A bisphenol based fluorescence chemosensor for the selective detection of Zn2+ and PPi ions and its bioluminescence imaging

Article abstract of DOI:10.1016/j.saa.2020.118730

A bisphenol based fluorescence “turn-on” chemosensor 4,4′-(propane-2,2-diyl)bis(2-((E)-(2-(benzo[d]thiazol-2-yl)hydrazineyldene)methyl)phenol) (BHMP) has been synthesized and its sensing behavior was tested towards various ionic species. The chemo-sensing behavior of BHMP has been established through absorption, fluorescence, NMR, and mass spectroscopic techniques. The probe BHMP selectively detects zinc ions over other metal ions and the resulting BHMP + Zn²⁺ ensemble serves as a secondary probe for the detection of pyrophosphate (PPi) anion specifically over other anions. The spectroscopic studies reveal the fluorescence enhancement of BHMP in association with Zn²⁺ ions was quenched in the presence of pyrophosphate (PPi) anions. A probable mechanism of this selective sensing behavior was described on the basis of “OFF-ON-OFF” strategy for detection of both cations and anions. Moreover, the biological applicability of the chemosensor BHMP was examined via cell imaging studies.

Full text of DOI:10.1016/j.saa.2020.118730

Journal Pre-proof
A bisphenol based fluorescence chemosensor for the selective
detection of Zn2+ and PPi ions and its bioluminescence imaging
Rajasekaran Dhivya, Venkatachalam Kavitha, Periasamy
Viswanathamurthi
PII:
S1386-1425(20)30709-5
https://doi.org/10.1016/j.saa.2020.118730
SAA 118730
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 3 February 2020
Revised date:
4 July 2020
4 July 2020
Accepted
date:
Please cite this article as: R. Dhivya, V. Kavitha and P. Viswanathamurthi, A bisphenol
based fluorescence chemosensor for the selective detection of Zn2+ and PPi ions and
its bioluminescence imaging, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy (2020), https://doi.org/10.1016/j.saa.2020.118730
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
© 2020 Published by Elsevier.

Journal Pre-proof
A bisphenol based fluorescence chemosensor for the selective detection of Zn2+ and PPi ions
and its bioluminescence imaging
Rajasekaran Dhivya, Venkatachalam Kavitha, Periasamy Viswanathamurthi*.
Department of Chemistry, Periyar University, Salem-636 011, India.
*To whom correspondence should be addressed, e-mail: viswanathamurthi@gmail.com
Fax: +91 427 2345124

Journal Pre-proof
Abstract
A bisphenol based fluorescence “turn-on” chemosensor 4,4’-(propane-2,2-diyl)bis(2-
((E)-(2-(benzo[d]thiazol-2-yl)hydrazineyldene)methyl)phenol) (BHMP) has been synthesized
and its sensing behavior was tested towards various ionic species. The chemo-sensing behavior
of BHMP has been established through absorption, fluorescence, NMR, and mass spectroscopic
techniques. The probe BHMP selectively detects zinc ions over other metal ions and the resulting
BHMP+Zn2+ ensemble serves as a secondary probe for the detection of pyrophosphate (PPi)
anion specifically over other anions. The spectroscopic studies reveal the fluorescence
enhancement of BHMP in association with Zn2+ ions was quenched in the presence of
pyrophosphate (PPi) anions. A probable mechanism of this selective sensing behavior was
described on the basis of “OFF-ON-OFF” strategy for detection of both cations and anions.
Moreover, the biological applicability of the chemosensor BHMP was examined via cell imaging
studies.
Key words: Fluorescence probe, zinc ions, pyrophosphate anions, live cell imaging.

Journal Pre-proof
1. Introduction
There is a growing interest in the design and syntheses of organic molecules to recognize
environmentally and biologically important species and it became a necessary research topic in
life science area [1-6]. Among different recognizing techniques, photosensitive measurements
using fluorescent chemosensors become suitable methodology for the detection of various
analytes due to the fact that they are sensitive, cheap, convenient, non-destructive and are having
high selectivity towards detection of analytes [7-10]. Generally, fluorescent sensors were
composed of subunits (fluorophore and ionophore) in their structure and the subunits were
connected through a linker/spacer.
The metal cations and anions serve essential roles in environment and are generally
important to lead the metabolic processes in biological systems. But, their presence in both
excess and deficient level may leads to severe problems in ecological and biological systems [11-
14]. Among the essential metal cations, the trace quantity of the zinc metal ions in human body
involved many physiological processes such like gene transcription, enzyme regulation and
neural signal transmission. Moreover, the zinc metal is having an excellent potential activity
towards therapeutic drug designing and was extensively investigated during nowadays. However,
the action of zinc metal cation towards the biological and therapeutic mechanisms are not yet
well understood due to the hindrance of d10 configuration of Zn2+ ion by usual spectroscopic
techniques [15-18]. Furthermore, due to modern industrial growth, there is a vast usage and
release of Zn2+ metal ions to the environment, which in turn leads to toxicity by causing severe
problems to soil as well as plants by inhibiting their growth and biological systems by causing
severe diseases like Alzheimer’s disease, ischemic stroke and epilepsy [19-25]. Therefore, due to
the above mentioned toxicity, the detection of zinc ions even in trace level with high reliability
and effective response became essential. In this sense, the development of fluorescent
chemosensors for the detection of zinc ions constitutes a very active field of research, because of
its simplicity, sensitivity and selectivity.
On the other hand, among the anions which are playing vital roles in both biotic and
natural systems [26,27], pyrophosphate (PPi) anion is significant due to its essential role in
various metabolic processes such as DNA, RNA polymerizations, transduction in organisms and

Journal Pre-proof
cellular ATP hydrolysis [28,29]. The presence of PPi in excess or lower level in biological
system may leads to several diseases including chondrocalcinosis, calcium pyrophosphate
deposition disease (CPPD) and rheumatologic disorder [30-37]. So, the development of
chemosensors to detect PPi anions is also gets importance.
Up-to-date, countless numbers of chemosensors bearing different types of fluorophores to
detect zinc ions have been reported including fluorescein [38], rhodamine [39], BODIPY [40],
coumarin [41] and PPi anions [42]. But nowadays, the design and synthesis of new molecular
probes for sequential detection of two analytes becomes an emerging area. Hence, a new probe is
synthesized and utilized to detect Zn2+ ions and the resulting ensemble is applied to sense PPi
anions since PPi having strong binding affinity towards the zinc complexes.
In this work, bisphenol A was chosen as fluorophore with hydrazinobenzothazole
ionophores units in the design of new chemosensor 4,4’-(propane-2,2-diyl)bis(2-((E)-(2-
(benzo[d]thiazol-2-yl)hydrazineyldene)methyl)phenol) (BHMP) to detect metal ions. Initially,
the probe was non fluorescent because of excited-state intramolecular proton transfer (ESIPT)
involving efficient photoelectron transfer (PET) process. However, on complexation with zinc
metal ions, the fluorescence property enhances strongly because of the restriction in the C=N
bond rotation and suppression of PET process. The BHMP+Zn2+ complex selectively detects PPi
anions with quenching in the fluorescence intensity.
2. Experimental section
2.1. Materials and methods
All chemicals taken for the syntheses were analar pure grade and are brought from
Sigma-Aldrich. The solvents were delivered from commercial suppliers and are used without any
further purification. The double distilled water was used all through the experiments. Technico
micro heating table was used to measure melting points in an open capillary tube and was
uncorrected. Vario EL III Elemental analyzer was used to acquire the analytical data. UV-Visible
spectra and fluorescence spectra were recorded on Shimadzu 1800 UV-Visible
spectrophotometer and JASCO FP-8200 spectrophotometer respectively at room temperature
using a 1cm2 quartz cuvette (made of pure silica) at pH 7.4. Bruker AMX-500 spectrometer was
used to obtain 1H NMR spectra in dimethyl sulfoxide-d6 or duterated chloroform.

Journal Pre-proof
Tetramethylsilane (TMS) is used as a reference to record the chemical shifts (δ) in ppm. By
using a high voltage electro spray ionization probe (aerosol) in an advanced Q-TOF micro™
mass spectrometer, the mass spectrum was recorded. By using fluorescence microscope (Japan,
Eclipse Ti-U, Nikon,) imaging studies were carried out. The bisphenol A dialdehyde (1) was
synthesized according to the method reported in the previous work [43].
2.2. Synthesis of probe BHMP
The probe BHMP was synthesized from bisphenol A dialdehyde (0.28 ml, 1 mmol) on
reaction with hydrazinobenzothiozole (0.3304 g, 2 mmol) in acetonitrile (25 ml) for about 4 h.
After completion of the reaction confirmed by Thin Layer Chromotography (TLC), the yellow
color precipitate formed was separated out, washed with ethanol and dried to afford the probe
BHMP.
Yield: 78%, 0.46 g; M.pt: 170-173 °C. Anal. Calc. for C31H26N6O2S2: C, 64.34; H, 4.53;
N, 14.52; S, 11.08 %. Found: C, 63.84; H, 4.03; N, 14. 02; S, 10.58 %. UV-vis (DMSO, λmax
,
nm): 335, 370. 1H NMR (DMSO-d6, ppm): 12.22 (s, 2H) 8.52 (s, 2H) 7.69-7.61 (t, Ar 4H) 8.08-
8.02 (d, Ar 4H) 7.31-8.28 (d, Ar 2H) 6.98-6.96 (d, Ar 4 H) 4.49 (s, NH 2H) 1.80 (s,CH3 6H). 13C
NMR (DMSO-d6, ppm): 168.47, 157.08 (C-OH), 132.50 (Ar), 127.18 (Ar), 126.21 (Ar), 124.42
(Ar), 123.53 (Ar), 122.61 (Ar), 119.52 (Ar), 114.3(Ar), 56.20 (Ar), 33.67 (CH3). (Fig. S1 and
S2). MS (ESI, m/z) 579.5 [M+H]+ (Fig. S3).
2.3. Synthesis of BHMP-Zn complex
The probe BHMP (0.578 g, 1 mmol) was dissolved in ethanol-chloroform mixture (25
ml, 1:1 v/v) and zinc acetate (0.438 g, 2 mmol) was added and stirred for 3 h at 68 °C. The
greenish yellow color precipitate formed was separated and washed with cold ethanol.
Yield: 82%, 0.73 g; M.pt: 250-254 °C; Anal. Calc. for C34H29N6O6S2Zn2: C, 50.26; H,
3.60; N, 10.34; S, 7.77 %. Found: C, 50.76; H, 4.10; N, 10.84; S, 7.27 %. UV-vis (DMSO, λmax,
1
nm): 338, 385, 440. H NMR (DMSO-d6, ppm): 8.87 (s, 2H) 7.69-7.61 (t, Ar 4H) 8.08-8.02 (d,
Ar 4H) 7.31-8.28 (d, Ar 2H) 6.98-6.96 (d, Ar 4 H) 4.47 (s, NH 2H) 1.80 (s, CH3 6H) (Fig. S4)
MS (ESI, m/z) 826.2 [M+H]+ (Fig. S5).
3. Results and discussion

Journal Pre-proof
3.1 Synthesis and spectral characterization
The ligand was synthesized by two step reaction sequences and it was displayed in
scheme 1. The formation of the ligand was confirmed by 1H & 13C NMR and mass spectroscopic
analysis. The 1H NMR spectrum shows some important signals of the ligand at 12.22, 8.52, and
4.49 due to the presence of OH, azomethane and NH protons respectively. The ESI-MS spectrum
of BHMP displays molecular ion peak with m/z 579.5 ([M + H]+) in accordance with calculated
molecular masses 578.7.
Scheme 1. Synthesis of probe BHMP
3.2. Absorption spectroscopic studies
To study the sensing behavior of probe as a preliminary study, the absorption properties
of the probe BHMP alone and with various metal ions like K+, Na+, Zn2+, Ni2+, Pb2+, Cd2+, Mg2+,
Hg2+, Fe3+, Cu2+, Ag+, Mn2+, Fe3+ and Al3+ were examined in DMSO-H2O (1:9 v/v) in HEPES
buffer at pH 7.4 and the spectra were shown in Fig. 1. The probe BHMP shows peak at 335 and
370 nm. Upon the addition of Zn2+ ions to the probe BHMP, the peaks show slight red shift and
appeared in the region 338 and 385 nm. Moreover, a new peak was also appeared at 440 nm due
to the inhibition of ESIPT (excited state intramolecular proton transfer) and PET process [44].
The addition of other ions do not show any characteristic change in the absorption spectrum.
< Insert Fig. 1>
In addition, the sensing ability of the probe with Zn2+ ions was studied by UV-vis
titration experiments (Fig. 2). Upon incremental addition of Zn2+ ions (0-100 μM) to BHMP

Journal Pre-proof
solution, the absorption band intensity at 370 nm gets decreased and at 440 nm the intensity of
the absorption band increases gradually. The appearance of isosbestic points at 405 nm shows
the formation of new product.
< Insert Fig. 2>
3.3. Emission spectroscopic studies
The emission spectra were carried out to confirm selectivity of the probe BHMP with the
various metal ions in DMSO-H2O (1:9 v/v) in HEPES buffer at pH 7.4. The probe BHMP shows
weak emission at 515 nm, after the addition of Zn2+ ions the fluorescence emission intensity was
suddenly increased. However, the other analytes such as Pb2+, Cr3+, Cu2+, K+, Ni2+, Cd2+, Cu2+,
Na+, Mg2+, Hg2+, Mn2+, Fe3+ and Al3+ do not induce any change in the fluorescence emission
intensity (Fig. 3). The emission results indicate that the probe BHMP selectively detects the Zn2+
ions over other metal ions.
< Insert Fig. 3>
After confirming the selectivity of the probe, fluorescence titrations experiments were
carried out to analyze the binding strength and detection limit of BHMP towards Zn2+ ions. The
addition of increasing concentration of Zn2+ ions (0 - 100 µM) to the probe (BHMP) solution, the
fluorescence intensity was gradually increased at 515 nm and reached the saturation when 100
µM of Zn2+ ions was added (Fig. 4). Fig. S6 shows the result of fluorescence intensity vs
concentration of Zn2+ ions (1–100 µM), and the spectra exhibits the good linear relationship. The
detection limit was calculated as 0.28 nM using the equation 3σ/K (σ is the standard deviation of
the blank measurement and k is the slope between the intensity versus Zn2+ ions concentration).
The high association constant value and low detection limit reveal that the probe BHMP can be
an effective fluorescence probe for the detection of zinc metal ions. The binding constant was
calculated by using Benesi−Hildebrand equation [45] and it was found to be 9.3 × 107 M−1 (Fig.
S7). The quantum yields (Ф) of BHMP and BHMP+Zn2+ complex were found to be 0.016 and
0.713, respectively. Moreover, the color change occurs while addition of Zn2+ ions (100 µM) to
the probe solution can be seen through naked eye under day light and UV lamp (Fig. S8). But,
addition of other ions such as K+, Na+, Cu2+, Ni2+, Pb2+, Cd2+, Mg2+, Hg2+, Ag+, Mn2+, Fe3+ and
Al3+ do not exhibit any notable color change.

Journal Pre-proof
< Insert Fig. 4>
In addition, the binding stoichiometry between the probe BHMP and Zn2+ ions was
1
established by the Job’s experiment, H NMR titration and mass spectral analysis. Job’s
fluorescence emission experiment was performed with various mole fractions of probe BHMP
and Zn2+ ions and the results denote the formation of 1:2 binding between the BHMP with the
Zn2+ ions (Fig. S9). The 1H NMR spectrum (Fig. 5) of BHMP displays peaks at 12.22, 4.49 and
8.52 ppm due to the presence of OH, NH and imine protons respectively. The addition of 1
equivalent Zn2+ ions to the probe, the intensity of the OH peak decreased and the imine proton
peak was shifted to downfield. While, addition of 2 equivalent Zn2+ ions, the OH peak
completely disappeared and the imine proton peak was shifted to downfield further. This
confirms the 1:2 binding ratio between the BHMP with the Zn2+ ions. Moreover, the ESI mass
spectrum of BHMP + Zn2+ complex displays molecular ion peak at m/z 826.2 which corresponds
to 1:2 stoichiometry.
< Insert Fig. 5>
In order to evaluate the specificity of BHMP, competitive experiments were performed.
In the competitive experiments, Zn2+ ions mixed with other interfering cations including Mg2+,
Ca2+, Fe3+, Co2+, Li+, Ni2+, Na+, Cu2+, Pb2+, Cd2+, K+, Hg2+ and Ag+ ions (2 equiv. Zn2+ and 2
equiv. other metal ions) and the emission spectra were recorded in presence of the probe BHMP.
As shown in Fig. S10, addition of Zn2+ ions into BHMP lead to effective enhancement in the
fluorescence intensity. The presence of other competitive metal ions along with Zn2+ do not
interfere the emission spectra. The above-mentioned results reveal that the probe BHMP
possesses suitable selectivity for the system containing Zn2+ ions over other analytes.
3.4. Anion sensing studies
After confirming the selectivity of the probe for the recognition of Zn2+ ions, the
detection ability of the BHMP + Zn2+ complex has been analyzed for the selective detection of
anions through displacement approach. Initially absorption spectra were recorded for BHMP +
Zn2+ complex alone and with various anions using DMSO-H2O (1:9 v/v) in HEPES buffer at pH
7.4. From the Fig. 6, it is observed that the addition of PPi to the BHMP+Zn2+ ensemble retrieve

Journal Pre-proof
2−
spectral pattern of free probe whereas other anions (F−, Cl−, I-, S2-, NO3−, ClO3−, SO4 , HSO3−
and Br-) do not change any spectral pattern.
< Insert Fig. 6>
The sensing property of the BHMP+ Zn2+ ensemble was further analyzed by emission
spectra. The emission spectra were recorded for BHMP + Zn2+ complex with various anions such
2−
as F−, Cl−, CN−, I-, S2-, NO3−, ClO3−, ClO4−, SO4 , HSO3−, PPi and CH3COO- in DMSO-H2O
(1:9 v/v) in HEPES buffer at pH 7.4. Fig. S11 shows that the above mentioned anions do not
alter the fluorescence intensity except PPi anion. The addition of PPi anions quenches the
fluorescence intensity of BHMP+Zn2+ complex. To further extend, photoluminescence titrations
were carried out for the BHMP+Zn2+ complex solution (1 equiv) with 0-100 μM concentration of
PPi anions (Fig. 7). The stepwise addition of the PPi anions to the BHMP+Zn2+ complex
solution, the emission intensity of the BHMP + Zn2+ensemble decreases gradually at 515 nm and
it remains stable when it gets saturated. The Zn2+ ions on interaction with the PPi anion, more
stable precipitate was formed with release of free probe BHMP. The LOD (calculated using
formula 3σ/K) for PPi ions was found to be 0.87 nM (Fig. S12). In addition, the 1:2 binding
stoichiometry for BHMP + Zn2+ and PPi anions was found from Job’s plot (Fig. S13).
< Insert Fig. 7>
Moreover, competitive experiments were done to evaluate the response of BHMP+Zn2+
ensemble with other anions in addition to PPi anions using the emission spectra. The results
show that the PPi anions quench the emission intensity even in presence of other anions (Fig.
S14). Therefore, it is concluded that the BHMP + Zn2+ ensemble shows good selectivity towards
PPi anions. Besides, color change of the BHMP + Zn2+ ensemble for addition of PPi ions (100
µM) was examined which describe that it can be seen through nacked eye under day light and
2−
UV lamp. But, addition of other ions such as F−, Cl−, I-, S2-, NO3−, ClO4−, SO4 , Br−, PPi and
CH3COO- do not exhibit any notable color change (Fig. S15).
3.5. pH study
In order to explore the application of probe in physiological medium, the fluorescence
responses of BHMP, BHMP+Zn2+ and BHMP+Zn2++PPi were examined under different pH

Journal Pre-proof
conditions (2-14) (Fig. S16). The results reveal that the probe can be applicable to detect Zn2+
and PPi ions in the environment and biotic system with the pH range of 5-10.
3.6. Time resolved fluorescence study
The fluorescence lifetime decay experiment was done and the lifetime of the BHMP was
found to be 1.56 ns (at λem= 440 nm). There is an increase in the life time from 1.56 to 1.98 was
observed on adding Zn2+ ions to the probe BHMP solution. Due to the suppression of PET and
ESIPT process the average life gets enhanced. While subsequent addition of PPi anions to the
BHMP+Zn2+ ensemble, there is a significant quenching in the lifetime to 1.61 ns (Fig. 8).
< Insert Fig. 8>
Based on the above spectroscopic results, a mechanism was proposed for detection of
Zn2+ cations and PPi anions in scheme 2.
Scheme 2. Plausible mechanism for the interaction of Zn2+ and PPi ions with probe BHMP.
3.7. Application in Bio imaging
Before entering the bio imaging studies, the probe BHMP, BHMP+Zn2+ and
BHMP+Zn2+ with PPi were tested their cytotoxicity against A549 cells using MTT assay. For

Journal Pre-proof
this assay, primarily the cells were incubated with different concentrations (0, 5, 10, 15, 20, 25,
0
30, 40 and 50 μM) of the BHMP, BHMP+Zn2+ and BHMP+Zn2++PPi for 48 h at 37 C. The
results show that the probe BHMP has no toxicity against the tested cells upto 50 μM. The
BHMP+Zn2+ and BHMP+Zn2++PPi also do not show toxicity upto 20 μM (Fig. S17). The
toxicity results reveal that the probe BHMP, BHMP+Zn2+ and BHMP+Zn2++PPi can have the
capability to use in live cell imaging. In order to prove the detection capacity of the probe BHMP
towards Zn2+ and PPi ions in the live A549 cell, the cells were incubated with 10 μM of BHMP
solution and examined with fluorescence microscopy. Initially no fluorescence was observed but
on adding 20 μM of Zn2+ ions solution to the cells incubated with BHMP solution, fluorescence
enhancement takes place. After that, the 20 μM of PPi ions solution was added to the cells
containing BHMP- Zn2+ complex, the fluorescence property gets quenched. The obtained results
were shown in Fig. 9. From this result, it is proved that the OFF-ON-OFF mechanism was taking
place and the observation from the imaging studies shows that the probe BHMP possesses cell
permeability and excellent selectivity to detect both Zn2+ and PPi ions.
< Insert Fig. 9>
The present sensor BHMP was compared with the already reported chemosensors
towards zinc ions detection (Table S1). Although the reported chemosensors have some
advantages such as easy synthesis and sensitivity of detection, the present chemosensor BHMP
possesses merits like high binding constant, low detection limit and naked eye detection.
Therefore, the reported chemosensor BHMP has the greater ability to monitor Zn2+ and PPi ions
in both quantitatively and qualitatively.
4. Conclusions
We have successfully synthesized the luminescent chemosensor BHMP which shows
high selective and sensitive response for the sequential detection of Zn2+ and PPi ions in DMSO-
H2O (1:9 v/v) in HEPES buffer at pH 7.4. The probe BHMP has low detection limit (LOD = 0.28
nM) and high binding affinity (9.3×107) towards Zn2+ ions. In addition, the resultant
BHMP+Zn2+ complex detect the PPi anions over the other anions in the similar physiological
condition with detection limit of 0.87 nM. The fluorescent enhancement occurs due to inhibition
of photoelectron transfer (PET) process after formation of BHMP+Zn2+ complex. While the
addition of PPi anions into the solution of BHMP+Zn2+ quenches the fluorescence intensity due

Journal Pre-proof
to restoration of PET process in the free BHMP. Moreover, the probe BHMP was effectively
applied for the detection of Zn2+ and PPi ions in live cells.
Acknowledgements
The authors express their sincere thanks to Council of Scientific and Industrial Research
(CSIR), New Delhi, India [Grant No. 01 (2907)/17/EMR-II] and University Grants Commission
(UGC), India [SAP grant No. 540/20/DRS-I/2016(SAP-I)] for financial assistance.

Journal Pre-proof
References
[1] M. E. Moragues, R. Martinez-Manez, F. Sancanon, Chromogenic and fluorogenic
chemosensors and reagents for anions, Chem. Soc. Rev. (2011) 40 2593-2643.
[2] X. B. Yang, B. X. Yang, J. F. Ge, Y. J. Xu, Q. F. Xu, J. Liang, J. M. Lu,
Benzo[a]phenoxazinium-Based Red-Emitting Chemosensor for Zinc Ions in Biological
Media, Org. Lett. 13 (2011) 2710-2713.
[3] M. C. L. Yeung, V. W. W. Yam, Luminescent cation sensors: from host–guest chemistry,
supramolecular chemistry to reaction-based mechanisms, Chem. Soc. Rev., 44 (2015)
4192-4202.
[4] M. H. Lee, J. S. Kim, J. L. Sessler, Small molecule-based ratiometric fluorescence probes
for cations, anions, and biomolecules, Chem. Soc. Rev., 44 (2015) 4185-4191.
[5] P. A. Gale, Anion receptor chemistry, ChemComm. 47 (2011) 82-86.
[6] L.Y. Zhao, Q.L. Mi, G.K. Wang, J.H. Chen, J.F. Zhang, Q.H. Zhao and Y. Zhou,
1,8Naphthalimide-based “turn-on” fluorescent sensor for the detection of zinc ion in
aqueous media and its applications for bioimaging, Tetrahedron 54 (2013) 3353–3358.
[7] T. Kambe, T. Tsuji, A. Hashimoto, N. Itsumura, The Physiological, Biochemical, and
Molecular Roles of Zinc Transporters in Zinc Homeostasis and Metabolism, Physiol.
Rev. 95 (2015) 749–784.
[8] K. Wechakorn, K. Suksen, P. Piyachaturawat, P. Kongsaeree, Rhodamine-based
fluorescent and colorimetric sensor for zinc and its application in bioimaging, Sens.
Actuators B. 228 (2016) 270–277.
[9] P. Das, N. B. Chandar, S. Chourey, H. Agarwalla, B. Ganguly, A. Das, Role of Metal
Ion in Specific Recognition of Pyrophosphate Ion under Physiological Conditions and
Hydrolysis of the Phosphoester Linkage by Alkaline Phosphatase, Inorg. Chem. 52
(2013) 11034-11041.
[10] M. Sohrabi, M. Amirnasr, S. Meghdadi, M. Lutz, M. B. Torbati, H. Farrokhpour, A
highly selective fluorescence turn-on chemosensor for Zn2+, and its application in live
cell imaging, and as a colorimetric sensor for Co2+: experimental and TD-DFT
calculations, New. J. Chem., 42 (2018) 12595-12606.

Journal Pre-proof
[11] S. Gharami, K. Aich, L. Patra, T.K. Mondal, Detection and discrimination of Zn2+ and
Hg2+ using a single molecular fluorescent probe, New J. Chem. 42 (2018) 8646-8652.
[12] J.Y. Choi, D. Kim, J. Yoon, A highly selective “turn-on” fluorescent chemosensor based
on hydroxy pyrene–hydrazone derivative for Zn2+, Dyes Pigm. 96 (2013) 176-179.
[13] X. Zhou, Y. Lu, J.F. Zhu, W.H. Chan, A.W. Lee, P.S. Chan, R.N. Wong and N. Mak,
Ratiometric fluorescent Zn2+ chemosensor constructed by appending a pair of
carboxamidoquinoline on 1,2-diaminocyclohexane scaffold, Tetrahedron, 67 (2011)
3412-3419.
[14] S. Ullmann, R. Schnorr, C. Laube, B. Abel, B. Kersting, Photoluminescence properties
of tetrahedral zinc (II) complexes supported by calix [4] arene-based salicylaldiminato
ligands, Dalton Trans., 47 (2018) 5801-5811.
[15] C. Zhang, Z. Liu, Y. Li, W. He, X. Gao, Z. Guo, In vitro and in vivo imaging application
of a 1,8-naphthalimide-derived Zn2+ fluorescent sensor with nuclear envelope
penetrability, Chem. Commun. 49 (2013) 11430-11432.
[16] H.-Y. Lin, P.-Y. Cheng, C.-F. Wan, A.-T. Wu, A turn-on and reversible fluorescence
sensor for zinc ion, Analyst. 137 (2012) 4415–4417.
[17] X. Yan, J. J Kim, H. S Jeong, Y. K Moon, Y. K Cho, S. Ahn, S. B. Jun, H. Kim, Y. You,
Low-affinity zinc sensor showing fluorescence responses with minimal artifacts, Inorg.
Chem. 56 (2017) 4332-4346.
[18] J. Wang, W. Lin, W. Li, Single fluorescent probe displays a distinct response to Zn2+
and Cd2+, Chem. Eur. J. 18 (2012) 13629-13632.
[19] C. Gao, X. Jin, X. Yan, P. An, Y. Zhang, L. Liu, H. Tian, W. Liu, X. Yao, Y. Tang, A
small molecular fluorescent sensor for highly selectivity of zinc ion, Sens. Actuators B.
176 (2013) 775-781.
[20] J. H. Kang, C. Kim, Colorimetric detection of iron and fluorescence detection of zinc
and cadmium by a chemosensor containing a bio-friendly octopamine, Photochem.
Photobiol. Sci., 17 (2018) 442-452.
[21] C. Rivas, G. J. Stasiuk, M. S. Henga and N. J. Long, Towards understanding the design
of dual-modal MR/fluorescent probes to sense zinc ions, Dalton Trans. 44 (2015) 4976-
4985.

Journal Pre-proof
[22] S. Zhang, R. Adhikari, M. Fang, N. Dorh, C. Li, M. Jaishi, J. Zhang, A. Tiwari, R. Pati,
F.-T. Luo, H. Liu, Near-Infrared Fluorescent Probes with Large Stokes Shifts for
Sensing Zn(II) Ions in Living Cells, ACS Sens. 1 (2016) 1408-1415.
[23] X. Yan, J. J. Kim, H. S. Jeong, Y. K. Moon, Y. K. Cho, S. Ahn, S. B. Jun, H. Kim, Y.
You, Low-Affinity Zinc Sensor Showing Fluorescence Responses with Minimal
Artifacts, Inorg. Chem. 56 (2017) 4332-4346.
[24] L. Yan, R. Li, F. Ma and Z. Qi, A simple salicylaldehyde-based fluorescent “turn-on”
probe for selective detection of Zn2+ in water solution and its application in live cell
imaging, Anal. Methods, 9 (2017) 1119-1124.
[25] (a) M. Shyamal, P. Mazumdar, S. Maity, S. Samanta, G. P. Sahoo, A. Misra, Highly
Selective Turn-On Fluorogenic Chemosensor for Robust Quantification of Zn(II) Based
on Aggregation Induced Emission Enhancement Feature, ACS Sens. 1 (2016) 739-747;
(b) A.Gomathi, M. Vasanthi, P. Viswanathamurthi, S. Suresh, R. Nandhakumar, A
Simple Perceptive Diphenyl‐Imidazole‐Based Dipodal Schiff‐Base Chemosensor for
Zn2+ and PPi ions and Its Live‐Cell Imaging Applications, chemistryselect, 3 (2019)
11809-11815 (c) A. Manna, K. Jana, N. Guchhait, S. Goswami, Discrimination of tabun
mimic diethyl cyanophosphonate from sarin mimic diethyl chlorophosphate via Zn(ii)-
triggered photoinduced electron transfer-decoupled excited state intramolecular proton
transfer processes, New J. Chem., 41 (2017) 6661-6666.
[26] B. K. Datta, D. Thiyagarajan, S. Samanta, A. Ramesh, G. Das, A novel chemosensor
with visible light excitability for sensing Zn2+ in physiological medium and in HeLa cells,
Org. Biomol. Chem. 12 (2014) 4975-4982.
[27] H. T. Ngo, X. Liu, K. A. Jolliffe, H. Lee, Anion recognition and sensing with Zn(ii)–
dipicolylamine complexes, Chem. Soc. Rev. 41 (2012) 4928-4965.
[28] C. Chang, F. Wang, T. Wei, X. Chen, Benzothiazole-based fluorescent sensor for
ratiometric detection of zn(II) ions and secondary sensing PPi and its applications for
biological imaging and PPase catalysis assays, Ind. Eng. Chem. Res. 56 (2017) 8797-
8805.
[29] L. Lin, X. Song, Y. Chen, M. Rong, T. Zhao, Y. Jiang, Y. Wang, X. Chen, One-pot
synthesis of highly greenish-yellow fluorescent nitrogen-doped graphene quantum dots

Journal Pre-proof
for pyrophosphate sensing via competitive coordination with Eu3+ ions, Nanoscale, 7
(2015) 15427-15433.
[30] D. Chao, Y. Zhang, Anion-induced emissive nanoparticles for tunable fluorescence
detection of pyrophosphate and bioimaging application, Sens. Actuators B, 242 (2017)
253-259.
[31] A.Gomathi, P. Viswanathamurthi, K. Natarajan, Fluorescence sensing response of
zinc(II) and pyrophosphate ions by benzoxazole appended dipodal Schiff base, J.
photochem.photobiol. A, 370 (2019) 75-83.
[32] (a) K. Kim, Y. Ha, L. Kaufman, D. G. Churchill, Labile Zinc-Assisted Biological
Phosphate Chemosensing and Related Molecular Logic Gating Interpretations Inorg.
Chem., 51 (2012) 928-938. (b) O. G. Tsay, S. T. Manjare, H. Kim, K. M. Lee, Y. S. Lee,
D. G. Churchill, Novel Reversible Zn2+-Assisted Biological Phosphate “Turn-On”
Probing through Stable Aryl-hydrazone Salicylaldimine Conjugation That Attenuates
Ligand Hydrolysis, Inorg. Chem. 52 (2013) 10052-10061.
[33] K. A. Jolliffe, Pyrophosphate Recognition and Sensing in Water Using
Bis[zinc(II)dipicolylamino]-Functionalized Peptides, Acc. Chem. Res. 50 (2017) 2254-
2263.
[34] S. Watchasit, P. Suktanarak, C. Suksai, V. Ruangpornvisuti, T. Tuntulani, Discriminate
sensing of pyrophosphate using a new tripodal tetramine-based dinuclear Zn(ii) complex
under an indicator displacement assay approach, Dalton Trans. 43 (2014) 14701-14709.
[35] J. Wang, X. Liua, Y. Pang, A benzothiazole-based sensor for pyrophosphate (PPi) and
ATP: mechanistic insight for anion-induced ESIPT turn-on, J. Mater. Chem. B, 2 (2014)
6634-6638
[36] W. H. Chen, Y. Xing, Y. Pang, A Highly Selective Pyrophosphate Sensor Based on
ESIPT Turn-On in Water, Org. Lett. 13 (2011) 1362-1365.
[37] (a) V. E. Zwicker, B. M. Long, K. A. Jolliffe, Selective sensing of pyrophosphate in
physiological media using zinc(ii)dipicolylamino-functionalised peptides, Org. Biomol.
Chem. 13 (2015) 7822-7829.
[38] L. M. Mesquita, V. Andre, C. V. Esteves, T. Palmeira, M. N. Berberan-Santos, P.
Mateus, R. Delgado, Dinuclear Zinc(II) Macrocyclic Complex as Receptor for Selective
Fluorescence Sensing of Pyrophosphate, Inorg. Chem. 55 (2016) 2212-2219.

Journal Pre-proof
[39] X. Liu, D. G. Smith, K. A. Jolliffe, Are two better than one? Comparing intermolecular
and intramolecular indicator displacement assays in pyrophosphate sensors, ChemComm.
52 (2016) 8463-8466.
[40] I. Roy, J.Y. Shin, D. Shetty, J.K. Khedkar, J.H. Park, K. Kim, E-Bodipy fluorescent
chemosensor for Zn2+ ion, J Photochem. Photobiol. A Chem. 331 (2016) 233-239.
[41] Y. Gao, H. Liu, .P Li, Q. Liu, W. Wang, B. Zhao, Coumarin-based fluorescent
chemosensor for the selective quantification of Zn2+ and AcO− in an aqueous solution and
living cells, Tetrahedron Lett. 58 (2017) 2193-2198.
[42] (a) T. Cheng, T. Wang, W. Zhu, X. Chen, Y. Yang, Y. Xu, X. Qian, Red-Emission
Fluorescent Probe Sensing Cadmium and Pyrophosphate Selectively in Aqueous
Solution, Org. Lett. 13 (2011) 3656-3659. (b) H. T. Ngo, X. Liu, K. A. Jolliffe, Anion
recognition and sensing with Zn(ii)–dipicolylamine complexes, Chem. Soc. Rev. 41
(2012) 4928-4965.
[43] S. Erdemir, O. Kocyigit, S. Malkondu, Fluorogenic Recognition of Zn2+, Al3+ and
F− Ions by a New Multi-Analyte Chemosensor Based Bisphenol A-Quinoline, J.
Fluoresc. 25 (2015) 719-727.
[44] B. Bag, A. Pal, Water induced chromogenic and fluorogenic signal modulation in a bi-
fluorophore appended acyclic amino-receptor system, Org. Biomol. Chem. 9 (2011) 915-
925.
[45] J.Y. Choi, D. Kim, J. Yoon, A highly selective “turn-on” fluorescent chemosensor based
on hydroxy pyrene–hydrazone derivative for Zn2+. Dyes Pigm. 96 (2013) 176 -179.

Journal Pre-proof
Fig. 1. UV-Vis spectra of probe BHMP (10 μM) in DMSO/H2O (1:9 (v/v), 50 mM HEPES,
pH=7.4) with 100 μM of metal ions (Na+, Cd2+, Mg2+, Co2+, Zn2+, Cu2+, Hg2+, Pb2+, Fe3+, K+,
Ni2+, and Al3+).

Journal Pre-proof
Fig. 2. UV-Vis titration spectra of the probe BHMP (10 μM) with 0-100 μM of Zn2+ ions.

Journal Pre-proof
Fig. 3. Fluorescence response of probe BHMP (10 µM) in DMSO/H2O (1:9 (v/v),
HEPES=50mM, pH=7.4) with 100 µM of metal ions (K+, Na+, Zn2+, Ni2+, Cd2+, Cu2+, Mg2+,
Hg2+, Co2+, Fe3+ and Al3+) (λex=440 nm).

Journal Pre-proof
Fig. 4. Fluorescence titration spectra of the probe BHMP (10 µM) with 0 - 100 µM of Zn2+.

Journal Pre-proof
Fig. 5. 1H NMR titration spectra of BHMP with Zn2+ ions.

Journal Pre-proof
Fig. 6. UV-Vis spectra of probe BHMP+Zn2+ (10 μM) in DMSO/H2O (1:9 (v/v), 50 mM
2−
HEPES, pH=7.4) with 100 μM of varies anions (F−, Cl−, PPi, I-, S2-, NO3−, ClO3−, SO4 , HSO3−
and Br-).

Journal Pre-proof
Fig. 7. Fluorescence response of the probe BHMP + Zn2+ (10 µM) with 0 - 100 µM of PPi anion.

Journal Pre-proof
Fig. 8. Fluorescence lifetime decay profiles of probe BHMP, BHMP+Zn2+ and
BHMP+Zn2++PPi.

Journal Pre-proof
Fig. 9. Fluorescence cell imaging studies of cells (A549) treated with probe BHMP (left
column), BHMP + Zn2+ (middle column) and BHMP + Zn2+ + PPi (right column).

Journal Pre-proof
Credit author statement
Rajasekaran Dhivya: Conceptualization Investigation, Writing - Original Draft.
Venkatachalam Kavitha: Verification, whether as a part of the activity or separate, of the overall
replication/ reproducibility of results/experiments and other research outputs.
Periasamy Viswanathamurthi: Methodology, Validation, Supervision, Project administration.

Journal Pre-proof
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

Journal Pre-proof
Graphical abstract
A bisphenol based fluorescence “turn-on” chemosensor (BHMP) has been synthesized and its
sensing behavior was tested towards various ionic species. The probe BHMP selectively detects
zinc ions over other metal ions and the resulting Zn2+-BHMP ensemble serves as a secondary
probe for the detection of PPi anion. The biological applicability of the chemosensor BHMP was
examined via cell imaging studies.