English

• Cu²⁺ is an essential transition metal ion in human body, which plays critical roles in various physiological processes and normal development of tissues^{[1, 2]}because of its redox pro-perties^[3]. However, the imbalance of Cu²⁺ in cellular homeostasis, both excess and deficiency, can cause serious neurodegenerative diseases. For example, epileptic seizures^[4], Parkinson's, Alzheimer's^[5], Wilson's and Menke's disease^{[6, 7]} are induced by the excess intake of Cu²⁺ ion^[8], whereas Men-kessyndrome is induced by Cu²⁺ deficiency. Further-more, Cu²⁺ is an important environmental pollutant because of its widespread application^{[9, 10]}. Therefore, it is very important to detect Cu²⁺ both in biological and environmental systems.

  Unlike traditional analytical assay, fluorescent probes of specific ions have many advantages such as high sensitivity, excellent selectivity, instantaneous response, easy operation, low cost, low toxicity in vivo, and cumulative signaling behavior^[11~14]. Recent studies have shown that acylhydrazone group is a fascinating metal ion recognition site in chemical sensors because they are rich in nitrogen and oxygen atoms^{[15, 16]} and can selectively bind metal cations. Additionally, some acylamide group will change to the enol-form when forming a complex with metal ions, which could conjugate small π systems to a bigger one^[17].

  In this paper, probes 1 and 2 with acylhy-drazones as the copper ion detection unit and coumarins as the fluorescent signal group were synthesized (Scheme 1). They could selectively detect Cu²⁺ among various ions through a turn-off fluorescence signaling mechanism, and the detection process is accompanied by a significant color change, which can be directly observed by the naked eyes.

  Scheme 1

  

  Scheme 1.  探针1和2的合成路线

  Scheme 1.  Synthesis routes for probes 1 and 2

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  Scheme 2

  

  Scheme 2.  探针1、2对Cu²⁺的识别机理

  Scheme 2.  Proposed binding mode of probes 1 and 2 with Cu²⁺

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  1.   Experimental

  1.1   Materials and instruments

  Commercially available analytical grade solvents and reagents were used directly in all reactions unless otherwise stated. 1, 2-Dichloroethane was used after purification by standard methods.

  ¹H NMR spectra were obtained on Varian Mercury Plus 400MHz and Agilent Technologies DDZ 600MHz Spectrometer with tetramethylsilane as the internal standard. Absorption spectra and photoluminescence spectra were recorded by a Shimadzu UV-2550 spectrometer and a Perkin-Elmer LS-55 spectrometer, respectively. MS was obtained from a Thermo Scientific Orbitrap Elite mass spectrometer.

  1.2   Syntheses and characterizations

  1.2.1   Preparation of 3-formyl-7-N, N-diethylam-inocoumarin (FDC)

  Under N₂, anhydrous DMF (13.30 mL, 172.5 mmol) was dropped into POCl₃(3.16 mL, 34.5 mmol) with stirring for 6 h in an ice bath. The solution of 7-N, N-diethylaminocoumarin (3.00 g, 13.8 mmol) in anhydrous 1, 2-dichloroethane was added to the above solution and the mixture was stirred at 60 ℃ for 12 h. Subsequently, the mixture was poured into ice water and neutralized with NaOH solution (20%) to pH 7~8. The formed precipitate was filtered off and washed three times with water. The residue was chromatographed on silica, eluting with petroleum ether /CH₂Cl₂ (2:1, v/v) to give the product as an orange solid. Yield: 76%.¹H NMR (400 MHz, CDCl₃) δ: 10.10 (s, 1H, CHO), 8.25(s, 1H, coumarin H), 7.40~7.42 (s, 1H, coumarin H), 6.63~6.65(d, 1H, coumarin H), 6.49 (s, 1H, coumarin H), 3.45~3.51(q, J=21.3Hz, 4H, CH₂), 0.96~0.97(t, J=3.2Hz, 6H, CH₃).

  1.2.2   Preparation of (N¹′E, N³′E)-N¹′, N³′-bis((7-N, N-diethylaminocoumarin-3-yl)methylene) malonohydrazide (probe 1)

  Under N₂, a solution of malonohydrazide (0.2 g, 1.5 mmol) in 30 mL DMSO was added to a solution of synthesized FDC (1.1 g, 4.5 mmol) in 50 mL ethanol, and then the mixture was heated to 80℃ and stirred for 4 h. After cooling to room temperature, the mixture was filtered, and the solid was washed with methanol (3×10 mL) to obtain an orange solid with a yield of 79%. m.p.: >250 ℃. MS (ESI) m/z: 587.26 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ: 11.54(s, 2H, N-H), 8.36~8.24 (m, 2H, C-H), 8.15 (s, 2H, N=C-H), 8.06(s, 2H, coumarin H), 7.40 (d, 2H, coumarin H), 6.70 (d, 2H, coumarin H), 6.55~6.50 (m, 2H, coumarin H), 3.43 (m, 8H, CH₂), 1.13~1.10 (t, 12 H, CH₃). ¹³C NMR(101MHz, DMSO-d₆) δ: 169.73, 161.15, 156.85, 151.64, 138.35, 137.77, 130.465, 113.10, 110.09, 108.05, 96.96, 44.67, 29.55, 12.90.

  1.2.3   Preparation of (N¹′E, N⁴′E)-N¹′, N⁴′-bis((7-N, N-diethylaminocoumarin-3-yl)methylene) succinohydrazide (probe 2)

  Under N₂, a solution of succinohydrazide (0.3 g, 2.0 mmol) in 20 mL DMSO was added to a solution of FDC (1.0 g, 4.0 mmol) in 50 mL methanol, and then the mixture was heated to 70 ℃ and stirred for 24 h. After cooling to room temperature, the mixture was filtered, and the solid was washed with methanol (3×10 mL) to obtain an orange solid with a yield of 79%. m.p.: >250 ℃. MS (ESI) m/z: 601.28 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ: 11.3(s, 2H, N-H), 8.31~8.22 (m, 4H, CH₂), 8.21~8.17 (m, 2H, N=C-H), 8.04~8.03 (d, 2H, coumarin H), 7.64~7.57 (m, 2H, coumarin H), 7.56~7.52 (m, 2H, coumarin H), 6.75~6.71 (d, 2H, coumarin H), 3.47~3.44 (m, 8H, CH₂), 1.14~1.12 (t, 12 H, CH₃). ¹³C NMR(101MHz, DMSO-d₆)δ: 173.79, 169.23, 161.19, 156.79, 151.58, 140.60, 138.77, 138.101, 137.77, 130.77, 113.28, 110.08, 108.50, 96.72, 44.58, 29.25, 27.98, 27.19, 12.83.

  2.   Results and Discussion

  2.1   Selectivity and interference study of probes 1 and 2 to metal ions

  To detect the selectivity of probes for metal ions, the fluorescence spectra of probes after addition of various metal species, such as Ag⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Cd²⁺, Hg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Pb²⁺, Zn²⁺, Al³⁺ and Fe³⁺, were recorded under the same conditions. Probes 1 and 2 in DMSO/H₂O (v/v, 9/1) solution showed yellow-green fluorescence under a UV-lamp. After adding Cu²⁺(1.0 eq) to the solution, the fluorescence of probe 1 is quenched immediately, while other metal ions can slightly increase the fluorescence of probe 1 (Fig. 1a). Both Ni²⁺ and Cu²⁺ can quench the fluorescence emission of probe 2, but the quenching effect of Cu²⁺ is much stronger than that of Ni²⁺ (Fig. 2a). In addition, the remarkable change in the color of the probe solution could be observed with the naked-eye under UV lamp (inset of Fig. 1a and 2a).

  Figure 1

  

  图 1.  (a)探针1对不同金属离子的响应情况，插图为探针1对Cu²⁺(1eq)的荧光响应; (b)探针1的离子竞争实验

  (溶剂：DMSO/H₂O (v/v, 9/1)，浓度：1.0×10^-6 mol·L^-1，激发波长:445nm, 激发狭缝宽度:5.0nm, 发射狭缝宽度:10.0nm, 室温)

  Figure 1.  (a) Fluorescence spectra of 1 in the absence and presence of common metal ions. Inset: Photograph of 1 in the absence and presence of Cu²⁺ (1 eq); (b) Fluorescence response of 1 towards Cu²⁺ in the presence of other co-existed metal ions (1 eq)

  (Solvent: DMSO/H₂O (v/v, 9/1), c(probe 1):1.0×10^-6 mol·L^-1, λ_Ex: 445nm, Ex-slit: 5.0nm, Em-slit:10.0nm, T=room temperature)

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  Figure 2

  

  图 2.  (a)探针2对不同金属离子的响应情况，插图为探针2对Cu²⁺(1eq)的荧光响应; (b)探针2的离子竞争实验

  (溶剂：DMSO/H₂O (v/v, 9/1)，浓度：1.0×10^-6mol·L^-1，激发波长:445nm, 激发狭缝宽度:10.0nm, 发射狭缝宽度:20.0nm, 室温)

  Figure 2.  (a) Fluorescence spectra of 2 in the absence and presence of common metal ions. Inset: Photograph of 2 in the absence and presence of Cu²⁺ (1 eq); (b) Fluorescence response of 2 towards Cu²⁺ in the presence of other co-existed metal ions (1 eq)

  (Solvent: DMSO/H₂O (v/v, 9/1), c(probe 1):1.0×10^-6 mol·L^-1, λ_ex:445nm, Ex-slit:10.0nm, Em-slit: 20.0nm, T=room temperature)

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  Furthermore, fluorescence experiments of probes toward Cu²⁺ in the presence of other competitive metal ions were performed to study the interference immunity of probes in the sensing process. From Fig. 2a and Fig. 2b, it can be seen that the coexistence of equal amount of other cations did not bring out any notable interference on the Cu²⁺ recognition.

  2.2   Sensitivity of probes 1 and 2 to Cu²⁺

  To determine the interaction mode of Cu²⁺ with probes, absorption titration was employed to monitor the changes in absorbance upon increasing the Cu²⁺ concentration. Upon the addition of Cu²⁺, the absorption band of each probe showed obvious bathochromic shift and an increase in absorption intensities, which indicates the interactions between probes and Cu²⁺ (Fig. 3). The λ_max of probes 1 and 2 exhibited a bathochromic shift from 445 to 479 nm and 451 to 475 nm, respectively (Fig. 3a and 3b).

  Figure 3

  

  图 3.  (a)探针1和(b)探针2对Cu²⁺的紫外吸收滴定光谱[DMSO/H₂O (v/v, 9/1), c:1.0×10^-6 mol·L^-1)]

  Figure 3.  UV-Vis spectra of (a) probe 1 and (b) probe 2 in DMSO/H₂O (v/v, 9/1, c:1.0×10^-6 mol·L^-1) upon addition increasing amount of Cu²⁺

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  The quantitative nature of the sensing of Cu²⁺ by probes was elucidated by fluorescence titration as described in Fig. 4. With the increase of Cu²⁺ concentration, the fluorescence emission peaks at 529 nm of probes 1 (Fig. 4a) and 2 (Fig. 4b) decreased significantly, and the fluorescence was completely quenched when 1.0 eq Cu²⁺ was added.

  Figure 4

  

  图 4.  (a)探针1和(b)探针2对Cu²⁺(0~1.0eq)的荧光滴定光谱[DMSO/H₂O (v/v, 9/1), c:1.0×10^-6 mol·L^-1)]

  Figure 4.  Fluorescence spectra of (a) probe 1 and (b) probe 2 in DMSO/H₂O (v/v, 9/1, c: 1.0×10^-6 mol·L^-1) responding to various concentrations of Cu²⁺ (0~1.0 eq)

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  2.3   Reversibility

  To examine the reversibility of the binding of the probe to Cu²⁺ in aqueous solution, EDTA was added to the probe 1-Cu²⁺ system. As shown in Fig. 5, the fluorescence signal upon the addition of Cu²⁺ was entirely reversed with the addition of EDTA. These results indicated that probe 1 was a reversible sensor for Cu²⁺. Probe 2 has the similar reversibility.

  Figure 5

  

  图 5.  探针1加入Cu²⁺前后及1-Cu²⁺体系中加入EDTA后的荧光光谱

  Figure 5.  Fluorescence intensity of probe 1 before and after adding Cu²⁺ and that after treatment with EDTA (EDTA: Cu²⁺ 1 :1 M ratio)

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  2.4   Sensing mechanism

  To gain insight into the sensing mechanism of probe towards Cu²⁺, Job's plot experiments were conducted by using fluorescence emission intensity at 529 nm, and the molar ratio of Cu²⁺ in probe 1 + Cu²⁺ complex varied from 0 to 1.0. As illustrated in Fig. 6a, the fluorescence intensity at 529 nm decreased to the minimum when the molar ratio of Cu²⁺ in complex was 0.668, confirming that the stoichiometric ratio of the binding of probe 1 to Cu²⁺ in DMSO/H₂O (v/v, 9/1) solution is 1:2. The stoichiometric ratio of the binding of probe 2 to Cu²⁺ is also 1:2 (see Fig. 6b).

  Figure 6

  

  图 6.  (a)探针1和(b)探针2与Cu²⁺的Job’s曲线

  Figure 6.  Job's for the determination of the stoichiometry of (a) probe 1 and (b) probe 2 with Cu²⁺ in the complexes

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  The ¹H NMR titration spectra (Fig. 7) of probes 1 and 2 in the absence and presence of varying stoichiometry of CuCl₂·2H₂O in DMSO-d₆ solution were carried out to further validate the binding behavior of probe with Cu²⁺. Upon addition of 2.0 eq Cu²⁺, the signal at δ 11.5 (N-H) in probe 1 disappeared, the signal (C-H) in acylhydrazone group shifted from δ 8.15 to δ 8.30 and the hydrogen atoms in coumarins showed a significant downfield shift (Fig. 7a), which confirmed that probe 1 may bind to Cu²⁺ in the enol-form, and in this way the H atom moves from the N atom to the O atom. The NMR titration test of probe 2 has similar results (Fig. 7b).

  Figure 7

  

  图 7.  在探针1(a)和探针2(b)与Cu²⁺的核磁滴定图谱

  Figure 7.  The ¹H NMR titration spectra of (a) probe 1 and (b) probe 2 in the absence and presence of varying stoichiometry Cu²⁺ in DMSO-d₆ solution

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  From the above data, the binding mode of probes 1 and 2 with Cu²⁺ in Fig. 8 is deduced.

  2.5   The limit of detection (LOD) deter-mination of probes 1 and 2 toward Cu²⁺

  The LOD is very important to detect the analyte at low concentrations for practical purposes, which was calculated using the equation 3σ/κ^{[18, 19]}. Fig. 9 showed that probes 1 and 2 can be applied to detect Cu²⁺ at an extremely low concentration level of 10^-9 mol/L, which may fully meet the detection needs.

  Figure 9

  

  图 9.  探针1(a)和探针2(b)对Cu²⁺检测限的测定

  Figure 9.  Fluorescence detection limit of (a) probes 1 and (b) probe 2

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  3.   Conclusions

  In this work, we have developed two acylhy-drazone based fluorescent probes for Cu²⁺. These probes display highly selective and sensitive recog-nition for Cu²⁺ over other metal ions through the "turn-off" effect of fluorescence intensity in DMSO/H₂O (v/v, 9/1) solution. The fluorogenic response is based on cation-mediated reductive photo induced electron transfer (PET). These probes interact with Cu²⁺ through 1:2 binding ratio with detection limit of 10^-9 mol/L. Therefore, the probes should potentially be used to monitor Cu²⁺ in environmental and biological systems.

  
  
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• 

  Scheme 1  Synthesis routes for probes 1 and 2

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  Scheme 2  Proposed binding mode of probes 1 and 2 with Cu²⁺

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  Figure 1  (a) Fluorescence spectra of 1 in the absence and presence of common metal ions. Inset: Photograph of 1 in the absence and presence of Cu²⁺ (1 eq); (b) Fluorescence response of 1 towards Cu²⁺ in the presence of other co-existed metal ions (1 eq)

  (溶剂：DMSO/H₂O (v/v, 9/1)，浓度：1.0×10^-6 mol·L^-1，激发波长:445nm, 激发狭缝宽度:5.0nm, 发射狭缝宽度:10.0nm, 室温)

  (Solvent: DMSO/H₂O (v/v, 9/1), c(probe 1):1.0×10^-6 mol·L^-1, λ_Ex: 445nm, Ex-slit: 5.0nm, Em-slit:10.0nm, T=room temperature)

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  Figure 2  (a) Fluorescence spectra of 2 in the absence and presence of common metal ions. Inset: Photograph of 2 in the absence and presence of Cu²⁺ (1 eq); (b) Fluorescence response of 2 towards Cu²⁺ in the presence of other co-existed metal ions (1 eq)

  (溶剂：DMSO/H₂O (v/v, 9/1)，浓度：1.0×10^-6mol·L^-1，激发波长:445nm, 激发狭缝宽度:10.0nm, 发射狭缝宽度:20.0nm, 室温)

  (Solvent: DMSO/H₂O (v/v, 9/1), c(probe 1):1.0×10^-6 mol·L^-1, λ_ex:445nm, Ex-slit:10.0nm, Em-slit: 20.0nm, T=room temperature)

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  Figure 3  UV-Vis spectra of (a) probe 1 and (b) probe 2 in DMSO/H₂O (v/v, 9/1, c:1.0×10^-6 mol·L^-1) upon addition increasing amount of Cu²⁺

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  Figure 4  Fluorescence spectra of (a) probe 1 and (b) probe 2 in DMSO/H₂O (v/v, 9/1, c: 1.0×10^-6 mol·L^-1) responding to various concentrations of Cu²⁺ (0~1.0 eq)

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  Figure 5  Fluorescence intensity of probe 1 before and after adding Cu²⁺ and that after treatment with EDTA (EDTA: Cu²⁺ 1 :1 M ratio)

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  Figure 6  Job's for the determination of the stoichiometry of (a) probe 1 and (b) probe 2 with Cu²⁺ in the complexes

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  Figure 7  The ¹H NMR titration spectra of (a) probe 1 and (b) probe 2 in the absence and presence of varying stoichiometry Cu²⁺ in DMSO-d₆ solution

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  Figure 9  Fluorescence detection limit of (a) probes 1 and (b) probe 2

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•