A Benzothiazole-Based Fluorescence Turn-on Sensor for Copper(II)

A new benzothiazole-based chemosensor BTN (1-((Z)-(((E)-3-methylbenzo[d]thiazol-2(3H)-ylidene)hydrazono)methyl)naphthalen-2-ol) was synthesized for the detection of Cu2+. BTN could detect Cu2+ with “off-on” fluorescent response from colorless to yellow irrespective of presence of other cations. Limit of detection for Cu2+ was determined to be 3.3 μM. Binding ratio of BTN and Cu2+ turned out to be a 1:1 with the analysis of Job plot and ESI-MS. Sensing feature of Cu2+ by BTN was explained with theoretical calculations, which might be owing to internal charge transfer and chelation-enhanced fluorescence processes.


Introduction
Copper is one of the pivotal transition metals in human body [1][2][3]. It exists diverse enzymes like cytochrome oxidase and tyrosine, and plays an essential key role in vital metabolisms, such as redox system and physiological response [4]. However, excessive level of copper can cause neurodegenerative troubles like Parkinson's, Wilson's and Alzheimer's disease [5][6][7]. Furthermore, copper can also be a major source of environmental pollutants because it is widely used in industrial and agricultural practices [8]. Accordingly, it is essential to develop probes that can selectively detect copper with high sensitivity, selectivity and quick response.
Till now, a number of fluorescence chemosensors detecting Cu 2+ have been developed because of the features such as great selectivity, versatility, sensitivity and relatively simple handling [9][10][11][12][13]. However, many fluorescent sensors for detecting Cu 2+ are based on "turn-off" system, owing to the paramagnetic character of Cu 2+ [14]. Therefore, less examples were reported for fluorescence "turn-on" for detecting Cu 2+ [15]. Fluorescence sensors which have "turn-on" system shows better sensing properties at their sensitivity, selectivity and easy observation than "turn-off" system [16][17][18]. Moreover, the fluorescence "turn-off" system induced by a fluorescence quenching may give false results from other quenchers in practical samples and then, is less suitable for analytical applications [19]. Thus, the development of "turnon" chemosensors for Cu 2+ has been receiving substantial attention.
Herein, we demonstrated the design and application of a "turn-on" fluorescence probe (BTN) for detecting Cu 2+ . In particular, BTN can sense selectively Cu 2+ in samples with metal ions like Hg 2+ , Fe 3+ , Co 2+ and Ni 2+ having a fluorescent quenching property. The sensing mechanism and binding structure of BTN toward Cu 2+ were explained by using Job plot, FT-IR, ESI-MS analysis and theoretical calculations.
an ACQUITY QDa. FT-IR spectra were recorded on Agilent Cary 670 spectrometer.

Quantum Yield
To compare the quantum yields of BTN and BTN-Cu 2+ , fluoresceine (Φ = 0.54 in 100 mM H 2 SO 4 solution) was used as reference fluorophore [42]. With the following equation, quantum yield was calculated [42]. 19.98 mL of CH 3 CN. Then, 0.3-2.7 mL of BTN were added to each quartz. 0.3-2.7 mL of Cu 2+ was added into the quartz to give a total 3 mL volume. After stirring them for 3 s, fluorescence measurements were executed.

Theoretical Studies
Calculations were executed with Gaussian 16 program [43]. DFT method was applied for geometry optimizations [44,45]. B3LYP was used for the hybrid functional, and the 6-31G (d,p) basis set was applied to all atoms except for Cu 2+ [46,47]. For BTN-Cu 2+ complex, the LANL2DZ basis set was employed for applying effective core potential to Cu 2+ [48][49][50]. Imaginary frequencies were not found for both BTN and BTN-Cu 2+ , representing the local minima of the structures. The solvent effect of acetonitrile was regarded with IEFPCM [51]. With energy-optimized forms of BTN and BTN-Cu 2+ , the possible UV-vis transition states were calculated by using TD-DFT method with twenty lowest singlet states.

Fluorescent Sensing of BTN to Cu 2+
The fluorescence spectral measurements to varied metal ions were executed to confirm the fluorescence sensing capability of BTN (Fig. 1). BTN showed little fluorescence emission. After the addition of Cu 2+ to BTN, the fluorescence emission at 539.5 nm remarkably increased (λ ex = 397 nm) and its solution color varied to yellow under UV light. Quantum yields of BTN and BTN-Cu 2+ were given to be 0.0778 and 0.7919, respectively. By contrast, the presence of other cations with BTN did not show any variation. On the other hand, the photo-stability of BTN was examined (Fig. S4). Sensor BTN was stable enough for 24 h.
To find out the sensing process of BTN towards Cu 2+ , fluorescent titration was carried out (Fig. 2). With addition of Cu 2+ , the fluorescence at 539.5 nm was consistently enhanced. UV-vis spectral tests were carried out to demonstrate the binding property of BTN and Cu 2+ (Fig. 3). The absorbance at 300 and 500 nm significantly increased and that at 380 nm gradually decreased. An obvious isosbestic point observed at 420 nm meant that the binding of BTN with Cu 2+ produced a single product.
The Job plot was executed to investigate the complexation ratio of BTN and Cu 2+ (Fig. 4). It displayed a 1:1 complexation ratio of BTN and Cu 2+ , which was verified by ESI-MS (Fig. S5)  Additionally, to illustrate the binding structure of BTN with Cu 2+ , the FT-IR investigation was performed (Fig.  S6). The broad O-H peak (2800-3200 cm −1 ) disappeared after the complexation of BTN-Cu 2+ , demonstrating that the hydroxy group in BTN was deprotonated. The C=N peak at 1614 cm −1 shifted to 1574 cm −1 after BTN was bound to Cu 2+ . These outcomes illustrated that the sulfur, the nitrogen and the deprotonated oxygen of BTN might coordinate to Cu 2+ (Scheme 2). An association constant of BTN with Cu 2+ was analyzed to be 2.0 × 10 3 M −1 (R 2 = 0.9906) with the equation of Benesi-Hildebrand [52] (Fig.  S7). With the fluorescence titration, detection of limit for Cu 2+ turned out to be 3.3 μM using 3σ/K (Fig. 5) [53]. This is only the fourth benzothiazole-based fluorescent "turn-on" probe for detecting Cu 2+ , while the number of benzothiazole-based fluorescent "turn-off" sensors have been reported (Table S1).
Competitive test was executed to confirm a sensing capability of BTN (Fig. 6). BTN was not interfered by other cations and exhibited the constant fluorescence emission at 539.5 nm. This indicated that BTN can detect Cu 2+ without being disturbed by other metal ions, resulting in high selectivity. Therefore, BTN could be a very effective fluorescence sensor for sensing Cu 2+ in samples containing other metal ions.

Calculations
Optimized forms of BTN and BTN-Cu 2+ were calculated, based on the Job plot, ESI-mass and IR data (Fig. 7). BTN has a distorted structure with bending of the naphthol group, while BTN-Cu 2+ exhibits a nearly planar structure. With the structural change during the complex formation, the dihedral angle (1S, 2 N, 3 N, 4O) changed from 154.190°to 18.894°.
With energy-optimized forms of BTN-Cu 2+ and BTN, TD-DFT calculations were executed to investigate molecular orbitals and transition energies. For BTN, the major absorption band stemmed from the HOMO → LUMO transition (380.48 nm, Figs. S8 and S9), which meant intramolecular charge transfer (ICT) from the benzothiazole group to the naphthol one. With BTN-Cu 2+ , the main transition at 427.12 nm stemmed from the HOMO → LUMO (α) and HOMO → LUMO+1 (β), which showed ICT characteristics ( Fig. S10 and S11). Given the experiment and calculation results, the fluorescent turn-on process of BTN to Cu 2+ may be CHEF effect [54,55]. Free vibration and rotation of BTN, non-radiative transitions, were restricted due to the complex formation with Cu 2+ . The red shift shown in the experimental UV-vis spectra was consistent with the decreased energy gap. Based on calculations and experimental results, proper structure of BTN-Cu 2+ was proposed (Scheme 2). Scheme 2 Response process of BTN to Cu 2+

Conclusion
We presented a great efficient fluorescent turn-on chemosensor BTN synthesized from the combination of 3-methyl-2-benzothiazolinone hydrazone and 2-hydroxy-1-naphthaldehyde. BTN can work as one of a few benzothiazole-based fluorescent "turn-on" probes for detecting Cu 2+ , while the number of benzothiazole-based fluorescent "turn-off" sensors has been addressed. Limit of detection for Cu 2+ was 3.3 μM. In particular, in samples with metal ions like Hg 2+ , Fe 3+ , Co 2+ and Ni 2+ having a fluorescent quenching property, BTN can selectively sense Cu 2+ . Detection process of Cu 2+ by BTN was demonstrated to be CHEF and ICT processes through theoretical calculations.
Acknowledgments The National Research Foundation of Korea (NRF) (2018R1A2B6001686 and NRF-2020R1A6A1A03042742) is gratefully acknowledged.
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Declarations
Conflict of interest 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.