Abstract
A highly selective chemosensor BHC ((E)-N-benzhydryl-2-((2-hydroxynaphthalen-1-yl)methylene)hydrazine-1-carbothioamide) for detecting indium(III) was synthesized. Sensor BHC can detect In(III) by a fluorescence turn-on method. The detection limit was analyzed to be 0.89 μM. Importantly, this value is the lowest among those previously known for fluorescent turn-on In(III) chemosensors. Based on the analytical methods like ESI-mass, Job plot, and theoretical calculations, the detection mechanism for In(III) was illustrated to be chelation-enhanced fluorescence (CHEF) effect. Additionally, sensor BHC was successfully applied to test strips.
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Introduction
Indium is one of the elements of group 13 and its consumption has been gradually increased [1]. Most usage of indium is in semiconductor-related applications [2]. Apart from these applications, the pollution from it can affect severe health problems [3]. Although it has no biological role in human body, its effects have been reported to be toxic to humans, causing kidney disease and interference towards iron metabolism [4]. Therefore, it is needed to develop efficient detecting strategies for indium [5,6,7].
There are several analytical tools for the detection of a broad range of metal ions like AAS, ICP-AES (inductively coupled plasma atomic emission spectrometry), and other electrochemical methods (Absence of Gradients and Nernstian Equilibrium Stripping) [8,9,10,11]. However, they need complex procedures and sample pre-treatment and the costs are relatively high [12]. In contrast, chemosensors have been noted for its easy usage, fast response and cost-effective advantages [13,14,15,16,17].
Owing to similar properties of the 13 group elements, Al3+ and Ga3+, it is a challenge to distinguish In3+ from them. Until now, many chemosensors for Al3+ and Ga3+ were developed, but a few for In3+ [18,19,20,21,22]. Moreover, some of the In3+ chemosensors have difficulty in detecting In3+ because they are inhibited by Al3+ and Ga3+ or detected via quenching response which is a less preferred method [2, 4, 5, 7]. Thus, the chemosensor capable of sensing In3+ without interferences especially from Al3+ and Ga3+ with a turn-on response is highly demanded.
The benzhydryl isothiocyanate with hydrazine moiety could offer binding site to metal ions as well as act as a linker. A naphthol moiety is widely used as a fluorophore because of its unique photophysical property [23]. Therefore, we expected that the linkage of the two functional moieties can induce a unique optical change towards a specific metal ion.
Herein, we present a highly selective and sensitive fluorescence probe BHC, which can detect In3+ via a fluorescence turn-on. Importantly, it can distinguish In3+ from the same group metals, Al3+ and Ga3+, without interferences. In addition, the binding mode and sensing mechanism for In3+ were explained, based on the spectroscopic studies and theoretical calculations.
Experimental Section
General Information
Chemicals were provided commercially. Stock solutions of the cations (Cd2+, Al3+, K+, Ga3+, Ca2+, In3+, Zn2+, Na+, Cu2+, Ni2+, Fe3+, Co2+, Hg2+, Mg2+, Cr3+, Pb2+, Mn2+ and Ag+) were prepared using nitrate salts in dimethylsulfoxide (20 mM). Perchlorate salt was used for the Fe2+ stock solution. NMR data were measured using a Varian spectrometer (400 MHz). A Perkin Elmer spectrometer (Lambda 2S UV/Vis) was used for absorption spectra. ESI-mass data were gained on a Thermo Finnigan LCQTM instrument. A Perkin-Elmer spectrometer (LS45) was used for fluorescence data, and the slit width for excitation and emission was 10 nm.
Synthesis and Characterization of BHT (1-benzhydrylthiourea)
Benzhydryl isothiocyanate (1.12 g, 5.0 mmol) and hydrazine monohydrate (334 μL, 5.5 mmol) were dissolved in 10 mL of absolute ethanol and stirred for 6 h until white precipitate formed. It was filtered and washed with chilly ethanol and diethylether. Yield: 0.87 g (60%). 1H NMR (DMSO-d6, 400 MHz, ppm): δ 8.96 (s, 1H), 8.37 (s, 1H), 7.28 (m, 10H), 6.76 (s, 1H), 4.64 (s, 2H).
Synthesis and Characterization of BHC ((E)-N-benzhydryl-2-((2-hydroxynaphthalen-1-yl)methylene)hydrazine-1-carbothioamide)
BHT (480 mg, 2.0 mmol) and 2-hydroxy-1-naphthaldehyde (360 mg, 2.1 mmol) were dissolved in 5 mL of absolute ethanol and stirred for 1 day until pale yellow powder formed. It was filtered and washed with chilly ethanol and diethylether. Yield: 0.46 g (56%). 1H NMR (CDCl3, 400 MHz, ppm): δ 10.62 (s, 1H), 10.23 (s, 1H), 9.00 (s, 1H), 7.92 (d, J = 8.4, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.4 (t, J = 7.6 Hz, 1H), 7.32 (m, 11H), 7.17 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 13C NMR (DMSO-d6, 100 MHz, ppm): δ = 176.73 (1C), 156.70 (1C), 142.51 (1C), 141.90 (2C), 132.50 (1C), 131.40 (1C), 128.82 (1C), 128.55 (4C), 128.14 (1C), 127.61 (1C), 127.37 (4C), 127.23 (2C), 123.47 (1C), 122.84 (1C), 118.42 (1C), 110,11 (1C), 60.70 (1C). Positive-ion ESI-MS: m/z calcd, for [2·BHC + Na+]+, C50H42N6O2S2 + Na+, 845.27; found, 845.18.
Fluorescence and UV-vis Titrations of BHC with In3+
A stock solution of BHC was prepared in dimethylsulfoxide (DMSO, 1 × 10−2 M). 3 μL of it was diluted to 3 mL of DMSO for 10 μM concentration. A stock solution (2 × 10−2 M) of In(NO3)3 was prepared in DMSO. For the fluorescence titration, 1.5–37.5 μL of the In3+ solution was taken and mixed with BHC. 1.5–19.5 μL of the In3+ solution was added to the solution of BHC for UV-vis titration. Both fluorescence and UV-vis spectra were measured.
Quantum Yield of BHC and BHC-In3+
Quantum yield (Ф) was calculated by using fluorescein (ФF = 0.92 in basic ethanol) as a standard fluorophore [24]. The equation of quantum yield is as follows [25]:
Where the meaning of each abbreviation is
- ФF:
-
fluorescence quantum yield
- A :
-
Absorbance
- F :
-
The area of fluorescence emission curve
- n :
-
Refractive index of the solvent
- s :
-
standard
- x :
-
unknown
Job Plot Measurement of BHC with In3+
160 μL of a BHC stock solution (DMSO, 1 × 10−2 M) was diluted to 39.84 mL DMSO for 40 μM concentration. 80 μL of an In3+ stock solution (DMSO, 2 × 10−2 M) was diluted to 39.92 mL DMSO for 40 μM concentration. Both solutions were mixed from the molar fractions of 0.1 to 0.9 while maintaining a constant overall concentration (40 μM). The emission spectrum of each solution was measured.
Competition Experiment
34.5 μL of various metal-ion stock solutions dissolved in DMSO (Cd2+, Al3+, K+, Ga3+, Ca2+, In3+, Zn2+, Na+, Cu2+, Ni2+, Fe3+, Co2+, Hg2+, Mg2+, Cr3+, Pb2+, Mn2+ and Ag+, 20 mM) were diluted to 3 mL of DMSO (23 equiv). The same amount of an In3+ stock solution was added to each solution. A stock solution of BHC (10 mM, 3 μL) was added to them and mixed. The emission spectrum of each solution was measured.
Fluorescence Test Kit
Filter papers were immersed to 700 mM of BHC solution (1 mL, DMSO). After they were dried in the oven, various amounts (10, 20, 50, 100, and 200 μM) of an In3+ stock solution were applied to them for determining the lowest visible detection limit. The test kit prepared above was also applied to 20 μM of various metal solutions (Cd2+, Al3+, K+, Ga3+, Ca2+, In3+, Zn2+, Na+, Cu2+, Ni2+, Fe3+, Co2+, Hg2+, Mg2+, Cr3+, Pb2+, Mn2+ and Ag+).
Theoretical Studies
Energy-optimized structures of BHC and BHC-In3+ complex were calculated by density functional theory (DFT) using Gaussian 09 W program [26]. The hybrid functional was Becke, 3-parameter, Lee-Yang-Parr (B3LYP) and the basis set was 6-31G(d,p) [27,28,29,30]. All atoms except In3+ were applied to 6-31G(d,p) while LANL2DZ basis set was used as effective core potential (ECP) for In3+ [31,32,33]. Since imaginary frequency was not found in optimized structures of BHC and BHC-In3+, their geometries represented local minima. CPCM was used for considering solvent effect of DMSO [34, 35]. According to energy-optimized structures of sensor BHC and BHC-In3+ complex, the UV-vis transition studies were confirmed using TD-DFT (time-dependent DFT) method with thirty lowest singlet states.
Results and Discussion
By the nucleophilic addition reaction of benzhydryl isothiocyanate and hydrazine, compound BHT was synthesized. Compound BHC was obtained from the condensation reaction of BHT and 2-hydroxy-1-naphthalaldehyde (Scheme 1). It was fully characterized through 1H and 13C NMR and ESI-MS analyses (Figs. S1-S3).
In order to study the sensing ability of compound BHC towards various metals, fluorescence spectra were measured with the excitation wavelength of 416 nm (Fig. 1). Most metals did not show critical fluorescence change. In contrast, only In3+ displayed a remarkable increase of the fluorescence emission at 490 nm. This obvious change indicated that sensor BHC could detect In3+ by fluorescence turn-on. For investigating the counter-anion effect, we also used In2(SO4)3 instead of indium nitrate. Indium sulfate also showed nearly identical fluorescence enhancement as done with indium nitrate.
To investigate binding properties, fluorescence titration was achieved (Fig. 2). As the amount of In3+ increased, fluorescence emission at 490 nm was constantly increased. Quantum yields (Ф) of BHC and BHC-In3+ were calculated to be 0.0563 and 0.147. The binding interaction between BHC and In3+ was further studied with UV-vis titration (Fig. 3). The increase of In3+ induced absorption spectral changes with two defined isosbestic points at 300 nm and 392 nm, and it implies that only one species is present at the isosbestic point.
For the determination of binding stoichiometry of sensor BHC and In3+, Job plot experiment was achieved (Fig. S4). The highest fluorescence intensity appeared at the point where the mole fraction was 0.5. It indicated that sensor BHC and In3+ were combined in a 1 to 1 ratio. To support the binding interaction between BHC and In3+, positive-ion ESI-MS experiment was executed (Fig. 4). The peak of m/z = 679.99 was suggestive of BHC-2H++In3+ (calcd, m/z = 680.05). Its isotope pattern was well matched with the calculated value, supporting the 1: 1 binding stoichiometry of BHC and In3+. Job plot and ESI-MS analyses drove us to propose the plausible binding mode of BHC-In3+ in Scheme 2.
With the result of fluorescence titration, detection limit using 3σ/slope was analyzed to be 0.89 μM (R2 = 0.9996) (Fig. 5) [36]. Significantly, the detection limit is the lowest value among those previously known for fluorescent turn-on In3+ chemosensors, to date. (Table S1). Also, the association constant (K) based on Benesi-Hildebrand equation was turned out to be 4.3 × 103 M−1 (Fig. S5) [37].
For the practical application, the selectivity of compound BHC for In3+ was tested in the existence of other cations (Fig. S6). Hg2+ and Cu2+ inhibited the fluorescence of sensor BHC, and Fe3+ and Fe2+ displayed about half reduction of the fluorescence. Nevertheless, group 13 metals, Al3+ and Ga3+, didn’t show any fluorescence interferences.
Moreover, sensor BHC was applied to test strips. As shown in Fig. 6a, the obvious fluorescent emission appeared above 20 μM of In3+. On the contrary, the same concentration of other cations did not show fluorescence emission (Fig. 6b). Therefore, it demonstrated that sensor BHC could be also used for detecting In3+ in the test strip.
To comprehend the detection mechanism of BHC towards In3+, theoretical calculations were achieved. Based on the 1 to 1 binding stoichiometry between BHC and In3+, energy-optimized structures and molecular orbital contributions of BHC and BHC-In3+ complex were calculated. As shown in Fig. 7a, sensor BHC displayed a bent form with the dihedral angle 33.384° for 1 N, 2 N, 3C, and 4O. Upon chelating to In3+, its structure was flattened to 0.125° (Fig. 7b).
Based on these structures, molecular orbitals and transition energies were obtained by using TD-DFT calculation with the singlet excited states of BHC and BHC-In3+. Thirty singlet states having non-zero oscillator strength were considered as allowed-transition. For BHC, the main absorption band was originated from the HOMO → LUMO transition (382.26 nm, Fig. S7), indicating ICT (intramolecular charge transfer) transition from the naphthol group to the thiocyanate. In case of BHC-In3+, the main absorption band was originated from the HOMO-1 → LUMO+1 and HOMO → LUMO+1 transitions (414.62 nm, Fig. S8). The electrons of both HOMO and HOMO-1 were mainly localized in the dibenzene ring, whereas those of LUMO+1 were localized in the naphthol moiety (Fig. S9). Their transitions indicated ICT and LMCT (ligand-to-metal charge-transfer). The decrease of the energy gap between HOMO and LUMO corresponded to red shift of the experimental UV-vis spectra.
From these results, the sensing mechanism of BHC towards In3+ maybe due to chelation-enhanced fluorescence (CHEF) effect. As In3+ bound to BHC, the rotation of imine (-C=N) was inhibited [38]. Therefore, the rigid structure and inhibited non-radiative transition could induce fluorescence enhancement.
Conclusion
In conclusion, we synthesized a fluorescence chemosensor BHC for detecting In3+ by a fluorescence turn-on method. It can obviously discriminate In3+ from the same group metals, Al3+ and Ga3+, with no interferences. The detection limit for In3+ was 0.89 μM, which is the lowest among those previously known for fluorescent turn-on In3+ chemosensors, to date. Sensor BHC was also successfully applied to test strips. Moreover, fluorescence turn-on mechanism was proposed as chelation-enhanced fluorescence (CHEF) effect using DFT/TD-DFT calculation.
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The National Research Foundation of Korea (NRF) (NRF-2018R1A2B6001686) is thankfully acknowledged.
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Kim, C., Chae, J.B. A Highly Selective Fluorescent Chemosensor for Detecting Indium(III) with a Low Detection Limit and its Application. J Fluoresc 28, 1363–1370 (2018). https://doi.org/10.1007/s10895-018-2299-z
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DOI: https://doi.org/10.1007/s10895-018-2299-z