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

A fluorogenic probe based on a coumarin-derivative for Cu2+ sensing in CH3CN/H2O 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.


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 of concern, the Cu 2+ is the third most plentiful trace cation in the clay as well as in numerous livings, and it acts an essential role in numerous biological reactions. Cu 2+ 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 mostly in the brain [3]. However, it has been reported that excess intake of Cu 2+ causes adverse health effects in the body such as gastrointestinal diseases and it damages the kidneys and liver [1,4]. Also, Cu 2+ catalyzes the creation of reactive oxygen species which can harm basic biological molecules. According to various literatures, the toxicity of Cu 2+ has linked to severe neurological diseases [5], Indian childhood cirrhosis (ICC) and prion diseases [2,4,6,7]. World Health Organization (WHO) has reported that the optimum intake of Cu 2+ for adults * Correspondence: iyilmaz@kmu.edu.tr This work is licensed under a Creative Commons Attribution 4.0 International License. SAVRAN et al./Turk J Chem should not go above the level of 10-12 mg.day −1 [8]. For this reason, the monitoring of Cu 2+ in a variety of drinking and food samples has been a vital issue for the protection of human health [7]. Thus, the development of reliable analytical procedures for Cu 2+ sensing is still a critical research topic [9].
The reported methods for monitoring Cu 2+ include inductively coupled mass-atomic emission spectrometry (ICM-AES), capillary electrophoresis (CE), atomic absorption spectrometry (AAS), voltammetry and inductively coupled plasma-mass spectrometry (ICP-MS) [10]. These techniques involve expensive analysis systems, troublous analysis procedures, and also need excessive use of samples [11]. In contrast, the fluorescence spectroscopy is currently taken into account as a powerful method for the recognition of Cu 2+ owing to its real time, low cost, rapid and non destructive sensing with high selectivity and sensitivity [12] and easy operation.
Herein, a "turn-off" fluorogenic probe based on a coumarin-derivative, 3-(4-chlorophenyl)-6,7-dihydroxycoumarin, probe MCPC, for Cu 2+ sensing in CH 3 CN/H 2 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 such as 1 H-NMR and 13 C-ATP-NMR, as well as FT-IR. The theoretical computations were also done for the optimized geometric features.
A Bruker DPX 400 MHz spectrometer (Bruker Corp., Billerica, MA, USA) was employed to measure 13 C-APT-NMR and 1 H-NMR spectra. A Mass spectrometer (Bruker Daltonics-Microflex T M , 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 method, the amount of Cu 2+ 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.

Fabrication of the probe MCPC
Probe MCPC, 6,7-dihydroxy-3-(4-chlorophenyl)coumarin, was successfully synthesized under microwave irradiation procedure with solvent free conditions according to the reported method in the literatures [25][26][27]. The synthesis protocols and characterization data were presented in Supplementary Information file in detail.

Fluorescence spectroscopic studies of the probe MCPC towards Cu 2+ 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 CH 3 CN/H 2 O (v/v, 95/5). The titration experiments were performed to achieve the relationship between emission intensities and Cu 2+ . 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 λ em = 456 nm ( λ ex = 366 nm, 5 nm slit width).
The LOD of the probe MCPC was calculated from the equation of 3 σ /k (slope value) [28][29][30][31]. To analyze the effect of the possible competing cations on the sensing ability of probe MCPC to Cu 2+ , suitable concentration of Cu 2+ (3.0 equiv) was added into probe MCPC solution to generate MCPC-Cu 2+ complex, afterwards each of the other cation solutions was added into the MCPC-Cu 2+ , respectively. The emission intensities of the MCPC-Cu 2+ complex were measured before and after adding other cations. The possible stoichiometric ratio between the probe MCPC and Cu 2+ in MCPC-Cu 2+ system was found by using Job's method calculation [32].

Studies based on DFT
Theoretical calculations were performed for the probe MCPC and MCPC-Cu 2+ 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][34][35][36][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.

Detecting Cu 2+ 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. with a buffer solution (NH 3 /NH 4 Cl), and just after 15 min of waiting time, all teas were centrifuged at 10.000 rpm (~5 min). 0.5 mL of d-HNO 3 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 µ m 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.

Design of the probe MCPC
The probe MCPC was prepared under microwave irradiation with solvent free condition by the reaction of compound (3) with pyridinium hydrochloride (85% yield) (Scheme 1), and it was well-verified by 1 H-NMR, FT-IR and 13 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 are observed at 9.56 and 10.34 ppm belong to H 7 and H 8 . Characteristic functional group conversions show that the formation of probe MCPC was successfully achieved.

Fluorogenic response of the probe MCPC towards Cu 2+ sensing
To study the fluorogenic sensing ability, the response of probe MCPC towards a series of various cations (Hg 2+ ,

Competitive experiments
To determine the practical efficacy of probe MCPC as a fluorogenic chemosensor for Cu 2+ monitoring, the competitive experiments were performed with various cations (3.0 equiv) (Hg The emission intensity of probe MCPC was quenched with Cu 2+ in the presence of other ions and there was no interference with other cations except Fe 3+ (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 2+ . That is to say, probe MCPC could be employed as an excellent selective sensor for the monitoring of Cu 2+ .

Sensivity experiments
Fluorescence titration study was performed to investigate the quantitative interaction features of probe MCPC toward Cu 2+ (Figure 3). Upon the adding of Cu 2+ (0 to 3.0 equiv), probe MCPC showed a gradual emission  decreasement up to the constant equiv value, 3.0 equiv. Due to the complexation reaction between the probe MCPC and Cu 2+ , 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 as an outstanding "turn-off" fluorogenic sensor for Cu 2+ monitoring.

Response time experiments
As well, the time dependence of the probe MCPC (5.0 µ M) to Cu 2+ (3.0 equiv) was studied ( Figure S7

ESI-MS and Job's plot results
To consider the binding mode/stoichiometry of probe MCPC toward Cu 2+ , 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 of these analyses showed the stoichiometric ratio of MCPC-Cu 2 + complex as 1:1.
As illustrated in Figure 5 demonstrated that probe MCPC complexed with Cu 2+ in a ratio of 1:1. The content of water in the MCPC-Cu 2+ system can be seen from the ESI-MS, as well from FT-IR spectrum ( Figure S8). The complexation process was recognized between Cu 2+ and two protons belong to the hydroxyl groups (Scheme 2).
On the basis of 1:1 stoichiometry, the binding constant of the MCPC-Cu 2+ system was computed from the Benesi-Hildebrand equation [40], and it was found to be 1.23 ×10 4 M −1 , which inferred that the stability of MCPC-Cu 2+ complex was very high (Figure 6a). The calibration curve of the quantitative association between the intensity and amount of Cu 2+ was acquired with an excellent linear range (y = 5.92 ×10 7 x − 905.04, R 2 = 0.9957) at nanomolar levels ( Figure 6b). The detection limit value of probe MCPC for Cu 2+ was calculated as 1.47 nM (3 σ / slope) [28,29]. The sensing properties of the probe MCPC are comparable to those of other reported literatures which are include cumarin-based fluorescent probes for Cu 2+ (Table 1). When this study is   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 of the toxic Cu 2+ at lower concentrations. The electron-donating ability of F 3 C − , H 3 C − , Cl − is examined, Scheme 2. Possible binding mechanism of the probe MCPC upon adding of Cu 2+ .
it is found that F 3 C − <H 3 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 had an excellent sensitivity toward Cu 2+ sensing, as well as a good linearity for quantitative detections.

DFT calculations of the probe MCPC and MCPC-Cu 2+ complex
To determine the fluorometric sensing mechanism of probe MCPC binding to Cu 2+ , 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][34][35][36][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 and MCPC-Cu 2+ complex. As presented in Figure 7, the energy gap ( ∆ E) of probe MCPC was calculated to be 3.80 eV, and after Cu 2+ addition, the ∆ E decreased remarkably to 1.30 eV. We offered the stability of  MCPC-Cu 2+ complex was better than probe MCPC based on the theoretical calculations. The increase in the stability indicates that the route of the reaction tends to the formation of MCPC-Cu 2+ complex, and therefore the fluorescence quenching occurs.

Fluorometric sensing and ICP-OES analysis of the tea samples
To assess the potential usage of probe MCPC for practical applications, Cu 2+ levels were measured in various herbal tea samples, as well as black tea samples ( Table 2). The samples were analyzed by the calibration plot    The validity of the proposed sensing method for Cu 2+ detection was checked by employing ICP-OES analysis. Likewise the proposed fluorescence method, samples were spiked with recognized quantities of Cu 2+ (0.01-0.02 mgL −1 ) (Table S3). The calibration graphs were constructed for the determination of Cu 2+ quantitatively (0.01-1.00 mgL −1 ) by ICP-OES method. Recoveries of Cu 2+ were between 94.58 and 105.66% values, and the good agreements were found between the obtained and added quantities of Cu 2+ . 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