A novel
fluorescent probe based on isocoumarin for Hg
2
þ
and Fe
3
þ
ions and its application in live-cell imaging
Sukriye Nihan Karuk Elmas
a, Zeynep Emine Dincer
a, Ali Serol Erturk
b, Aykut Bostanci
c,
Abdurrahman Karagoz
a, Murat Koca
d, G€okhan Sadi
c, Ibrahim Yilmaz
a,*aKaramanoglu Mehmetbey University, Kamil Ozdag Science Faculty, Department of Chemistry, Karaman 70100, Turkey bAdıyaman University, Faculty of Pharmacy, Department of Analytical Chemistry, Adiyaman, Turkey
cKaramanoglu Mehmetbey University, Kamil Ozdag Science Faculty, Department of Biology, Karaman 70100, Turkey dAdıyaman University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Adiyaman, Turkey
a r t i c l e i n f o
Article history: Received 8 June 2019 Received in revised form 12 July 2019
Accepted 17 July 2019 Available online 18 July 2019 Keywords: Isocoumarin Fluorescence sensor Living cell Cytotoxicity Mercury Iron
a b s t r a c t
Synthesis of the 2-amino-4-phenyl-6- (isocoumarin-3-yl) -3-cyanopyridine (APICP) containing both isocoumarin and pyridine ring in its structure was carried out, and this compound was characterized by ATR-FTIR,1H NMR, and13C NMR spectral techniques. Afluorescence sensor determining Hg2þand Fe3þ
ions in DMSO/HEPES buffer solution (9/1 v/v, 5mM, pH 7.0) was developed using the synthesized com-pound, and the detection limits of the sensor with exquisite selectivity were calculated as 8.12 nM and 5.51 nM for Hg2þand Fe3þions, respectively. Jobs plot method was used to determine the stoichiometry of APICP-Hg2þ/Fe3þcomplexes as 2:1 and FT-IR and ESI-MS methods confirmed the results. Besides, cell growth inhibitory potentials of the sensor over HepG2 cells and in vivofluorescent cell imaging ex-periments were conducted. Findings revealed the relatively low cytotoxic effects of the synthesized sensor (IC50: 0.541± 0.039 mM), and it could be utilized as an intracellular imaging agent for the
determination of Fe3þand Hg2þions in biological systems.
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
Fluorescence spectroscopy is an analytical tool used in many scientific fields such as environmental and clinical chemistry, drug, genetic biomolecular analysis, and it provides fast accurate and reliable results [1]. It is possible to determine fats, nutrients, drugs, and air-water pollutants with the direct spectrofluorimetric method if an analyte hasfluorescent properties. However, for the substances not having intrinsic fluorescence properties, indirect fluorimetric methods such as derivatization, fluorescent complex formation, andfluorescence quenching [2e4] could be operated.
Isocoumarins, constitute an essential part of the natural prod-ucts, are used in many scientific and technological fields, especially in medicinal and drug chemistry due to their pharmacological and optical properties. Isocoumarins have wide ranges of pharmaco-logical activities including antifungal [5], anti-inflammatory [6], antimicrobial [7], phytotoxic [8], cytotoxic [9] and some other essential properties [10]. Pyridine is one of the most critical het-erocyclic compounds in organic chemistry and 3-cyanopyridine
derivatives having cyanopyridine ring in its structure are present in some natural products and synthetic compounds. Their anti-tumor, analgesic, anti-inflammatory, antimicrobial, antiviral, fungicidal, and cardiotonic activity have been demonstrated pre-viously [11,12], and therefore, they could play an essential role in the pharmacological industry. Among the pyrimidine derivatives, heterocyclic compounds involving pyridine or coumarin ring have attracted considerable interest in sensory applications for metal ions' detections [13,14].
Iron has many critical functions in biological systems [15], and its elemental or compound form has beneficial or detrimental ef-fects on various biological systems. The ones present in the stage of complex compounds play significant roles in the plant, animal, and human metabolism. For instance, it transfers electrons among the molecules, regulates the activities of several enzymes, and stabi-lizes the structure of complex biological compounds such as he-moglobin and myoglobin [16]. Elevated levels of iron cause an actual health hazard by damaging the essential tissues such as liver and kidneys, and its scarcity would give rise to anemia. Therefore, there is still a need for sensitive methods to detect Fe3þin medical, environmental, and industrial samples [17,18]. Among the metal ions, mercury has been recognized as the third most toxic element
* Corresponding author.
E-mail address:iyilmaz@kmu.edu.tr(I. Yilmaz).
Contents lists available atScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
j o u rn a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s a a
https://doi.org/10.1016/j.saa.2019.117402
Hg and Fe ions, tested its cytotoxic properties over hepatocel-lular carcinoma cell lines, in vivo detection properties.
2. Experimental 2.1. General information
All reagents were purchased from Merck and Aldrich with the highest purity available and used without purification. The IR (ATR) spectrums (4000e650 cm1) were recorded with a Perkin Elmer
spectrum 100 FT-IR and1H,13C NMR spectrums were recorded on a (tetramethylsilane as standard), Bruker 600 MHz spectrometers. UVeVis absorption spectra were obtained using a spectropho-tometer (Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific). Fluorescence spectra were taken with Agilent Cary Eclipse spectrometer using quartz cells of 1.0 cm path length. Mass spectra were obtained using a Bruker Daltonics Microflex Mass Spectrometer. Cellular fluorescence images were recorded by an invertedfluorescence microscope (ZOE, Bio-Bad, Germany). 2.2. Synthesis of APICP
Compounds 3-acetyl isocoumarin (1) and 2-benzylidene malo-nonitrile (2) were synthesized according to the literature [31,32]. Synthesis of APICP was performed with a method adopted from the literature [33] and summarized inScheme 1.
The reagent 3-acetyl isocoumarin (0.188 g, 1 mmol), ammonium acetate (0.154 g, 2 mmol) and 10 ml methanol were placed in a two-neck reactionflask and then stirred for about 1 h at room temper-ature. To this mixture, benzylidene malononitrile (0.154 g, 1 mmol) dissolved in a small amount of methanol was added and stirred for 8 h while refluxing. After cooling to the room temperature, the resultant product wasfiltered and crystallized from the alcohol. The yield was calculated as 70%.
temperature. Forfluorescence experiments, the excitation wave-length was determined as 355 nm. UVeVis measurements were recorded from 200 to 600 nm at room temperature.
2.4. Computational details
The computational studies were performed using GAUSSIAN 09W and GaussView 5.0 molecular visualization software. The ge-ometry optimization of the APICP, APICPeFe3þ and APICPeHg2þ were carried out with the DFT/B3LYP method, the LANL2DZ basis set. All calculations were conducted with aqueous solutions. 2.5. Determination of cell growth inhibitory potential of APICP on HepG2 cells
The HepG2 cells obtained from ATCC (ATCC® HB-8065) were grown in DMEM media supplemented with 10% FBS,L-glutamine
and penicillin/streptomycin at 37C with 5% CO2. Cytotoxic
prop-erties of APICP over HepG2 cells were determined with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) in vitro cellular toxicity assay [34]. Accordingly, 5 104cells
were seeded in a 96-well cell culture plate and incubated for 4 h for cell attachment. Afterward, different amounts of APICP (0.025e1.0 mM) in growing media were applied to the cells, and they were incubated for 48 h at 37C in 5% CO2. After the addition
of 25
m
L of activated XTT solution (1 mg/ml) and 2-h incubation period, the intensities of the formazan were measured at 455 nm with Multiskan™ GO Microplate Spectrophotometer (Thermo Sci-entific, USA).2.6. Cell imaging studies
HepG2 cells were grown in a 24-well plate with a culture me-dium overnight. The cells were then incubated with 400
m
M ofAPICP for 30 min. Subsequently, they were washed with PBS solu-tion for three times and further incubated with Fe3þ and Hg2þ solutions (100
m
M each) for 30 min, separately. Following another washing step with PBS, a brightfield and fluorescent images of the cells were taken before and after metal ion applications with a fluorescent cell imaging system (ZOE, Bio-Bad, Germany). 3. Results and discussion3.1. Synthesis and structural characteristics of APICP
The isocoumarin and pyridine ring-based chemosensor were obtained by the method described in the experimental part. Its structure was clarified by 1H NMR,13C NMR, FT-IR, and ESI-MS
techniques (Figs. S1eS3, S8). All data obtained from these tech-niques indicate the pure synthesis of APICP sensor.
3.2. UVeVis spectral responses of APICP
Absorption studies were conducted in DMSO/HEPES (9/1, v/v, 5
m
M, pH 7.0) solution after adding 5.0 equivalence of various metal ions (Agþ, Al3þ, Ba2þ, Ca2þ, Co2þ, Kþ, Zn2þ, Hg2þ, Fe3þ, Cd2þ, Cr3þ, Mg2þ, Mn2þ, Naþ, Ni2þ, Pb2þ, Cu2þ). Apart from Hg2þand Fe3þions, the addition of other cations constitutes any considerable changes. As shown in Fig. S4, only in the presence of Fe3þand Hg2þions, the chemosensor APICP exerts a change in the UV/Vis spectra with theenhancement band at 280 nm for APICP-Hg2þ/Fe3þcomplexes. UV/ Vis titration study of APICP was performed by the addition of a various amount of Fe3þand Hg2þ(0e2 eq.), and upon the addition of these two metal ions, the absorption band at 280 nm gradually increased as shown in Figs. S5 and S6.
3.3. Fluorescence spectral responses of APICP
The sensing abilities of APICP have been evaluated using fluo-rescence spectra in the presence of 5-fold excess metal ions (Agþ, Al3þ, Ba2þ, Ca2þ, Co2þ, Kþ, Zn2þ, Hg2þ, Fe3þ, Cd2þ, Cr3þ, Mg2þ, Mn2þ, Naþ, Ni2þ, Pb2þ, Cu2þ) in DMSO/HEPES (9/1, v/v, 5
m
M, pH 7.0) so-lution. The fluorescence spectra were recorded at 455 nm (l
ex: 355 nm). Upon the addition of an increasing amount of Fe3þ or Hg2þsolution to the 5m
M probe solution, the emission intensity decreased gradually. The mechanism of thefluorescence quenching can be considered as the result of the complexation reaction of APICP with Fe3þor Hg2þ ion in the ground state. However, the addition of other metal ions did not cause any changes, indicating the selectivefluorescence quenching of APICP with Fe3þor Hg2þ ions (Fig. 1) Furthermore, a selectivefluorescence change with the addition of different ions to APICP has been observed.To observe sensing ability of the synthesized compound, the fluorescence titration was also conducted with the addition of increased amounts of Fe3þ or Hg2þ (0e5 equiv.) to the APICP
Fig. 1. Fluorescence spectral changes of APICP in the presence of various cations in DMSO/HEPES buffer solution (9/1 v/v, 5mM, pH 7.0) (lex¼ 355 nm,lem¼ 455 nm).
Fig. 2. Thefluorescence spectra of APICP with of increasing concentration of Fe3þ/Hg2þin DMSO/HEPES buffer solution (9/1, v/v, 5mM, pH 7.0) (lex¼ 355 nm,lem¼ 455 nm).
solution (5
m
M), and thefluorescence decreased notably. The fluo-rescence intensity of APICP decreased as the amount of Fe3þ or Hg2þwas raised. When 2.0 equivalent of Fe3þor Hg2þwas added to the probe solution, the fluorescence intensity almost completely quenched (Fig. 2).3.4. Effect of pH
It is known that the pH of the solutions would affect the response mechanisms of fluorescence sensors. Therefore, mea-surements were made in the presence and absence of the Hg2þand Fe3þions in DMSO/H2O (9/1, v/v) solutions having a pH range of
4.0e10.0 to determine the optimum pH (Fig. S7). Accordingly, UVeVis and fluorescence studies were performed at pH 7.0 in 5
m
M HEPES solution.3.5. Job's plot of APICP with Hg2þand Fe3þ
A Job's plot was utilized to estimate the binding stoichiometry of APICP-Fe3þand APICP-Hg2þcomplexes, and results indicated a 2:1 stoichiometric ratio for Hg2þ and Fe3þ, as shown in Fig. 4. The stoichiometry of APICP-Fe3þ/Hg2þ was also verified by ESI-MS technique and a peak at 767.8 m/z in the ESI-MS spectra of APICP-Fe3þcomplex corresponding to the [APICP-Fe3þþ 2H2O] indicates
2:1 ratio as shown in Fig. S8. Besides, a peak at 875.4 m/z ESI-MS
spectra of APICP-Hg2þcomplex regarding the APICP-Hg2þ shows a 2:1 ratio which is demonstrated inFig. 3.
Using thefluorescence titration data, the binding constant of APICP-Fe3þand APICPeHg2þwere found to be 1.7 105M1and
1.2 105 M1 according to Benesi Hildeberg plot, respectively (Fig. 5). Fluorometric titration data was also utilized to obtain the limit of the detection value of APICP for Fe3þand Hg2þ. The con-centration of Fe3þor Hg2þ was plotted against thefluorescence intensity at 455 nm. The detection limit of APICP towards Hg2þand Fe3þions was 8.12 nM and 5.51 nM, respectively (based on S/N¼ 3). To better clarify the mechanism of the complexation of the sensor with Fe3þ and Hg2þ ions, IR spectroscopy was used. IR spectra of the APICP and complexes were measured with ATR-FTIR apparatus. Accordingly, one of the peaks of NH2in the spectrum of
the APICP, 3321 cm1, disappeared in the spectra of the complexes indicating the involvement of the NH2group in the coordination.
Upon APICP complexation with Fe3þor Hg2þions, the nitrile peak at 2202 cm1was shifted to 2214 cm1and decreased, supporting the coordination through the nitrile group in the complexes. Be-sides, there was any change at the vibration of the carbonyl at 1729 cm1and the vibration of the C-O-C at 1074 cm1, indicating that carbonyl and etheric oxygen were not involved in the coordi-nation (Figs. S9 and S10). When the results of all characterization studies are evaluated together, the complexation mechanism of APICP-Hg2þ/Fe3þis concluded, as shown inScheme 2.
Fig. 4. Job plots of APICP-Fe3þ/Hg2þcomplexes in DMSO/HEPES buffer solution (9/1 v/v, 5mM, pH 7.0) at 445 nm (lex¼ 355 nm).
3.6. Interference studies
The interference studies of APICP towards the various compet-itive cations (2.00 eq.) in the presence of Fe3þand Hg2þ(2.00 eq.) were investigated (Figs. 6 and 7). The results demonstrate any interference by other competitive cations and our probe has good selectivity towards Hg2þand Fe3þions. The presence of other metal cations displayed insignificant trouble for the determination of Hg2þbut not for Fe3þ. Only if Hg2þwere transferred to the solution of probe and Fe3þ, the emission intensity of APICP-Fe3þ was decreased (Fig. 7). It is understood that Hg2þcould replace Fe3þin its complex system and APICPeHg2þcomplex system is more stable
than APICPeFe3þ complex. Therefore, our probe could be
confidingly used as a selective and sensitive turn off fluorescence sensor for the determination of Hg2þand Fe3þions in the presence of other cations.
3.7. Response time studies
As it is well known, the response time for many sensor systems is a significant point for practical applications. Thus, the reaction time of the APICP and Fe3þor Hg2þsensor system was evaluated and illustrated in Figs. S11 and S12. After the addition of the Fe3þor Hg2þto the probe, the emission intensity of the complexes ach-ieved a stable level within 60 s. It is an essential behavior for the robust and real-time monitoring of Fe3þand Hg2þions.
Fig. 6. Selectivity of the APICP-Fe3þsensor over the various cations in DMSO/HEPES buffer solution (9/1, v/v, 5mM, pH 7.0) at 445 nm (lex¼ 355 nm).
Fig. 7. Selectivity of the APICP-Hg2þsensor over the various cations in DMSO/HEPES buffer solution (9/1, v/v, 5mM, pH 7.0) at 445 nm (lex¼ 355 nm).
3.8. Theoretical calculations
In order to further understand the fluorescence quenching mechanism of APICP in the presence and absence of Fe3þand Hg2þ, DFT calculations were performed. The creation of inputfiles and analysis of the results was done by GaussView 5.0.8 software [35]. The geometry optimization of APICP, APICPeFe3þ, and APICPeHg2þ
was carried out utilizing the B3LYP hybrid function [36] 6-311G (d) base set, and the energy-optimized structures were shown inFig. 8.
The energy gaps between HOMO and LUMO were 6587
and 2.346 eV for APICP, 7802 and 7329 eV for APICPeFe3þ and 15,278 and 14,841 eV for APICPeHg2þ, respectively. The energy gaps of APICPeFe3þ and APICPeHg2þ complexes were
smaller than that of APICP alone, which indicates that the com-plexes were more stable than the ligand. This emission quenching behavior of the sensor was interpreted as the strong interaction between copper ions and hydroxyl groups of APICP due to charge transfer process of the variation of the electronic structure of APICP [37e39].
3.9. Cytotoxic effects
Cancer is accepted as one of the most deadly diseases in the world, and hepatocellular carcinoma is the most common type of liver cancers [30]. Liver hepatocellular carcinoma (HepG2) cells are the model cell lines to study liver functions in vitro since they maintain several liver properties. Findings from our study demonstrated that the cytotoxic effects of APICP were augmented with increasing concentrations (Fig. 9). Non-linear regression
analysis was utilized to calculate IC50values of APICP, which were
0.541± 0.039 mM. Notably, at 0.800 mM and higher concentra-tions, cell viability decreased to near to zero. Considering this cytotoxic property, we have incubated HepG2 cells with 0.400 mM of APICP in further cell imaging experiments at which almost 80% of cells remain alive.
Fig. 9. Cytotoxic effects of APICP on HepG2 cells. Data presented the mean of at least triplicate measurements and given as mean± standard deviation.
3.10. Cell imaging studies
To further evaluate the practical applicability of APICP for Hg2þ and Fe3þsensing,fluorescent imaging experiments were carried out using HepG2 cell lines (Fig. 10). To do this, we have incubated HepG2 cells with 400
m
M APICP for 30 min. As demonstrated inFig. 10b, APICP alone increased the intracellularfluorescence in HepG2 cells, suggesting that APICP could penetrate the cellular membranes and be effective in intracellular imaging applications. On the other hand, if the cells were treated with Hg2þ ions after APICP, a dramatic decrease in intracellular greenfluorescence was observed (Fig. 10d). Similarly, green fluorescence was also quenched with Fe3þ ions (Fig. 10f). These results demonstrate the diffusion of these two metal ions into the cells and repression of the intracellularfluorescence of APICP. Moreover, Hg2þ was found to be more effective in APICP quenching. In this study, an equimolar amount of Hg2þand Fe3þto APICP concentration is not preferred due to high cytotoxic effects of these two metal ions (data not shown).4. Conclusion
In summary, we have successfully developed a newfluorescent sensor based on isocoumrine which is highly sensitive and selective for the detection of Hg2þ and Fe3þ ions in DMSO/HEPES buffer solution (9/1 v/v, 5
m
M, pH 7.0) and living cells. The stoichiometry of the complexes between APICP and Fe3þ/Hg2þwere determined by the Job's method, ESI-MS techniques, and found to be 2:1 ratio. DFT studies were carried out to verify the experimental data. Finally, cell growth inhibitory potentials of APICP over HepG2 cells were investigated in this study, which is not reported previously, and fluorescent imaging experiments using HepG2 cell were carried out successfully. The results offluorescent imaging experiments indi-cated that the probe APICP could be used as an intracellular im-aging agent for the determination of Fe3þ and Hg2þ ions in biological systems. Besides, as seen in Table S1, the detection limits (LOD) of APICP are comparable or better than that of previously developed Fe3þ and Hg2þ probes. These results presented that APICP can be utilized as a sensor for the detection of submicromolar concentrations of Fe3þand Hg2þions both in vitro and in vivo.Acknowledgment
The authors are 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 Gaussview 5.0.8 programs. Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2019.117402. References
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![Fig. 5. Benesi-Hildebrand plot ( l em¼ 445 nm) of 1/(I-I 0 ) vs 1/[M nþ ] based on 2.1 association stoichiometry between APICP-Hg 2þ and APICP-Fe 3þ .](https://thumb-eu.123doks.com/thumbv2/9libnet/4550208.82688/4.892.101.781.826.1086/fig-benesi-hildebrand-based-association-stoichiometry-apicp-apicp.webp)
