Spirooxazine-Based Dual-Sensing Probe for Colorimetric Detection of Cu2+ and Fe3+ and Its Application in Drinking Water and Rice Quality Monitoring.

A spirooxazine derivative, PheSPO (3,3-dimethyl-1-phenethylspiro[indoline-2,3'-naphtho[2,1-b][1,4]oxazine]), as a dual-sensing probe for Cu2+ and Fe3+ was synthesized, and its structure was confirmed by 1H NMR, 13C NMR, HRMS, and single-crystal X-ray diffraction. The results reveal that the PheSPO probe is selective to both Cu2+ and Fe3+ through distinct colorimetric responses in acetonitrile. The sensing performance of PheSPO toward Cu2+ was investigated, and upon addition of Cu2+, an instant change in color from colorless to bright yellow with a strong absorption band at 467 nm was observed. Due to a dual-sensing behavior, PheSPO also exhibits a unique response toward Fe3+ that can be discovered from a color change from colorless to red at an absorption wavelength of 514 nm. Based on spectroscopic analyses and density functional theory calculations, the 1:1 stoichiometric complexation of PheSPO with the targeted metal ions was proposed and the binding constants of 1.95 × 103 M-1 for Cu2+ and 1.29 × 103 M-1 for Fe3+ were obtained. In addition, the detection limits of PheSPO for Cu2+ and Fe3+ were 0.94 and 2.01 μM, respectively. To verify its applicability in real samples, PheSPO was further explored for quantitative determination of both Cu2+ and Fe3+ in spiked drinking water. The results showed that the recoveries of Cu2+ and Fe3+ examined using the PheSPO probe were found comparable to those obtained from atomic absorption spectroscopy. Moreover, the PheSPO strip test was developed, and its utilization for qualitative detection of Fe3+ in real rice samples was demonstrated.

Introduction

Among essential transition metal ions, Cu2+ and Fe3+ are vital for biological processes including catalysis, metabolism, and signaling.[1−3] Under physiological imbalance, these metal ions can lead to diverse health problems.[4−7] Although Cu2+ plays a crucial role in ATP production, catecholamine biosynthesis, and protecting the cells from oxygen-free radicals,[8−10] disturbance in homeostasis of Cu2+ can be highly poisonous to cells and has been linked to the predominance of neurodegenerative diseases such as Menkes,[11] Wilson’s,[12] Alzheimer’s,[13] and Parkinson’s diseases.[14] Moreover, these chronic diseases can originate from both the deficiency and excess of Fe3+ despite its necessity for enzyme catalysis in cellular metabolism.[15,16] As a consequence, the US EPA has recommended that the dietary intake of Cu2+ and Fe3+ should not exceed the maximum allowable concentrations in food (Cu2+, 1.0–1.3 mg/day for adults and Fe3+, 19.3–20.5 mg/day in men and 17.0–18.9 mg/day in women) and water (Cu2+, 1.3 mg/L and Fe3+, 0.3 mg/L).[17−19] Ordinarily, the capability of measuring the quantity of Cu2+ and Fe3+ in biological and environmental samples is exemplified by the conventional methods, including atomic absorption spectroscopy (AAS),[20,21] inductively coupled plasma mass spectrometry (MS),[22,23] and ion chromatography.[24,25] These methods, however, are rather complicated, time-consuming, and costly, especially for inexperienced users. Therefore, many researchers have focused on the development of an applicable and reliable approach for the detection of Cu2+ and Fe3+ by using a chemosensor.[26−28]

A chemosensor is a molecular probe that empowers the transformation of analyte information into a measurable signal of colorimetric or fluorescent responses.[29] Much effort has been drawn to develop chemosensors with efficient sensing performance for rapid and accurate detection.[30] To obtain an improved selectivity and sensitivity for the analysis of metal ions, a particular part of the chemosensors is designed for specific binding with the metal-ion analyte. This subsequently leads to a spectral change in their signals and sometimes a structural change can be observed in some chemosensors.[31] Several organic molecules, for example, rhodamine, anthracene, benzothiadiazole, squaraine, and phenothiazine, have been studied as potential chemosensors to detect a wide range of metal ions.[32−36] Moreover, their sensing mechanism in response to metal ions was also proposed based on the metal–ligand coordination and chemical reactions, such as bond cleavage, bond formation, rearrangement, and cyclization.[37] To date, several chemosensors as colorimetric probes with high selectivity and sensitivity as a facile and rapid tool for on-site analysis of metal ions have been reported.[38−42]

Owing to its unique optical property, spirooxazine has shown the capability as a chemosensor in response to metal ions.[43] The specific ion recognition modulated by spirooxazine occurs via ring opening together with metal–ligand complexation. Typically, the ring-opening reaction of spirooxazine proceeds through bond cleavage at the spiro carbon (Cspiro–O), which is induced by either electromagnetic radiation or metal ion stimuli.[44,45] This process results in the formation of an open-ring form, also known as merocyanine, which can serve as an active ligand to selectively coordinate with a metal ion and produce a merocyanine–metal complex.[46] Recently, some spirooxazine probes showed high selectivity for the detection of metal ions, including Mg2+, Al3+, Fe3+, Co2+, Zn2+, Hg2+, and CH3Hg+.[47−53] However, few studies of spirooxazine probes for Cu2+ detection have been described, and to the best of our knowledge, the spirooxazine as a dual probe for Cu2+ and Fe3+ detection has not yet been reported.

Herein, we demonstrated the utilization of a spirooxazine derivative, 3,3-dimethyl-1-phenethylspiro[indoline-2,3′-naphtho[2,1-b][1,4]oxazine] (PheSPO), as a dual-sensing probe that possessed high selectivity and sensitivity toward Cu2+ and Fe3+ in acetonitrile. Its synthesis is presented in Schemes and 2 in three steps of the longest linear sequence. The sensing performance of PheSPO against Cu2+ and Fe3+ was determined by a distinct change in color at the micromolar level. To prove that PheSPO can be applied in practical application, the probe was further used to detect the trace amount of Cu2+ and Fe3+ in spiked drinking water. Moreover, the test strips of PheSPO were also fabricated for qualitative detection of Fe3+ in rice samples.

Scheme 1

Preparation of Zinc Complex 2

Scheme 2

Synthesis of PheSPO

Experimental Section

Materials and General Information

1-Nitroso-2-naphthol, zinc chloride, (2-bromoethyl)benzene, 2,3,3-trimethylindolenine, and triethylamine were purchased from Tokyo Chemical Industry (TCI). Tetrahydrofuran, acetonitrile, dichloromethane, and ethanol were obtained from Honeywell Burdick & Jackson (B&J). Metal ions including Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+, Sn2+, Pb2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+ were obtained from Sigma-Aldrich as chloride salts. All reagents were of analytical grade and used as received unless stated otherwise. Deionized water (DI) was used for all experiments. Analytical thin-layer chromatography (TLC) was performed on Kieselgel F254 pre-coated aluminum TLC plates obtained from EM Science. Visualization was performed with a 254 nm ultraviolet lamp. Column chromatography was carried out with Merck silica gel 60 (230–400 mesh ASTM). UV/vis absorption spectra were measured on a Shimadzu (UV-1800) spectrophotometer at ambient temperature. The path length of a quartz cell was 1 cm. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra with entire proton decoupling were recorded on a Bruker AVANCE 500 NMR spectrometer, and chemical shifts in ppm were quoted relative to the residual signals of deuterated solvents. High-resolution mass spectra were recorded using a Bruker micrOTOF mass spectrometer (ESI-TOF) and reported with ion mass/charge (m/z) ratios as values in atomic mass units.

Synthesis of PheSPO

1-Nitroso-2-naphthol Zinc Salt (2)

To a stirred solution of 1-nitroso-2-naphthol (1) (5.00 g, 28.87 mmol) in a mixture of tetrahydrofuran and water (1:1 v/v) (130 mL) was added zinc chloride (1.64 g, 12.03 mmol) in one portion, and the resulting mixture was heated to 100 °C and stirred at this temperature for 2 h. The reaction mixture was cooled to room temperature, and the suspension was filtered. The precipitate was washed with cold water and dried under a vacuum for 24 h to give 1-nitroso-2-naphthol zinc salt (2) as a brown solid (4.58 g). This crude product was used in the next step without purification.

3,3-Dimethyl-2-methylene-1-phenethylindoline (6)

To a stirred solution of (2-bromoethyl)benzene (4.60 g, 24.87 mmol) in acetonitrile (120 mL) under an Ar atmosphere was added 2,3,3-trimethylindolenine (3.96 g, 24.87 mmol). The reaction mixture was heated to reflux with stirring for 48 h. The mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The resulting viscous oil was washed with diethyl ether (2 × 60 mL) and dried under a vacuum for 12 h to give indolium salt 5, which was dissolved in dichloromethane (120 mL). To the resulting solution was added triethylamine (7.55 g, 74.60 mmol), and the mixture was stirred at room temperature for 8 h. The reaction mixture was washed with water (2 × 75 mL) and the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (5% ethyl acetate in hexane) to afford the title product (6) as a red oil (5.24 g, 80%). 1H NMR (500 MHz, CDCl3): δ 7.31–7.20 (m, 5H), 7.10–7.07 (m, 2H), 6.74 (t, J = 7.4 Hz, 1H), 6.46 (d, J = 8.0 Hz, 1H), 3.92 (s, 1H), 3.86 (d, J = 2.0 Hz, 1H) 3.70 (t, J = 7.8 Hz, 2H), 2.89 (t, J = 7.8 Hz, 2H), 1.33 (s, 6H); HRMS (ESI) m/z: calcd for C19H22N [M + H]+, 264.3847; found, 264.1752.

3,3-Dimethyl-1-phenethylspiro[indoline-2,3′-naphtho[2,1-b][1,4]oxazine] (7, PheSPO)

To a stirred solution of 1-nitroso-2-naphthol zinc salt 2 (2.14 g, about 9 mmol) in ethanol (70 mL) under an Ar atmosphere was added indoline 6 (2.00 g, 7.6 mmol), and the resulting mixture was heated to reflux for 8 h. The mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (40% dichloromethane in hexane) to afford PheSPO (1.18 g, 37%) as a green solid. 1H NMR (500 MHz, CD3OD): δ 8.44 (d, J = 10.5 Hz, 1H), 7.76 (d, J = 10.2 Hz, 1H), 7.70 (d, J = 11.1 Hz, 1H), 7.54 (dd, J = 8.6, 1.4 Hz, 1H), 7.38 (dd, J = 10.1, 1.4 Hz, 1H), 7.25–7.17 (m, 4H), 7.07 (dd, J = 9.1, 0.9 Hz, 1H), 7.04–7.02 (m, 2H), 6.99 (s, 1H), 6.97 (d, J = 11.1 Hz, 1H), 6.86 (t, J = 9.2 Hz, 1H), 6.71 (d, J = 9.7 Hz, 1H), 3.44–3.35 (m, 2H), 3.07–2.99 (m, 1H), 2.82–2.76 (m, 1H), 1.25 (s, 3H), 1.19 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 151.3, 146.5, 143.9, 139.6, 135.6, 130.5, 130.0, 129.4, 128.9, 128.0, 127.6, 127.5, 126.7, 126.0, 123.8, 122.6, 121.1, 121.0, 119.3, 116.4, 106.5, 98.8, 51.8, 46.3, 34.5, 24.4, 19.6; HRMS (ESI) m/z: calcd for C29H26N2ONa [M + Na]+, 441.1943; found, 441.1937.

Single-Crystal Analysis

20.9 mg of PheSPO was gently dissolved in 5 mL of 1,4-dioxane with the assistance of sonication for 5 min at 40 °C. Then, 3 mL of DI water was slowly dropped into the solution. After slow evaporation of the solvent under ambient temperature for 2 weeks, a colorless single crystal of PheSPO was obtained for analysis. The X-ray diffraction intensity data were collected on a Bruker D8 Venture geometry diffractometer with Cu Kα radiation (λ = 1.54178 Å) at room temperature. A complete structure solution of the PheSPO single crystal was performed on Olex2 software.

UV–Visible Absorption Study

The stock solutions of PheSPO (0.1 mM) and metal ions (0.1 mM), including Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+, Sn2+, Pb2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+, were freshly prepared in acetonitrile and stored in dark for further use. The spectral change of the mixed solutions of PheSPO (50 μM) and metal ions (50 μM) was monitored on a Shimadzu UV-1800 spectrophotometer operated at room temperature. The quartz cuvettes with 1 cm path length were used.

DFT Calculations

The ground-state geometries of PheSPO in its open form and its complexation with cationic species in an implicit solvent model of acetonitrile were fully optimized at the density functional theory (DFT) level of theory using the B3LYP hybrid functional[54,55] with the DFT-D3 dispersion correction.[56,57] The 6-311+G(d,p) and def2-tzvp basis sets were used to describe the electronic configurations of nonmetal and metal atoms, respectively. The solvent effects of acetonitrile (with a dielectric constant ε = 35.688) were accounted for using the polarizable continuum model.[58,59] The optimized geometries and frontier molecular orbitals were visualized with ChemCraft software.[60] All calculations were performed using the Gaussian 09 suite of programs.[61]

Analysis of Cu2+ and Fe3+ in Drinking Water

To determine the optimal conditions for PheSPO in detecting Cu2+ and Fe3+ in drinking water, the effect of solvent polarity was studied in detail, and the results are discussed in the Supporting Information (Figure S1).

In brief, 5 mL of drinking water obtained from a water dispenser was spiked with known concentrations of Cu2+ and Fe3+. The spiked solution was made up to 10 mL with DI water in a volumetric flask. Then, 2 mL of the spiked solution was thoroughly mixed with 2 mL of 20 μM PheSPO in acetonitrile. The mixed solution was irradiated with 395 nm UV light for 5 min. The colorimetric response of the solution was monitored by UV–visible spectroscopy.

To evaluate the efficiency and accuracy of the PheSPO probe, the concentrations of Cu2+ and Fe3+ in the spiked sample were also analyzed by standard flame AAS operated on a PerkinElmer AAnalyst 200 system.

Strip Test for Fe3+ Detection in Rice

The test strips of the PheSPO probe for Fe3+ detection were prepared by immersing TLC plates (1 × 1 cm2) into a solution of PheSPO (1 mM) in acetonitrile for 5 min, and the resulting wet strips were dried in air. To optimize the analysis conditions for the strip test, the sensing performance of PheSPO coated on a TLC plate in detecting Cu2+ and Fe3+ was investigated under various pH conditions, and the results are shown in Figure S2.

The rice sample was prepared as follows: 5 g of ground rice (Khao Dawk Mali 105) was added to a 50 mL block digestion tube, which contained 6 mL of a mixture of 37% HCl and 70% HClO4 (2:1, v/v). The resulting mixture was heated at 180 °C for 6 h. After digestion was completed, the clear solution was transferred into a volumetric flask and made up to 10 mL with ultrapure water. The stock solution of the digested rice sample was kept in dark for further Fe3+ analysis.

To evaluate the presence of Fe3+ in the rice, a drop of the digested rice sample was cast on the PheSPO-treated strips, and the change in color was observed by the naked eye.

Results and Discussion

Single Crystal of PheSPO

The single crystal of PheSPO was grown through slow evaporation of solvents, and it crystallized in the monoclinic space group P21/c. The crystallographic data are reported in Table S1 and deposited at CCDC (no. 2154731). As shown in Figure , the molecular structure of PheSPO contains two heterocyclic rings of indoline and oxazine fragments that are mutually orthogonal to each other and connected through the sp3-hybridized spiro carbon (C8). The O1–C8 bond length is 1.4578 Å, which is slightly longer than that of typical oxygen-containing heterocycles (1.41–1.43 Å). Upon exposure to the external stimuli, the cleavage of the O1–C8 bond in PheSPO via ring-opening reaction is activated. This subsequently leads to the formation of an open-form merocyanine.

Figure 1

ORTEP diagram of PheSPO at 50% probability displacement of the ellipsoids.

UV–Visible Absorption Study

The selectivity of PheSPO was investigated against various metal ions, including Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+, Sn2+, Pb2+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+ in acetonitrile solutions. In Figure , the results clearly show the change in color of PheSPO solutions from colorless to red for Fe3+ treatment and from colorless to yellow for Cu2+ treatment. On the contrary, the mixed solutions remained colorless upon the treatment with other metal ions. This indicates that PheSPO can provide a selective response against Cu2+ and Fe3+ with a distinct change in color that can be seen with the naked eye. In addition, the spectral change of PheSPO upon addition of metal ions was further evaluated by UV–visible absorption. As shown in Figure , free PheSPO exhibits two main absorption peaks at 317 and 349 nm due to the π → π* transition of the naphthooxazine ring.[62] Addition of Cu2+ into the PheSPO solution caused an emergence of a relatively strong absorption band at λmax 467 nm. Meanwhile, the PheSPO solution mixed with Fe3+ displays a new absorption band at λmax 514 nm. These two bands of absorption in the visible region are mainly ascribed to the formation of the open-form merocyanine with extended π-conjugation induced by the complexation with Cu2+ and Fe3+. In the case of other metal ions, no significant change in the absorption spectra was observed. These results suggest that PheSPO can act as a dual-sensing probe for the detection of Cu2+ and Fe3+.

Figure 2

Photograph of colorimetric responses of PheSPO (50 μM) in acetonitrile in the presence of various metal ions (50 μM).

Figure 3

UV–visible absorption spectra of PheSPO (50 μM) in acetonitrile in the presence of various metal ions (50 μM).

To examine the selectivity of the PheSPO probe toward Cu2+ and Fe3+ detection, competitive experiments in acetonitrile solutions were carried out in the presence of other interfering metal ions. As shown in Figure a where the selectivity of PheSPO toward Cu2+ is investigated, the absorbance change at 467 nm of other cations was negligible when compared to that of PheSPO mixed with Cu2+. This suggests that the coexistence of other metal ions has insignificant effect on the sensing performance of PheSPO toward Cu2+. In the case of PheSPO and Fe3+, Cu2+ was the only metal ion that exhibited significant interference to the absorbance at 514 nm (Figure b). A marked decrease in absorbance at 514 nm when Cu2+ was added to the solution of PheSPO and Fe3+ might be the result from the replacement of Fe3+ in the Fe3+–PheSPO complex with Cu2+. To confirm our proposal, the spectral change of PheSPO and Fe3+ solution was monitored with increasing addition of Cu2+, and the results in Figure show an increase in absorbance at 467 nm (Cu2+–PheSPO) along with a simultaneous decrease in absorbance at 514 nm (Fe3+–PheSPO). This suggests that Cu2+ could generate considerable interference against Fe3+ detection with the PheSPO probe in mixed metal-ion solutions.

Figure 4

Selectivity of PheSPO (50 μM) in acetonitrile toward (a) Cu2+ and (b) Fe3+ (5 equiv) in the presence of other interfering metal ions (5 equiv).

Figure 5

Spectral change of the solution of PheSPO (2 mM) and Fe3+ (50 μM) in acetonitrile upon increasing addition of Cu2+ (50–300 μM).

The complexation stoichiometry of PheSPO and metal ions (Cu2+ and Fe3+) was studied by using Job’s method. The equimolar solutions of PheSPO and metal ions were prepared with different mole fractions, and Job’s plots were established using the absorbance of 467 nm for Cu2+ and 514 nm for Fe3+ as shown in Figure a,b, respectively. The maximum absorbance at a mole fraction of 0.5 in both cases suggests that the metal–PheSPO complex occurs at a 1:1 stoichiometric ratio. Therefore, the reaction mechanism for the ring opening of PheSPO in the presence of Cu2+ or Fe3+ (represented as M) was proposed based on the 1:1 complex formation as shown in Figure . This metal ion-induced ring opening of PheSPO takes place via bond cleavage at the spiro carbon and liberates the phenolate oxygen (Ph–O–), which subsequently coordinates to metal ions through the vacancy site. This process also causes a unique change in the optical behavior of PheSPO due to the effect of extended π-conjugation of open-form merocyanine after bond breaking reaction and metal complexation. In addition, the MS spectra of metal–PheSPO complexes in Figure S3 also show the molecular peaks at 497.1338 m/z and 509.1144 m/z, which correspond to the presence of [PheSPO–2H+ + Cu2+ + H2O] and [PheSPO–H+ + Fe3+ + 2H2O], respectively. These results clearly confirm the complex formation of PheSPO with the targeted metal ions (Cu2+ and Fe3+).

Figure 6

Job’s plots for the determination of complexation stoichiometry of acetonitrile solutions of (a) PheSPO and Cu2+ and (b) PheSPO and Fe3+. The total concentration was fixed at 10 μM.

Figure 7

Proposed metal ion-induced ring-opening reaction of PheSPO in the presence of the targeted metal ions (M = Cu2+ or Fe3+).

The sensitivity of PheSPO for the detection of Cu2+ and Fe3+ was also examined to evaluate the detection limits. This was conducted by the absorption titration with the concentration of metal ions ranging from 0 to 1 equiv. The results in Figure a,b reveal that the absorbance at the wavelength corresponding to the complexation gradually increased with increasing metal-ion concentrations. Moreover, the absorbance changes of PheSPO versus Cu2+ and Fe3+ concentrations exhibit a good linear relationship with R2 > 0.99 as shown in the insets. Based on the linear response observed, the detection limits derived from 3σ/m, where σ is the standard deviation of blank measurements and m is the slope of a plot between absorbance versus metal-ion concentration, were found to be 0.94 μM for Cu2+ and 2.01 μM for Fe3+. This demonstrates that the PheSPO dual-sensing probe possesses high sensitivity toward Cu2+ and Fe3+ detection when compared to the previously reported dual-sensing probes (see Table S2).

Figure 8

Spectral changes of PheSPO (50 μM) in acetonitrile with increasing addition (0–1 equiv) of (a) Cu2+ and (b) Fe3+. The insets show a linear response with the increase in Cu2+ and Fe3+ concentrations.

According to the 1:1 reaction stoichiometry, the binding constant (Kα) was evaluated by using the Benesi–Hildebrand equation: , where A and A0 are the absorbance of PheSPO in the presence and absence of metal ions, respectively, Amax is the saturated absorbance of PheSPO in the presence of an excess amount of metal ions, and [C] is the concentration of metal ions. The resulting plots in Figure a,b show the best fit of the linear function with R2 > 0.99, and the Kα values of the complexes were found to be 1.95 × 103 M–1 for Cu2+ and 1.29 × 103 M–1 for Fe3+.

Figure 9

Benesi–Hildebrand plots of the 1:1 stoichiometric ratio of (a) PheSPO and Cu2+ and (b) PheSPO and Fe3+.

Computational Study

To gain insight into the structures and absorption behaviors of PheSPO and its 1:1 complex with metal ions, DFT calculations were performed at the B3LYP-D3 level with hybrid basis sets of 6-311+G(d,p) and def2-tzvp. The optimized structures of free PheSPO and the resulting complexes with Cu2+ and Fe3+ are shown in Figure a. The result suggests that in the absence of metal ions, the free PheSPO remains stable in a closed form in which the oxazine ring is arranged orthogonally with the indoline ring through a spiro carbon linkage. Upon complexation, the optimized geometry of PheSPO turned into open-form merocyanine with the planar TTC (trans–trans–cis) conformation, of which the oxygen phenolate anion plays an important role in binding with the metal-ion center. According to the DFT results, the optimized complex contains monodentate PheSPO together with water and chloride ligands in binding with Cu2+ in square planar and Fe3+ in octahedral coordination geometry.

Figure 10

(a) Optimized structures and (b) frontier molecular orbitals of free PheSPO, Cu2+–PheSPO, and Fe3+–PheSPO complexes calculated at the B3LYP-D3 level using hybrid basis sets 6-311+G(d,p) for H, C, N, O, and Cl and def2-tzvp for Cu and Fe.

In Figure b, the frontier molecular orbitals of free closed-form PheSPO exhibit the localization of π-electrons on the indoline fragment, and the calculated energy gap between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) was found to be 3.77 eV. On the contrary, in the case of the metal–PheSPO complex, the open-form merocyanine can facilitate π-electron delocalization throughout the molecule, giving rise to a significant decrease in energy gap for the electronic transition from the HOMO to LUMO, that is, 2.54 eV (488 nm) for Cu2+–PheSPO and 2.34 eV (530 nm) for Fe3+–PheSPO. These DFT calculation results are consistent with the absorption spectra and also confirm the proposed metal ion-induced ring-opening reaction of PheSPO in the presence of Cu2+ and Fe3+.

Analysis of Cu2+ and Fe3+ in Drinking Water

To verify that the PheSPO dual-sensing probe can be employed as a sensing tool in the practical application, it was used to determine the amounts of Cu2+ and Fe3+ in spiked drinking water. The results in Table show that %recovery of Cu2+ analyzed with PheSPO was in the range of 93–97% at the micromolar concentrations. Meanwhile, %recovery of Fe3+ exceeded 100%, which may result from the background concentration of Fe3+ existing in drinking water. Impressively, the results obtained from the PheSPO probe were comparable to those obtained from the standard AAS. Therefore, it is obvious that PheSPO can be practically used as a colorimetric probe for accurate detection of Cu2+ and Fe3+ in drinking water.

Table 1

% Recoveries of Cu2+ and Fe3+ in Drinking Water

		[Cu2+]found (μM)
sample	[Cu2+]added (μM)	PheSPO	% recovery	AAS	% recovery
1	3.00	2.81	93.63	2.88	96.08
2	7.00	6.83	97.55	6.99	99.84

Strip Test for Fe3+ Detection in Rice

The PheSPO test strip coated on a TLC plate was fabricated and used for qualitative detection of Fe3+ in the digested solution of the rice sample. In Figure , the PheSPO test strip shows a distinct color change from pale greenish blue to red when treated with the sample solution. In the case of the acid control, the PheSPO test strip remains unchanged in color. This confirms the colorimetric response of PheSPO to the existence of Fe3+ in rice, in which the actual amount of Fe3+ in the sample solution was 87.62 μM as determined by AAS. Thus, the PheSPO test strip is apparently applicable for qualitative detection of Fe3+ in rice.

Figure 11

Photographs of (a) PheSPO test strip, (b) PheSPO test strip treated with the digested solution of the rice sample, and (c) PheSPO test strip treated with the acid control solution.

Conclusions

In summary, the sensing performance of our spirooxazine derivative, PheSPO, was successfully demonstrated through its applications in drinking water and rice. Among various metal ions, PheSPO showed high selectivity for the detection toward Cu2+ and Fe3+ with distinct color and spectral changes in acetonitrile. The binding mechanism of PheSPO with the targeted metal ions was proposed to be 1:1 stoichiometric complexation and evaluated by means of spectroscopic experiments and DFT calculations. The results showed that the detection limits of the PheSPO probe were 0.94 μM for Cu2+ and 2.01 μM for Fe3+. Moreover, PheSPO was evaluated for its applicability for the analysis of Cu2+ and Fe3+ in spiked drinking water, and its sensing performance was comparable to that of the standard AAS. Additionally, the strip test of PheSPO could also provide a unique colorimetric response when the strip was treated with the digested solution of the rice sample containing Fe3+.