Synthesis and photochemical properties of BODIPY-functionalized silica nanoparticles for imaging Cu²⁺ in living cells

Abstract

A highly selective copper(II) sensor based on BODIPY-functionalized silica nanoparticles, BODIPY–DPA@SN, is designed and synthesized. Its absorption and fluorescence maxima in dry organic solvents are red-shifted by ∼75 and ∼50 nm compared with those of the BODIPY fluorophore and are blue-shifted by ∼55 and ∼15 nm, respectively, in aqueous–organic (1 : 1, v/v) media with fluorescence enhancement. The fluorescence intensity almost increases linearly as a function of water concentration (below 5%, v/v). BODIPY–DPA@SN exhibits high specificity for Cu²⁺ over other transition metal ions in aqueous–organic media, resulting in notable fluorescence quenching and a visible pink-to-yellow color change. Confocal microscopy experiments successfully proved that BODIPY–DPA@SN can be a biosensor for copper in living cells.

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Introduction

Imaging techniques in living cells are now feasible and effective for the observation of events at a molecular level. Fluorescence is widely applied to monitor the intracellular consistency of important ions and molecules, and advances are remarkable.^1,2 Of major importance are fluorophores targeting transition and heavy metal ions (such as Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺). A considerable amount of fluorescent labeling reagents are organic fluorophores with flexible applications and versatile chemical properties. However, there are still many problems to be solved to effectively target at the intracellular level, especially the toxicity of the fluorophore.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) (2)^3–5 derivatives are fluorescent dyes with noteworthy advantages such as high fluorescence quantum yields, large molar absorption coefficients, high stability towards chemicals and light, and negligible intersystem crossing.⁶ Moreover, BODIPY dyes are excitable with visible light, have narrow emission bandwidths with high peak intensities, and are amenable to structural modification, providing tunable spectral characteristics.

Since paramagnetic Cu²⁺ is a notorious fluorescence quencher, very few ratiometric fluorescent chemosensors for Cu²⁺ are available in the literature.⁷ The first fluorescent chemosensor for Cu²⁺ based on BODIPY as a reporter subunit (K_d = 3 μM) was published in 2006.⁸ It had 8-hydroxyquinoline as a receptor and showed significant fluorescence quenching in the presence of Cu²⁺ and Hg²⁺ with markedly higher selectivity for Cu²⁺ than Hg²⁺. A fluorescent off/on BODIPY-based chemosensor that displayed a chelation-enhanced fluorescence effect with Cu²⁺ (K_d = 20 μM), Pb²⁺ (K_d = 0.1 mM) and Zn²⁺ (K_d = 2 mM) was described by Yoon et al.⁹ Bis(pyridin-2-ylmethyl)amine [commonly known as di(2-picolyl)amine, DPA] is a chelator of several metal ions, including Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺ and Pb²⁺.¹⁰ A colorimetric and NIR fluorescent turn-on BODIPY-based probe with DPA as a chelator with high selectivity for Cu²⁺ among many transition metal ions has been reported by Boens.¹¹ Furthermore, many sensors for Cu(II) detection based on nanoparticles with excellent selectivity and efficiency have been developed. Jiang described an optical sensor of copper with upconverting luminescent nanoparticles as an excitation source.¹² A highly luminescent colloidal Eu³⁺-doped KZnF³ nanoparticle for the detection of Cu(II) ions was reported by Mahalingam.¹³ Silica nanoparticles are an environmentally friendly non-toxic material with additional properties beneficial for optical imaging applications in biological systems, including chemical inertness, transparency, and the ability to act as stabilizers in protecting embedded dyes from the outside environment.¹⁴

In a previous paper, we reported the preparation and spectroscopic properties of BODIPY–DPA (2)-functionalized hydroxyapatite (HA) nanoparticles.¹⁵ BODIPY–DPA@THA nanoparticles are not well soluble in aqueous solution due to the low solubility and agglomeration of nano-hydroxyapatite. Based on the above research results, we developed a turn-off probe 1 of copper(II) ions based on BODIPY-functionalized nanoparticles. It had di(2-picolyl)amine (DPA) modified at the 3-position of BODIPY as the ion recognition subunit and silica NPs coupled at the 5-position as the hydrophilic matrix to amplify the optical signal and improve sensitivity. Solvent-dependent photophysical properties and the response to Cu²⁺ ions were investigated.

Experimental section

Materials and methods

Di-(2-picolyl) amine (DPA) and p-tolualdehyde were purchased from HEOWNS Company; trifluoroacetic acid, N-chlorosuccinimide, chloranil, tetraethyl orthosilicate (TEOS) and 3-aminopropyltriethoxysilane were obtained from TCI Company. Boron trifluoride diethyl etherate was obtained from Alfa Aesar. Metal ions solutions were prepared from their perchlorate salts. Other materials for the synthesis were acquired from commercial companies and used without further purification.

Sensor 1 was characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), thermal gravimetric analysis (TG) test, UV-vis absorption and fluorescence spectroscopy. Fig. 1a showed the TEM image of SN, with a spherical structure and average size of 200 nm. Fig. 1b revealed the nanocrystalline appearance of 1. Dynamic light scattering (DLS) analysis at room temperature shows that the average hydrodynamic diameter of 1 is 347 nm (Fig. 1c), which is due to the interaction between the dye (2)-functionalized surface and solvent. The thermal behavior of 1 was studied by thermal gravimetric analysis (TG) in nitrogen environments (Fig. 1d). A constant mass flow of nitrogen at a rate of 60 mL min⁻¹ was set for the test. 4.69% of 1 disappeared at around 106 °C, which is due to the very low water content. Then, 1 began to lose surface-modified 2 (mp 182–183 °C) at 180 °C, and 15.71% of it totally disappeared at 535 °C. The loss of 11.02% of the weight is due to the organic dye 2.

Fig. 1 TEM images of (a) silica NPs, (b) 1, (c) particle size distribution of 1 and (d) TG curve of 1.

In the infrared spectrum (Fig. S1†) of SN, Si–O main bands are at 1098 (ν_as) cm⁻¹, 800 (ν_s) and 956 (δ) cm⁻¹ and hydroxide bands are at 3431 (ν_s) and 1633 (δ) cm⁻¹. The amino and –CH₂– groups are at 1570 (δ) cm⁻¹ and 2922 (ν_s) cm⁻¹. In the IR spectrum of 1, new strong bands appear at 2965, 2852, 1869 and 1535 cm⁻¹ in accordance with 2 bonding covalently to the SN nanoparticles.

Apparatus

¹H and ¹³C NMR spectra were taken on a Bruker DRX-400 spectrometer with TMS as the internal standard and CDCl₃ as the solvent. Mass spectra were recorded in E.I. mode. Melting points were measured on an X-4 micro-melting point apparatus (Gongyi Yuhua Instrument Co.). Transmission electron microscopy images (TEM) were obtained on a JEM-2100 transmission electron microscope at an acceleration voltage of 120 kV. The hydrodynamic diameter and size distribution were measured with dynamic light scattering (BI-200SM) at room temperature using ethanol–water as the solvent. The thermal gravimetric analysis (TG) test was measured by an STA PT1600 instrument from Linseis, Germany. UV-vis absorption spectra were recorded on a Varian UV-Cary100 spectrophotometer and for the corrected steady-state excitation and emission spectra, a Hitachi F-4500 and an FLS920 spectrofluorometers were employed. For the determination of fluorescence quantum yields Φ_f of 1 and 2, only dilute solutions with absorbance below 0.1 at the excitation wavelength (λ_ex = 480 or 500 nm) were used. Rhodamine 6 G in water (Φ_f = 0.92) and rhodamine B in water (Φ_f = 0.31) were used as the fluorescence standards.¹⁶ Fluorescence decay histograms were obtained on an Edinburgh instrument FLS920 spectrometer equipped with a supercontinuum white laser (400–700 nm), using the time-correlated single photon counting technique in 2048 channels. Histograms of instrument response functions (using a LUDOX scatterer) and sample decays were recorded until they typically reached 5 × 10³ counts in the peak channel.

Synthesis of BODIPY–DPA

Compound 2 was synthesized according to a literature procedure,¹⁷ resulting in an orange powder. Yield: 72%; mp 182–183 °C. ¹H NMR (CDCl₃, 400 MHz): δ = 2.42 (s, 3H, tolyl-CH₃), 5.24 (s, 4H, –CH₂–), 6.21 (d, 1H, J = 4.0 Hz, H-2), 6.28 (d, 1H, J = 4.8 Hz, H-1), 6.33 (d, 1H, J = 4.0 Hz, H-7), 6.81 (d, 1H, J = 5.2 Hz, H-6), 7.19 (dd, 2H, J = 5.2 Hz, J = 2.0 Hz, 2H, H-5′), 7.24 (d, 2H, J = 8.0 Hz, H-3′), 7.33 (d, 2H, J = 8.0 Hz, aromatic), 7.41 (d, 2H, J = 8.0 Hz, aromatic), 7.67 (td, 2H, J = 7.6 Hz, J = 1.6 Hz, H-4′), 8.55 (d, 2H, J = 4.8 Hz, H-6′); ¹³C NMR (CDCl₃, 100 MHz): δ 21.28, 57.93, 113.18, 115.02, 119.61, 122.00, 122.63, 128.82, 130.22, 130.36, 131.29, 132.48, 135.13, 135.43, 137.02, 139.22, 149.37, 156.23, 163.87; IR (KBr) ν_max/cm⁻¹ 995–700 (m), 1059 (s), 1111 (s), 1158 (m), 1261 (m), 1305 (m), 1416 (s), 1435 (s), 1535 (m), 1585 (s), 1627 (s), 1869 (m), 2852 (m), 2924 (m), 2965 (m), 3437 (s); MS (ESI) m/z calcd. for C₂₈H₂₃BClF₂N₅ 513.2; found 514.3 (M + 1, 100%).

Synthesis of BODIPY–DPA@SN

The synthetic route is depicted in Scheme 1. APTES-modified silica nanoparticles (1.0 g) were dispersed in 30 mL of acetonitrile by ultrasonication, 1.0 mL of triethylamine was added, and the mixture was stirred for 30 min. Next, 30 mL of a BODIPY–DPA (500 mg) acetonitrile solution was injected into the mixture, and the reaction mixture was stirred and refluxed for 12 h in the dark. The mixture was then washed with and dispersed in DI water and ethanol for several times. BODIPY–DPA@SN was obtained by centrifugation and dried overnight, followed by dialysis for 5 days to remove the free dyes.

Scheme 1 Synthesis of BODIPY–DPA@SN (1).

Binding copper

Titration experiments with copper were carried out by adding small quantities of a stock ethanol–water (1 : 1, v/v) solution of metal perchlorate salts to a larger volume (25 mL) of a solution of 1. The ground-state dissociation constant K_d of the BODIPY–DPA@SN–Cu complex was determined in an ethanol–water solution (1 : 1, v/v) by fluorometric titration. The nonlinear fitting of eqn (1) (ref. 18) to the steady-state fluorescence data F recorded as a function of [Cu²⁺] yields values of K_d. The fluorescence signals F_min and F_max at minimal and maximal [Cu²⁺] correspond to the free and Cu²⁺ bound forms of the nano-probe, respectively, and n stands for the number of copper ions bound per probe.

Fluorescence decay

Fluorescence lifetime was measured by an FLSP920 spectrofluorometer at room temperature. 1 was dissolved in aqueous–organic media (1 : 1, v/v), and the concentration was adjusted to make the optical densities <0.1 at the excitation wavelength. The monitored wavelengths were 580 nm, 590 nm and 600 nm, and the excitation wavelength was 480 nm.

Cell culture

SMMC-7721 cells were provided by the Institute of Biology (Lanzhou University). To determine the cell permeability of 1, the cells were incubated with 1 mg L⁻¹ of 1 (0.1% ethanol) for 30 min at 37 °C and washed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) to remove the remaining 1. To observe the fluorescence changes of the medium, 0.5 μM of Cu²⁺ was added into 1-loaded cells to preincubate for 0.5 h at 37 °C, followed by further incubation with 0.1 mM Na₂S for 0.5 h. Confocal fluorescence imaging was performed with a Leica DM-4000D microscope.

Results and discussion

The absorption spectrum of 2 is similar to that of BODIPY dyes,^19–24 showing an intense absorption band with maximum λ_abs located at 505 nm, assigned to the 0–0 band of the S₁ ← S₀ transition, and a shoulder peak (480 nm) on the high-energy side, attributed to the 0–1 vibrational band of the same transition. In addition, a weaker broad absorption band attributed to the S₂ ← S₀ transition is found around 350 nm. The maximum emission wavelength λ_em is 556 nm. The absorption of 1 shows a well-known pattern for BODIPY derivatives, and λ_abs is 75 nm red-shifted compared to 2 (from 505 to 580 nm) in ethanol. The fluorescence spectrum of 1 displays a narrow, slightly Stokes-shifted emission band at 605 nm and is 50 nm red-shifted compared to 2 (Fig. 2). The silica NPs show neither absorption nor fluorescence under the same conditions (Fig. S2†), which demonstrates that there is no energy transfer between silica NPs and BODIPY–DPA dye but the N-substituents at 3- and 5-positions that contribute to the special spectroscopic change between 1 and 2.

Fig. 2 Normalized absorption (dash curves) and fluorescence spectra (solid curves) of 1 (red) and 2 (black). Measured in absolute ethanol and the excitation wavelength λ_ex = 500 nm.

Compared to classic BODIPY derivatives, nonsymmetrically substituted (mono-substituted) BODIPY derivatives with an N-substituent at the 3-position have wider absorption and emission bands, red-shifted λ_em(max) and larger Stokes shifts than symmetric BODIPY dyes.²⁵ Moreover, there is a large difference in photophysical properties between the mono- and bis-substituted (symmetrically substituted) aminophenyl BODIPY derivatives. The emission wavelength λ_em(max) of bis-substituted derivatives is shifted further to red in comparison to the nonsymmetrically substituted derivatives.²⁶ The differences above suggest that the spectroscopic properties of these BODIPY derivatives are mainly affected by the aminophenyl group at the 3- (and 5-) position(s).²⁷

Water has been the most widely used solvent for proton transfer studies due to its proton accepting and conducting properties and being recognized as an active participant, rather than just a passive medium in the initial deprotonation step and in the transport mechanism of the proton^28,29 The effects of water on the emission spectra of two cupreidine derivatives in methanol–water mixed solutions have already been reported by Brouwer et al.; complexation with water leads to enhanced excited-state proton transfer (ESPT), and a fully solvated ‘‘free’’ ion pair (FIP) model can explain this change.³⁰ With the increase of water content in various solvents, compounds show fluorescence enhancement by the suppression of ICT due to the formation of fluorescent ionic structures by hydrolysis (Scheme 2).³¹

Scheme 2 Simplified reaction scheme of the excited-state free ion pair formation of 1 in ethanol–water media.

Fig. 3 shows the normalized absorption and fluorescence spectra of 1 in aqueous–organic media (1 : 1, v/v). The maximum λ_abs is blue-shifted from 580 to 528 nm, and the maximum λ_em is also blue-shifted from 610 nm to 590 nm accompanied by large intensity enhancement.

Fig. 3 Normalized (a) absorbance and (b) fluorescence spectra of 1 in different solvents. The excitation wavelength λ_ex = 480 nm, and the vial shows the sensor 1 in different solvents under UV light (left to right: 1. toluene; 2. ethanol; 3. ethanol–water; 4. THF; 5. THF–water; 6. MeCN; 7. MeCN–water).

Absorption (Fig. S3†) and fluorescence spectra of 1 (Fig. 4, keeping the concentration of 1 constant) were measured in ethanol containing various concentrations of water. Further sonication for 30 min was applied to obtain homodispersed solutions. The main absorption band at 580 nm is blue-shifted and decreases, whereas the band at 525 nm becomes more prominent, revealing ground-state hydrogen bonding interactions. The fluorescence band maximum is slightly blue-shifted and accompanied by an increase in intensity. The intensity increases almost linearly with the water content lower than 5% (v/v) (Fig. S4†). However, the fluorescence levels off and is no longer dependent on the water content once it is higher than 25% (v/v) (Fig. S5†).

Fig. 4 Fluorescence spectra of 1 with different water content in ethanol solution. The excitation wavelength λ_ex = 480 nm.

Both hydroxyl groups that reside on the SN surface and the DPA moieties have a high affinity via hydrogen bonding interactions with water, especially in solvents with moderate polarity. The fluorescence spectra of 2 (with the same DPA group) exhibited no change with increasing water content in solvents. Therefore, the results indicate that the fluorescence enhancement of 1 in anhydrous ethanol may be attributed to the suppression of ICT by the hydrogen bonding between the hydroxyl group of ethanol and the tertiary amino group of 1 and the interaction between the hydroxyl groups that reside on SN, water and the tertiary amino group.³²

To examine the chelating ability of 1, UV-vis spectrophotometric and fluorometric titrations were carried out in ethanol–water media (1 : 1, v/v). Upon the addition of Cu²⁺, the absorption band of 1 exhibited a hypsochromic shift from 525 nm to 455 nm (Fig. S6†), and the fluorescence was strongly quenched: Φ_f of 1–Cu complex is only 0.02 versus 0.14 of 1. The average K_d value of the 1–Cu complex amounted to 3.30 ± 0.4 μM (Fig. 5). The addition of Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺ and Pb²⁺ to the ethanol–water (1 : 1, v/v) solutions of 1 caused no changes that could be detected in the UV-vis absorption and fluorescence spectra (Fig. S7†), which indicated that the detection of Cu²⁺ is hardly affected by these common coexistent metal ions.

Fig. 5 (a) Fluorescence spectra of 1 as a function of [Cu²⁺] in ethanol–water (1 : 1, v/v) solution and (b) Best fit to direct fluorimetric titration data. The concentration of 1 is 50 mg L⁻¹, λ_ex = 480 nm and λ_em = 590 nm.

A near–linear correlation between the fluorescence intensity and Cu²⁺ concentration (r = 0.993, n = 6) was obtained over the range of 0.05–0.72 μM Cu²⁺ (Fig. S8†). The detection limit was 0.1 nM through the calculation by multiplying the standard derivation of 10 blank measurements by 3 and dividing by the slope of the linear calibration of sensor between fluorescence intensity at 590 nm and copper ion concentration in a 1 : 1 ethanol–water (v/v, pH 7.2) solution. This demonstrates the potential utility of the sensor 1 for the determination of the copper ion concentration in aqueous solutions.

To verify if the counter anion corresponding to the Cu²⁺ cation affected detection, we performed detection experiments with copper(II) chloride dihydrate and copper(II) nitrate trihydrate as Cu²⁺ sources. The results were very analogous (Fig. S9†) and provided evidence that the counter anions have little influence on the detection of Cu²⁺ ions. Thus, we used copper(II) perchlorate in the following experiments.

To examine the reversibility of the processes, an excess amount of Na₂S was added to the 1–Cu complex solution. The fluorescence spectra of 1 as a function of Na₂S concentration are shown in Fig. 6. Upon the addition of Na₂S, S²⁻ reacted with Cu²⁺ to form precipitates, and the bright pink fluorescence of 1 immediately turned on. Then, we continued to add Cu²⁺ for induced quenching and Na₂S for recovery. After four cycles, fluorescence remained very weak and no longer changed with plenty of Na₂S (Fig. S10†). This result clearly implies that probe 1 binds reversibly with Cu²⁺.

Fig. 6 Fluorescence spectra of 1–Cu complex as a function of [Na₂S]. Measured in ethanol–water (1 : 1, v/v) solution, λ_ex = 480 nm.

For the further biological application of copper (II) detection, ethanol–water (1 : 1, v/v) solutions of 1 with different pH (3–10) were applied to confirm the perfect testing environment (Fig. 7). The detection of copper (II) ions is the most sensitive in neutral solution, which is due to the suppression of complex formation by acid environment and Cu²⁺ sedimentation induced by base environment. We tested the practical applicability of 1 for Cu²⁺detection in a neutral biological environment.

Fig. 7 Fluorescence intensity of 1–Cu in the absence (A) and presence (B) of Cu²⁺ at different pH values. λ_ex = 480 nm.

SMMC-7721 cells incubated with 1 initially displayed a strong fluorescence image (Fig. 8a), but the image became black in the presence of Cu²⁺ (Fig. 8c). However, after incubation with sulfide, fluorescence gradually recovered (Fig. 8e). The experiment clearly demonstrated that 1 is permeable to the membrane, and the fluorescence changes are due to the synchronous presence of 1 and Cu²⁺. Herein, we believe that 1 can be used to image intracellular Cu²⁺ in living cells.

Fig. 8 Confocal fluorescence images of SMMC-7721 cells in a red (570−620 nm) emission channel. (a) Fluorescence image and (b) bright field microscopy image of cells incubated with 1 mg L⁻¹ of 1 (0.1% ethanol) for 30 min; (c) fluorescence image and (d) bright field microscopy image of 1-stained cells exposed to 0.5 μM of Cu²⁺ for 30 min. (e) fluorescence image and (f) bright field microscopy image of cells preincubated with 1 and Cu²⁺ followed by incubation with Na₂S for 30 min.

To investigate the fluorescence dynamics of 1, fluorescence decay traces in different aqueous–organic (water–ethanol, water–THF and water–MeCN) media (1 : 1, v/v) were collected as a function of emission wavelength λ_ex (Fig. 9). The fluorescence lifetimes of 1 in pure solvents were too short and could not be determined. The results of the time-resolved fluorescence experiments are shown in Table 1 and Fig. S9.†

Fig. 9 Fluorescence decay curves of 1 in different aqueous–organic media.

Table 1 Photophysical data of 1, 2 and 1–Cu complex

Complex	Solvent	λ abs(max)/nm	λ em(max)/nm	Φ f
2	Cyclohexane	518	555	0.009
	Toluene	516	557	0.009
	THF	503	554	0.006
	EtOH	506	551	0.008
	MeCN	485	553	0.005
1	Toluene	579	610	0.006
	THF	580	605	0.004
	EtOH	580	605	0.004
	MeCN	580	605	<0.0005
	THF–H2O (1 : 1)	528	593	0.10
	EtOH–H2O (1 : 1)	525	592	0.14
	MeCN–H2O (1 : 1)	525	593	0.05
1–Cu	EtOH–H2O (1 : 1)	455	592	0.02

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The fluorescence decay of 1 in an ethanol–water (1 : 1, v/v) solution in the region of 580–600 nm was fitted to the bi-exponential profile with the decay times of 4.50 ns (3%) and 1.05 ns (97%). The fluorescence decay of 1 in a THF–water solution (1 : 1, v/v) and an MeCN–water solution (1 : 1, v/v) also revealed bi-exponential behavior, but the decays become shorter (Table S1†). Most probably, there is an equilibrium between monomers and aggregates in the ground state. The slow decay might be attributed to the lifetime of aggregates, while the fast decay may be attributed to the deactivation of the monomer molecules.

The complex formation between 1 and Cu²⁺ was also investigated by time-resolved fluorescence. Upon the addition of Cu²⁺ to 1, the longer decay time decreases (∼4.50 to ∼3.50 ns) along with an increase in the amplitude (∼3% to ∼25%). The shorter component remains constant (∼1.05 ns), and in general, the contribution from this component decreases (∼97% to ∼75%) (Table S1†).

Conclusions

A BODIPY-based fluorescent chemosensor 1 was synthesized by the nucleophilic substitution of BODIPY derivatives with silica nanoparticles. Unlike literature values for BODIPY–Fe₃O₄, Ni or gold nanoparticles and their corresponding BODIPY sensors, there is quite a large difference in the properties between the free BODIPY–DPA dye and BODIPY–DPA@SN. The absorption and fluorescence maxima of 1 in organic–water media are blue-shifted compared to in dry solvents and red-shifted compared to BOPIDY–DPA. The fluorescence intensity was greatly enhanced after the addition of water to organic solvents (polar, less polar, protic and aprotic solvents) and sonication, which is attributed to the suppression of ICT by the formation of the FIP structure. 1 formed a 1 : 1 complex with Cu²⁺ in an ethanol–water (1 : 1, v/v) solution, which resulted in a distinct fluorescence decrease that can be observed by naked eye. When Na₂S solution is added to the complex, copper ions were released, and fluorescence recovered. The fluorescence decay of 1 in aqueous–organic media (1 : 1, v/v) in the region of 580–600 nm is fitted to the bi-exponential profile. Confocal microscopy experiment shows the potential utilization of chemosensor 1 for the fluorescent imaging of Cu²⁺ levels in living cells.

Acknowledgements

This work was supported by the Chinese “Program for New Century Excellent Talents in University” (NCET-09-0444), the ”Fundamental Research Funds for the Central Universities” (lzujbky-2011-22 and lzujbky-2012-k13), the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant no. J1103307) and the “International Cooperation Program of Gansu Province” (1104WCGA182). The authors would like to thank the Natural Science Foundation of China (no. 21271094), and this study was supported in part by the “Key Program of National Natural Science Foundation of China” (20931003).

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Footnote

† Electronic supplementary information (ESI) available: Compound characterization data, absorption and fluorescence spectra can be found in the ESI. See DOI: 10.1039/c4ra03183k

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