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3+ 2+
A novel turn on fluorescent probe for the determination of Al and Zn ions and its
cells applications

Duygu Aydin

PII: S0039-9140(19)31248-2
DOI: https://doi.org/10.1016/j.talanta.2019.120615
Reference: TAL 120615

To appear in: Talanta

Received Date: 25 September 2019

Revised Date: 29 November 2019
Accepted Date: 2 December 2019

3+
Please cite this article as: D. Aydin, A novel turn on fluorescent probe for the determination of Al and
2+
Zn ions and its cells applications, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2019.120615.

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Graphical Abstract
A Novel Turn On Fluorescent Probe For The Determination of Al3+ and Zn2+ Ions
and Its Cells Applications

Duygu AYDIN

Karamanoglu Mehmetbey University, Kamil Ozdag Science Faculty, Department of Chemistry, Karaman,
70100 Turkey

*Corresponding Author: Dr. Duygu AYDIN, Kamil Ozdag Science Faculty, Department of Chemistry,
Karamanoglu Mehmetbey University, 70100 Karaman, Turkey
Tel.: +90 338 226 00 00/3860
E-mail: [email protected]

Abstract
Early detection of probes is very important for limiting toxic effects of various
transition metal ions for example aluminum and zinc. Monitoring of these metal ions
can be challenging via conventional methods since they are high cost instrumentations,
time consuming and so on. We report facile preparation of a fluorescence probe
containing biphenyl groups that effectively selective and superb sensitivity towards
aluminum (III) and zinc (II) in neutral solutions without interference from each other
and other ions. In neutral pH value, the probe FOB displayed the OFF-ON fluorescence
enhancement at 464 nm and 512 nm toward aluminum (III) and zinc (II), respectively.
The detection limit values of FOB for Al3+ and Zn2+ in neutral solutions were 1.27 and
1.02 nM, respectively and these values were significantly lower than permitted Al3+ and
Zn2+ concentrations in drinking water determined by the World Health Organization
(WHO) and European Water Quality. Also, fabricated cyano-biphenyl based probe is
effective for sensing for aluminum (III) and zinc (II) in living human colon cancer cells
even when employed at low concentrations (1µM). Overall, this work allows us to
obtain a great potential to be applied to detect Al3+ and Zn2+.

Keywords: Fluorescence sensor, aluminum and zinc ion.

1
1. Introduction

Utilizing of heavy metal ions in industry, environmental and biological sources and
several human activities causes various side effects such as contamination of
environment and several diseases [1–4]. Among the various metal ions, aluminum and
zinc ions exist widely in air, water, and soil because of acidic rain. These d block metal
ions have broad applications in daily life including clinical drugs, packing materials,
water treatment, cookie sheets and etc and so they are crucial agents for human life.
However, high concentration of aluminum and zinc ions may cause many serious
diseases [5–9]. Over the past decade, based on these metal ions, extensive research has
reported that aluminum (III) ions induces a serious threat to public health, when it is in
high concentration levels. The excessive aluminum (III) ions may cause several human
diseases like Alzheimer's disease, Wilson syndrome, Parkinson's disease, dementia and
impairment of memory, breast cancer and including dialysis encephalopathy. Moreover,
the adverse effect of zinc (II) ions can cause a serious detrimental health disorders
including prostate cancer, cerebral ischemia and epilepsy. Thus, the study of
determination of aluminum (III) and zinc (II) ions in solutions and environmental and
biological systems is valuable to check its effect on the human life and also
environment [10–13].

Compared with other analytical methodologies for example ions atomic absorption
spectrometry, inductively coupled mass atomic emission spectrometry (ICM–AES),
electrochemical assays, inductively coupled plasma optical emission spectroscopy
(ICP–OES), ion selective membrane and so on are currently available for regarding the
detection of aluminum (III) and zinc (II) ions [8,14–16]. Nevertheless, these methods
are not effective applied for the sensing and recognition of environmentally and
biologically pertinent metal ions because they are high cost instrumentations, need an
extremely trained person, complicated and time consuming procedures and lack of
selectivity. Therefore, fluorescence chemosensors have emerged as one of the most
widely utilized analytical tools because of their unique advantages such as excellent
temporal-spatial resolution, non-invasiveness, operational simplicity, high sensitivity,
good selectivity, low cost and rapid response time. Recently, various fluorescent
chemosensors including coumarin, rhodamine, naphalimide, pyrene, BODIPY and
fluorescein were exploited for detection of aluminum (III) and zinc (II) [13,17–20].
Cyano-biphenyl moiety has also been employed to identify various metal ions including

2
aluminum (III) and zinc (II). For instance, Erdemir et. al. reported a cyanobiphenyl
containing fluorescent chemosensor for the selective determination of aluminum (III)
and zinc (II) ions [21]. Also, hydrazides have been widely employed in many research
areas because of their easy preparation, tunable electronic properties and good chelating
capability simple [12,22]. Li et. al. developed an a novel pyrazine-derived fluorescent
chemosensor for monitoring Al3+ in ethanol [23].

Herein, we report a novel ultrasensitive and selective fluorescent ''turn on'' probe, FOB,
based on cyano-biphenyl and 2-furoic hydrazide derivative that could be used as dual
analyte fluorescent chemosensor to monitor aluminum (III) and zinc (II) ions in
ACN/H2O (v/v=80/20, 1µM, pH=7.0) solution. Moreover, the FOB sensor can monitor
Al3+ and Zn2+ without being affected by various metal ions. Additionally, the results
showed that fabricated probe could be employed in living cells for determination of Al3+
and Zn2+. Therefore, fabricated probe can be chosen as a tool for detection of Al3+ and
Zn2+.

2. Experimental Section

2.1. Materials and instrumentation

4′-Hydroxy-4-biphenylcarbonitrile, hexamethylenetetramine (HMTA) and 2-furoic

hydrazide were bought from Sigma-Aldrich. Other reagents and solvents were obtained
from commercial suppliers (Sigma-Aldrich and Merck). Unless otherwise stated, they
were utilized without further purification. Preperation of 3-formyl-4-hydroxy-4-
biphenylcarbonitrile (1) was undertaken according to a previously reported methods [9].
1
H NMR spectra was performed utilizing a Varian 300 MHz spectrometer in deuterated
dimethyl sulfoxide (DMSO-d6) at room temperature. Fluorescent spectra were taken
using on a Perkin Agilent Cary Eclipse spectrometer using quartz cells of 1.0 cm path
length. Elemental analysis data was obtained using a Leco CHNS-932 elemental
analyzer (Leco, USA).

2.2. Synthesis of the Fluorescent Probe (FOB)

To synthesize the ligand (FOB), 3-formyl-4-hydroxy-4-biphenylcarbonitrile (1) (0.5 g,

2.24 mmol) and 2-furoic hydrazide (0.31 g, 2.44 mmol) was refluxed in absolute
ethanol (30 mL) for 3 h under nitrogen atmosphere. After 3h, the solution was filtered to

3
obtain precipitated ligand and unreacted species were removed by washing fabricated
precipitate with excess EtOH. The precipitate was dried in vacuo and yield is 81%;
[Found: C, 73.60; H, 4.02; N, 9.35; O, 13.03. C46H31N5O6 requires C, 73.69; H, 4.17; N,
9.34; O, 12.80%]. nmax (solid, ATR) 3225, 3113, 3058, 2217, 1653, 1598, 1532, 1479,
1355, 1284, 1183, 1011, 960, 824; dh (300 MHz, DMSO-d6) 12.28 (1H, s), 11.49 (1H,
s), 8.70 (1H, s), 7.96 (2H, m), 7.87 (4H, dd, J 8.50 Hz), 7.71 (1H, dd, J 2.29 Hz, J 8.70
Hz), 7.33 (1H, d, J 3.40 Hz), 7.05 (1H, t, J 8.60 Hz), 6.71 (1H, m); dc (100 MHz,
DMSO-d6) 158.66, 154.74, 148.06, 146.86, 144.54, 133.51, 130.72, 130.06, 128.24,
127.53, 120.10, 119.69, 117.94, 116.14, 112.93, 109.89.

2.3. Fluorescence experiments

For fluorescence studies, the stock solution of FOB was prepared in DMSO (1 mM) and
diluted with the mixture of ACN/H2O (80/20) (1 µM, 25 mL) and 3 mL probe solution
was added to a cuvette to evaluate detection of various cations. Perchlorate salts of
metals were used and solved in ACN for preparing cation solutions (1mM).
Corresponding volume of perchlorate salts were put into solution of FOB and then the
spectra of mixtures were recorded after mixing for 10 min by the help of the
fluorescence spectroscopy utilizing λex=355 nm, λem=464 nm for Al(III) and λex=355
nm, λem=512 nm for Zn(II). Also, slit widths=10 nm, 10 nm and all titration
experiments were repeated three times.

2.4. Cell Culturing and Cytotoxicity Testing

Human prostate cancer PC-3 (CRL 1435) cell lines were obtained from ATCC
(American Type Culture Collection, Rockville, MD, USA). The PC-3 cells were
maintained in Ham's F12K Medium supplemented with 10 % fotal bovine serum, 1% L-
glutamine, and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere and 95%
humidity. The stock solutions of FOB, and its complexes were prepared in DMSO and
diluted with growth medium. The DMSO concentration was kept under 0.1%. To
further investigate the biosensor properties of compound, the cells were incubated 1 µM
of FOB and incubated 30 min then to remove excess FOB, the cells were washed three
times with 10 mM PBS. Cells were then treated with 10 µM of Al3+ and Zn2+ and
reincubated for 30 min. After the washing in PBS, the fluorescence signals showing
aluminum and zinc ions was monitored with Bio-Rad ZOE fluorescence microscope.

4
3. Results and Discussion

3.1. Synthesis

An aldehyde derivative of cyano-biphenyl was prepared according to previous reports

and then, the designed fluorescent sensor was prepared by condensation reaction
between the fabricated aldehyde and 2-furoic hydrazide in EtOH with high yields
(scheme 1). The fabricated compound was characterized by the help of 1H-NMR (Fig.
13
S2), C-NMR (Fig. S3), APT (Fig. S4), and FT-IR spectroscopy (Fig. S1) and
elemental analyzer.

Scheme 1.

3.2. Fluorescence study of ligand FOB towards metal ions

The photophysical property of the fabricated fluorescent chemosensor in ACN/H2O

(v/v=80/20, 1 µM, pH=7.0) was investigated in the presence of the various metal ions
(Ba2+, Cs+, Na+, Li+, Ag+, Ni2+, Pb2+, Zn2+, Mn2+, Cu2+, Fe2+, Sr2+, Fe3+, Cd2+, Cr3+, Co2+,
Al3+, Hg2+ and Mg2+) and utilizing fluorescence spectroscopy. As depicted in Figure 1,
free probe (FOB) demonstrated very weak fluorescence at about 464 and 512 nm
because of efficient the effect of PET from the amine and the excited-stated
intermolecular proton transfer (ESIPT) process. Upon the adding of 5.0 equiv different
metal ions to FOB probe solution in ACN/H2O (v/v=80/20, 1 µM, pH=7.0), the
fluorescence emission increment at 464 and 512 nm remained unchanged except Al3+
and Zn2+ ions. Addition of 5.0 equiv of aluminum (III) and zinc (II) ions causes
remarkable enhancement of fluorescent intensity.

Figure 1.

However, fluorescent enhancements were easily observed in different emission

wavelengths and two different colors: blue for complex between aluminum (III) ions
and probe and green for complex between zinc (II) ions and probe (FOB). Although the
probe FOB exhibited remarkable fluorescent respond toward aluminum (III) ions at 464
nm, FOB displayed significant binding with zinc (II) at 512 nm. An obvious

5
fluorescence enhancement was associated with the complexation between Al3+ and FOB
and also zinc (II) ion and probe FOB and formed complexes might be prevented the
PET effect and the excited-stated intermolecular proton transfer in the probe and so the
formation of the stable chelation between chemosensor FOB and ions (aluminum (III)
and zinc (II)) resulted in CHEF (chelation enhanced fluorescence). While the FOB and
aluminum (III) solution displayed blue fluorescence with the maximum wavelength (λem
≈ 464 nm upon excitation at λex= 355 nm), formed complex between the probe FOB
and zinc (II) showed a rapid ‘’turn-on’’ fluorescence respond toward zinc (II) at λem ≈
512 nm (Fig. S5a and b). Consequently, these results suggest that strong fluorescence
turn on emissions in different emission wavelengths were a result of complexation
between the probe FOB and the metal ions (Al3+ and Zn2+).

To verify the quantitative analysis of probe FOB towards aluminum (III) and zinc (II)
ions, fluorescence signals of the probe solution were performed in the presence of
aluminum (III) and zinc (II) perchlorate at various concentrations. According to the
fluorescence titration profiles, “OFF-ON” fluorescence sensing behaviors toward both
metal ions were observed. Furthermore, the intensity ratio of the FOB solution at 464
nm raised gradually with the adding of increasing amount of aluminum (III) ions
concentration in the range from 0.0-5.0 equiv and stabilized a maximum value in the
presence of 1 equiv. of aluminum (III) (Fig. 2a). Similarly, gradual addition of Zn2+ into
the FOB solution induced an increase the emission intensity at 512 nm because of the
prevention of PET effect and the intensity of probe remained stable about 1-fold (Fig.
2b).

Figure 2.

The Job’s plot was conducted to display the complexation ratios of FOB with both
metal ions. The mole fraction of FOB with the both metal ions was 0.5 indicating that
FOB and both metal ions (aluminum (III) and zinc (II)) were coordinated with a 1:1
binding stoichiometry (Fig. 3a and b). Furthermore, the binding constant values of the
probe both Al(III) and Zn(II) (Ka) have been calculated utilizing Benesi-Hildebrand
equation and determined to be 12.8x105 and 5.05x105 M-1 for Al3+ and Zn2+,
respectively (Fig. S6a-b) [25].

Figure 3.

6
To determine the detection limits (LOD) of the probe FOB for the analysis of Al3+ and
Zn2+, S/N=3 equation was utilized [9,16,19]. Where, S is the standard deviation of
fabricated FOB measurement in the absence Al3+ and Zn2+ and K is the slope from
plotting the relative fluorescence intensity versus the concentration of Al3+ and Zn2+. As
shown in Fig. S7a and b, detection limits values of probe FOB have been found 1.27
nM for Al3+ and 1.02 nM for Zn2+. These values were below the World Health
Organization (WHO) guideline values for drinking water (7.41 µM and 76 µM for Al
(III) and Zn (II), respectively) [22,24,27]. Also, there are many probes with µM levels
of LOD values in the literature [7,22,26], but LOD values of the probe FOB in this
study are nM values for both metal ions. These results shows that this probe has
excellent sensitive for the determination of aluminum (III) and zinc (II). Moreover, the
reliability and accuracy of the proposed fluorescence application was confirmed as an
external standard method (Table S1).

Furthermore, the selectivity of the probe FOB has been performed adding 5.0 equiv of
various metal ions into the system ACN/H2O (v/v=80/20, 1µM, pH=7.0) at room
temperature. When the intensity of the FOB rises at 464 nm as a result of adding of Al
(III) ion (λex = 355 nm), we check at 464 nm to understand whether other metal ions
have any impact on the intensity of the sensor FOB. As shown in Fig. 4a, only
aluminum (III) shows a large fluorescence increment in the FOB solution, whereas
other metals excepting Zn2+ exhibited only minimal changing in the intensity of the
FOB. Similarly, our probe at 512 nm has good selectivity for zinc (II) ions over other
cations, only Fe2+, Ca2+, Cu2+ and Fe3+ ions make a drastic decrease of the intensity of
FOB solution with zinc(II) in small magnitude (Fig. 4b). This data indicates that the
FOB could be employed to monitor aluminum and zinc ions even in the presence of
other competitive metal ions.

Figure 4

The effect of pH on the emission intensity of probe FOB both Al (III) and Zn (II) ions
has been tested in the pH range of 2.0-10.0 separately due to it is well known that pH is
especially important for many biological applications. It is clear from Fig. S8a and b
that the intensity of FOB in the presence of both aluminum (III) and Zn(II) has
decreased until pH 6.0 and altered significantly high at pH 7.0. The insensitivity of the
probe both Al3+ and Zn2+ at high pH values decreased probably since the competition of

7
OH- ions interacted with Al3+ and Zn2+ coordination. In addition, the nitrogen groups in
the -C=N- groups in FOB at low pH values (pH: 2.0 and 6.5) were protonated and
coordination with both the metal ions does not formed. Obtained pH-dependent studies
display that both aluminum (III) and zinc (II) ions can produce remarkable turn on
fluorescence signals at different emission wavelengths with the FOB in the
physiological pH region. Therefore, these results highlight that the FOB could be
utilized as a candidate for applications in living systems.

3.3. 1H NMR/FTIR studies and DFT calculations

1
H NMR spectroscopy in DMSO-d6 was used to highlight the nature of complexation
between probe FOB and both the metal ions (aluminum (III) and zinc (II) ions). FOB in
the presence and absence of both of the metal ions was analyzed in DMSO-d6. It was
clear that the phenolic proton and the NH proton signal of the probe FOB appear at
12.28 and at 11.49 ppm, respectively. In the presence of 1.0 equiv of aluminum (III) and
zinc (II) ions, the phenolic OH and NH peaks from ligand at 12.28 and 11.49 ppm
decrease and especially disappeared for Zn2+ due to deprotonation of the phenolic and
NH proton. After addition of aluminum (III) and Zn (II), the C=N moiety was shifted
downfield by 0.04 and 0.05 ppm, respectively (Fig S9a and b). The possible
complexation formed between both the metal ions is shown in Scheme 2 according to
above results.

Scheme 2.

In order to support the possibility of chelation process, the electron density of FOB and
its metal complexations with Al3+ and Zn2+ were performed with density functional
theory (DFT) calculations by GaussView 5.0.8 software [28,29]. As depicted in Figure
8, the differences of energy gaps between highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) were 3.661 for FOB, 1.344 eV
for FOB-Al3+ and 3.329 eV for FOB-Zn2+. This results exhibit that the HOMO-LUMO
energy gaps of the complexations of each metal (Al+3 and Zn2+) to FOB is less than
only the energy gaps of FOB molecule and the result supported that system stabilize
(Figure 5).

Figure 5.

3.4. Cell studies and fluorescence imaging of FOB-Metals

8
Additionally, human colon cancer cells could be employed in fluorescence imaging to
determine the intracellular Zn2+ and Al3+ imaging behavior of probe FOB. The cells
display no fluorescence after it has been incubated with FOB alone. Also, probe FOB
deposited PC-3 cells were incubated with 10 µM Zn2+ and Al3+ for 60 min at 37 °C. It
was clear from the micrographs that probe FOB exhibited similar turn on sensing
fluorescent behaviors in PC-3 cells to Zn2+ and Al3+ as in the solutions (Figure 6).

Figure 6.

4. Conclusion

Overall, the synthesis of cyano-biphenyl aldehyde was achieved according to previous

literature and then, it is reacted with an amine containing 2-furoic hydrazide. Probe
FOB was prepared with high conversions and utilized as an ultrasensitive and selective
fluorescence turn on for the determination of in ACN/H2O (v/v=80/20, 1 µM, pH=7.0).
Moreover, the sensor shows superb response and high sensitivity toward aluminum (III)
ion and zinc (II) through a turn on fluorescence intensity over other relevant competing
metal ions. The high Al3+ chelation-enhanced fluorescence (CHEF) is probably because
of including prevention of PET and ESIPT. Probe FOB was employed in practical pH
values and in living cells for monitoring of aluminum (III) ion and zinc (II). In
summary, the facile preparation of FOB, low detection value of FOB for Al3+ and Zn2+
and FOB is applicable for detecting Al3+ and Zn2+ in living cells warrant its ability to
utilize as a tool for detection of aluminum (III) and zinc (II) ions.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgment
The author is grateful to the KMU Scientific Research Project Center for their support
with the project numbers 30-M-16 and to provide the Gaussian 09W and Gauss
view 5.0.8 programs. Also, author thanks Serdar Karakaya for help with in vitro studies.

Appendix A. Supplementary data

9
Supplementary data to this article can be found online at https://doi.

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 1

1 FOB

Scheme 1. Synthesis routes of 1 and FOB.

Scheme 2. Fluorescence increment mechanism of the FOB-Metal complex.

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Figure captions

Figure 1. Fluorescence spectra of FOB ACN/H2O (v/v=80/20, 1 µM, pH=7.0) solution

before and after addition of 5.0 equiv of different metal ions (Na+, Li+, Ag+, Ni2+, Ba2+, Mn2+,
Mg2+, Sr2+, Fe2+, Cr3+, Co2+, Cu2+, Fe3+, Zn2+, Cd2+, Hg2+, Al3+, Cs+ and Pb2+). The spectra
were studied after 5 min mixing with λex = 355 nm, λex =464 nm for Al3+ and λex =512 nm for
Zn2+, slit: 10-10.

Figure 1. Fluorescence spectra of FOB (1 µM) after addition of increment concentrations of

(a) Al3+ and (b) Zn2+ in ACN/H2O (v/v=50/50). The spectra were provided after mixing for 5
min for Al3+ and 3 min for Zn2+ (sensors) using λex = 355 nm.

Figure 3. Job’s plots of the complexation between FOB with (a) Al3+ and (b) Zn2+.
Figure 4. Relative fluorescence of (a) FOB in ACN/H2O (v/v=80/20, 1µM, pH=7.0) in the
presence of (a) Al (III) and (b) Zn(II) (5.0 eqv) with another interfering metal ion tested.

Figure 5. Electron density in HOMO and LUMO of FOB and its complexes with Al3+ and
Zn2+.
Figure 6. Fluorescence images of (a) Al (III) and (d) Zn (II) ions utilizing sensor FOB in
Human prostate cancer PC-3 (CRL 1435) cells: (a and d) bright field image of living Human
epithelium Lovo cells treated with sensor FOB (1 µM) for 30 min, then threated with Al(III)
and Zn(II) ions for 30 min; (b and e). (c and f) Merged fluorescence images of (a) and (b), and
also (d) and (e). Scale bar = 100 µm.

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Figure 1.

17
Figure 2.

Figure 3.

18
Figure 4.

Figure 5.

19
Figure 6.

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Highlights

• A novel turn on fluorescent probe (FOB) for Al3+ and Zn2+ was fabricated.
• The fluorescent of FOB can be enhanced in the presence of Al3+ and Zn2+.
• The limits of detection of the fabricated sensor was 1.27 nM for Al3+ and 1.02
nM for Zn2+.
• This fluorescent sensor can be used for detection of aluminum and zinc ion in
biological applications
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: