A Highly Selective Fluorescent Chemosensor for Detecting Indium(III) with a Low Detection Limit and its Application

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.

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 (Ф_F = 0.92 in basic ethanol) as a standard fluorophore [24]. The equation of quantum yield is as follows [25]:

$$ {\Phi}_{\mathrm{F}\left(\mathrm{X}\right)}={\Phi}_{\mathrm{F}\left(\mathrm{S}\right)}\ \left({A}_S{F}_X/{A}_X{F}_S\right)\ {\left({n}_X/{n}_S\right)}^2 $$

Where the meaning of each abbreviation is

Ф_F:

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).

Scheme 1

Synthesis of compound BHC

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.

Fig. 1

Fluorescence spectra of BHC (10 μM) with various metal ions (23 equiv). Excitation wavelength: 416 nm

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.

Fig. 2

Changes in fluorescence emission spectra when In³⁺ was added into sensor BHC (10 μM). Inset: fluorescence intensity at 490 nm (0–25 equiv). Excitation wavelength: 416 nm. Error bars represent standard deviations from three repeated experiments

Fig. 3

Changes in UV-vis spectra when In³⁺ was added into sensor BHC solution (10 μM)

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.

Fig. 4

Positive-ion ESI-MS spectrum of BHC-In³⁺ (100 μM, 1 equiv. of In³⁺)

Scheme 2

Fluorescence turn-on mechanism and proposed binding structure of BHC-In³⁺

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].

Fig. 5

Detection limit of BHC toward In³⁺ based on 3σ/slope. Error bars represent standard deviations from three repeated experiments

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.

Fig. 6

Photographs of the test strips coated with sensor BHC. a Sensor BHC-test strips immersed in various concentrations of In³⁺ (0–200 μM). b Sensor BHC-test strips immersed in 20 μM of various metal ion solutions

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).

Fig. 7

Energy-optimized structure of (a) sensor BHC and (b) BHC-In³⁺ complex

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.