A novel displacement-type colorimetric chemosensor for the detection of Cu²⁺ and GSH in aqueous solution

Abstract

A new and simple colorimetric chemosensor 1 was developed for the sequential detection of Cu²⁺ and glutathione (GSH) in aqueous solution. Receptor 1 detected Cu²⁺ ions by changing its color from colorless to yellow. Based on UV-vis titrations, Job plot, and ESI-mass spectrometry analysis, the sensing mechanism for Cu²⁺ was proposed to be the enhancement of the intramolecular charge transfer band, which was further explained by theoretical calculations. The detection limit of 1 for Cu²⁺ (3.89 μM) was below the World Health Organization (WHO) guideline for drinking water (31.5 μM). Moreover, the resulting 1–Cu²⁺ complex could sequentially sense GSH, showing recovery of 1 from the complex.

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1. Introduction

Selective recognition and detection for transition metal ions have received a great deal of attention in the past years due to their significant importance in chemical, biological, and environmental processes.^1–9 Copper is the third essential transition metal nutrient of the human body after iron and zinc, and plays a variety of fundamental roles in the physiological processes in organisms raging from bacteria to mammals.^10–14 Due to its unique redox-active nature copper serves as an important catalytic cofactor in a variety of enzymes such as superoxide dismutase, cytochrome c oxidase and tyrosinase, and also has importance in controlling tuberculosis.^15–18 Apart from its biological importance copper is the most useful material for making alloys, fertilizers, machine parts, batteries and electrical wires because of its relatively high availability, low cost, malleability, and electrical and thermal conductivity. However, unregulated concentration of copper can catalyze the production of reactive oxygen species (ROS), that can damage lipids, nucleic acids, and proteins. The disturbance in Cu²⁺ ion homeostasis in neuronal cytoplasm may result neurodegenerative disorders like, Alzheimer's diseases,¹⁹ Indian childhood cirrhosis (ICC),²⁰ prion disease,²¹ and Menkes and Wilson diseases.²² Therefore, the World Health Organization (WHO) has set the safe limit of copper in drinking water at 2 ppm (31.5 μM).²³ Many measurement technologies such as atomic absorption spectrometry,²⁴ fluorescence techniques,^25–30 and electrochemical methods³¹ have been developed to sensitively and reliably detect Cu²⁺. However, the expensive equipment, highly trained operators and complicated pre-treatment make them unsuitable for routine monitoring and application. For this reason, the development of colorimetric probe has attracted considerable attention due to its several outstanding advantages such as low cost, simplicity and high sensitivity.^32–35

Among many biological molecules, biothiols such as glutathione (GSH) and cysteine (Cys), are essential for biological systems^36–38 and processes^39,40 and the maintenance of the redox balance of organisms, proteins, and cells.⁴¹ Especially, GSH is the most abundant cellular thiols and plays fundamental roles in antioxidant defense, gene regulation and intracellular signal transduction.^42,43 However, an abnormal level of GSH causes a variety of severe diseases, including Alzheimer's disease, Parkinson's disease and liver disease.⁴⁴ Therefore, the development of selective probes for the detection of biothiols has attracted much attention.^45–47 For example, various specific detection methods have been developed such as Michael addition reaction,^48–50 the cleavage reaction,⁵¹ the cyclization with aldehyde,⁵² metal-complex displacement reaction⁵³ and thiol-halogen nucleophilic substitution reaction.^45,54 Among them, the metal-complex displacement reaction has been attractive due to its excellent advantages of good selectivity and high sensitivity.^32,55 Especially, Cu²⁺ complexes were used to devise chemosensors for detecting thiol-containing amino acid and peptide through the high affinity between Cu²⁺ and thiol group.^56–58 However, there is no report on the metal-complex displacement-based colorimetric chemosensor discriminating GSH from other biothiols.

Herein, we report on the development of a displacement-type sensor, entitled (E)-5-(diethylamino)-2-(((2-(methylthio)phenyl)imino)methyl)phenol, that could sequentially recognize Cu²⁺ and GSH. The sensor 1 showed the colorimetric response toward Cu²⁺ with remarkably high selectivity. In addition, the resulting 1–Cu²⁺ complex could selectively respond to GSH in the presence of other amino acids via naked-eye.

2. Experimental

2.1. Materials and equipment

All the solvents and reagents (analytical and spectroscopic grade) were purchased from Sigma-Aldrich. Absorption spectra were recorded at room temperature using a Perkin Elmer model Lambda 25 UV/Vis spectrometer. ¹H and ¹³C NMR spectra were recorded on a Varian 400 MHz and 100 MHz spectrometer and chemical shifts (δ) were recorded in ppm. Electro spray ionization mass spectra (ESI-MS) were collected on a Thermo Finnigan (San Jose, CA, USA) LCQ™ Advantage MAX quadrupole ion trap instrument by infusing samples directly into the source using a manual method. Spray voltage was set at 4.2 kV, and the capillary temperature was at 80 °C. Elemental analysis for carbon, nitrogen, and hydrogen was carried out using a Flash EA 1112 elemental analyzer (thermo) at the Organic Chemistry Research Center of Sogang University, Korea.

2.2. Synthesis of receptor 1

An ethanolic solution of 2-(methylthio)benzenamine (0.14 g, 1 mmol) was added to 4-(diethylamino)-2-hydroxybenzaldehyde (0.21 g, 1.1 mmol) in absolute ethanol (5 mL). Two drops of HCl were added into the reaction solution, which was stirred for 6 h at room temperature. A yellow precipitate was filtered, washed several times with ethanol and diethyl ether, and dried in vacuum to obtain the pure yellow solid. Yield: 0.23 g (72%) and mp: 150–152 °C. ¹H NMR (400 MHz DMSO-d₆, ppm): δ 13.28 (s, 1H), 8.67 (s, 1H), 7.33 (m, 2H), 7.23 (m, 3H), 6.33 (d, 1H), 6.08 (s, 1H), 3.40 (m, 4H), 2.43 (s, 3H), 1.13 (t, 6H); ¹³C NMR (100 MHz, DMSO-d₆, 25 °C): δ = 178.79, 155.91, 150.11, 148.05, 131.15, 115.81, 109.24, 108.79, 52.15, 51.60, 29.33, 24.33, 23.87, 23.55, 23.30. ESI-MS: m/z [1 + H⁺]⁺ calcd 315.10, found, 315.15 anal. calc. for C₁₈H₂₂N₂OS: C, 68.75; H, 7.05; N, 8.91; found: C, 68.97; H, 7.38; N, 9.33.

2.3. UV-vis titration

For Cu²⁺, 1 (3.1 mg, 0.01 mmol) was dissolved in dimethylformamide (DMF, 1 mL) and 9 μL of this solution (10 mM) was diluted with 2.991 mL of DMF/bis-tris buffer (7/3, 10 mM bis-tris, pH = 7.0) to make the final concentration of 30 μM. Cu(NO₃)₂·2.5H₂O (4.65 mg, 0.02 mmol) was dissolved in DMF (1 mL) and 4.5–22.5 μL of this Cu²⁺ solutions (20 mM) were transferred to the receptor 1 solution (30 μM) prepared above. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

For GSH, 1 (3.1 mg, 0.01 mmol) was dissolved in DMF (1 mL) and 9 μL of this solution (10 mM) was diluted with 2.991 mL of DMF/bis-tris buffer (7/3, 10 mM bis-tris, pH = 7.0) to make the final concentration of 30 μM. 22.5 μL of Cu²⁺ solution (20 mM) was transferred to each receptor solution (30 μM) to give 5 equiv. Then, GSH (6.1 mg, 0.02 mmol) was dissolved in bis-tris (10 mM, 1 mL) and 4.5–31.5 μL of this GSH solutions (20 mM) were transferred to 1–Cu²⁺ solution (30 μM) to give 7 equiv. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

2.4. Job plot measurements

For Cu²⁺, 1 (3.1 mg, 0.01 mmol) was dissolved in DMF (1 mL). 9.0, 8.1, 7.2, 6.3, 5.4, 4.5, 3.6, 2.7, 1.8, 0.9 and 0 μL of the 1 solutions were taken and transferred to vials. Each vial was diluted with 2.982 mL of DMF/bis-tris buffer (7/3, 10 mM bis-tris, pH = 7.0). Cu(NO₃)₂·2.5H₂O (6.1 mg, 0.02 mmol) was dissolved in DMF (1 mL). 0, 0.45, 0.9, 1.35, 1.8, 2.25, 2.7, 3.15, 3.6, 4.05 and 4.5 μL of the Cu(NO₃)₂ solutions were added to each diluted 1 solution. Each vial had a total volume of 3 mL. After reacting them for a few seconds, UV-vis spectra were taken at room temperature.

For GSH, 1 (3.1 mg, 0.01 mmol) was dissolved in DMF (1 mL) and Cu(NO₃)₂·2.5H₂O (4.65 mg, 0.02 mmol) was dissolved in DMF (1 mL). The two solutions were mixed to make 1–Cu²⁺ complex. 18, 16.2, 14.4, 12.6, 10.8, 9.0, 7.2, 5.4, 3.6, 1.8 and 0 μL of the 1–Cu²⁺ complex solution were taken and transferred to vials. Each vial was diluted with 2.982 mL of DMF/bis-tris buffer (7/3, 10 mM bis-tris, pH = 7.0). GSH (6.1 mg, 0.02 mmol) was dissolved in 10 mM bis-tris buffer (1 mL). 0, 0.45, 0.9, 1.35, 1.8, 2.25, 2.7, 3.15, 3.6, 4.05 and 4.5 μL of the GSH solutions were added to each diluted 1–Cu²⁺ solution. Each vial had a total volume of 3 mL. After reacting them for a few seconds, UV-vis spectra were taken at room temperature.

2.5. Competition experiments

For Cu²⁺, 1 (3.1 mg, 0.01 mmol) was dissolved in DMF (1 mL) and 9 μL of this solution (10 mM) was diluted with 2.991 mL of DMF/bis-tris buffer (7/3, v/v) to make the final concentration of 30 μM. MNO₃ (M = Na, K, Ag, 0.02 mmol) or M(NO₃)₂ (M = Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, Mg, Ca, Pb, 0.02 mmol) or M(NO₃)₃ (M = Fe, Cr, Al, Ga, In, 0.02 mmol) were separately dissolved in DMF (1 mL). 22.5 μL of each metal solution (20 mM) was taken and added to 3 mL of the solution of receptor 1 (30 μM) to give 5 equiv. of metal ions. Then, 22.5 μL of Cu²⁺ solution (20 mM) was added into the mixed solution of each metal ion and 1 to make 5 equiv. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

For GSH, 1 (3.1 mg, 0.01 mmol) was dissolved in DMF (1 mL) and 9 μL of this solution (10 mM) was diluted with 2.991 mL of DMF/bis-tris buffer (7/3, v/v) to make the final concentration of 30 μM. Cu(NO₃)₂·2.5H₂O (4.65 mg, 0.02 mmol) was dissolved in DMF (1 mL). 22.5 μL of this Cu²⁺ solution (20 mM) was transferred to the 1 solution (30 μM) to make copper complex. Then, various amino acids and peptide such as Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Val and glutathione (GSH) (0.02 mmol) were separately dissolved in bis-tris buffer (10 mM, 1 mL). 31.5 mL of each amino acid and peptide solution (20 mM) was taken and added into each copper complex solution prepared above to make 7 equiv. Then, 31.5 μL of the GSH solution (20 mM) was added into the mixed solution of each amino acid or peptide and copper complex to make 7 equiv. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

2.6. ¹H NMR titration

Four NMR tubes of 1 (3.1 mg, 0.01 mmol) dissolved in DMF-d₇ (0.5 mL) were prepared, and four different equivalents (0, 0.25, 0.5, and 1.0 equiv.) of copper nitrate were added into the solutions separately. After shaking them for a few seconds, their ¹H NMR spectra were taken.

2.7. pH effect test

For Cu²⁺, a series of solutions with pH values ranging from 2 to 12 was prepared by mixing sodium hydroxide solution and hydrochloric acid. After the solution with a desired pH was achieved, receptor 1 (3.1 mg, 0.01 mmol) was dissolved in DMF (1 mL), and then 9 μL of the receptor (10 mM) was diluted with 2.991 mL of DMF/bis-tris buffer (7/3, v/v) to make the final concentration of 30 μM. Cu(NO₃)₂·2.5H₂O (6.1 mg, 0.02 mmol) was dissolved in bis-tris (1 mL). 22.5 μL of the Cu²⁺ solution (20 mM) was transferred to each receptor solution (30 μM) prepared above. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

For GSH, a series of solutions with pH values ranging from 2 to 12 was prepared by mixing sodium hydroxide solution and hydrochloric acid. After the solution with a desired pH was achieved, 9 μL of the 1 solution (30 μM) and 22.5 μL of the Cu(NO₃) solution (20 mM) were dissolved in DMF/bis-tris buffer (7/3, v/v, pH 2–12), respectively. Then, 31.5 μL of the GSH solution (20 mM) was transferred to 1–Cu²⁺ complex solution prepared above. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

2.8. Determination of Cu²⁺ in real samples

UV-vis spectral measurements of water samples containing Cu²⁺ were carried by adding 9 μL solution of the sensor 1 (10 mM) and 0.18 mL of 50 mM bis-tris buffer stock solution to 2.811 mL sample solutions. After mixing them for a few seconds, UV-vis spectra were taken at room temperature.

2.9. Theoretical calculations

All DFT/TDDFT calculations based on the hybrid exchange correlation functional B3LYP^59,60 were carried out using Gaussian 03 program.⁶¹ The 6-31G** basis set^62,63 was used for the main group elements, whereas the Lanl2DZ effective core potential (ECP)^64,65 was employed for Cu. In vibrational frequency calculations, there was no imaginary frequency for the optimized geometries of 1 and 1–Cu²⁺, suggesting that these geometries represented local minima. For all calculations, the solvent effect of DMF was considered by using the Cossi and Barone's CPCM (conductor-like polarizable continuum model).^66,67 To investigate the electronic properties of singlet excited states, time-dependent DFT (TDDFT) was performed in the ground state geometries of 1 and 1–Cu²⁺. Twenty lowest singlet states were calculated and analyzed. The GaussSum 2.1 (ref. 68) was used to calculate the contributions of molecular orbitals in electronic transitions.

3. Results and discussion

Receptor 1 was obtained by the combination of 2-(methylthio)benzenamine and 4-(diethylamino)-2-hydroxybenzaldehyde with 72% yield in ethanol (Scheme 1), and characterized by ¹H NMR and ¹³C NMR, ESI-mass spectrometry, and elemental analysis.

Scheme 1 Synthetic procedure of 1.

3.1. Colorimetric and spectral response of 1 toward Cu²⁺

The colorimetric sensing abilities of 1 were primarily investigated in DMF/bis-tris buffer (7/3, v/v; 10 mM bis-tris, pH = 7.0) upon addition of various metal ions (Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Ga³⁺, In³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺) as their nitrate salts. Upon the addition of 5 equiv. of each cation, 1 showed almost no change in absorption peak in the presence of Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Fe²⁺, Fe³⁺, Ga³⁺, In³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺, while only the presence of Cu²⁺ showed a distinct spectral change (Fig. 1a) and a color change from colorless to yellow (Fig. 1b). Hg²⁺ and Ag⁺ ions precipitated out in the presence of sensor 1. These results indicated that receptor 1 can serve as a potential candidate of a “naked-eye” chemosensor for Cu²⁺ in aqueous solution. Moreover, to check the anion effect of various copper salts such as Cu(ClO₄)₂, CuCl₂, and Cu(OAc)₂, the sensing abilities of 1 were examined with them. They showed the same results as did the copper nitrate (Fig. S1†).

Fig. 1 (a) Absorption spectral changes of 1 (30 μM) upon the addition of 5 equiv. of various metal ions in DMF/bis-tris buffer (7/3; v/v. 10 mM bis-tris, pH = 7.0). (b) The color changes of 1 (30 μM) upon the addition of 5 equiv. of various metal ions.

The binding property of 1 with Cu²⁺ was studied by UV-vis titration experiment (Fig. 2). Upon addition of Cu²⁺ into 1, the absorption band at 392 nm decreased and a new absorption band at 416 nm steadily increased. An isosbestic point was observed at 401 nm, suggesting the formation of only one UV-vis active species. This bathochromic shift of the absorption band led us to propose the change of intramolecular charge transfer (ICT) band through the binding between 1 and Cu²⁺. The donor chromophore –NEt₂ and the acceptor imine might create a ‘push–pull’ interaction which causes the enhancement of ICT (Scheme 2).⁶⁹ As a result, a new absorption band at 416 nm appeared, resulting in the formation of a yellow color.

Fig. 2 Absorption spectral changes of 1 (30 μM) after addition of increasing amounts of Cu²⁺ in DMF/bis-tris buffer (7/3, v/v) at room temperature. Inset: absorption at 416 nm versus the number of 8 equiv. of Cu²⁺ added.

Scheme 2 Proposed sensing mechanism of Cu²⁺ by 1 and binding mode of 1–Cu²⁺ complex.

The Job plot analysis for the binding between 1 and Cu²⁺ exhibited a 1 : 1 stoichiometry (Fig. S2†).⁷⁰ Moreover, a positive-ion ESI-mass spectrum provided an additional evidence for the formation of a 1 : 1 complex between 1 and Cu²⁺ (Fig. 3). A peak at m/z 376.00 was assigned to [1 − H⁺ + Cu²⁺]⁺ [calcd 376.07]. Based on the Job plot and the ESI-mass spectrometry analysis, we propose the structure of a 1 : 1 complex of 1 and Cu²⁺, as shown in Scheme 2. The association constant was calculated to be 1.0 × 10⁴ M⁻¹ from a Benesi–Hildebrand plot (Fig. S3†).⁷¹ The obtained value was within the range of those (10³ to 10¹²) reported for Cu²⁺ chemosensors.^72–76 The detection limit (3σ/k) of receptor 1 as a colorimetric sensor for the analysis of Cu²⁺ ions was found to be 3.89 × 10⁻⁶ M (Fig. S4†).⁷⁷ Importantly, the value (3.89 μM) for Cu²⁺ is much below the World Health Organization (WHO) guideline (31.5 μM) in the drinking water,²³ which means that 1 could be a practical chemosensor for the detection of copper in the drinking water. For comparison, some examples of detection limits previously reported for Cu²⁺ chemosensors are shown in Table. S1.†

Fig. 3 Positive-ion electrospray ionization mass spectrum of 1 (10 μM) upon addition of 1 equiv. of Cu²⁺.

To further check the practical applicability of 1 as a copper ion chemosensor, competitive experiments have been conducted by the addition of copper ions (5 equiv.) to the solution of 1 containing interfering cations viz. Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Fe²⁺, Fe³⁺, Ga³⁺, In³⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺, and Zn²⁺ (5 equiv., Fig. 4). The presence of interfering cations did not result in any significant change in the UV-vis as achieved by the addition of only Cu²⁺ to the solution of 1. However, in the presence of 5-time excess (25 equiv.) of interfering cations, Ga³⁺ and Mn²⁺ inhibited some interference with the sensing of Cu²⁺ (Fig. S5†). Thus, receptor 1 could be used as an excellent selective colorimetric sensor for Cu²⁺ in the presence of most competing metal ions.

Fig. 4 (a) Absorption spectral changes of competitive selectivity of 1 (30 μM) toward Cu²⁺ (5 equiv.) in the presence of other metal ions (5 equiv.) in DMF/bis-tris buffer (7/3, v/v). (b) The color changes of competitive selectivity of 1 (30 μM) toward Cu²⁺ (5 equiv.) in the presence of other metal ions (5 equiv.).

The ¹H NMR titration experiments were studied to further examine the binding mode 1 and Cu²⁺ (Fig. S6†). Upon addition of 1.0 equiv. of Cu²⁺, the phenolic–OH signal (H₁₂) at 13.4 ppm completely disappeared and the aromatic protons showed small or large shifts to downfield. These results indicate that the oxygen atom of the phenol group, the nitrogen atom of the imine moiety, and the sulfur atom might coordinate to Cu²⁺ (Scheme 2).

For environmental applications, the pH dependence of the 1–Cu²⁺ complex was investigated. Over the pH range tested, the absorbance intensity of the complex displayed a strong pH dependence (Fig. S7†). The intense and stable absorption intensity of 1–Cu²⁺ complex in the pH range of 7.0–10.0 warrants its application in monitoring Cu²⁺ by naked-eye without its being affected by changes in physiological pH values.

We conducted the construction of a calibration curve for quantitative analysis of Cu²⁺ by 1 (Fig. S8†). 1 exhibited a good linear relationship between the absorbance of 1 and the Cu²⁺ concentration (0.0–48.0 μM) with a correlation coefficient of R² = 0.9921 (n = 3). Based on the calibration curve, the chemosensor 1 was applied for the determination of Cu²⁺ in the tap and artificial polluted water samples (Table 1). The satisfactory recoveries and R.S.D. values were obtained.

Table 1 Determination of Cu²⁺ in water samples^a

Sample	Cu(II) added (μmol L^−1)	Cu(II) found (μmol L^−1)	Recovery (%)	R.S.D (n = 3) (%)
a Condition: [1] = 30 μmol L^−1 in DMF/bis-tris buffer (7/3, v/v).b Prepared by deionized water 12.0 μmol L^−1 Cu(II), 9.0 μmol L^−1 Zn(II), Cd(II), Pb(II), Hg(II) and 24 μmol L^−1 Na(I), K(I), Ca(II), Mg(II).
Tap water	0.00	0.00	—	—
	6.00	6.13	102.2	0.2
Artificial polluted water^b	0.00	11.56	96.3	0.1
	9.00	20.46	98.9	0.3

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3.2. Theoretical calculations

To understand spectral behaviors and chemical transformation of 1 and 1–Cu²⁺ complex, theoretical calculations were performed in parallel to the experimental studies. Based on Job plot and ESI-mass spectrometry analysis, all theoretical calculations were performed with the 1 : 1 stoichiometry for the 1–Cu²⁺ complex. Under these conditions, 1 and 1–Cu²⁺ species were optimized to obtain their energy-minimized molecular geometries (S = 1/2, DFT/B3LYP/main group atom: 6-31G** and Cu: Lanl2DZ/ECP) (Fig. 5). The energy-minimized structure of 1 showed a distorted structure with the dihedral angle of 1C, 2C, 3N, 4C = −33.917° (Fig. 5a). 1–Cu²⁺ complex has a planar structure with the dihedral angle of 1C, 2C, 3N, 4C = −11.014°, and it showed that 1 coordinated with Cu²⁺ via the N atom in the Schiff-base, the O atom in the phenol group and the S atom in the –SCH₃ group. We also calculated the singlet excited states of 1 and 1–Cu²⁺ complex using the TD-DFT (time dependent-density functional theory) methods. In case of 1, the main molecular orbital (MO) contribution of the first lowest excited state was determined for HOMO → LUMO transition (375.23 nm, Fig. S9†), which indicated intramolecular charge transfer (ICT) band. For 1–Cu²⁺ complex, radiative-allowed transition was determined for the thirteenth lowest excited state. The MO contributions of the thirteenth excited state were mainly determined for HOMO (α) → LUMO (α) and HOMO (β) → LUMO+1 (β) transitions with predominant ICT (389.95 nm, Fig. S10†). These results are well consistent with the experimental absorption wavelengths. Also, it showed no obvious change in the electronic transitions between 1 and 1–Cu²⁺ complex. Only, the energy gap decreased upon chelating of 1 with Cu²⁺ (Fig. S11†). Thus, the chelation of Cu²⁺ with 1 induced the enhancement of ICT transitions (375.23 to 389.95 nm), which caused the hypochromic shift with color change from colorless to yellow.

Fig. 5 Energy-minimized structures of (a) 1 and (b) 1–Cu²⁺ complex from B3LYP level.

3.3. Colorimetric and spectral response of 1–Cu²⁺ complex toward GSH

It has been well known that the thiol-containing amino acid and peptide have a high affinity toward Cu²⁺ ions.^56–58 This propensity led us to examine the absorbance variation of 1–Cu²⁺ with addition of 20 different amino acids and peptide, Cys, Gly, Ile, Ala, Met, Val, Ser, Thr, Phe, Asp, Gln, Asn, Leu, Arg, Pro, Lys, His, Trp, Glu, and glutathione (GSH) in bis-tris DMF/buffer (7 : 3, v/v). Upon the addition of 7 equiv. of each amino acid and peptide to 1–Cu²⁺, only GSH showed a color change from yellow to colorless and caused an obvious spectral change (Fig. 6). The final UV-vis spectrum was almost identical to the original absorption spectrum of 1. The absorption recovery indicates that 1 might be released from the 1–Cu²⁺ complex, resulting in the chelation of GSH with copper (Scheme 3). Therefore, these results demonstrated that 1–Cu²⁺ complex could be a selective chemosensor for GSH over other sulfur-containing amino acid, such as Met and Cys via naked eye. We assume that the preference of GSH by 1 over the sulfur-containing amino acids might be due to the presence of the secondary amine in GSH, while Met and Cys do not contain it. Importantly, the 1–Cu²⁺ complex is the first colorimetric chemosensor for GSH by using copper complex as a receptor, to the best of our knowledge.^53,57,78,79

Fig. 6 (a) Absorption spectral changes of 1–Cu²⁺ complex (30 μM) upon the addition of 7 equiv. of various amino acids and peptide in DMF/bis-tris buffer (7/3; v/v. 10 mM bis-tris, pH = 7.0). (b) The color changes of 1–Cu²⁺ complex (30 μM) upon the addition of 7 equiv. of various amino acids and peptide.

Scheme 3 Proposed sensing mechanism of GSH by 1–Cu²⁺ complex.

We conducted the UV-vis titration experiments to understand the binding properties of 1–Cu²⁺ with GSH. Upon incremental addition of GSH to the solution of 1–Cu²⁺, the absorption band at 416 nm significantly decreased, and a new band at 392 nm gradually reached a maximum at 7 equiv. of GSH (Fig. 7). Two isosbestic points were also observed at 286 nm and 401 nm, indicating that only one species was generated from the interaction of 1–Cu²⁺ with GSH. The binding stoichiometry of GSH and 1–Cu²⁺ was determined by Job plot analysis, which revealed a 1 : 1 stoichiometric ratio (Fig. S12†).⁷⁰ In addition, we conducted an ESI-mass spectrometry analysis to confirm the demetallation of 1–Cu²⁺ complex by GSH (Fig. S13†). When GSH was added into 1–Cu²⁺ solution, the positive ion mass spectrum showed that a peak at m/z 315.10 was assigned to [1 + H⁺]⁺ [calcd 315.15], indicating recovery of 1 from 1–Cu²⁺ complex by GSH. Based on UV-vis titrations, Job plot and ESI-mass spectrometry analysis, we proposed the sensing mechanism of the 1–Cu²⁺ complex toward GSH (Scheme 3). The association constant was calculated to be 6.35 × 10³ M⁻¹ from a Benesi–Hildebrand plot (Fig. S14†).⁷¹ The detection limit (3σ/k) for GSH was found to be 5.86 μM (Fig. S15†).⁷⁷

Fig. 7 Absorption spectral changes of 1–Cu²⁺ complex (30 μM) after addition of increasing amounts of GSH in DMF/bis-tris buffer (7/3, v/v) at room temperature. Inset: absorption at 416 nm versus the number of 7.5 equiv. of GSH added.

To examine the practical applicability of 1–Cu²⁺ complex as a GSH-selective receptor, competitive experiments were carried out in the presence of GSH (7 equiv.) with competing amino acid (7 equiv.) (Fig. 8). There was no interference for the detection of GSH by 1–Cu²⁺. These results indicate that the detection of GSH by 1–Cu²⁺ was not disturbed from various amino acids, especially sulfur-containing substances such as Cys and Met. In order to investigate the pH dependence of 1–Cu²⁺ toward GSH, the pH effect test was conducted in a wide range of pH. The optimal range for the colorimetric sensing of GSH by 1–Cu²⁺ was turned out to be between pH 6 and pH 10 (Fig. S16†).

Fig. 8 (a) Absorption spectral changes of competitive selectivity of 1–Cu²⁺ (30 μM) toward GSH (7 equiv.) in the presence of other amino acids and peptide (7 equiv.) in DMF/bis-tris buffer (7/3, v/v). (b) The color changes of competitive selectivity of 1–Cu²⁺ (30 μM) toward GSH (7 equiv.) in the presence of other amino acids and peptide (7 equiv.).

4. Conclusion

We have developed a new selective and sensitive chemosensor 1 for the sequential detection of Cu²⁺ and GSH via naked-eye. 1 showed selectivity toward Cu²⁺ in a 1 : 1 stoichiometric manner, which induces an obvious color change from colorless to yellow, accompanied with a remarkable bathochromic shift in UV spectrum. Based on the theoretical calculations, the sensing mechanism of 1 toward Cu²⁺ was explained. Also, the detection limit (3.89 μM) for Cu²⁺ is much lower than the WHO acceptable limit (31.5 μM) in drinking water. In addition, 1 could successfully monitor Cu²⁺ in real water samples, which means that 1 could be a practical probe. Moreover, 1–Cu²⁺ complex could selectively detect GSH without any inhibition in the presence of other various amino acids by using the property of the copper–sulfur affinity. Most importantly, 1–Cu²⁺ complex was the first colorimetric chemosensor that could selectively detect GSH via naked-eye using the copper complex. We believe that the chemosensor 1 could be a guidance to the development of a new type for the colorimetric sequential recognition of Cu²⁺ and GSH.

Acknowledgements

Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A2A1A11051794 and NRF-2015R1A2A2A09001301) are gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures and additional experimental data. See DOI: 10.1039/c6ra12368f

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