An investigation of some Schiff base derivatives as chemosensors for Zn(II): The performance characteristics and potential applications

Article abstract of DOI:10.1016/j.saa.2018.05.116

The fluorescence properties of four simple Schiff bases (LH₂, LDMH₂, LH₂ ^H and LDM^HH₂) and their potential application as chemosensors for the detection of zinc ion in aqueous solution have been investigated. While LH₂ and LDMH₂ have displayed specific recognition to Zn(II), the reduced derivatives (LH₂ ^H and LDM^HH₂) of these ligands have shown no fluorescence response due to the lack of C[dbnd]N group. The Job plots, fluorescence titration experiments and ESI-MS results indicate the formation of 1:1 complexes between sensors and Zn(II). The analytic methods based on LH₂ and LDMH₂ as chemosensors have been proposed and optimized to detect Zn(II) ions in aqueous solution. The optimized methods have shown a good range of linearity, high precision, good accuracy and low detection limit. As an alternative to these methods, LH₂ and LDMH₂ have the capability to detect Zn(II) ions by naked eye under UV lamp. Moreover, LH₂-Zn and LDMH₂-Zn complexes have the ability to be a staining agent for identifying the radiation treatment of food by DNA comet assay.

Full text of DOI:10.1016/j.saa.2018.05.116

Accepted Manuscript
An investigation of some Schiff base derivatives as chemosensors
for Zn(II): The performance characteristics and potential
applications
Ece Ergun, Ümit Ergun, Özgür İleri, Muhammed Fatih
Küçükmüzevir
PII:
DOI:
Reference:
S1386-1425(18)30516-X
doi:10.1016/j.saa.2018.05.116
SAA 16150
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date: 28 December 2017
Revised date:
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29 May 2018
Please cite this article as: Ece Ergun, Ümit Ergun, Özgür İleri, Muhammed Fatih
Küçükmüzevir , An investigation of some Schiff base derivatives as chemosensors for
Zn(II): The performance characteristics and potential applications. Saa (2017),
doi:10.1016/j.saa.2018.05.116
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ACCEPTED MANUSCRIPT
An investigation of some Schiff base derivatives as chemosensors for Zn(II):
The performance characteristics and potential applications
Ece Ergun a*, Ümit Ergun b, Özgür İleri b, Muhammed Fatih Küçükmüzevir b
* corresponding author
e-mail:ece.ergun@taek.gov.tr
phone:+90.312.8101591
fax:+90.312.8154307
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a
TAEA, Sarayköy Nuclear Research and Training Center, Kazan, 06983, Ankara, Turkey
Department of Chemistry, Faculty of Art and Sciences, Düzce University, Düzce 81620, Turkey
b
Abstract
H
The fluorescence properties of four simple Schiff bases (LH2, LDMH2, LH2 and LDMHH2)
and their potential application as chemosensors for the detection of zinc ion in aqueous
solution have been investigated. While LH2 and LDMH2 have displayed specific recognition
H
to Zn(II), the reduced derivatives (LH2 and LDMHH2) of these ligands have shown no
fluorescence response due to the lack of C=N group. The Job plots, fluorescence titration
experiments and ESI-MS results indicate the formation of 1:1 complexes between sensors and
Zn(II). The analytic methods based on LH2 and LDMH2 as chemosensors have been proposed
and optimized to detect Zn(II) ions in aqueous solution. The optimized methods have shown a
good range of linearity, high precision, good accuracy and low detection limit. As an
alternative to these methods, LH2 and LDMH2 have the capability to detect Zn(II) ions by
naked eye under UV lamp. Moreover, LH2-Zn and LDMH2-Zn complexes have the ability to
be astaining agent for identifying the radiation treatment of food by DNA comet assay.
Keywords: Zinc ion; Chemosensor; Fluorescent probe; Schiff bases; Aqueous solution
1. Introduction
Zinc is one of the most common element in the Earth’ crust. Zinc is found in the air, soil and
water and is present in all foods [1]. Zinc concentrations, however, are rising unnaturally, due
to anthropogenic additions. Most zinc is added during industrial activities, such as mining,
coal, and waste combustion and steel processing. Industrial sources or toxic waste sites may
cause the concentrations of Zn(II) in drinking water to reach levels that can cause health
problems [2]. Zinc is an essential trace element in biological systems and has been found to
play an important role in survival and functioning of all living organisms. It is only second to
iron among the trace elements found in humans [3]. In total, the adult human body contains
2–3 g zinc. Probably thousands of proteins contain zinc and over 200 zinc-containing
enzymes have now been characterized [4]. Zinc can be relatively easily determined by
standard analytical techniques such as AAS or ICP-MS, but the determination of the “free” or
“available” Zn(II) concentrations in some samples such as biological systems has proved
difficult using classic techniques. So, this has led to the emergence of zinc specific molecular
sensors.
Among the various chemosensors, fluorescent chemosensors present many advantages, which
are high sensitivity, low cost, easy detection, and suitability as a diagnostic tool for ion
recognition and biological concern [5, 6, 7, 8, 9]. Because Zn(II) is spectroscopically silent
due to its d10 electron configuration, numerical fluorescent chemosensors for the detection of
Zn(II) have been studied intensively [10-16]. The nitrogen of a Schiff base exhibits a strong
affinity for zinc, therefore, the Schiff base has also been used to develop zinc chemosensors
[10, 17-24]. It has been reported that two mechanisms, the C=N isomerization and the
suppression of this isomerization, are responsible for fluorescence emission behavior of the
Schiff bases [10, 17, 22]. The binding of metal ions by the C=N group would stop the
isomerization, and a significant fluorescence enhancement could be achieved. However, the
main drawbacks of Schiff-base type receptors are the poor solubility and the instability of the
Schiff-base in aqueous solutions. In addition, many available fluorescent chemosensors for
Zn(II) encountered difficulty in distinguishing Zn(II) from other metal ions (especially from
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Cd(II), Ni(II) and Cu(II)), because of their similar properties or paramagnetic nature [11-15,
18, 20, 22-25]. Most of these chemosensors have been used particularly for live-cell imaging
because interfering ions are present in much lower concentrations compared with Zn(II) in
biological samples. However, it is important to testify the potential use of the chemosensors
for the monitoring of Zn(II) concentrations in water samples containing significant amounts
of heavy metal ions. Besides, the synthesis of some chemosensors requires complicated
pathways which can be time-consuming with low yields and costly.
Contamination of foods, especially of those of animal origin, with microorganisms,
particularly pathogenic non-spore-forming bacteria, parasitic helminths and protozoa is an
enormous public health problem and important cause of human suffering all over the world.
Considering the tremendous importance of microbial and parasitic diseases related to foods,
food safety should be guaranteed. The chemical sanitizing procedures have inherent problems
concerning residues and environmental pollution. This is where food irradiation among the
other intervention alternatives comes into the picture [26]. A number of analytical methods
have been investigated for the detection of radiation treatment of foods. In 1996, the European
Committee for Standardization (CEN) adapted several methods based on Electron Spin
Resonance (ESR) spectroscopy, Gas Chromatography (GC) and Thermoluminescence (TL).
Since all these techniques for the detection of irradiated foods are expensive, time consuming
and need special expertise, therefore, there is a need to find out rapid and simple tests, which
should be versatile and require inexpensive instrumentation. Since the large molecule of DNA
is a sensitive target to ionizing radiation, radiation-induced changes in DNA could be a basis
for detection of irradiation in a number of foods. A sensitive technique to detect DNA
fragmentation is the microgel electrophoresis of single cells, commonly called ‘Comet Assay’
[27].
DNA Comet Assay has been described as a rapid and inexpensive screening test to identify
radiation treatment of food. This method involves lysis with detergent and high salt, after
embedding cells in agarose so that the DNA is immobilized for subsequent electrophoresis.
The DNA fragments migrate out of the cells during the electrophoresis, forming a long ‘tail’
in the direction of the anode and giving the damaged cells the appearance of a comet. In
contrast, non-irradiated DNA samples appear as discrete dots on the gel, lacking a ‘tail’ or
with only slight ‘tails’. The most common way to visualize the comets is staining with a
fluorescent dye such as ethidium bromide, DAPI, acridine orange and propidium iodide and
then examining by a fluorescent microscope [28-30]. At this point, the fluorescent compounds
in our study have been investigated for their potentiality as DNA staining agents.
In this study, two symmetric ONNO type Schiff base N-N'-bis(salicylidene)-1,3-
propanediamine (LH2), bis-N,N'-(salicylidene)-2,2'-dimethyl-1,3-propanediamine (LDMH2),
H
and the reduced derivatives (LH2 and LDMHH2) of this Schiff bases (Fig. 1) have been
synthesized and examined for their potential use as fluorescent chemosensors for the detection
of Zn(II) in water. These are not novel ligands but will provide convenience and flexibility for
researchers who need to analyze zinc due to their simple synthesis and even the commercial
availability of one (LH2). Fluorescence is known to be very sensitive to the environment, so
the parameters which play an important role in regulating the fluorescent property of the
studied compounds have also been optimized. The results show that LH2 and LDMH2 are
highly selective and sensitive probes for Zn(II) in aqueous solution.
2. Experimental section
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2.1. Reagents and apparatus
All chemicals were obtained commercially (analytical grade and spectroscopic grade) and
used without further purification. Absorption spectra were obtained by an Agilent Cary 100
UV-Vis spectrophotometer. Fluorescence spectra measurements were performed on a Cary
Eclipse fluorescence spectrophotometer. The pH values were determined using a Mettler
Toledo pH probe. Electrospray ionization mass spectra (ESI-MS) were carried out on a
Waters Micromass ZQ 2000 spectrometer.
2.2. Synthesis of chemosensors
Bis-N,N'-(salicylidene)-1,3-propanediamine (LH2), bis-N,N'-(salicylidene)-2,2'-dimethyl-1,3-
propanediamine (LDMH2), bis-N,N'-(2-hydroxybenzyl)-1,3-propanediamine (LHH2), and bis-
N,N'-(2-hydroxybenzyl)-2,2'-dimethyl-1,3-propanediamine (LDMHH2) were prepared
according to the reported procedure [31, 32]. According to the procedure, LH2 and LDMH2
were prepared by the condensation of salicylaldehyde with 1,3-diaminopropane and 2,2'-
dimethyl-1,3-propanediamine in ethanol, respectively. After the isolation, these Schiff bases
were reduced with sodium borohydride to obtain LHH2 and LDMHH2.
2.3. Spectroscopic studies
The stock solutions of chemosensors (LH2, LDMH2, LHH2 and LDMHH2) (2.010-3 M) were
prepared in ethanol. Stock solutions (2.010-3 M) of Fe(III), Al(III), Cr(III), Mn(II), Cd(II),
Co(II), Cu(II), Ni(II), Pb(II), Hg(II), Ca(II), Mg(II), K(I), Na(I) and Zn(II) were prepared
from their nitrate or chloride salts in deionized water. Spectral data were recorded at room
temperature. For fluorescence measurements, both the excitation and emission slit widths
were 5.0 nm unless otherwise stated.
ESI-MS studies were performed in the positive ion mode with a quadrupole mass analyzer.
The major conditions were as follows: scan range, m/z 50–800; capillary voltage, 3.5kV; cone
voltage, 30V; source temperature, 150 °C; desolvation temperature, 300 °C.
2.4. Irradiation and DNA comet assay
The gamma irradiation of chicken breast meat samples was carried out in a Co-60 laboratory
irradiator (Ob-Servo Sanguis, Institute of Isotopes, Hu), at dose of 3.00 kGy (dose rate 1.83
kGy/h) at Saraykoy Nuclear Research and Training Center. The effect of gamma-ray radiation
on DNA in chicken breast was examined by DNA comet assay. The DNA Comet Assay for
the cell suspensions was carried out as described in the European Standard EN 13784 [33].
The coated slides were immersed in lysis buffer (0.045MTBE, pH 8.4, containing 2.5% SDS)
for 7 min. Using the same buffer but devoid of SDS, electrophoresis (Thermo Electrophoresis
System) was performed at 2 V/cm for 2 min. The mixtures of LH2-Zn and LDMH2-Zn ([L] =
1×10-4, [Zn] = 1×10-4) was employed as staining agent to visualize DNA in irradiated and
non-irradiated samples. Slides were examined using a microscope (Olympus BX51 model
with fluorescence, DIC attachments and filter U-DM-DA/FI/TX2E) at magnification
20×(Objective) by digital color video camera (Penguin PIXERA 600 CL).
3. Results and Discussion
3.1. UV–vis and fluorescence studies of chemosensors with various metal ions
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UV–Vis absorption spectra of LH2 and LHH2 (1.010-5 M in ethanol) are shown in Fig. 2a. It
is known that the isolated benzene ring exhibits three characteristic -* absorption bands;
two primary bands at 184 and 202 nm and a secondary band at 255 nm. However, these bands
shift to longer wavelengths depending on the nature of the substituents, i.e. their electron
donating or withdrawing nature [34]. Schiff bases, in this study, contain two benzene ring,
however, absorption bands of the benzene are expected to be somewhat red-shifted in the UV
spectra of these ligands due to the substitution. This can be easily observed in UV-Vis
spectrum of LHH2 (Fig. 2a), which exhibits only three absorption bands of benzene (201, 218
as a shoulder, and 281 nm) that are red-shifted compared to isolated benzene. On the other
hand, in the case of an electron-withdrawing group substitution such as C=N, the primary
absorption bands are shifted to a longer wavelengths (especially second primary band), but
the position of secondary absorption band does not significantly change [34]. These effects
can be seen in the spectrum of LH2; two primary bands were shifted to longer wavelengths
(216 and 257 nm) compared to the bands obtained in LHH2 spectrum and the position of
secondary absorption band did not significantly change (282 nm). Another difference
observed in the electronic spectrum of LH2 was associated with the appearance of two new
bands at 320 and 405 nm due to the presence of azomethine group. These bands were
attributed to the -* and n-* transitions of the C=N chromophore. It should be noted that
LDMH2 and LDMHH2 ligands, containing only two additional methyl groups compared to
LH2 and LHH2, gave similar absorption spectra (data not shown).
The binding ability of the ligands in ethanol (2.010-5 M) with cations in deionized water
(2.010-3 M), such as Na(I), K(I), Mg(II), Ca(II), Al(III), Cr(III), Mn(II), Fe(III), Co(II),
Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II), was examined. Upon addition of Na(I), K(I), Mg(II),
Ca(II), Al(III), Cr(III), Mn(II), Fe(III), Cd(II) and Pb(II) no significant changes in absorption
bands were observed (the inset of Fig. 2b). By contrast, addition of Co(II), Ni(II), Cu(II) and
Zn(II) resulted in a redshift of C=N band indicating the formation of metal complexes (Fig.
2b). It should be indicated that in all ligands studied, -* transitions of benzene exhibit
maximum absorption at the same wavelength but somewhat broadened. LDMH2 has showed
similar binding affinity toward the ions mentioned above, therefore, only the absorption
spectra of LH2 and LH2-cation mixtures are given in Fig. 2b to illustrate these interactions. On
the other hand, since the binding affinity is determined by the redshift of the C=N band, it is
not possible to observe a change in the spectra of LHH2 and LDMHH2 due to the absence of
this bond.
The complexation of LH2 and LDMH2 with Co(II), Ni(II), Cu(II) and Zn(II) ions was
monitored using UV–visible spectrophotometric studies. The absorption spectra shown in Fig.
S1, depict the spectral changes upon titrating a fixed concentration of LH2 and LDMH2
(1.010-4 M) against increasing concentrations of Zn2+ ions (0–1.010-4 M) in ethanol:water
= 1:1 (v/v). It is noteworthy that, the intensity of -* transition of the C=N chromophore
gradually increased along with redshift on increasing the concentrations of Co(II), Ni(II),
Cu(II) and Zn(II) ions. In addition, the intensity of absorbance bands corresponding to the n-
* transition also decreased, indicating ligands coordinate to the metal ions through
azomethine nitrogen. The appearance of clear isosbestic points suggests that an equilibrium
was established between ligands and metal ions.
Fluorescence studies were further performed in order to evaluate the potential use of the
ligands as fluorescent chemosensors for identification of metal ions. First, preliminary
emission spectra were scanned to find emission peak wavelength of free ligands in ethanol
(1.010-5 M), ligand-deionized water (1:1 v/v, stock ligand solution in ethanol 2.010-5 M)
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and ligand-cation (stock cation solution in water 2.010-3 M) mixtures (1:1 v/v). As can be
seen in Fig. 3a and b, free LH2 and LDMH2 emitted only one fluorescence peak appeared at
446 and 451 nm, respectively. The excitation spectra obtained from emission at 446 and 451
nm for LH2 and LDMH2 gave three peaks (the insets of Fig. 3a and b) at 235, 268, 349 nm
(LH2) and 238, 271, 356 nm (LDMH2). On the other hand, no emission peak was observed for
the reduced ligands (data not shown). This result is an evidence that the C=N bond is required
for fluorescence emission of these ligands. Upon the addition of deionized water to LH2 and
LDMH2 solutions (2.010-5 M) in ethanol (1:1, v/v), the observed emission band decreased,
probably due to decrease in solubility of ligands, increase in solvent polarity and hydrolysis of
carbon-nitrogen double bond (Fig. 3a and b). These suggestion were examined by UV-Vis
studies (Fig. S2). It was observed that UV-Vis spectra of ligands in ethanol and in
ethanol:water (1:1) mixture showed different absorption characteristics. In ethanol:water
mixture, the intensity of -* transitions were decreased while the intensity of n-* transition
was increased accompanied with a blue shift (λmax=410 nm in ethanol; λmax=400 nm in
ethanol:water) (the inset of Fig. S2a and S2b). It is known that position (λmax) and intensity
(εmax) of the absorption bands are affected by the surrounding molecular environment due to
the intermolecular interactions and/or change in solubility. Therefore, the spectral
modifications mentioned above appeared as a result of change in solvent composition.
Consequently, the decrease in intensity of -* absorptions in ethanol:water environment
resulted in a decrease in fluorescence emission of the ligands. Another factor that contributed
the observed decrease in intensity can be the hydrolysis of the ligands. It is well known that
Schiff bases undergo hydrolysis in acidic, neutral and basic conditions by different rate
limiting pathways [35, 36]. The spectral studies clearly indicated that intensity of -*
transition of the C=N chromophore decreased accompanied with a redshift for a definite
period of time after mixing the ligand solutions in ethanol with water (Fig. S2a and S2b). It
can be suggested that the observed decrease occurs because of the hydrolysis of ligands by the
attack of H2O to the imine linkage. In addition, the increase in solvent polarity (due to the
presence of water) decreases the energy of -* transitions. Therefore, the observed redshift
in the absorption maxima in a time range (0-40 min for LH2, Fig. S2a; 0-120 min for LDMH2,
Fig. S2b) was the result of this phenomenon. Furthermore, hydrolysis reaction caused also a
decrease in the intensity of n-* absorption over the same period of time. As the result of
hydrolysis reaction, the lone pair of nitrogen becomes a bonding pair and C=N double bond
content is reduced. So, less intense n-* absorption bands were detected as time progressed.
At this point, it is considered that hydrogen bonding also plays an important role. The
presence of water enhanced the number of hydrogen bonds, leading an increase in the energy
of n-* transitions and absorption maximum appeared blue shifted over the definite time
range mentioned above. As a consequence, it can be suggested that all mentioned interactions
modify position, intensity and shape of absorption bands, resulting in a decrease in the
intensity of fluorescence emission of the ligands in ethanol:water media. For better
elucidation, effect of hydrolysis was examined by mass spectrometry studies and obtained
results are given in Section 3.4.
The fluorescence responses of LH2 and LDMH2 with the common metal ions were
investigated to evaluate whether these ligands could serve as a cation-selective chemosensor.
Interestingly, a significant fluorescence emission was observed only for Zn(II) and Al(III)
upon the treatment with LH2 and LDMH2. These peaks appeared at 446 nm for Zn(II) and at
484 nm for Al(III) in the emission scan spectra of both ligands (Fig. 4). In order to determine
the best excitation wavelength for Zn(II)-ligand solutions, the emission wavelength was fixed
at 446 nm. The most symmetrical and ideal gaussian-shape excitation peak was obtained at
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350 nm for both LH2 and LDMH2, so this wavelength was used as the excitation wavelength
during analysis.
On the other hand, no significant fluorescence emission was observed when LH2 and LDMH2
were treated with other cations (Fig. 4). In particular, the absence of any significant peak in
the emission scan spectra of the ligands treated with Co(II), Ni(II), Cu(II) and Cd(II), the most
frequently described as interfering ions for Zn(II) sensing in the literature, is one of the most
remarkable result at this point. In addition, the red shift of Al(III)-ligand emission peak
compared with Zn(II)-ligand peak will allow the fluorometric determination of Zn(II) without
any significant interference (selectivity of ligands to zinc ion over other metal ions were
investigated in Section 3.5). Finally, the reduced derivatives (LHH2 and LDMHH2) did not
give any fluorescence signal even with Zn(II) as expected (data not shown). Therefore, it
should be noted that further optimization studies were performed with LH2 and LDMH2.
The fluorescence associated with the reaction of ligands with Zn(II) can readily detectable
under a UV lamp. Under 365 nm UV light, a bright fluorescence was clearly visible to naked
eyes only for LH2-Zn2+ and LDMH2-Zn2+ solutions, whereas no significant fluorescence could
be observed for the ligand solutions with other metal ions (Fig. 5). This feature is useful for
the fast visual Zn(II) detection.
The observed fluorescence enhancement was attributed to occurrence of the strong
complexation via addition of Zn(II) to LH2 and LDMH2 ligands (Fig. S3). Based on the
obtained results it can be suggested that the complexation of ligands with Zn(II) makes the
Schiff bases more rigid, restricts the C=N isomerization, prevents the rapid hydrolysis and
produces a large chelation-enhanced fluorescence (CHEF) effect [37]. However, although
complex formation in Co(II)-ligand, Ni(II)-ligand, Cu(II)-ligand and Cd(II)-ligand solutions
was detected by UV-Vis spectrometer, these complexes did not exhibit fluorescence emission.
This can be explained by electron occupancy of d orbitals of the metal ions. Usually transition
metals with partially filled d-orbital often quench the fluorescence of the fluorophore through
a highly efficient non-radiative pathway while transition metals like Zn(II) with fully filled d-
orbital do not possess any low energy metal centered orbitals and hence such charge transfer
through non radiative pathway is prohibited [38-40].
3.2. Optimization studies
The parameters such as solvent, ligand concentration, pH, response time and stability of
ligand-metal complexes, and presence of interfering ions were optimized to achieve the best
fluorescence performance for the studied compounds.
3.2.1. Solvent effect
It is well known that the solvents play significant role in the optical properties of ligand-metal
complexes. In order to describe the fluorescence emission behavior of the LH2-Zn and
LDMH2-Zn complexes in different solvents, ligands were dissolved in ethanol, methanol,
acetonitrile, acetone, N,N dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and
isopropanol (210-5 M). The fluorescence scan spectra were recorded after mixing these
ligand solutions with Zn(II) solution (1:1 v/v, stock [Zn2+] = 2.010-3 M in water),
respectively. The fluorescence emission was observed with all of the above mentioned
solvents for the ligands in the presence of Zn(II). It was obtained that although the change in
wavelength of excitation and emission was not pronounced, different solvents showed
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enhancing and quenching effect on the emission intensity. As can be seen in Fig. 6 there was a
significant enhancement in the fluorescence intensity by addition of Zn(II) to the ethanol,
DMF and DMSO solutions of LH2 and LDMH2. Taking into account of the economic cost,
practical application, and environmental factors subsequent experiments were carried out with
the ligands dissolved in ethanol.
3.2.2. Ligand concentration
It is known that there is an equilibrium between the chemosensor/analyte in free form and the
chemosensor-analyte complex. An increase in the ligand concentration will drive this
equilibrium reaction to the right, which can lead to an improvement in fluorescence signal.
Therefore, fresh LH2 and LDMH2 solutions, each at 2.010-4 and 2.010-3 M concentrations,
were prepared in ethanol in order to examine the fluorescence response. After adding Zn(II)
solution (2.010-3 M in water) to each sensor solution (1:1 v/v), fluorescence spectra were
recorded (Ex = 350 nm). It was observed that addition of 2.010-3 M Zn(II) to 2.010-4 M
ligand solutions caused fluorescence enhancement approximately 2.5 fold for LH2 and 2 fold
for LDMH2 compared to the signals obtained from 2.010-5 M ligand-2.010-3 M Zn(II)
mixtures. On the other hand, after about 20 minutes following the addition of 2.010-3 M
Zn(II) to 2.010-3 M LH2, a white precipitate was observed. Such precipitation was observed
after 3 days for LDMH2-Zn mixture at same concentrations, however, in much lower
amounts. Under this conditions, 2.010-4 M ligand solutions were used in further studies.
3.2.3. Response time and stability
In this study, response time is defined as the time at which the complex formation reaction
reaches an equilibrium and the detected fluorescence signal becomes almost constant. Fig. 7
shows the fluorescence intensities of LH2-Zn and LDMH2-Zn complexes versus time at
different detection intervals. As can be seen in Fig. 7a the fluorescence intensity of LH2-Zn
showed a substantial increase over the time interval from 0 min to 30 min following mixing of
2.010-4 M ligand and 2.010-3 M zinc solutions. On the other hand, a much slower
fluorescence increase was observed in 2.010-4 M LDMH2 and 2.010-3 M Zn(II) mixture
(Fig. 7b), indicating that LDMH2-Zn complex formation was slower than LH2-Zn formation.
However, the fluorescence response of the LH2-Zn complex began to decrease after 60
minutes, while the fluorescence intensity of the LDMH2-Zn complex remained almost
constant after about 270th minute until 2 days. This result indicates that the interaction of
Zn(II) with LDMH2 forms a complex with much more stable fluorescent response than LH2-
Zn. Nevertheless, it should be pointed out that a decrease in fluorescence intensity of
LDMH2-Zn complex was also observed after 2 days. This study was repeated with the
solutions prepared by lower zinc concentration (2.010-5 M) than the ligand concentration
(2.010-4 M) and similar results were obtained (Fig. 7).
The decrease in the fluorescence response of LH2-Zn complex can be explained by the
alterations in its molecular structure. It has been reported that the versatile coordination
properties of the Zn(II) ion allow for a large variety of structures ranging from monomers to
oligomers or polymers and networks resulting from the self-assembly of Zn(II) with
polydentate ligands [41]. Such changes in chemical composition over time can quench the
fluorescence property or cause shifts in the absorption and emission maxima of LH2-Zn,
which results in a decrease in the emission at 446 nm. On the other hand, the decrease in
fluorescence response of LDMH2-Zn complex was observed after a very long time (after 2
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days). This result may be attributed to the flexibility of the backbone. Since 2,2'-dimethyl-1,3-
propanediamine is less flexible than 1,3-diaminopropane, the structural changes mentioned
above may take place over a long time span. As a result, it is more convenient to perform zinc
analysis within 30-60 minutes after the addition of LH2, however, LDMH2 and zinc mixture
should be left for approximately 5 hours prior to take the spectrum.
3.2.4. pH effect
The effect of pH on the fluorescence response was investigated to determine a suitable pH
condition for Zn(II) detection. For this purpose, a series of zinc solutions (blank, 2.010-3 M,
2.010-4 M and 2.010-5 M) each with a pH of 2-9 were prepared as representatives of Zn(II)
samples with different concentrations. The pH of the solutions was not adjusted to values
above 9 in order to avoid the precipitation of Zn(OH)2 totally. After the ligand solutions
(2.010-4 M) were added to these solutions, fluorescence spectra were recorded (Ex = 350
nm) after 45 minutes for LH2 and 5 hours for LDMH2 (Fig. 8).
No significant change in the fluorescence emission intensity of the blank solutions (ligand-
water) was observed in the mentioned pH range, indicating that Zn(II)-free ligand solutions
are pH-insensitive. However, in presence of all concentrations of Zn(II), solutions had a very
weak fluorescence response in the acidic environment (pH 4-5) due to protonation [42].
Protonation of active sites on the ligands inhibits their coordination with metals. It should be
indicated that no fluorescence emission was obtained in strong acid medium (pH 2-4),
probably due to acidic decomposition of the Schiff base complexes. This suggestion was
examined by ESI-MS and the results were reported in Section 3.4. As the pH increased (pH 5-
6), a gradual increase in fluorescence intensity was observed because of deprotonation.
However, a decrease in fluorescence intensity occurred when the pH value exceeded 7, which
can be explained by the onset of the formation of the colloidal Zn(OH)2 species. It is worth
noting that the largest fluorescence enhancement was achieved at about pH 7. Since pH value
of Zn(II)-ligand solutions is about 7 after mixing, Zn(II) can be detected by using LH2 and
LDMH2 without pH adjustment or buffering. This finding has some important outcomes, such
as reducing the time and chemical consumption and improving the practical application of the
method.
3.3. Job’s plot, fluorescence titration and binding constant
In order to determine the stoichiometry of LH2-Zn and LDMH2-Zn complexes, the Job’s plots
were obtained by the fluorescence method. The intensity of fluorescence emission was
measured by varying the mole fraction of Zn(II) (the ratio of Zn(II) and ligands varies in
increments from 0:10 up to 10:0). As shown in Fig. 9a and 9b, the fluorescence emission
intensity showed a maximum when the molar fractions of Zn(II) were very close to 0.5 for
both ligands. These results demonstrates the formation of 1:1ratio of ligands with Zn(II).
For better elucidation of the binding mode of ligands with Zn(II), fluorescence titration
experiments were performed. As clearly shown, increasing the concentration of Zn(II) results
in a gradual increase in the fluorescence intensity of LH2 (1×10-4 M) and LDMH2 (0.5×10-4
M) until saturation is achieved (Fig. S4). The saturation of 1x10-4 M LH2 and 0.5x10-4 M
LDMH2, evident in Fig. S4, has occurred almost with the same Zn(II) concentrations, which
corresponds to a 1:1 interaction. However, the exact position of the saturation point was
determined by linear regression equations obtained from the linear sections of the saturated
and non-saturated part of the curve (Fig. S4). The x-positions of the sections (corresponding
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to the Zn(II) concentrations at which saturation occurs) were calculated to be 0.91×10-4 M for
LH2-Zn and 0.47×10-4 M for LDMH2-Zn titration curves (Eq. S1), which is in good
agreement with the 1:1 interaction. Based on the fluorescence titration experiments, the
binding constants of 1:1 LH2-Zn and LDMH2-Zn complexes were calculated by Benesi–
Hildebrand plots and found to be 1.02×104 M-1 and 3.96×104 M-1, respectively (Fig. S5, Eq.
S2).
To further confirm the binding stoichiometry of ligands with Zn(II), ESI-MS experiments
were also carried out (Section 3.4).
3.4. ESI-MS binding studies
Electrospray ionization mass spectrometry (ESI-MS) was used to characterize the ligand-
metal binding properties. The ESI-MS spectra of LH2 and LDMH2 in ethanol (Fig. 10a and
10e) were dominated by the [M+H]+ ions at m/z 283 and 311 that corresponds to the
molecular weight of the Schiff bases. The peaks at m/z 305 (Fig. 10a) and m/z 333 (Fig. 10e)
were identified as the sodium adduct ions.
As mentioned in Section 3.1, the decrease in fluorescence intensity observed in ligand-water
mixtures was also examined by mass spectrometer. It was found that both ligands underwent
hydrolysis when they were mixed with DI water. As can be seen in Fig. 10b and 10f,
intensities of the molecular ion peaks of the ligands are relatively low and new ions are
observed in ESI-MS spectra, corresponding to hydrolysis products of the ligands. The ions at
m/z 179 (Fig. 10b) and m/z 207 (Fig. 10f) were assigned to [C10H15N2O]+ for LH2 and
[C12H19N2O]+ for LDMH2, respectively. It is proposed that these ions are generated by loss of
a hydrolysis product (salicylaldehyde) from the ligand structure. The ions at m/z 136, 107, 75
and 58, observed for both ligands, were attributed to [C8H10NO]+, [C7H7O]+, [C3H11N2]+ and
[C3H8N]+, respectively. The product ion at m/z 136 could be explained by loss of C2H5N
(ethanimine) group from m/z 179 and elimination of C4H9N (methallylamine) moiety from
m/z 207. Loss of CH3N (methenamine) group from m/z 136 led to the formation of an ion at
m/z 107. The peak at m/z 75 was assigned to molecular ion peak of 1,3-propanediamine and
direct elimination of NH3 from this molecule gave the peak observed at m/z 58 (allylamine).
It should be also indicated that the ion at m/z 103 was observed only in the spectra related to
LDMH2 and assigned to protonated N,N′-dimethyl-1,3-propanediamine. Although the ion at
m/z 75 was attributed to protonated 1,3-propanediamine, this peak can be also seen in
LDMH2 spectra due to direct loss of etilen from m/z 103.
The mass spectra of the LH2-Zn(II) and LDMH2-Zn(II) show molecular ion peaks at m/z 345
and m/z 373, respectively (Fig. 10c and 10g). These peaks correspond to the molecular weight
of the respective compounds and demonstrate that the binding ratio of ligands and Zn(II) is a
1:1 stoichiometry. These spectra also indicate that the ions of hydrolysis products are less
pronounced, which means Zn(II)-ligand complexes are relatively stable towards hydrolysis
and this coordination largely prevents premature ligand dissociation.
In Section 3.2.4, it was proposed that acidic decomposition occurred at pH 2-4. As can be
seen in Fig. 10d and 10h, no peak corresponding to the metal complexes or protonated free
ligands is observed, indicating these compounds were fully decomposed. The peaks found in
the spectra were resulted from the fragmentation of the main compounds. The peak at m/z 121
was attributed to [C7H5O2]+ and produced by loss of a labile hydrogen atom from
salicylaldehyde. The product ion at m/z 93 observed for both ligand-Zn complexes was
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generated by elimination of CO group from m/z 121. The ion m/z 86, like the ion m/z 103,
was found only in the mass spectrum associated with LDMH2 and formed by the loss of NH3
group from m/z 103. In addition to these ions, the ions at m/z 75 and 58 structural information
of which were given above were also seen in the spectrum.
Finally, the decrease in fluorescence response observed for the LH2-Zn complex solution after
60 minutes was mentioned in Section 3.2.3, and it was suggested that this decrease might be
due to alterations in molecular structure over time. This suggestion was confirmed by ESI-MS
studies and found that new peaks were appeared at m/z 690, 692 and 694 in the mass
spectrum of LH2-Zn solution after 60 minutes (data not shown). It should be also pointed out
that intensity of these peaks were increased over time. The ions at m/z 690 [M] +, 692 [M+2]+
and 694 [M+4]+ were indicative of new dimeric Zn(II) complex formation. Furthermore, the
white precipitate obtained after the addition of 2.010-3 M Zn(II) to 2.010-3 M LH2 (Section
3.2.2) probably occurred due to the formation such dimeric or oligomeric etc. structures.
3.5. Selectivity of ligands to zinc ion over other metal ions
The selectivity of LH2 and LDMH2 for Zn(II) over other metal ions, such as Na(I), K(I),
Mg(II), Ca(II), Al(III), Cr(III), Mn(II), Hg(II), Fe(III), Co(II), Ni(II), Cu(II), Cd(II) and
Pb(II), was investigated. Based on the data obtained from the optimization studies, firstly each
ion (210-3 M, in water) was added to zinc solution (210-4 M, in ethanol) (1:1, v/v) and then
each metal ion solution was mixed with LH2 and LDMH2 (210-4 M) (1:1 v/v). The pH
adjustment was not required because the pH of the solutions were in neutral conditions after
mixing. Fluorescence spectra of these mixtures were recorded (Ex = 350 nm) after 45 minutes
for LH2 and 5 hours for LDMH2 and fluorescence intensity of each solution was given in Fig.
11. LH2 and LDMH2 were exhibited very good selectivity of zinc over other metals. While
Al(III) ion slightly enhanced the fluorescence signal, Fe(III), Ni(II) and Cu(II) ions showed
very little quenching effect. However, it should be noted that total interference of the metal
ions had not practical significance when the fluorescence intensity of [Zn + all metal ions + L]
was compared with that of [Zn + blank + L] solution.
The fluorescence response of the ligands toward individual metal ions has been previously
discussed in Section 3.1. LH2 and LDMH2 have shown high selectivity for Zn(II) over other
metal ions due to similar reasons such as close-shelled orbitals of zinc, appropriate ion radius,
and binding affinity. It should be indicated that some metal ions such as Co(II), Ni(II), Cu(II)
quenched the fluorescence significantly when the ligand concentrations were 110-5 M (data
not shown). This can be explained by the fact that ligands have a higher affinity for these
interfering cations than Zn(II) and probably only a small fraction of the ligand is available for
binding to Zn(II) due to low ligand concentration. Thus, using a ligand at 110-4
M
concentration does not only enhance the fluorescence response mentioned in Section 3.2.2,
but also minimizes the quenching effect of certain cations by providing free ligand for Zn(II).
3.6. Analytical figures of merit
Under optimal conditions, individual calibration curves were constructed with several points
for the determination of Zn(II). As shown in Fig. 12a, LH2-Zn exhibited a linear response for
the Zn(II) concentration range of 0.8  6.010-5 M with correlation coefficient of R2  0.9992
(n  3). Limit of detection was calculated to be 0.510-6 M based on the definition by IUPAC
(CDL  3Sb/m, where Sb is the standard deviation from 10 blank solutions while m
represents the slope of the calibration curve). On the other hand, the fluorescence response
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observed for LDMH2-Zn was linear for a wider range of Zn(II) concentrations (0.3  5.010-5
M, R2  0.9995) compared to LH2-Zn. Moreover, with a limit of detection down to 0.110-6
M, LDMH2 was found to be more sensitive to Zn(II) ions at low concentrations than LH2. For
this reason, LDMH2 is recommended as the preferred sensor for identification of low Zn(II)
concentrations. In addition, the calibration curve of LH2-Zn exhibited a saturation level
beyond 810-5 M, so Zn(II) concentrations higher than 810-5 M could not be detected by
using LH2. However, the plateau observed for the Zn-LDMH2 calibration curve originated
from the fact that the fluorescence intensities of Zn(II) concentrations above 510-5 M were
higher than the maximum setting value (1000 a.u) of the y-axis of the spectra. It should be
indicated that linearity was obtained in the range of 7×10-5  0.8×10-5 M Zn (II)
concentrations by reducing the emission slit width to 2.5 and creating a new calibration curve
(the inset of Fig. 12b) based on this setting.
For the purpose of demonstrating the practical usefulness of the procedure, the analysis of
Zn(II) in a certified ICP multielement (68 element) standard solution ICP-MS-68A was done
by the proposed method under optimal conditions. Each metal concentration in this solution is
10 mg/L and standard is acidified with 2% HNO3 to prevent hydrolysis of these metals.
Therefore, appropriate dilutions were made to ensure that the Zn(II) concentration fell within
the linear portion of the calibration curve and then pH of the sample was adjusted to
approximately 7 (neutral pH). After the addition of LH2 and LDMH2 (210-4 M in ethanol,
1:1 v/v), samples were left for 45 minutes for LH2 and 5 hours for LDMH2 prior to take the
spectrum. Fluorescence analysis of each sample was repeated 5 times (Ex = 350 nm). The
results were calculated by taking into account the dilution factor (Table 1).
In summary, according to the analytical performance parameters of the proposed method, LH2
and LDMH2 were adequate for the detection and quantification of Zn(II) in water samples,
containing various cations. In this study, the sensitivity of the method is described in the terms
of the limit of detection and the linearity evaluation experiments. With the methods based on
LH2 and LDMH2 discussed above, sufficiently low LODs were achieved which allow the
determination of low Zn(II) concentrations. The correlation coefficients (R2) of the analytical
curves were greater than 0.999, which is evidence of a fit of the data to the regression line.
Since certified reference material was used, accuracy was expressed in terms of bias % in this
study. It should be noted that proposed methods for both ligands provided bias less than 10%.
The precision of the analyses was evaluated by determining the relative standard deviation
(RSD %) under repeatability conditions and the methods based on both ligands were reported
to be highly precise with an RSD of <1%.
3.7. DNA comet imaging
The effect of gamma-ray on DNA of chicken breast meat was examined by using a DNA
comet assay. Fig. 1a and 1d show the color of LH2-Zn and LDMH2-Zn complexes, revealed
as green under the microscope with U-DM-DA/FI/TX2E filter. After staining with LH2-Zn
and LDMH2-Zn solutions and waiting at about 10 min, the non-irradiated and irradiated meat
samples of chicken were clearly distinguishable just by naked eye inspection at the slide
under the microscope as yellow-orange color (Fig 13b, 13c, 13e, 13f). Non-irradiated meat
samples of chicken showed no comets because there was no DNA damage at 0.0 kGy as seen
in Fig. 13b and 13e. In this assay, strand breaks of individual cells due to the gamma-ray
irradiation at 3 kGy appear like tails of a comet (Fig. 13c and 13f). According to the obtained
results, LH2-Zn and LDMH2-Zn solutions can serve as an alternative staining agent for
identifying the radiation treatment of food by DNA comet assay. Future study will be focused
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on the implementation of LH2-Zn and LDMH2-Zn complexes as staining agents on several
food and biological samples. Moreover, fluorescence imaging experiments will be carried out
in living cells.
4. Conclusion
In this study, four simple structure Schiff bases were synthesized and absorption, excitation
and fluorescence properties of their zinc complexes were examined with the purpose of
obtaining information about the usability of these ligands as a chemosensor for Zn(II) in
aqueous solution. In conclusion, we successfully developed two “turn on” fluorescent
chemosensors for the detection of Zn(II). The results obtained from this study suggest several
important conclusions.
1. LH2 and LDMH2 showing good selectivity toward Zn(II) can be synthesized by a facile
one-step Schiff base reaction, moreover LH2 is commercially available. We think that this
will provide a great convenience and flexibility to researchers who need to analyze zinc
ion.
2. Although these are not new ligands, we have reported for the first time their applicability
as fluorescent chemosensors. In addition, new methods have been developed for the rapid
detection of Zn(II) using these ligands.
3. The important role of the C=N group in the fluorescent sensing mechanism has been
demonstrated by studying with reduced derivatives of the ligands (LHH2 and LDMHH2).
4. The optimized method allows the determination of zinc in aqueous solutions. Zinc can be
identified and quantified in aqueous neutral samples without pH adjustment.
5. The time required to reach the complex formation equilibrium is not very short (especially
for LDMH2-Zn), but most importantly, ligands are able to differentiate Zn2+ from other
cations.
6. The optimized methods based on LH2 and LDMH2 show a good range of linearity, high
precision, good accuracy and low detection limits.
7. These ligands can also be used as chemosensors for the detection of Zn(II) by the naked
eye under UV lamp and thus allows for an extremely simple, rapid, and low-cost Zn(II)
analysis by obviating the requirement of sophisticated equipments.
8. Beside the Zn(II) detection in water samples, LH2-Zn and LDMH2-Zn complexes offer an
promising alternative as staining agent for identifying the radiation treatment of food by
DNA comet assay.
Acknowledgements
We would like to thank Prof. Dr. Orhan Atakol for his guidance and support during ligand
synthesis.
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References
[1] S. Soni, A. Salhotra, M. Suar, Handbook of Research on Diverse Applications of
Nanotechnology in Biomedicine, Chemistry, and Engineering. Chapter 24.
Functionalized Magnetic Nanoparticles for Environmental Remediation, Sources of
Heavy Metals in the Environment, IGI Global, (2015) p. 521.
[2] R.A. Wuana, F.E. Okieimen, Heavy Metals in Contaminated Soils: A Review of
Sources, Chemistry, Risks and Best Available Strategies for Remediation, ISRN Ecol.
402647 (2011) 20 pages.
doi: 10.5402/2011/402647
[3] A. Dutta, Regulation of Zinc Homeostasis in Cultured Cells. Chapter 1. Introduction,
ProQuest LLC, (2008) p. 1.
[4] N.C. Lim, H.C. Freake, C. Brückner, Illuminating Zinc in Biological Systems, Chem.
Eur. J. 11 (2005) 38-49.
doi: 10.1002/chem.200400599
[5] Y. Guo, X. Tang, F. Hou, J. Wu, W. Dou, W. Qin, J. Ru, G. Zhang, W. Liu, X. Yao, A
reversible fluorescent chemosensor for cyanide in 100% aqueous solution, Sens.
Actuators B Chem. 181 (2013) 202-208.
http://dx.doi.org/10.1016/j.snb.2013.01.053
[6] Q. Lin, T.T. Lu, X. Zhu, T.B. Wei, H. Li, Y.M. Zhang, Rationally introduce multi-
competitive binding interactions in supramolecular gels: a simple and efficient
approach to develop multi-analyte sensor array, Chem. Sci. 7 (2016) 5341-5346.
http://dx.doi.org/10.1039/C6SC00955G
[7] Q. Lin, K.P. Zhong, J.H. Zhu, L. Ding, J.X. Su, H. Yao, T.B. Wei, Y.M. Zhang, Iodine
Controlled Pillar[5]arene-Based Multiresponsive Supramolecular Polymer for
Fluorescence Detection of Cyanide, Mercury, and Cysteine, Macromolecules 50
(2017) 7863-7871.
DOI: 10.1021/acs.macromol.7b01835
[8] Q. Lin, P.P. Mao, Y.Q. Fan, L. Liu, J. Liu, Y.M. Zhang, H. Yao, T.B. Wei, A novel
supramolecular polymer gel based on naphthalimide functionalized-pillar[5]arene for
the fluorescence detection of Hg2+ and I− and recyclable removal of Hg2+via cation–π
interactions, Soft Matter 13 (2017) 7085-7089.
DOI: 10.1039/C7SM01447C
[9] Q. Lin, Y.Q. Fan, P.P. Mao, L. Liu, J. Liu, Y.M. Zhang, H. Yao, T.B. Wei,
Pillar[5]arene-Based Supramolecular Organic Framework with Multi-Guest Detection
and Recyclable Separation Properties, Chem. Eur. J. 24 (2018) 777-783.
DOI: 10.1002/chem.201705107
[10]Z. Xu, J. Yoon, D.R. Spring, Fluorescent chemosensors for Zn2+, Chem Soc Rev. 39
(2010) 1996-2006.
doi: 10.1039/B916287A
14

ACCEPTED MANUSCRIPT
[11]T. Liu, S. Liu, Responsive Polymers-Based Dual Fluorescent Chemosensors for Zn2+
Ions and Temperatures Working in Purely Aqueous Media, Anal. Chem. 83 (2011) (7)
2775-2785.
dx.doi.org/10.1021/ac200095f
[12]S.A. Ingale, F. Seela, A Ratiometric Fluorescent On–Off Zn2+ Chemosensor Based on
a Tripropargylamine Pyrene Azide Click Adduct, J. Org. Chem. 77 (2012) 9352-9356.
doi:10.1021/jo3014319
[13]Z. Mao, L. Hu, X. Dong, C. Zhong, B. Liu, Z. Liu, Highly Sensitive Quinoline-Based
Two-Photon Fluorescent Probe for Monitoring Intracellular Free Zinc Ions, Anal.
Chem. 86 (2014) 6548-6554.
doi: 10.1021/ac501947v
[14]Y. Tang, Y. Ding, X. Li, H. Ågren, T. Li, W. Zhang, Y. Xie, Acylation of
dipyrromethanes at the α and β positions and further development of fluorescent Zn2+
probes,
Sens.
Actuators
B
Chem.
206
(2015)
291-302.
http://dx.doi.org/10.1016/j.snb.2014.09.060
[15]H. Yu, T. Yu, M. Sun, J. Sun, S. Zhang, S. Wang, H. Jiang, A symmetric pseudo salen
based turn-on fluorescent probe for sensitive detection and visual analysis of zinc ion,
Talanta 125 (2014) 301-305.
http://dx.doi.org/10.1016/j.talanta.2014.03.011
[16]K. Akutsu, S. Mori, K. Shinmei, H. Iwase, Y. Nakano, Y. Fujii, Investigation of
substitution effect on fluorescence properties of Zn2+-selective ratiometric fluorescent
compounds: 2-(2′-Hydroxyphenyl)benzimidazole derivatives, Talanta 146 (2016) 575-
584.
http://dx.doi.org/10.1016/j.talanta.2015.09.001
[17]L. Li, Y. Dang, H. Li, B. Wang, Y. Wu, Fluorescent chemosensor based on Schiff
base for selective detection of zinc(II) in aqueous solution, Tetrahedron Lett. 51
(2010) 618–621.
http://dx.doi.org/10.1016/j.tetlet.2009.11.070
[18]M. Hosseini, Z. Vaezi, M.R. Ganjali, F. Faridbod, S.D. Abkenar, K. Alizadeh, M.
Salavati-Niasari, Fluorescence turn-on chemosensor for the selective detection of
zinc ion based on Schiff-base derivative, Spectrochim. Acta Part A 75 (2010) 978-982.
http://dx.doi.org/10.1016/j.saa.2009.12.016
[19]M. Yan, T. Li, Z. Yang, A novel coumarin Schiff-base as a Zn(II) ion fluorescent
sensor, Inorg. Chem. Commun. 14 (2011) 463-465.
doi:10.1016/j.inoche.2010.12.027
[20]K. Li, X. Wang, A. Tong. A “turn-on” fluorescent chemosensor for zinc ion with
facile synthesis and application in live cell imaging, Anal. Chim. Acta 776 (2013) 69-
73. http://dx.doi.org/10.1016/j.aca.2013.03.033
15

ACCEPTED MANUSCRIPT
[21]N. Roy, H.A.R. Pramanik, P.C. Paul, S.T. Singh, A Sensitive Schiff-Base Fluorescent
Chemosensor for the Selective Detection of Zn2+, J. Fluoresc. 24 (2014) 1099-1106.
doi:10.1007/s10895-014-1390-3
[22]J. Yan, L. Fan, J. Qin, C. Li, Z. Yang, A novel and resumable Schiff-base fluorescent
chemosensor
for
Zn(II),
Tetrahedron
Lett.
57
(2016)
2910-2914.
http://dx.doi.org/10.1016/j.tetlet.2016.05.079
[23]V.K. Gupta, A.K. Singh, L.K. Kumawat, A turn-on fluorescent chemosensor for Zn2+
ions based on antipyrine schiff base, Sens. Actuators B Chem. 204 (2014) 507-514.
http://dx.doi.org/10.1016/j.snb.2014.07.128
[24]Y. Li, K. Li, J. He. A “turn-on” fluorescent chemosensor for the detection of Zn(II) in
aqueous solution at neutral pH and its application in live cells imaging, Talanta 153
(2016) 381-385.
http://dx.doi.org/10.1016/j.talanta.2016.03.040
[25]G.J. Park, J.J. Lee, G.R. You, L. Nguyen, I. Noh, C. Kim, A dual chemosensor for
Zn2+ and Co2+ in aqueous media and living cells: Experimental and theoretical studies,
Sens. Actuators B Chem. 223 (2016) 509-519.
http://dx.doi.org/10.1016/j.snb.2015.09.129
[26]J. Farkas, Irradiation as a method for decontaminating food. A review, Int. J. Food
Microbiol. 1998 44(3):189-204.
https://doi.org/10.1016/S0168-1605(98)00132-9
[27]H.M. Khan, A.A. Khan, S. Khan, Application of DNA comet assay for detection of
radiation treatment of grams and pulses, J. Food Sci. Technol. (2011) 48(6):718-723.
DOI 10.1007/s13197-010-0169-z
[28]Andrew R. Collins, The comet assay for DNA damage and repair: Principles,
applications, and limitations, Mol. Biotechnol. (2004) 26: 249-261.
https://doi.org/10.1385/MB:26:3:249
[29]Y. Jo, B. Sanyal, K. Ameer, J.H. Kwon, Characterization of DNA comet and cellulose
radical signal in Valencia oranges treated with different forms of ionizing radiation,
Postharvest Biology and Technology 135 (2018) 68-76.
http://dx.doi.org/10.1016/j.postharvbio.2017.08.004
[30]T. Gichner, A. Mukherjee, J. Velemínský, DNA staining with the fluorochromes EtBr,
DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl
methanesulphonate, hyperthermia and DNase-I, Mutat. Res. 605 (2006) 17-21.
doi:10.1016/j.mrgentox.2006.01.005
[31]S. Durmuş, Ü. Ergun, J.C. Jaud, K.C. Emregül, H. Fuess, O. Atakol, Thermal
decomposition of some linear trinuclear Schiff base complexes with acetate bridges, J.
Therm. Anal. Calorim. 86 (2006) 337–346.
https://doi.org/10.1007/s10973-005-7282-7
16

ACCEPTED MANUSCRIPT
[32]B.M. Ateş, B. Zeybek, M. Aksu, Ü. Ergun, F. Ercan, M.L. Aksu, O. Atakol, Thermal
decomposition of new mononuclear NiII complexes with ONNO type reduced Schiff
bases and pseudo halogens, Z. Anorg. Allg. Chem. 636 (2010) 840–845.
doi: 10.1002/zaac.200900499
[33]CEN, 2001. EN 13784 Foodstuffs – DNA Comet Assay for the Detection of Irradiated
Foodstuffs – Screening Method. European Committee for Standardization, Brussles.
[34]D.L. Pavia, G.M. Lampman, G.S. Kriz, J.A. Vyvyan, Introduction to Spectroscopy.
Chapter 10 Ultraviolet Spectroscopy, Cengage Learning, 5th edition, USA, (2014) p.
602.
[35] E.H. Cordes, W.P. Jencks, The mechanism of hydrolysis of Schiff bases derived from
aliphatic amines, J. Am. Chem. Soc. 85 (1963) 2843-2848.
DOI: 10.1021/ja00901a037
[36]A.C. Dash, B. Dash, P.K. Mahapatra, Hydrolysis of imines. Part 2. Kinetics and
mechanism of hydrolysis of N-Salicylidene-2-aminopyridine in the presence and
absence of copper (II) Ion. A study of the catalytic effect of some mixed-ligand
complexes of copper (II), J. Chem. Soc., Dalton Trans. 8 (1983) 1503-1509.
DOI: 10.1039/DT9830001503
[37]L. Yang, W. Zhu, M. Fang, Q. Zhang, C. Li, A new carbazole-based Schiff-base as
fluorescent chemosensor for selective detection of Fe3+ and Cu2+, Spectrochim. Acta
Part A 109 (2013) 186-192.
http://dx.doi.org/10.1016/j.saa.2013.02.043
[38]L. Wang, H. Li, D. Cao, A new photoresponsive coumarin-derived Schiff base:
Chemosensor selectively for Al3+ and Fe3+ and fluorescence turn-on under room
light, Sens. Actuators B Chem. 181 (2013) 749-755.
http://dx.doi.org/10.1016/j.snb.2013.01.090
[39]D. Sarkar, A.K. Pramanik, T.K. Mondal, Coumarin based fluorescent ‘turn-on’
chemosensor for Zn2+: An experimental and theoretical study, J. Lumin. 146 (2014)
480-485.
http://dx.doi.org/10.1016/j.jlumin.2013.09.080
[40]J. Wu, W. Liu, X. Zhuang, F. Wang, P. Wang, S. Tao, X. Zhang, S. Wu, S. Lee,
Fluorescence turn on of Coumarin derivatives by metal cations: A new signaling
mechanism based on C=N isomerization, Org. Lett. 9 (2007) 33-36.
doi:10.1021/ol062518z
[41]A. Erxleben, Structures and properties of Zn(II) coordination polymers, Coord. Chem.
Rev. 246 (2003) 203-228.
http://dx.doi.org/10.1016/S0010-8545(03)00117-6
[42]A.A. El-Sherif, M.S. Aljahdali, Review: protonation, complex-formation equilibria,
and metal–ligand interaction of salicylaldehyde Schiff bases, J. Coord. Chem. 66
(2013) 3423-3468. http://dx.doi.org/10.1080/00958972.2013.839027
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Figure Captions
Fig. 1. Molecular structure of a) N-N'-bis(salicylidene)-1,3-propanediamine (LH2), b) bis-
N,N'-(salicylidene)-2,2'-dimethyl-1,3-propanediamine
(LDMH2),
c)
bis-N,N'-(2-
hydroxybenzyl)-1,3-propanediamine (LHH2), d) bis-N,N'-(2-hydroxybenzyl)-2,2'-dimethyl-
1,3-propanediamine (LDMHH2)
Fig. 2. UV–Vis spectra of a) LH2 (1.010-5 M in ethanol) and LHH2 (1.010-5 M in ethanol) b)
LH2 (1.010-5 M in ethanol) and LH2-cation mixtures (1:1 v/v, stock [LH2] = 2.010-5 M in
ethanol, stock [cation] = 2.010-3 M in DI water)
Fig. 3. Fluorescence excitation (inset) and emission spectra of a) LH2 (1.010-5 M in ethanol)
and LH2-DI water mixture (1:1 v/v, stock [LH2] = 2.010-5 M in ethanol) b) LDMH2 (1.010-
5
M in ethanol) and LDMH2-DI water mixture (1:1 v/v, stock [LDMH2] = 2.010-5 M in
ethanol)
Fig 4. Fluorescence emission spectra of LH2 and LDMH2 (inset) in the presence of different
cations (1:1 v/v, stock [ligand] = 2.010-5 M in ethanol, stock [cation] = 2.010-3 M in DI
water)
Fig 5. Photographs of a) LH2 and b) LDMH2 with various metal ions under UV light (λEx =
365 nm).
Fig. 6. Fluorescence intensity changes of LH2 and LDMH2 (1.010-5 M) in presence of Zn2+
(1.010-3 M) in different solvents: 1- ethanol; 2- methanol; 3- acetonitrile; 4- acetone; 5-
DMF; 6- DMSO; 7- isopropanol at room temperature, respectively
Fig. 7. The influence of response time on the fluorescent intensity of a) LH2 b) LDMH2 in the
presence of Zn(II) (1:1 v/v, stock [LH2] = [LDMH2] = 2.010-4 M in ethanol, stock [Zn2+] =
2.010-3 M in DI water) (Ex = 350 nm)
Fig. 8. The influence of pH on the fluorescent intensity of LH2 and LDMH2 in the presence of
Zn(II) (1:1 v/v, stock [LH2] = [LDMH2] = 2.010-4 M in ethanol, stock [Zn2+] = 2.010-5 M
in DI water) (Ex = 350 nm)
Fig. 9. Job's plot data for evaluating the stoichiometry of a) LH2-Zn and b) LDMH2-Zn
complexes. The total concentrations of LH2+Zn(II) and LDMH2+Zn(II) were kept at 1.010-4
M and 0.510-4 M, respectively (Ex = 350 nm)
Fig. 10. ESI-MS spectra of a) LH2 in ethanol (1.010-4 M) b) LH2 in ethanol + DI water
mixture (1:1, v/v) c) LH2 in the presence of 1 equiv. Zn(II) pH  7 d) LH2 in the presence of 1
equiv. Zn(II) pH = 3 e) LDMH2 in ethanol (1.010-4 M) f) LDMH2 in ethanol + DI water
mixture (1:1, v/v) g) LDMH2 in the presence of 1 equiv. Zn(II) pH  7 h)LDMH2 in the
presence of 1 equiv. Zn(II) pH = 3
Fig. 11. Selectivity of LH2 and LDMH2 for Zn(II) in the absence or presence of other metal
ions (stock [LH2] = [LDMH2] = 2.010-4 M in ethanol, stock [Zn2+] = 2.010-5 M in DI water,
stock [Na+] = [K+] = [Mg2+] = [Ca2+] = [Al3+] = [Cr3+] = [Mn2+] = [Hg2+] = [Fe3+] = [Co2+] =
[Ni2+] = [Cu2+] = [Cd2+] = [Pb2+] = 2.010-3 M in DI water, Ex = 350 nm, pH  7)
Fig. 12. Fluorescence intensity of a) LH2 b) LDMH2 as a function of Zn(II) concentration (1:1
v/v, stock [ligand] = 2.010-5 M in ethanol, stock [Zn2+] = 2.010-4 M in DI water, Ex = 350
nm, Em = 446 nm)
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Fig. 13. The color of a) LH2-Zn and d)LDMH2-Zn complexes under the fluorescence
microscope with U-DM-DA/FI/TX2E filter. DNA comet assay images after electrophoresis of
cells from chicken breast meat irradiated at b) 0.0 kGy LH2-Zn staining c) 3.0 kGy LH2-Zn
staining e) 0.0 kGy LDMH2-Zn staining and f) 3.0 kGy LDMH2-Zn staining
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Highlights
 Four Schiff base derivatives were investigated as a fluorescent sensor for Zn(II)
 The structure of the sensors are quite simple, one of them is commercially available
 Two chemosensors can selectively recognize Zn(II) ions over other important cations
 Optimized methods show good linearity, precision, accuracy and low detection
limits
 Two chemosensors can be used as fluorescent DNA staining agents
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Graphics Abstract

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