A Coumarin-based Fluorescent Sensor for Selective Detection of Copper (II)

Abstract

Cu (II) detection is of great importance owing to its significant function in various biological processes. In this report, we developed a novel coumarin-based chemosensor bearing the salicylaldimine unit (2) for $Cu^{2+}$ selective detection. The results from fluorescence spectra demonstrated that the sensor could selectively recognize $Cu^{2+}$ over other metal cations and the detection limit is as low as $0.2{\mu}M$. Moreover, the confocal fluorescence imaging in HepG2 cells illustrated its potential for biological applications.

Introduction

Copper is one of the transition metal abundant in the human body and actively participates in various biological processes.1-4 It mainly binds to metallothionein in cytoplasm and involves in cellular respiration, antioxidant defence and neurotransmitter.5,6 And the abnormal levels of copper were proposed to be related to certain diseases such as cardiovascular, diabetes, cancer and neurodegenerative diseases.7-11 Therefore, it is of great importance in developing efficient methods for copper detection in physiological conditions.

Recently, Fluorescent probes have become a powerful tool for Cu2+ detection in living systems owing to its high sensitivity, selectivity and especially, non-destructive intracellular detection. To date, a variety of fluorescent Cu2+ sensors have been reported based on the mechanism of Cu2+ induced chemical reactions or Cu2+ coordination.12-14 And most of these reported chemosensors decorate different fluorophores, including rhodamine,15-17 fluoroscein,18 naphthalimide19 and BODIPY.20 However, there are only a few sensors for Cu2+ detection based on the coumarin fluorophore so far. For example, Kim et al. reported a coumarin-based fluorogenic probe bearing the 2-picolyl unit as Cu2+ coordination site.21 Yoon et al. constructed the coumarin-Cu2+ complex employing dipicolylamine as binding sites and demonstrated the application in pyrophosphate detection through a Cu-desorption procedure.22 Leung and co-workers very recently developed a novel coumarin-DPA-Cu2+ chemosensing ensemble for selective detection of histidine in biological conditions.23 In fact, the coumarin-based chemosensors are identified as a special class of sensitive fluorophores for their excellent fluorescence characteristics, favourable membrane permeability and good solubility.24-26 Therefore, the development of new coumarin-chemosensors able to efficiently and selectively detect Cu2+ is of great importance.

Herein, we present a relatively simple chemosensor (2, Scheme 1), based on the coumarin scaffold bearing an aryl schiff base moiety in which the salicylaldimine framework will provide the coordination site for selective binding of Cu2+.27 Due to the intrinsic paramagnetic properties, Cu2+ has the propensity to quench the fluorescence of metal complex.28 Accordingly, the potential of Cu2+ selective detection was examined both in organic aqueous solution and in living systems. The chemosensor 2 was synthesized as shown in Scheme 1.

Scheme 1.Synthetic route of chemosensor 2.

 

Experimental

All reagents and solvents were commercially available and used without further purification. Column chromatography was carried out using silica gel (300-400 mesh). Thin layer chromatography (TLC) was performed with silica gel 60 F254 indicator. 1H NMR spectra were recorded in CDCl3 or DMSO on Bruker AV-400 spectrometer with TMS as internal standards. High resolution mass data were collected on Bruker ultrafleXtreme MALDI-TOF-TOF mass instrument. The fluorescence spectra were measured using a Varian Cary Eclipse Fluorescence spectrophotometer. The living cell imaging was performed using Zeiss LSM710 confocal fluorescence microscopy. All stock solutions of metal ions were prepared from analytical grade nitrate salts which were dissolved in acetonitrile. The HEPES buffer solutions were prepared as standard procedure.

Compound 1 was prepared following the method in the literature.29

Synthesis of (3-(4-(Diethylamino)-2-hydroxybenzylidene-amino)-7-hydroxy-coumarin (2). 3-Amino-7-hydroxy-coumarin 1 (1.77 g, 10.0 mmol) and 4-diethylamino-salicylaldehyde (1.93 g, 10.0 mmol) were added into anhydrous ethanol (60 mL), then a drop of glacial acetic acid was added. The reaction mixture was stirred at 78 ℃ for overnight under N2 protection. After cooling to room temperature, the solvent was evaporated under reduced pressure, then the residue was recrystallized from ethanol to afford a yellow solid 2. 1H NMR (400 MHz, CDCl3) δ 13.54 (s, 1H), 8.90 (s, 1H), 7.83 (s, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 8.9 Hz, 1H), 6.75 (dd, J = 8.5, 1.8 Hz, 1H), 6.67 (s, 1H), 6.31 (dd, J = 8.9, 2.2 Hz, 1H), 6.05 (d, J = 2.1 Hz, 1H), 3.41-3.36 (m, 4H), 1.11 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO) δ 163.6, 162.2, 161.5, 158.6, 153.5, 151.9, 134.1, 129.1, 128.9, 128.5, 114.3, 111.1, 108.8, 104.1, 102.1, 96.8, 44.1, 12.6. ESI-HRMS (m/z): Calcd. For C20H20N2O4 ([M+H]+): 353.1496; Found 353.1495.

 

Results and Discussion

The selective response of 2 toward Cu2+ was evaluated by addition of 1 equiv. of various metal ions in HEPES buffer (0.1 M, pH 7.4, containing 0.2% CH3CN) at 25 ℃. As shown in Figure 1, free 2 displayed strong fluorescence band centered at 518 nm. The addition of metal cations including Fe3+, Cr3+, Mn2+, Cd2+, Pb2+, Hg2+, Mg2+, K+, Ag+, and Na+, respectively, did not alter the fluorescence intensity of 2. In contrast, the Fe2+, Co2+ induced fluorescence quenching to a certain degree. However, it is worth noting that the fluorescence intensity (518 nm) of 2 was highly sensitive to Cu2+, which was reduced by approximate 100% upon the addition of 1 equiv. of Cu2+. The results obviously indicate sensor 2 has an excellent selectivity for Cu2+ over other metal cations. To further examine the sensitivity of 2 binding to Cu2+, Cu2+ titration experiment was performed. As shown in Figure 2, upon addition of increasing amounts of Cu2+, fluorescence intensity of 2 gradually decreases. When 1.0 equiv of Cu2+ was present, the total fluorescence intensity at 518 nm of 2 almost completely quenched. Using the fluorescence titration data, we then found that there exists a good linear correlation between the fluorescence intensity of 2 and Cu2+ in the range of 0-10 μM (Fig. 2, inset). Moreover, the detection limit is as low as 0.2 μM. The linear dependence of the intensity ratio within the equivalent range of Cu2+ demonstrated that 2 generate a 1:1 complex with Cu2+. In addition, the Job’s plot with fluorescence titrations further confirmed the 1:1 binding between 2 and Cu2+ (data not shown). Considering the salicylaldimine unit existing in 2, the recognition mechanism can be reasonably concluded to the formation of Cu2+ coordinating with salicylaldimine framework of 2.

Figure 1.Fluorescence emission spectra of 2 (10 μM) in HEPES buffer (0.1 M, pH 7.4, 0.2% CH3CN) in the presence of different metal cations (100 μM). Slite 5, λex = 420 nm.

Figure 2.Emission spectra of 2 (10 μM) in HEPES buffer (0.1 M, pH 7.4, 0.2% CH3CN) upon addition of various concentrations of Cu2+ (0-2 equiv.). Excitation wavelength was 420 nm. Inset: the plot of emission intensity at 518 nm as a function of Cu2+ concentration.

Based on the highly selective and sensitive Cu2+ detection profiles of chemosensor 2 in HEPES buffer, we further test the ability of 2 for intracellular Cu2+ imaging by using confocal fluorescence microscopy. HepG2 cells, incubated with probe 2 (10 μM) in culture medium for 30 min at 37 ℃, exhibited strong fluorescence (Fig. 3(a)). Upon addition of Cu2+ (10 μM), the fluorescence intensity of 2 was immediately quenched (Fig. 3(b)), indicating the potential application of sensor 2 in visualizing intracellular Cu2+ levels in HepG2 cells.

Figure 3.(a) Confocal fluorescence images of HepG2 cells treated with 2 (10 μM) for 30 min. (b) Fluorescence images of HepG2 cells treated with Cu2+ (10 mM). (c) Bright field images of (a). (d) Bright field images of (b).

 

Conclusion

In summary, a novel coumarin-based chemosensor 2 for Cu2+ selective detection was developed. The sensor exhibits highly sensitive and selective “turn-off” fluorescence detection towards Cu2+ over other metal cations. Moreover, the intracellular imaging in HepG2 cells demonstrates its potential for biological applications. Thus, the present results would broaden design strategies for Cu2+ detection.