Fluorescent sensing platform for low-cost detection of Cu2+ by coumarin derivative: DFT calculation and practical application in herbal and black tea samples

Article abstract of DOI:10.3906/KIM-2004-63

A fluorogenic probe based on a coumarin-derivative for Cu ²⁺ sensing in CH 3 CN/H 2 O media (v/v, 95/5, 5.0 μM) was developed and applied in real samples. 3-(4-chlorophenyl)-6,7-dihydroxy-coumarin (MCPC) probe was obtained by synthetic methodologies and identified by spectral techniques. The probe MCPC showed remarkable changes with a “turn-off” fluorogenic sensing approach for the monitoring of Cu²⁺ at 456 nm under an excitation wavelength of 366 nm. The response time of the probe MCPC was founded as only 1 min. The detection limit of the probe MCPC was recorded to be 1.47 nM. The binding constant and possible stoichiometric ratio (1:1) values were determined by Benesi-Hildebrand and Job’s plot systems, respectively. The mechanism of the probe MCPC with Cu²⁺ was further confirmed by ESI-MS and FT-IR analyses, as well as supported by theoretical calculations. Furthermore, the probe MCPC was successfully employed for the practical applications to sense Cu²⁺ in different herbal and black tea samples. The proposed sensing method was also verified by ICP-OES method.

Full text of DOI:10.3906/KIM-2004-63

Turk J Chem
Turkish Journal of Chemistry
(2020) 44: 1148 – 1163
© TÜBİTAK
http://journals.tubitak.gov.tr/chem/
doi:10.3906/kim-2004-63
Research Article
2+
Fluorescent sensing platform for low-cost detection of Cu
by coumarin
derivative: DFT calculation and practical application in herbal and black tea
samples
1
1
1
Tahir SAVRAN , Abdurrahman KARAGÖZ , Şükriye Nihan KARUK ELMAS ,
1
2
3
1
Duygu AYDIN , Furkan ÖZEN , Kenan KORAN , Fatma Nur ARSLAN ,
Ahmet Orhan GÖRGÜLÜ , İbrahim YILMAZ1,∗
3
1
Department of Chemistry, Kamil Özdağ Science Faculty, Karamanoğlu Mehmetbey University, Karaman, Turkey
2
Department of Mathematics and Science, Faculty of Education, Akdeniz University, Antalya, Turkey
3
Department of Chemistry, Faculty of Science, Fırat University, Elazığ, Turkey
Received: 22.04.2020
•
Accepted/Published Online: 26.06.2020
•
Final Version: 18.08.2020
2+
Abstract: A fluorogenic probe based on a coumarin-derivative for Cu
sensing in CH3 CN/H2 O media (v/v, 95/5, 5.0
µM) was developed and applied in real samples. 3-(4-chlorophenyl)-6,7-dihydroxy-coumarin (MCPC) probe was obtained
by synthetic methodologies and identified by spectral techniques. The probe MCPC showed remarkable changes with a
2+
“turn-off” fluorogenic sensing approach for the monitoring of Cu
at 456 nm under an excitation wavelength of 366 nm.
The response time of the probe MCPC was founded as only 1 min. The detection limit of the probe MCPC was recorded
to be 1.47 nM. The binding constant and possible stoichiometric ratio (1:1) values were determined by Benesi-Hildebrand
2+
and Job’s plot systems, respectively. The mechanism of the probe MCPC with Cu
was further confirmed by ESI-MS
and FT-IR analyses, as well as supported by theoretical calculations. Furthermore, the probe MCPC was successfully
2+
employed for the practical applications to sense Cu
method was also verified by ICP-OES method.
in different herbal and black tea samples. The proposed sensing
Key words: Coumarin, fluorescent sensor, copper, DFT, tea samples
1. Introduction
The rapid industrialization has caused heavy metal pollution which is gradually becoming a critical issue over
the past three decades. This pollution is considered as a major threat, because heavy metal ions lead to serious
problems for the ecological environment and human health [1]. Among the commonly encountered heavy metals
2+
of concern, the Cu
is the third most plentiful trace cation in the clay as well as in numerous livings, and it acts
2+
an essential role in numerous biological reactions. Cu
acts a vital role in the connective tissue growth, bone
and blood formation as one of the physiological processes of organisms [2], it is also placed in the cornea and
2+
mostly in the brain [3]. However, it has been reported that excess intake of Cu
causes adverse health effects in
2+
the body such as gastrointestinal diseases and it damages the kidneys and liver [1,4]. Also, Cu
catalyzes the
creation of reactive oxygen species which can harm basic biological molecules. According to various literatures,
2+
the toxicity of Cu has linked to severe neurological diseases [5], Indian childhood cirrhosis (ICC) and prion
2+
diseases [2,4,6,7]. World Health Organization (WHO) has reported that the optimum intake of Cu
for adults
∗Correspondence: iyilmaz@kmu.edu.tr
1148
This work is licensed under a Creative Commons Attribution 4.0 International License.

SAVRAN et al./Turk J Chem
2+
should not go above the level of 10–12 mg.day−1 [8]. For this reason, the monitoring of Cu
in a variety of
drinking and food samples has been a vital issue for the protection of human health [7]. Thus, the development
2+
of reliable analytical procedures for Cu
sensing is still a critical research topic [9].
2+
The reported methods for monitoring Cu
include inductively coupled mass-atomic emission spec-
trometry (ICM-AES), capillary electrophoresis (CE), atomic absorption spectrometry (AAS), voltammetry and
inductively coupled plasma-mass spectrometry (ICP-MS) [10]. These techniques involve expensive analysis sys-
tems, troublous analysis procedures, and also need excessive use of samples [11]. In contrast, the fluorescence
2+
spectroscopy is currently taken into account as a powerful method for the recognition of Cu
owing to its real
time, low cost, rapid and non destructive sensing with high selectivity and sensitivity [12] and easy operation.
2+
Recently, many fluorescent chemosensor studies for the recognition of Cu have been reported [5,13,14]. To
this end, various fluorescent probes based on rhodamine [15], naphthalene [16], BODIPY [17], fluorescein [18],
N-aminophthalimide [19], thiophene [20], indole [21], pyrene [22], carbazole [23], cyanine [24] and coumarin [15]
2+
derivatives have employed to determine Cu
in several samples. Among these derivatives, coumarin-based
molecules are powerful and highly adjustable fluorescent platforms for the selective and precise measurements
2+
of Cu
in drinking water, food and beverage samples [12].
Herein, a “turn-off” fluorogenic probe based on a coumarin-derivative, 3-(4-chlorophenyl)-6,7-dihydroxy-
2+
coumarin, probe MCPC, for Cu
sensing in CH3 CN/H2 O media (v/v, 95/5, 5.0 µM) was prepared and
employed to various herbal and black tea samples. Coumarin compounds have been commonly used to be
a florescent sensor thanks to its moderately high water solubility, high fluorescence quantum (Φ) yield and
chemical stability, and also its large Stoke’s shift. In addition, coumarin derivatives are less toxic to the livings
and eco-friendly compounds [9]. The synthesized probe MPCP was successfully identified by spectral techniques
1
13
such as H-NMR and C-ATP-NMR, as well as FT-IR. The theoretical computations were also done for the
optimized geometric features.
2. Materials and methods
2.1. Chemicals and instrumentation
All chemicals used herein were of spectroscopic grade and obtained from Merck KGaA (Darmstadt, Ger-
many) and VWR International B.V. (Amsterdam, Netherlands). Ultrapure water was acquired from Milli-
®
Q
7003/05/10/15 machine (Water purification system, Merck KGaA, Darmstadt, Germany). Solutions of
2+
+
2+
2+
2+
2+
2+
2+
2+
2+
+
2+
2+
different metal ions (Hg , K , Mn , Cu , Ba , Cd , Co , Ca , Zn , Mg , Na , Fe , Sr
Pb , Fe
,
2+
3+
and Al3+) (10 mM, perchlorate salts in acetonitrile) were prepared as a stock solution.
A Bruker DPX 400 MHz spectrometer (Bruker Corp., Billerica, MA, USA) was employed to measure
13
1
T M
C-APT-NMR and H-NMR spectra. A Mass spectrometer (Bruker Daltonics-Microflex
, Bruker Corp.)
was used to record electro spray ionization-mass spectra (ESI-MS). Infrared spectra were taken on a FT-IR
spectrometer (Spectrum 100, Perkin Elmer Inc., Wellesley, MA, USA). A fluorescence spectrophotometer (Cary
Eclipse, Agilent Tech., Santa Clara, CA, USA) was employed to record fluorescence spectra at rt. As a reference
2+
method, the amount of Cu
in herbal tea samples was analyzed by an inductively coupled plasma-optical
emission spectroscopy (ICP-OES) (720, Agilent Tech.). For the pH measurements and centrifugation procedures,
a pH meter (Mettler Toledo, Zaventem, Netherlands) and Universal 320R centrifuge system (Andreas Hettich
GmbH & Co. KG, Tuttlingen, Germany) were used, respectively.
1149

SAVRAN et al./Turk J Chem
2.2. Fabrication of the probe MCPC
Probe MCPC, 6,7-dihydroxy-3-(4-chlorophenyl)coumarin, was successfully synthesized under microwave irradi-
ation procedure with solvent free conditions according to the reported method in the literatures [25–27]. The
synthesis protocols and characterization data were presented in Supplementary Information file in detail.
2+
2.3. Fluorescence spectroscopic studies of the probe MCPC towards Cu sensing
Probe MCPC was dissolved in acetonitrile as a stock solution and 10 mM of this was adjusted to 5.0 µM
concentration with the mixture of CH3 CN/H2 O (v/v, 95/5). The titration experiments were performed to
2+
achieve the relationship between emission intensities and Cu . For the fluorescence titration experiments, the
different amounts of the cations (0–3.0 equiv) were added to 3000 µL of the probe solution. Spectra were taken
at room temperature and recorded at λem = 456 nm (λex = 366 nm, 5 nm slit width).
The LOD of the probe MCPC was calculated from the equation of 3σ/k (slope value) [28–31]. To
2+
analyze the effect of the possible competing cations on the sensing ability of probe MCPC to Cu , suitable
2+
2+
concentration of Cu
(3.0 equiv) was added into probe MCPC solution to generate MCPC-Cu
complex,
2+
afterwards each of the other cation solutions was added into the MCPC-Cu , respectively. The emission
intensities of the MCPC-Cu
stoichiometric ratio between the probe MCPC and Cu
method calculation [32].
2+
complex were measured before and after adding other cations. The possible
2+
2+
in MCPC-Cu system was found by using Job’s
2.4. Studies based on DFT
2+
Theoretical calculations were performed for the probe MCPC and MCPC-Cu
complex by the DFT /
B3LYP/6-31 g (d) method with Gaussian 09 software (Gaussian, Inc., Wallingford, CT, UK) and accompanying
graphical interface program GaussView 5.0.8 [33–37]. The LanL2DZ basis set for the effective potential set for
Cu, and the 6-311G basis set was used for C, H, O atoms. To obtain the HOMO and LUMO energy levels,
DFT analyses were performed based on the optimized geometries.
2+
2.5. Detecting Cu in tea samples by fluorometric sensing and ICP-OES
Eleven kinds of herbal tea leaves (green tea, white tea, sage tea, fennel tea, daisy tea, rose hip tea, ginger
tea, mint tea, apple tea, linden tea and green tea mixture with rose) and 5 kinds of black tea leaves (black
tea samples without aroma and black tea samples with bergamot aroma) were bought from super markets and
herbs shops. 2.0 g of each samples were homogenized, individually placed into the boiled ultrapure water (50.0
mL) and infused/waited approximately for 15 min. Prepared tea solutions were placed in a shaker for 20 min,
and then they were centrifuged at 10.000 rpm for 5 min. Final solutions were cleaned with a membrane filter
2+
(0.45 µm pore-size) and then the amounts of Cu in teas were identified by the probe MCPC and ICP-OES
systems. For fluorometric sensing analysis, probe MCPC solution was freshly prepared in CH3 CN/H2 O media
(v/v, 95/5, 5.0 µM). 3000 µL of probe MCPC solution was put into the quartz-cuvette, and then the spectra
were measured at rt (λex = 366 nm, λem = 456 nm, 5 nm slit width). Then, 15 µL of each tea samples
individually put into the probe MCPC, and the spectra were recorded by the same procedure. Finally, this
2+
solution (tea sample + probe MCPC) was spiked with 15 µL of diverse amounts of Cu
(0.1 and 0.2 µM)
(standard addition method) and spectra were obtained. For ICP-OES analysis, the solution of tea samples were
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SAVRAN et al./Turk J Chem
acidified with HNO3 (v/v, 1%) in the 10 mL of centrifuge tubes. pH value of these solutions were adjusted to 9
with a buffer solution (NH3 /NH4 Cl), and just after 15 min of waiting time, all teas were centrifuged at 10.000
rpm (~5 min). 0.5 mL of d-HNO3 solution was added to the bottom layer in the tubes and this layer was diluted
to 2500 µL with ultra-pure water. Before ICP-OES analysis, the sample solution was cleaned with a membrane
filter (0.45 µm pore-size) to eliminate possible contaminations. The operating conditions of ICP-OES method
were presented in Table S1. The analyses were done in triplicate and outcomes were illustrated as mean ±std.
3. Results and discussion
3.1. Design of the probe MCPC
The probe MCPC was prepared under microwave irradiation with solvent free condition by the reaction of
1
compound (3) with pyridinium hydrochloride (85% yield) (Scheme 1), and it was well-verified by H-NMR,
13
FT-IR and C-ATP-NMR analyses (Figures S1–S6).
Scheme 1. Synthetic scheme of the probe MCPC.
As seen in FT-IR spectrum of the probe MCPC, the peaks appeared at 3197 and 3398 cm−1 were
revealed the presence of -OH and also, -C = O peak in the coumarin ring was appeared at 1660 cm−1 (Figure
S4). Moreover, the proton NMR for 2, 5, and 9 protons gives singlet peaks at 6.80, 7.08, and 8.16 ppm,
respectively. In addition, protons 13 and 14 give doublet peaks at 7.51 and 7.74 ppm. The -OH proton peaks
7
8
are observed at 9.56 and 10.34 ppm belong to H and H . Characteristic functional group conversions show
that the formation of probe MCPC was successfully achieved.
2+
3.2. Fluorogenic response of the probe MCPC towards Cu sensing
2+
To study the fluorogenic sensing ability, the response of probe MCPC towards a series of various cations (Hg
K , Mn , Cu , Ba , Cd , Co , Ca , Zn , Mg , Na , Fe , Sr , Pb , Fe
,
and Al3+) was
+
2+
2+
2+
2+
2+
2+
2+
2+
+
2+
2+
2+
3+
1151

SAVRAN et al./Turk J Chem
investigated in CH3 CN/H2 O media (v/v, 95/5, 5.0 µM) (Figure 1). The sensing property of probe MCPC
was tested in the presence of possible competing ions at a fluorescence emission wavelength of 456 nm. As
2+
illustrated in Figure 1, only Cu
caused a diverse intensity change and emission intensity decreased sharply,
whereas the probe MCPC did not give fluorogenic response towards other cations. None of the other cations
caused remarkable impact on the intensity of probe MCPC. The probe MCPC exhibited only a small intensity
3+
3+
change in the presence of Al and Fe . Thus, we concluded that the probe MCPC has good selectivity
2+
toward Cu
compared to other potential cationic species.
Figure 1. (a) Spectra of probe MCPC in the presence of competing ions (3.0 equiv), and (b) fluorescence colors of
MCPC in presence of competing cations in CH3 CN/H2 O media (v/v, 95/5) (5.0 µM) (λex = 366 nm, λem = 456 nm).
3.2.1. Competitive experiments
2+
To determine the practical efficacy of probe MCPC as a fluorogenic chemosensor for Cu
monitoring, the
2+
+
2+
2+
2+
competitive experiments were performed with various cations (3.0 equiv) (Hg , K , Mn , Cu , Ba
,
2+
2+
2+
2+
2+
+
2+
2+
2+
3+
Cd , Co , Ca , Zn , Mg , Na , Fe , Sr , Pb , Fe
95/5, 5.0 µM) (Figure 2).
and Al3+) in CH3 CN/H2 O media (v/v,
2+
The emission intensity of probe MCPC was quenched with Cu
was no interference with other cations except Fe
in the presence of other ions and there
(Figure 2). This cation could be easily masked by potassium
thiocyanate (KSCN); hence it has a little impact on the selectivity of probe MCPC toward Cu . That is to
say, probe MCPC could be employed as an excellent selective sensor for the monitoring of Cu
3+
2+
2+
.
3.2.2. Sensivity experiments
Fluorescence titration study was performed to investigate the quantitative interaction features of probe MCPC
2+
2+
toward Cu
(Figure 3). Upon the adding of Cu
(0 to 3.0 equiv), probe MCPC showed a gradual emission
1152

SAVRAN et al./Turk J Chem
2+
Figure 2. Competitive selectivity of probe MCPC (5.0 µM) toward Cu
in the presence of competing ions in
CH3 CN/H2 O media (v/v, 95/5, 5.0 µM) (3.0 equiv, λex = 366 nm, λem = 456 nm).
decreasement up to the constant equiv value, 3.0 equiv. Due to the complexation reaction between the probe
2+
MCPC and Cu , the quenching phenomena could occur via the heavy atom & paramagnetic effect [38] and
CHEQ (chelation enhancement quenching effect) [39] mechanisms. Therefore, the probe MCPC could be used
2+
as an outstanding “turn-off” fluorogenic sensor for Cu
monitoring.
3.2.3. Response time experiments
2+
As well, the time dependence of the probe MCPC (5.0 µM) to Cu
(3.0 equiv) was studied (Figure S7) in
2+
CH3 CN/H2 O media (v/v, 95/5, 5.0 µM) to decide the reaction time for the MCPC-Cu . The intensity of
probe MCPC reached a maximum/stabile value within approximately 1 min; hence this result showed that
2+
probe MCPC could be used as a fluorogenic chemosensor for the rapid sensing of Cu
.
1153

SAVRAN et al./Turk J Chem
2+
Figure 3. Spectral changes of probe MCPC upon the adding of Cu
95/5, 5.0 µM) (λex = 366 nm, λem = 456 nm).
(0 to 3.0 equiv) in CH3 CN/H2 O media (v/v,
3.2.4. ESI-MS and Job’s plot results
2+
To consider the binding mode/stoichiometry of probe MCPC toward Cu , Job’s plot [32] (Figure 4), as well
as ESI-MS analysis recorded in different matrixes and modes (Figures 5a and 5b) were carried out. The results
2
of these analyses showed the stoichiometric ratio of MCPC-Cu + complex as 1:1.
As illustrated in Figure 5, the peaks at 288.00 [m/z], 351.50 [m/z] and 367.50 [m/z] can be attributed
2+
+
to [probe MCPC], [probe MCPC + Cu ] and [probe MCPC + Cu2++ H2 O - 2•H ], respectively, which
2+
demonstrated that probe MCPC complexed with Cu
in a ratio of 1:1. The content of water in the MCPC-
2+
Cu
system can be seen from the ESI-MS, as well from FT-IR spectrum (Figure S8). The complexation
2+
process was recognized between Cu
and two protons belong to the hydroxyl groups (Scheme 2).
2+
On the basis of 1:1 stoichiometry, the binding constant of the MCPC-Cu
system was computed from
the Benesi-Hildebrand equation [40], and it was found to be 1.23 ×10 M−1 , which inferred that the stability of
4
2+
MCPC-Cu
complex was very high (Figure 6a). The calibration curve of the quantitative association between
was acquired with an excellent linear range (y = 5.92 ×10 x − 905.04, R
0.9957) at nanomolar levels (Figure 6b). The detection limit value of probe MCPC for Cu
2+
7
2
the intensity and amount of Cu
=
2+
was calculated
as 1.47 nM (3σ / slope) [28,29]. The sensing properties of the probe MCPC are comparable to those of other
2+
reported literatures which are include cumarin-based fluorescent probes for Cu
(Table 1). When this study is
1154

SAVRAN et al./Turk J Chem
2+
Figure 4. Job’s plots of MCPC-Cu
456 nm).
complex in CH3 CN/H2 O media (v/v, 95/5, 5.0 µM) (λex = 366 nm, λem
=
2+
Figure 5. ESI-MS spectra of the probe MCPC and MCPC-Cu
system.
compared to our previous coumarin based fluorescent sensor studies [9,12], it is obvious that this study is more
sensitive than others in terms of the detection limit value as a result of the substituent effect in the structure.
Although the flour atom in the previous study is attached to the benzene ring through an aliphatic carbon, the
chlorine in this study is directly connected to the benzene and has greater ability to attract the shared pair of
electrons. Therefore, the acidic behaviors of the hydrogen of hydroxyl group increases, since chlorine in benzene
ring demonstrates as well as resonance and inductive effect withdraws electrons while the delocalization of lone
pair supplies electron density towards the ring. This system enables us the usability of this sensor for sensing
2+
of the toxic Cu
at lower concentrations. The electron-donating ability of F3 C− , H3 C− , Cl− is examined,
1155

SAVRAN et al./Turk J Chem
2+
Scheme 2. Possible binding mechanism of the probe MCPC upon adding of Cu
.
it is found that F3 C− <H3 C− <Cl− and the abilities are directly proportional to the obtained detection limit
values (24.5 nM <5.13 nM <1.47 nM). As a consequence, the obtained results showed that the probe MCPC
2+
had an excellent sensitivity toward Cu
sensing, as well as a good linearity for quantitative detections.
2+
Figure 6. (a) Benesi-Hildebrand curve of 1/[Cu ] vs 1/(I-I0), and (b) the calibration curve of the quantitative
2+
association between the intensities of probe MCPC and the amount of Cu
(λex = 366 nm, λem = 456 nm).
2+
3.3. DFT calculations of the probe MCPC and MCPC-Cu complex
2+
To determine the fluorometric sensing mechanism of probe MCPC binding to Cu , theoretical computations
were done by using the DFT / B3LYP/6-31 g (d,p) method with Gaussian-09 software package with and
accompanying graphical interface program GaussView 5.0.8 [33–37]. The LanL2DZ basis set for the effective
potential set for Cu, and the 6-311G basis set was employed for C, H, O atoms (Figure 7). The geometric
optimizations were performed in the excited states to obtain the energy minimized structures of probe MCPC
2+
and MCPC-Cu
complex. As presented in Figure 7, the energy gap (∆E) of probe MCPC was calculated
2+
to be 3.80 eV, and after Cu
addition, the ∆E decreased remarkably to 1.30 eV. We offered the stability of
1156

SAVRAN et al./Turk J Chem
Table 1. Relative work of the sensing performance of the probe MCPC with some coumarin-based probe studies.
Probe
Solvent system
LOD (M)
Binding
constant
(Ka, 1/M)
6
Sample
Ref.
application
Na2HPO4-citric
acid buffer
(10 mmol/L)
256.00 ×10−9 1.75 ×10
Yes
[41]
Buffered water
solution (10 mM
Tris-HCl,
35.00 ×10−9
62.00 ×10−9
-
-
Yes
Yes
[42]
[10]
containing 1%
DMSO)
PBS buffer
solution /DMSO
(v/v, 3/1)
4
CH3CN/H2O
(v/v, 95/5)
24.50 ×10−9
9.00 ×10
Yes
[9]
5
CH3CN/HEPES
(v/v, 95/5)
5.13 ×10−9
1.47 ×10−9
8.80 ×10
1.23 ×10
Yes
Yes
[12]
4
CH3CN/H2O
(v/v, 95/5)
this
work
2+
MCPC-Cu
complex was better than probe MCPC based on the theoretical calculations. The increase in the
2+
stability indicates that the route of the reaction tends to the formation of MCPC-Cu
the fluorescence quenching occurs.
complex, and therefore
3.4. Fluorometric sensing and ICP-OES analysis of the tea samples
2+
To assess the potential usage of probe MCPC for practical applications, Cu
levels were measured in various
herbal tea samples, as well as black tea samples (Table 2). The samples were analyzed by the calibration plot
2+
(Figure 6b) of the emission intensities of probe MCPC toward Cu . A standard addition method was also
1157

SAVRAN et al./Turk J Chem
2+
Figure 7. Energy level diagrams of the MCPC-Cu
complex and probe MCPC.
2+
employed by adding two different concentrations (0.1 and 0.2 µmol L−1) of Cu
indicated in Table S2, probe MCPC could be very practical for Cu
into the tea samples. As
2+
sensing with satisfactory accuracy and
precision values in tested tea samples. The fluorometric sensing method’s recovery values were calculated within
2+
a range from 90.22% to 109.19%. Thus, Cu
black tea samples.
can be identified with excellent recovery values in herbal and
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SAVRAN et al./Turk J Chem
1159

SAVRAN et al./Turk J Chem
2+
The validity of the proposed sensing method for Cu
detection was checked by employing ICP-OES anal-
2+
ysis. Likewise the proposed fluorescence method, samples were spiked with recognized quantities of Cu
(0.01–
0.02 mgL−1) (Table S3). The calibration graphs were constructed for the determination of Cu quantitatively
2+
(0.01–1.00 mgL−1) by ICP-OES method. Recoveries of Cu were between 94.58 and 105.66% values, and the
2+
2+
good agreements were found between the obtained and added quantities of Cu . The results of the fluorescence
and atomic spectroscopy methods were compared (Table 2), and the differences of the means of two methods
were not found significantly different from each others at the 0.05 level.
Conclusion
As a conclusion, we have developed a low-cost, highly selective and sensitive probe MCPC with quite-low
2+
detection limit for the fluorescent sensing of Cu . Probe MCPC demonstrated excellent fluorescence “turn-
2+
off” response to Cu
in CH3 CN/H2 O media (v/v, 95/5, 5.0 µM) at 456 nm with emission quenching, and it
revealed a rapid response time (∼60 s). The detection limit was found as 1.47 nM according to 3σ/slope, which
2+
indicated excellent sensitivity to Cu
sensing in nanomolar levels. The ∆E values of the probe MCPC and
its complex were calculated to be 3.80 eV and 1.30 eV, respectively. Studies on real samples, herbal and black
2+
teas, revealed that the probe MCPC can sensitively and selectively recognize Cu with good recovery values
(90.22%–109.19%). The outcomes of the probe MCPC and ICP-OES analyses were also compared and the
differences of the means of two methods were not found significantly different (P <0.05). Thus, these promising
2+
findings could make a great contribution to researchers studying Cu detection in different foodstuffs.
Acknowledgment
This work was supported by the Karamanoğlu Mehmetbey University (KMU) with the financial support to use
Gaussian-09 and GaussView-5.0.8 software packages (KMU BAP-Grant Number: 30-M-16 and 13-YL-18) and
by the Scientific and Technological Research and Council of Turkey (TÜBİITAK) (Grant Number: 110T652).
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2+
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2+
2+
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2+
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1163

Supplementary Information
MATERIALS AND METHODS
Microwave–assisted solvent free synthesis of probe MCPC
3.03 mmol Phenylacrylonitrile compound (3) [1–3] and pyridinium hydrochloride (5 g) were
reacted in silica gel and the reaction mixture was subjected to microwave irradiation at 320 watt
for 30 min. The reaction was quenched with 1.0 N HCl (200 mL) as soon as the reaction
completed. The reaction mixture filtered, the residue dissolved in acetone (20 mL) four times
and filtered over silica gel. The solvent was evaporated and the residue washed with water and
then dried. The obtained solid was precipitated with ethyl acetate (15 mL) and n–hexane (250
mL). The obtained crude product was separated by column chromatography.
A fawn colored solid compound, probe MCPC, was obtained (0.75 g, 85%). Anal. Calc. for
C15H9ClO4 (MW: 288.68): C, 62.41; H, 3.14; Found C, 62.44, H; 3.17%. FT–IR (KBr, cm–1):
1
3197, 3398νO–H, 3000–3090 νC–H(Ar), 1660 νC=O, 1563, 1571, and 1621 νC=C. H NMR (400
MHz, DMSO–d6): δ 6.80 (1H, s, H2), 7.08 (1H, s, H5), 7.51 (2H, d, J=8.4 Hz, H13), 7.74 (2H,
d, J=8.8 Hz, H14), 8.16 (1H, s, H9), 9.56 (1H, s, H7), 10.34 (1H, s, H8). 13C–NMR (400 MHz,
DMSO–d6): δ 143.59 C1, 102.69 C2, 151.21 C3, 148.65 C4, 112.92 C5, 111.88 C6, 141.92 C9,
121.27 C10, 160.66 C11, 132.97 C12, 128.59 C13,17, 130.48 C14,16, and 134.63 C15.
1

RESULTS AND DISCUSSION
Figure S1. FT–IR spectrum of the compound (3)
Figure S2. 1H NMR spectrum of the compound (3) in CDCl3–d1
2

Figure S3. 13C–APT NMR spectrum of the compound (3) in CDCl3–d1
Figure S4. FT–IR spectrum of the probe MCPC
3

Figure S5. 1H NMR spectrum of the probe MCPC in DMSO–d6
Figure S6. 13C–APT NMR spectrum of the probe MCPC in DMSO–d6
4

Figure S7. Response time of the MCPC–Cu2+ complex
Figure S8.
FT–IR spectrum of the MCPC–Cu2+ complex
5

Table S1. Operating parameters for the ICP–OES analysis
parameter
setting
standard one piece quartz axial, one–piece with 2.4
mm injector
Plasma torch:
Spray chamber type:
Supplied rf power:
glass cyclonic (single–pass)
1.0 kW
Plasma Ar gas flow:
10 L.min–1
Auxiliary Ar gas flow:
Nebulizer Ar gas flow:
Nebulizer type:
1.0 L.min–1
0.85 L.min–1
sea spray
white–white tabs (1.02 mm id) – Waste: blue–blue
tabs (1.65 mm id)
Pump tubing Rinse/Instrument pump:
Pump speed:
20 rpm
1 mL
15 sec
5
Total sample usage:
Replicate read time:
Number of replicates:
Sample uptake delay time:
Stabilization time:
Rinse time:
20 sec
10 sec
10 sec
on
Fast pump:
Cu 324.754 nm, Cd 228.8 nm, Hg 253.7 nm, Zn 213.8
nm, Pb 405.8 nm, Ag 328.1 nm, and Tl 377.6 nm
Wavelengths
from 167 to 785 nm
6

Table S2. Determination of Cu2+ in herbal and black tea samples by the probe MCPC
sample
Cu2+ added
(μmol L−1)
Cu2+ found
(μmol L−1)
recovery (%)
RSD (%) (n=3)
herbal tea samples
green tea
0.00
0.10
0.20
0.17 ±0.002
0.27 ±0.003
0.37 ±0.002
1.20
0.92
0.56
100.65
99.28
0.00
0.10
0.20
0.02 ±0.0004
0.12 ±0.002
0.23 ±0.003
1.75
1.31
1.38
0.80
0.90
0.81
1.22
0.61
0.73
green tea
(mixed with rose)
91.64
104.42
0.00
0.10
0.20
0.13 ±0.001
0.22 ±0.002
0.32 ±0.002
white tea
sage tea
93.41
99.52
0.00
0.10
0.20
0.07 ±0.0001
0.16 ±0.001
0.29 ±0.002
93.63
108.23
0.00
0.10
0.20
0.04 ±0.0004
0.13 ±0.001
0.24 ±0.003
1.04
0.59
0.98
fennel tea
daisy tea
rose hip tea
ginger tea
mint tea
90.22
101.25
0.00
0.10
0.20
0.08 ±0.001
0.18 ±0.003
0.27 ±0.004
1.06
1.41
1.29
98.36
96.10
0.00
0.10
0.20
0.07 ±0.001
0.17 ±0.002
0.25 ±0.003
1.48
0.88
1.00
105.78
91.70
0.00
0.10
0.20
0.02 ±0.0002
0.11 ±0.001
0.22 ±0.001
1.03
0.88
0.50
91.79
98.73
0.00
0.10
0.20
0.09 ±0.001
0.19 ±0.001
0.27 ±0.004
1.16
0.27
1.57
102.17
93.16
0.00
0.10
0.20
0.01 ±0.0001
0.12 ±0.002
0.21 ±0.001
0.97
1.31
0.47
apple tea
106.51
101.06
0.00
0.10
0.20
0.09 ±0.001
0.20 ±0.001
0.30 ±0.002
1.07
0.50
0.51
linden tea
109.19
104.17
black tea samples
black tea without aroma–A
0.00
0.10
0.20
0.08 ±0.001
0.19 ±0.001
0.29 ±0.001
1.19
0.40
0.35
100.80
102.63
0.00
0.10
0.20
0.02 ±0.0002
0.12 ±0.002
0.23 ±0.003
1.38
1.28
1.14
black tea without aroma–B
black tea without aroma–C
100.82
108.37
0.00
0.10
0.20
0.05 ±0.0002
0.14 ±0.002
0.27 ±0.003
0.65
1.39
0.93
90.47
108.92
0.00
0.10
0.20
0.13 ±0.001
0.23 ±0.002
0.34 ±0.003
0.65
0.65
0.90
black tea with bergamot
aroma–A
101.35
104.06
0.00
0.10
0.20
0.05 ±0.0003
0.15 ±0.001
0.23 ±0.003
0.53
0.77
1.29
black tea with bergamot
aroma–B
99.55
91.02
7

Table S3. Determination of Cu2+ in herbal and black tea samples by ICP–OES
sample
Cu2+ added
(μg L−1)
Cu2+ found
(μg L−1)
recovery (%)
RSD (%) (n=3)
herbal tea samples
green tea
0
10
20
10.96 ±0.11
20.55 ±0.16
30.72 ±0.13
0.96
0.78
0.43
95.96
98.83
0
10
20
1.16 ±0.04
11.46 ±0.10
21.67 ±0.38
2.44
0.85
1.74
green tea
(mixed with rose)
98.77
100.27
0
10
20
8.11 ±0.11
17.88 ±0.112
27.76 ±0.17
1.35
0.70
0.61
white tea
sage tea
97.67
98.28
0
10
20
4.34 ±0.09
14.45 ±0.06
24.23 ±0.13
1.98
0.44
0.55
101.11
99.48
0
10
20
2.45 ±0.05
12.16 ±0.05
22.48 ±0.15
1.96
0.40
0.67
fennel tea
daisy tea
rose hip tea
ginger tea
mint tea
97.08
100.14
0
10
20
5.02 ±0.06
15.15 ±0.16
25.52 ±0.22
1.28
1.05
0.87
101.39
102.50
0
10
20
4.21 ±0.08
14.11 ±0.10
24.42 ±0.46
1.87
0.69
1.88
99.08
101.09
0
10
20
1.40 ±0.07
11.25 ±0.06
20.90 ±0.07
4.97
0.56
0.33
98.52
97.50
0
10
20
5.43 ±0.11
16.00 ±0.03
25.39 ±0.27
2.00
0.20
1.07
105.66
99.81
0
10
20
0.65 ±0.04
10.44 ±0.10
19.56 ±0.06
5.76
0.93
0.32
apple tea
97.97
94.58
0
10
20
5.57 ±0.08
15.59 ±0.06
24.87 ±0.10
1.48
0.41
0.39
linden tea
100.27
96.53
black tea samples
black tea without aroma–A
0
10
20
5.37 ±0.14
14.82 ±0.05
25.34 ±0.06
2.68
0.32
0.25
94.46
99.84
0
10
20
1.08 ±0.04
11.44 ±0.10
20.93 ±0.17
3.35
0.83
0.80
black tea without aroma–B
black tea without aroma–C
103.66
99.30
0
10
20
3.41 ±0.08
13.30 ±0.13
23.50 ±0.30
2.43
0.96
1.29
98.84
100.43
0
10
20
8.38 ±0.19
17.91 ±0.10
27.78 ±0.19
2.32
0.54
0.70
black tea with bergamot
aroma–A
95.33
97.01
0
10
20
3.31 ±0.13
13.46 ±007
23.73 ±0.26
3.90
0.54
1.08
black tea with bergamot
aroma–B
101.41
102.05
8

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