English

• 0.   Introduction

  Fluorescent sensing of chemical species of biological and environmental significance by fluorescence spectroscopy has emerged as an attractive field for scientists, especially inspired by the development of confocal microscope and optical imaging techniques^[1-7]. As the third most abundant transition metal ions in the human body, copper ions play important roles in various biological processes, and maintaining its homeostasis is critical for development of living organisms^[8-12]. The disturbed homeostasis of copper ions can result in severe cell damage and further lead to many diseases such as Menkes syndrome, Wilson disease, and Alzheimer disease^[13-21]. Due to its essential toxic nature, long-term exposure to high levels of copper has been reported to induce the liver and kidney damage^[22]. According to the World Health Organization (WHO) International Standard for drinking water, the maximum acceptable level of copper in drinking water is 30 μmol·L^{-1 [23]}. Considering the important roles of copper in physiological processes and its high toxicity, development of specific fluorescent probes for sensitive copper detection, especially in environmental and physiological conditions is of great significance.

  To date lots of fluorescent probes have been reported for Cu²⁺ detection based on two main mechanisms: coordination of receptor with Cu^{2+ [24-34]} and Cu²⁺-triggered specific reactions^[35-47]. Due to the paramagnetic nature of Cu²⁺, Cu²⁺ coordination often results in fluorescence quenching. Fluorescence "turnoff" signals are usually less sensitive and generate false-positive results^{[24, 48]}, which is a disadvantage for practical application. Such obstacles can be eliminated by employing reaction-based probes, which achieve fluorescence "turn-on" response and offer high selectivity by reacting specifically with Cu²⁺ to generate fluorescent products. However, few fluorescence turn-on Cu²⁺ probes have been reported^[49-54], which make the development of new fluorescence turn-on Cu²⁺ probes highly desired.

  In this paper, Cou-P, a turn-on fluorescent probe for Cu²⁺ sensing based on a mechanism of Cu²⁺-coordination-induced hydrolysis of hydrazides was developed. Cou-P exhibits a selective and sensitive emission turn-on response to Cu²⁺ and features visible light excitation and emission profiles. Moreover, its turn-on imaging ability for intracellular Cu²⁺ has been confirmed in human breast adenocarcinoma cells (MCF-7 cells) using a confocal microscope.

  The synthesis route for Cou-P was depicted in Scheme 1. Cou-P was synthesized by reacting of 7-(diethylamino)-coumarin-3-carbonyl chloride and 2, 2′-(hydrazonomethylene)dipyridine in the presence of Et₃N in a high yield (85%). It was well characterized by ¹H, ¹³C NMR and ESI-MS (Supporting Information).

  Scheme 1

  

  Scheme 1.  Synthesis of Cou-P

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  1.   Experimental

  1.1   General procedures

  All chemicals and solvents were of analytical grade or spectroscopic grade and were used without further purification. Dichloromethane was refluxed with calcium hydride and distilled at ambient pressure. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker DRX-300 spectrometer or Bruker DRX-500 spectrometer with TMS as internal reference in CDCl₃. Mass spectrometric data were determined with LCQ ESI-MS Thermo Finnigan mass spectrometer. Absorption spectra were measured on a Shimadzu UV-3100 UV-Vis-NIR spectrophotometer. The fluorescence spectra were recorded with an AMINCO Bowman series 2 luminescence spectrophotometer (cuvette, 1 cm) with a xenon lamp as the light source.

  1.2   Synthesis of Cou-P

  A solution of compound 1 (200 mg, 0.72 mmol) in CH₂Cl₂ (5 mL) was slowly added to a solution of compound 2 (143 mg, 0.72 mmol) and Et₃N (80 mg, 0.68 mmol) in CH₂Cl₂ (10 mL). The reaction mixture was stirred at room temperature for 24 h. The mixture was washed with water (3×15 mL). The organic layer was dried on MgSO₄ and filtered, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography (silica gel, V_MeOH: V_CH2Cl2 =1:10, R_f=0.4), and pure compound Cou-P was obtained as an orange solid (270 mg) in 85% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.97 (d, 1H, J=4.0), 8.83 (s, 1H), 8.58 (d, 1H, J=4.0), 8.28 (d, 1H, J=8.0), 7.86~7.78 (m, 2H), 7.52 (d, 2H, J=8.0), 7.46~7.30 (m, 2H), 7.28~7.31 (t, 1H, J=4.0), 6.63~6.66 (dd, 1H, J₁=2.4, J₂=2.0), 6.47(s, 1H), 3.42~3.48(q, 4H, J₁=7.6, J₂=6.8), 1.21~1.253 (t, 6H, J=7.2); ¹³C NMR (100 MHz, CDCl₃): δ 161.92, 161.04, 158.12, 156.23, 153.04, 151.85, 149.45, 149.25, 149.07, 148.59, 136.86, 136.82, 131.53, 126.13, 124.18, 124.14, 123.70, 110.23, 110.16, 108.82, 96.84, 45.30, 12.59. ESI-MS (positive mode): Calcd. 905.90, Found: 905.08 for [2M+Na]⁺. Element analysis Calcd. for C₂₅H₂₃N₅O₃(%): C, 68.01; H, 5.25; N, 15.86. Found(%): C, 68.12; H, 5.46; N, 15.78.

  1.3   Spectroscopic measurements

  All the solvents were of analytic grade. The stock solution of Cou-P was prepared in CH₃OH (1.04 mmol ·L^-1). For spectroscopic determination, the stock solution was diluted with PBS to the desired concen-tration. The stock solutions of metal ions were prepared by dissolving Cu(NO₃)₂, CoCl₂·6H₂O, NiCl₂·6H₂O, HgCl₂, MnCl₂, Fe(NO₃)₃·9H₂O, FeSO₄·7H₂O, AgNO₃, PbCl₂, Zn(NO₃)₂·7H₂O, CdCl₂·2.5H₂O, KCl, CaCl₂, NaCl, MgCl₂·6H₂O with doubly distilled water. Fluorescence measurements of Cou-P (10 μmol·L^-1) were performed with 4 nm slit for excitation and 4 nm slit for emission. The UV-Vis titration of Cou-P was carried out by adding aliquots of 2.5 μL of Cu(NO₃)₂ aqueous solution (1.2 mmol·L^-1) to 3 mL of probe solution (10 μmol·L^-1) in a cuvette. The spectra were recorded after the solution was completely mixed. The fluorescence titration of Cou-P was also carried out by adding aliquots of 2.5 μL of Cu(NO₃)₂ aqueous solution (1.2 mmol·L^-1) to 3 mL of sample solution (10 μmol·L^-1) in a cuvette. The measurements were carried out in 2 min after the addition. All experiments were carried out at 298 K.

  1.4   Cell imaging

  MCF-7 cells were cultured in Dulbecco′s Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin (100 unit·mL^-1), streptomycin (100 mg·mL^-1) and 5%(V/V) CO₂ at 37 ℃. After removing the incubation media and rinsing with 1×PBS (0.01mol·L^-1) for three times. The MCF-7 cells were incubated with Cou-P (20 μmol·L^-1) for 30 min at 25 ℃. Then the cells were washed three times with 1×PBS and imaged with Zeiss LSM-710 microscope equipped with a 63× oil-immersion objective. For the imaging of MCF-7 cells upon incubation with exogenous Cu²⁺, the exogenous Cu²⁺ was introduced by incubating the cells with Cu(NO₃)₂ (200 μmol·L^-1) solution for 5 h at 25 ℃. The excitation wavelength of Cou-P used in experiment was 405 nm, while the filter was 420~520 nm.

  2.   Results and discussion

  2.1   Sensing properties

  The sensing ability of probe Cou-P for Cu²⁺ was investigated using fluorescence spectroscopy in HEPES buffer solution (50 mmol·L^-1, 0.1 mol·L^-1 KNO₃, pH=7.2). As shown in Fig. 1, the probe Cou-P itself showed relatively weak fluorescence at 497 nm (Ф=0.005) when excited by 460 nm. However, upon addition of 5 eq. of Cu²⁺, probe Cou-P exhibited a significant fluorescence enhancement at 464 nm (Ф=0.068), indicating that Cou-P could be efficiently responded in the presence of Cu²⁺.

  Figure 1

  

  Figure 1.  Fluorescence spectra of Cou-P (10 μmol·L^-1) and the corresponding hydrolysis product after adding 5 eq. Cu²⁺ in HEPES buffer (50 mmol·L^-1, 0.1 mol·L^-1 KNO₃, pH 7.2)

  Excitation wavelengths are 460 nm for Cou-P and 400 nm for adding 5 eq. Cu²⁺, respectively

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  2.2   Mechanism study

  Due to few reports on the reaction copper probes of hydrazide derivatives, the recognition mechanism of probe Cou-P for Cu²⁺ was investigated in detail. First, Cu²⁺ binding behaviour in Cou-P was investigated by fluorescent and UV-Vis titration experiments. The UV-Vis titration experiment demonstrates that free Cou-P has two main absorption bands centering at 454 (ε=2.8×10⁴ L·mol^-1·cm^-1) and 318 nm, which can be assigned to ICT and π-π* transition bands, respectively (Fig. 2a). When Cu²⁺ was added, distinct reduction of the former band can be observed accompanied by the evident bathochromic shift to 474 nm, suggesting the increased coplanarity of electrondonating group induced by Cu²⁺ binding. Similar change was also observed for the latter band, yet the band shift is much less pronounced. The clear isosbestic points at 471, 396 and 339 nm imply the undoubted conversion of free Cou-P to a copper complex. The titration profile based on the former band shows that the absorbance descends linearly with c_Cu²⁺ at the ratio of c_Cu²⁺/c_Cou-P≤1. Higher c_Cu²⁺ does not lead to any further evident change of isosbestic points at 471, 396 and 339 nm, suggesting a 1:1 stoichiometry for the copper complex. The result obtained from fluorescence Job′s plot also confirms the 1:1 Cu²⁺ binding stoichiometry (Supporting information, Fig.S1). Cou-P/Cu²⁺ complexes can remain stable for at least 120 min at pH 7.2 (Fig.S2). However, after the ratio of c_Cu²⁺/c_Cou-P attained 1, upon addition of another 0.1 eq. Cu²⁺ to the solution of Cou-P/Cu²⁺ complex, a new absorption band centered at 414 nm appeared and the intensity enhanced gradually in a time-dependent manner; at the same time, the absorption band at 474 nm exhibited decrease(Fig. 2b). The clear isosbestic points at 454 nm imply the undoubted conversion of Cou-P/Cu²⁺ complex to another compound. Normalized emission spectra of 3-(carboxylic acid)-7-(diethylamino)-coumarin (Cou-COOH) and Cou-P incubated with 1.1 eq. Cu(NO₃)₂ for 80 min overlaped well, Cou-P/Cu²⁺ complex may convert to Cou-COOH (Fig.S3).

  Figure 2

  

  Figure 2.  (a) Absorbance spectra of 10 μmol·L^-1 Cou-P in HEPES buffer obtained by adding 0~1.6 eq. Cu(NO₃)₂ (1.2 mmol·L^-1) solution; (b) Time-dependent UV-Vis absorption spectra of 20 μmol·L^-1 Cou-P in HEPES buffer obtained by adding excessive Cu(NO₃)₂ (100 μmol·L^-1) solution

  Inset: (a) titration profile based on the absorption ratio at 454 and 470 nm, A₄₅₄/A₄₇₀; (b) plot of A₄₁₄/A₄₇₄ as a function of time upon addition of Cu(NO₃)₂

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  Further fluorescence titration of Cou-P by Cu²⁺ shows similar phenomena. Titration of Cou-P by Cu²⁺ exhibits a linear emission decrease with c_Cu²⁺, and the fluorescence of Cou-P was quenched efficiently when the c_Cu²⁺/c_Cou-P ratio attained 1 (Fig. 3a). However, after the c_Cu²⁺/c_Cou-P ratio attained 1, upon addition of another 0.1 eq. Cu²⁺ to the solution of Cou-P/Cu²⁺ complex, a new excitation band centered at 403 nm appeared and the intensity enhanced gradually in a time-dependent manner (Fig. 3b). After 300 minutes, the fluorescence intensity at 403 nm (I₄₀₃) was reached its maximum. Further addition of EDTA could not eliminate the fluorescence (Fig.S4). This result demon-strates that Cou-P/Cu²⁺ complex might have undergone Cu²⁺-catalyzed chemical reactions.

  Figure 3

  

  Figure 3.  (a) Emission spectra of 10 μmol·L^-1 Cou-P in HEPES buffer obtained by adding aliquots of 2.5 μL Cu(NO₃)₂ (1.2 mmol·L^-1) solution; (b) Time-dependent fluorescent excitation spectra of 10 μmol·L^-1 Cou-P in HEPES buffer after addition of 1.1 eq. Cu(NO₃)₂

  Inset: (a) titration profile based on the emission intensity at 497 nm, I₄₉₇, λ_ex=460 nm; (b) time-dependent fluorescence at 403 nm, λ_em=510 nm, slit width=d_ex=d_em=4.0 nm, PMT voltage=950 V

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  To further verify the recognition mechanism of probe Cou-P for Cu²⁺, the reaction product of Cou-P with Cu²⁺ was characterized by the ESI-MS spectrometry. After addition of 1.1 eq Cu(NO₃)₂ for 60 min, ESI-MS of Cou-P shows a peak assigned to [Cou-P-H⁺+Cu²⁺+CH₃OH]⁺ (Fig.S5). After 300 min, there are three prominent peaks, 341.5 assigned to [3-H⁺+Cu²⁺+H₂O]⁺, 171.42 assigned to [(3+Cu²⁺+H₂O)/2]⁺, 251.33 assigned to [4+MeOH+Na⁺]⁺, respectively (Fig. 4). The isotopic distribution of peak at 341.5 matches with the results of ISOPRO 3.0 simulation, which further indicates the reaction product of Cou-P with Cu²⁺. This result is consistent with the experimental results of spectroscopy.

  Figure 4

  

  Figure 4.  (a) ESI-MS of Cou-P in MeOH/H₂O (1:1, V/V) after addition of 1.1 eq. Cu(NO₃)₂ for 5 h; (b) Corresponding simulated one for [3-H⁺+Cu²⁺+H₂O]⁺; (c) Determined isotopic distribution patterns of the peak with m/z of 341.50

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  Thus, the coordination of Cu²⁺ to picolinic ester in probe Cou-P is essential for the Cu²⁺-promoted hydrolytic cleavage of Cou-P to Cou-COOH (3). Based on the above results, a detailed recognition mechanism of probe Cou-P for Cu²⁺ was proposed as shown in Scheme 2.

  Scheme 2

  

  Scheme 2.  Proposed mechanism of Cu²⁺-catalysed reaction of Cou-P

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  2.3   Cu²⁺ detection selectivity

  Selectivity is a crucial parameter to assess the performance of the developed probes. Therefore, the selectivity experiments of Cou-P were extended to a variety of biological-related species including common cations (K⁺, Na⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mn²⁺, Zn²⁺, Cd²⁺, Pb²⁺ and Ag⁺). As shown in Fig. 5a, only introduction of Cu²⁺ could cause apparent fluorescence intensity changes of the probe Cou-P solution, most of the tested cation did not induce any emission enhancement at 403 nm. According to the value of I₄₀₃/I₄₆₀, Cu²⁺ can induce 13-fold fluorescence enhancement, and Co²⁺ and Ni²⁺ also induce very minor enhancement due to fluorescence quenching at 460 nm (Fig. 5b). Moreover, the abundant metal cations in natural water such as Na⁺, K⁺, Ca²⁺ and Mg²⁺ do not result in distinct emission either. This provides probe Cou-P the advantage in sense Cu²⁺ in samples containing abundant Na⁺, K⁺, Ca²⁺, and Mg²⁺, such as living cells.

  Figure 5

  

  Figure 5.  (a) Excitation spectra of Cou-P (10 μmol·L^-1) in the presence of various metal ions in HEPES buffer (50 mmol·L^-1, 0.1 mol·L^-1 KNO₃, pH 7.2); (b) Ratio of fluorescence intensity at 403 and 460 nm of 10 μmol·L^-1 Cou-P induced by different metal ions in HEPES buffer

  Final concentration for Cd²⁺, Zn²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mn²⁺, Ni²⁺ and Pd²⁺ is 20 μmol·L^-1, for Na⁺, K⁺, Ca²⁺ and Mg²⁺ is 10 mmol·L^-1

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  2.4   Cell cytotoxicity and confocal fluorescence imaging

  The cytotoxicity of probe Cou-P in MCF-7 cells was evaluated by MTT assays with the concentration of the probe ranging from 2 to 50 μmol·L^-1 (Fig.S6). The results show that Cou-P is of low cytotoxicity to cultured cells and should be safe when used for bioimaging of Cu²⁺.

  Based on the favorable features of Cou-P including visible emission, high selectivity for Cu²⁺ and low toxicity, the potential utility of Cou-P to visualize Cu²⁺ in living cells was investigated. As shown in Fig. 6, MCF-7 cells incubated with Cou-P (10 μmol·L^-1) alone for 30 min at 37 ℃ exhibited no observable fluorescence. However, when cells were further incubated with Cu²⁺ (200 μmol·L^-1) for 300 min at 37 ℃, remarkable intracellular fluorescence could be observed. These results indicate that probe Cou-P is permeable to cell membrane and suitable for imaging Cu²⁺ in living cells.

  Figure 6

  

  Figure 6.  Confocal fluorescence images of intracellular Cu²⁺ in MCF-7 cells with Cou-P-staining: (a) Bright-field transmission images; (b)MCF-7 cells incubated with Cou-P (10 μmol·L^-1) at 37 ℃ for 30 min; (c)Stained cells further exposed to 200 μmol·L^-1 Cu(NO₃)₂ solution at 37 ℃ for 300 min

  Band path: 420~520 nm, λ_ex=405 nm

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  3.   Conclusions

  In summary, we successfully developed a new reaction fluorescent probe for detecting Cu²⁺. Probe Cou-P exhibits a fluorescence "turn-on" recognition process for Cu²⁺. The recognition mechanism of probe Cou-P for Cu²⁺ was investigated by UV-Vis, fluorescence and ESI-MS spectrometry. In application, Cou-P shows low cytotoxicity and good membrane permeability. More importantly, Cou-P was successfully used for the detection of Cu²⁺ in living cells, indicating its great potential for Cu²⁺ detection in biological science.

  Supporting information is available at http://www.wjhxxb.cn

  
  
• 1. [1]

     Martínez-Máez R, Sancenón F. Chem. Rev., 2003, 103:4419-4476 doi: 10.1021/cr010421e

  2. [2]

     Thomas S W, Joly G D, Swager T M. Chem. Rev., 2007, 107:1339-1386 doi: 10.1021/cr0501339

  3. [3]

     Kim S K, Lee D H, Hong J I, et al. Acc. Chem. Res., 2009, 42:23-31 doi: 10.1021/ar800003f

  4. [4]

     Xu Z C, Chen X Q, Kim H N, et al. Chem. Soc. Rev., 2010, 39:127-137 doi: 10.1039/B907368J

  5. [5]

     Zhou Y, Xu Z C, Yoon J. Chem. Soc. Rev., 2011, 40:2222-2235 doi: 10.1039/c0cs00169d

  6. [6]

     Vendrell M, Zhai D T, Er J C, et al. Chem. Rev., 2012, 112:4391-4420 doi: 10.1021/cr200355j

  7. [7]

     Liu Z P, He W J, Guo Z J. Chem. Soc. Rev., 2013, 42:1568-1600 doi: 10.1039/c2cs35363f

  8. [8]

     Prohaska J R, Gybina A A. J. Nutr., 2004, 134:1003-1006 doi: 10.1093/jn/134.5.1003

  9. [9]

     Davis A V, O'Halloran T V. Nat. Chem. Biol., 2008, 4:148-151 doi: 10.1038/nchembio0308-148

  10. [10]

      Que E L, Domaille D W, Chang C J. Chem. Rev., 2008, 108:1517-1549 doi: 10.1021/cr078203u

  11. [11]

      Thiele D J, Gitlin J D. Nat. Chem. Biol., 2008, 4:145-147 doi: 10.1038/nchembio0308-145

  12. [12]

      Robinson N J, Winge D R. Annu. Rev. Biochem., 2010, 79:537-562 doi: 10.1146/annurev-biochem-030409-143539

  13. [13]

      Cuajungco M P, Lees G J. Neurobiol. Dis., 1997, 4:137-169 doi: 10.1006/nbdi.1997.0163

  14. [14]

      Bush A I. Curr. Opin. Chem. Biol., 2000, 4:184-191 doi: 10.1016/S1367-5931(99)00073-3

  15. [15]

      Valentine J S, Hart P J. Proc. Natl. Acad. Sci. USA, 2003, 100:3617-3622 doi: 10.1073/pnas.0730423100

  16. [16]

      Brown D R, Kozlowski H. Dalton Trans., 2004, 13:1907-1917

  17. [17]

      Millhauser G L. Acc. Chem. Res., 2004, 37:79-85 doi: 10.1021/ar0301678

  18. [18]

      Frederickson C J, Koh J Y, Bush A I. Nat. Rev. Neurosci., 2005, 6:449-462 doi: 10.1038/nrn1671

  19. [19]

      Gaggelli E, Kozlowski H, Valensin D, et al. Chem. Rev., 2006, 106:1995-2044 doi: 10.1021/cr040410w

  20. [20]

      Lutsenko S, Gupta A, Burkhead J L, et al. Arch. Biochem. Biophys., 2008, 476:22-32 doi: 10.1016/j.abb.2008.05.005

  21. [21]

      Kaler S G. Nat. Rev. Neurol., 2011, 7:15-29 doi: 10.1038/nrneurol.2010.180

  22. [22]

      Domaille D W, Que E L, Chang C J. Nat. Chem. Biol., 2008, 4:168-175 doi: 10.1038/nchembio.69

  23. [23]

      World Health Oganization. Guidelines for Drinking-Water Quality. 4th Ed. Geneva:World Health Organization, 2017:341

  24. [24]

      Wang X B, Ma X Y, Yang Z, et al. Chem. Commun., 2013, 49:11263-11265 doi: 10.1039/c3cc46585c

  25. [25]

      Kang D E, Lim C S, Kim J Y, et al. Anal. Chem., 2014, 86:5353-5359 doi: 10.1021/ac500329k

  26. [26]

      Sharma N, Reja S I, Bhalla V, et al. Dalton Trans., 2014, 43:15929-15936 doi: 10.1039/C4DT01676A

  27. [27]

      Han Y Y, Ding C Q, Zhou J, et al. Anal. Chem., 2015, 87:5333-5339 doi: 10.1021/acs.analchem.5b00628

  28. [28]

      Kaur M, Ahn Y H, Choi K, et al. Org. Biomol. Chem., 2015, 13:7149-7153 doi: 10.1039/C5OB00907C

  29. [29]

      Li S, Zhang D, Xie X Y, et al. Sens. Actuators B, 2016, 224:661-667 doi: 10.1016/j.snb.2015.10.086

  30. [30]

      Jiang H, Li Z J, Kang Y F, et al. Sens. Actuators B, 2017, 242:112-117 doi: 10.1016/j.snb.2016.11.033

  31. [31]

      Li Y Y, Sun M T, Zhang K, et al. Sens. Actuators B, 2017, 243:36-42 doi: 10.1016/j.snb.2016.11.118

  32. [32]

      Liu L L, Dan F J, Liu W J, et al. Sens. Actuators B, 2017, 247:445-450 doi: 10.1016/j.snb.2017.03.069

  33. [33]

      Yoon J W, Chang M J, Hong S, et al. Tetrahedron Lett., 2017, 58:3887-3893 doi: 10.1016/j.tetlet.2017.08.071

  34. [34]

      范方禄, 靖金球, 陈雪梅.无机化学学报, 2015, 31(3):548-554 http://www.wjhxxb.cn/wjhxxbcn/ch/reader/view_abstract.aspx?flag=1&file_no=20150318&journal_id=wjhxxbcnFAN Fang-Lu, JING Jin-Qiu, CHEN Xue-Mei. Chinese J. Inorg. Chem., 2015, 31(3):548-554 http://www.wjhxxb.cn/wjhxxbcn/ch/reader/view_abstract.aspx?flag=1&file_no=20150318&journal_id=wjhxxbcn

  35. [35]

      Lin W Y, Yuan L, Tan W, et al. Chem. Eur. J., 2009, 15:1030-1035 doi: 10.1002/chem.200801501

  36. [36]

      Li N, Xiang Y, Tong A. Chem. Commun., 2010, 46:3363-3365 doi: 10.1039/c001408g

  37. [37]

      Cheng J H, Zhang Y H, Ma X F, et al. Chem. Commun., 2013, 49:11791-11793 doi: 10.1039/c3cc47137c

  38. [38]

      Huang L Y, Gu B, Su W, et al. RSC Adv., 2015, 5:76296-76301 doi: 10.1039/C5RA14443D

  39. [39]

      Hu X X, Zheng X L, Fan X X, et al. Sens. Actuators B, 2016, 227:191-197 doi: 10.1016/j.snb.2015.12.037

  40. [40]

      Li D X, Sun X, Huang J M, et al. Dyes Pigm., 2016, 125:185-191 doi: 10.1016/j.dyepig.2015.10.016

  41. [41]

      Sun J, Wang B, Zhao X, et al. Anal. Chem., 2016, 88:1355-1361 doi: 10.1021/acs.analchem.5b03848

  42. [42]

      Tan W B, Leng T H, Lai G Q, et al. J. Photochem. Photobiol. A, 2016, 324:81-86 doi: 10.1016/j.jphotochem.2016.03.014

  43. [43]

      Tang J, Ma S G, Zhang D, et al. Sens. Actuators B, 2016, 236:109-115 doi: 10.1016/j.snb.2016.05.144

  44. [44]

      Wang B G, Cui X Y, Zhang Z Q, et al. Org. Biomol. Chem., 2016, 14:6720-6728 doi: 10.1039/C6OB00894A

  45. [45]

      Zhu D J, Luo Y H, Shuai L, et al. Tetrahedron Lett., 2016, 57:5326-5329 doi: 10.1016/j.tetlet.2016.10.056

  46. [46]

      Zheng X L, Ji R X, Cao X Q, et al. Anal. Chim. Acta, 2017, 978:48-54 doi: 10.1016/j.aca.2017.04.048

  47. [47]

      Gu B, Huang L Y, Xu Z F, et al. Sens. Actuators B, 2018, 273:118-125 doi: 10.1016/j.snb.2018.06.032

  48. [48]

      Sirilaksanapong S, Sukwattanasinitt M, Rashatasakhon P. Chem. Commun., 2012, 48:293-295 doi: 10.1039/C1CC16148B

  49. [49]

      Zhao C C, Feng P, Cao J, et al. Org. Biomol. Chem., 2012, 10:3104-3109 doi: 10.1039/c2ob06980f

  50. [50]

      Yang C Y, Chen Y, Wu K, et al. Anal. Methods, 2015, 7:3327-3330 doi: 10.1039/C5AY00224A

  51. [51]

      Tang L J, He P, Zhong K L, et al. Spectrochim. Acta Part A, 2016, 169:246-251 doi: 10.1016/j.saa.2016.06.045

  52. [52]

      Zhu D J, Luo Y H, Yan X W, et al. RSC Adv., 2016, 6:87110-87114 doi: 10.1039/C6RA18669F

  53. [53]

      Zhang H T, Feng L, Jiang Y, et al. Biosens. Bioelectron., 2017, 94:24-29 doi: 10.1016/j.bios.2017.02.037

• 

  Scheme 1  Synthesis of Cou-P

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  Figure 1  Fluorescence spectra of Cou-P (10 μmol·L^-1) and the corresponding hydrolysis product after adding 5 eq. Cu²⁺ in HEPES buffer (50 mmol·L^-1, 0.1 mol·L^-1 KNO₃, pH 7.2)

  Excitation wavelengths are 460 nm for Cou-P and 400 nm for adding 5 eq. Cu²⁺, respectively

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  Figure 2  (a) Absorbance spectra of 10 μmol·L^-1 Cou-P in HEPES buffer obtained by adding 0~1.6 eq. Cu(NO₃)₂ (1.2 mmol·L^-1) solution; (b) Time-dependent UV-Vis absorption spectra of 20 μmol·L^-1 Cou-P in HEPES buffer obtained by adding excessive Cu(NO₃)₂ (100 μmol·L^-1) solution

  Inset: (a) titration profile based on the absorption ratio at 454 and 470 nm, A₄₅₄/A₄₇₀; (b) plot of A₄₁₄/A₄₇₄ as a function of time upon addition of Cu(NO₃)₂

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  Figure 3  (a) Emission spectra of 10 μmol·L^-1 Cou-P in HEPES buffer obtained by adding aliquots of 2.5 μL Cu(NO₃)₂ (1.2 mmol·L^-1) solution; (b) Time-dependent fluorescent excitation spectra of 10 μmol·L^-1 Cou-P in HEPES buffer after addition of 1.1 eq. Cu(NO₃)₂

  Inset: (a) titration profile based on the emission intensity at 497 nm, I₄₉₇, λ_ex=460 nm; (b) time-dependent fluorescence at 403 nm, λ_em=510 nm, slit width=d_ex=d_em=4.0 nm, PMT voltage=950 V

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  Figure 4  (a) ESI-MS of Cou-P in MeOH/H₂O (1:1, V/V) after addition of 1.1 eq. Cu(NO₃)₂ for 5 h; (b) Corresponding simulated one for [3-H⁺+Cu²⁺+H₂O]⁺; (c) Determined isotopic distribution patterns of the peak with m/z of 341.50

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  Scheme 2  Proposed mechanism of Cu²⁺-catalysed reaction of Cou-P

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  Figure 5  (a) Excitation spectra of Cou-P (10 μmol·L^-1) in the presence of various metal ions in HEPES buffer (50 mmol·L^-1, 0.1 mol·L^-1 KNO₃, pH 7.2); (b) Ratio of fluorescence intensity at 403 and 460 nm of 10 μmol·L^-1 Cou-P induced by different metal ions in HEPES buffer

  Final concentration for Cd²⁺, Zn²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mn²⁺, Ni²⁺ and Pd²⁺ is 20 μmol·L^-1, for Na⁺, K⁺, Ca²⁺ and Mg²⁺ is 10 mmol·L^-1

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  Figure 6  Confocal fluorescence images of intracellular Cu²⁺ in MCF-7 cells with Cou-P-staining: (a) Bright-field transmission images; (b)MCF-7 cells incubated with Cou-P (10 μmol·L^-1) at 37 ℃ for 30 min; (c)Stained cells further exposed to 200 μmol·L^-1 Cu(NO₃)₂ solution at 37 ℃ for 300 min

  Band path: 420~520 nm, λ_ex=405 nm

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