

2
+
bond length of 2.3568 Å and 2.4439 Å respectively (Fig. 9 and Fig.13S). The L-Zn complex
exhibits square planar geometry and the coordinated nitrogen atoms are plane perpendicular to
the quinoline ring system. The bond length of Zn-N(amide) Zn-N(quinoline), and Zn-N(diethylamine) were
found to be 2.086 Å, 2.055 Å and 2.189 Å respectively. The interaction energy was found to be -
2
20.07 Kcal/mol.
2
+
The frontier molecular orbital analysis of HL and L-Zn shows significant information about
the electronic properties. As shown in Fig. 10, in free HL both HOMO and LUMO orbitals are
located on the quinoline ring with a bandgap of 4.13 eV. Whereas in the case of HL-Zn2+
complex, the HOMO orbitals are mainly located on quinoline ring system and the LUMO
orbitals are mainly located on Zn atom with the bandgap of 3.47 eV, which indicates the possible
electron transfer from ligand-to-metal (LMCT).
2
+
The excited state electronic property of HL and HL-Zn complex was calculated by employing
the time dependent density functional theory (TDDFT) analysis using the ground state optimized
structure. The predicted excitation energy, oscillation strength and orbital contributions are
tabulated in the Table 1S and the spectra was shown in Fig. 14S. The HL ligand exhibits two
absorbance bands at 342.96 nm and 234.42 nm with the oscillation strength of 0.0976 and 0.3078
2
+
respectively. In the case of HL-Zn complex show slightly redshifted band at 437.75 nm and
53 nm with the oscillation strength of 0.0613 and 0.1138 respectively. Overall, experimental
and theoretical spectral characteristics are well matched.
2
3
.6. Molecular Logic gate
As seen from literature reports that fluorescence sensors can act as logic gates. To constrict the
molecular logic gat, we have examined the fluorescence switching of HL by sequence-dependent
2
+
-
emission output at 496 nm, whereas Zn and H PO are the logical inputs. However, upon the
2
4
addition of all three inputs with a different sequence, the fluorescence intensity of HL was
changed differently. A two-input INHIBIT logic gate was successfully mimicked for L. As
2
+
shown in Fig. 11, the output is 1, it means the gate is ON and this occurs when only Zn is

present. If the output is 0, it means the gate is OFF and this occurs in all other cases. As a result,
2
+
-
Zn and H PO exhibits an OFF-ON-OFF switch. Hence, HL can act as INHIBIT logic gate.
2
4
3
.7. Test Paper Strip
2
+
-
Despite notable work on sensing of Zn and H PO as sequential detection, straight-forward
2
4
and cost-effective detection methods are still remaining a challenging one. Such an approach will
be beneficial for effective detection under various experimental conditions. In this context, test
strips were prepared by immersing the filter paper in the HEPES buffer solution of HL and L-
Zn2+ (containing 10% DMF, 10 mM, pH 7.2) followed by drying in air. Such test strips are
2
+
-
directly used by dipping them into an aqueous solution of Zn and H PO . As seen in Fig. 12a,
2
4
the HL contain test strip did not showed any fluorescence. Whereas, upon interaction of Zn2+
solution, it turn cyan colour fluorescence while other tested metal ions do not show such a
2
+
-
change under UV-light. When L-Zn containing test strips on interaction with H PO , it turns
2
4
off the fluorescence and other anions does not show such changes (Fig. 12b). It shows that these
2
+
-
test strips can be used for the qualitative analysis Zn and H PO in aqueous medium (Fig.
2
4
1
2c).
3
.8. Analytical Applications
2
+
The practical utility of HL was studied to estimate the Zn in different water samples such as
distilled water, tap water, and well water. In all these medium, a known amount ofZn2+ was
2
+
added and estimated Zn contents (Table 1). In addition, the ligand HL was applied for the
2
+
determination of Zn in different pharmaceutical samples such as multivitamin tablet and syrup
and results are compared with ICP-OES analysis. As it is observed from Table 1, there is a
2
+
satisfactory agreement between the Zn estimated by present sensor HL with standard ICP-OES
analysis.
The present work was compared with the already reported work in the Table 2[ 41–46]. In
comparison with various sequential detection ligands our HL shows more significant than other
ligands in terms of detection lime and various application studied.

4
. Conclusion
2
+
-
A new pincer type ligand HL was synthesized and applied to the detection of Zn and H PO .
2
4
2
+
This dual fluorescence receptor HL shows upon the interaction with Zn a distinct fluorescence
2
+
enhancement at 496 nm. The ligand HL was found to be selectively interacting with Zn witha
4
-1
1
:1 binding ratio and estimated formation constant of 3.28 ×10 M . The ligand HL could detect
2
+
the Zn down to be 8.8 nM without interfering other interfering fromother tested metal ions. The
2
+
-
in situ generated HL-Zn was found to be selectively uncomplex upon the addition of H PO
2
4
ions among the other tested ions, which resulting in the quenching of emission band at 496 nm
-
leads to detect H PO down to 55 nM.
2
4
Acknowledgment
Authors are thankful to DST-VIT-FIST for NMR, SIF-VIT for GC-MS. Vellore Institute of
Technology, Vellore for providing SEED Grant research facilities.
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Fig. 1. Visual test of HL with various metal ions in the presence of (a) visible (b) UV light in ACN:H O
2
(8:2, v/v) media
2
+
Fig. 2. UV-Vis spectral titration of HL with Zn in ACN:H O (8:2, v/v) media
2
Fig. 3. Fluorescence spectral response of HL with (a) various metal ions (b) Effect of pH
2
+
Fig. 4. (a) Fluorescence spectral titration of HL with Zn (b) Job’s plot (c) BH plot (d) Calibration plot
2
+
Fig. 5. (a) Dynamic response of L-Zn with EDTA (b) Interfering ions effect

2
+
Fig. 6. Colorimetric response of HL-Zn with various anions under (a) visible (b) UV light (c)
Fluorescence spectral response in ACN:H O (8:2, v/v) media
2
2
+
-
Fig. 7. (a) Fluorescence spectral titration of HL-Zn with H PO (b) Job’s plot (c) SV plot (d) Bindfit
2
4
plot
2
+
1
Fig. 8. Binding event studies (a) FTIR spectra of HL and L-Zn complex (b) H NMR titration
2
+
2+
of Zn to HL in DMSO-d (c) ESI-Mass spectra of L-Zn complex
6
2
+
Fig. 9. DFT optimized structure of HL and L-Zn complex using B3LYP method
2
+
Fig. 10. Frontier molecular orbitals of HL and HL-Zn complex
Fig. 11. Molecular circuit and truth table
Fig. 12. Cellulose test strip color change (a) with various metal ions (b) anions and (c) OFF-ON-
OFF response.
Scheme 1. Reagents and conditions to synthesis L
2
+
Table 1. Analysis of Zn in the different water systems and pharmaceutical samples using L
Zn2+
Added
mg/L)
2
+
Zn Found
Recovery
Samples
(mg /L)
(%)
(
1
0
9.87
98.7
98.5
DD water
2
0
19.71

1
2
1
2
0
0
0
0
9.80
19.52
9.64
98.0
97.6
96.4
96.2
97.9
96.6
Tap water
Well water
19.25
63.21
3.72
Tablet
Syrup
64.54*
3.85*
*
Estimated by ICP-OES analysis (mg/g)
Table 2. Comparison of present work with previously reported work
Analyte
LOD
[nM]
LOD
[nM]
Name of the Ligand
Type
Analyte-2
Applications
-1
Thiacalix[5]crown based di- Off-On-
Molecular
Logic Gate
Zn2+
13.8
H PO
2
-
17
4
topic receptor [41]
Off
5
-(hydroxymethyl)-2-
methyl-4-((pyren-1-
ylmethylene)amino)pyridin-
Off-On-
Off
Zn2+
2300
H PO
2
-
22
Cell Imaging
4
3
-ol[42]
Cell Imaging
/ Molecular
Logic Gate
Hydrazino-carbothioamide Off-On-
Zn2+
0.6
H PO
-
-
26000
2
4
4
[43]
Off
[
2,2'-bibenzoimidazole]-
On-Off-
On
1,1'-
Fe3+
Zn2+
Zn2+
1.1
35.6
6.7
H PO
2
10
29.5
5.1
Test Strip
diyl)dipropanenitrile[44]
Quinoline-based C3-
symmetric[45]
Off-On-
Off
PPi
PPi
NS
N ,N -bis(3-
methoxysalicylidene)
diethylenetriamine[46]
Molecular
Logic Gate/
Cell Imaging
Test Strip/
Molecular
Logic Gate/
Real Sample
Analysis
1
3
Off-On-
Off
2
-(diethylamino)-N-
quinolin-8-yl) acetamide
L)
Present Work
(
Off-On-
Off
Zn2+
8
H PO
2
-
55
4
(
*
PA- Picric acid, PPi-Pyrophosphate, NS-Not studied