A turn-on fluorescent pyrene-based chemosensor for Cu(II) with live cell application

Abstract

A pyrene-based fluorescent sensor (PHP) was synthesized for Cu(II) detection. It had high selectivity towards Cu²⁺ ions via photoinduced electron transfer (PET) based fluorescence enhancement. In the presence of Cu²⁺, PHP provided significant blue emission, while Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺, and Zn²⁺ metal ions produced only minor changes in the fluorescence spectra. The association constant (K_a) for Cu²⁺ binding to PHP had a value of 1.0 × 10⁴ M⁻¹. The maximum emission change induced by Cu²⁺ binding to the chemosensor PHP was observed over the pH range 5.0–10.0. Confocal fluorescence microscopy imaging using RAW264.7 cells showed that PHP can be used as an effective fluorescent probe for detecting Cu²⁺ in living cells.

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1. Introduction

The development of selective and sensitive chemosensors for biologically relevant cations and anions has gained much attention in the past decades because they play crucial roles in clinical and environmental analysis.^1–3 Among the essential metal ions, copper is the third most abundant essential transition metal ion in the human body. Copper ions are used as a cofactor for electron transport, or as a catalyst in oxido-reduction reactions in many proteins. As copper ions also react with dioxygen to form reactive oxygen species (ROS) that can damage lipids, nucleic acids and proteins, their cellular toxicity is connected to serious neurodegenerative diseases, including Menkes and Wilson disease,^4–7 Alzheimer's disease, and prion disease.⁸ Due to its extensive applications in our daily lives, copper is also a common metal pollutant. The limit for copper in drinking water, as set by the US Environmental Protection Agency (EPA) is 1.3 ppm (20 μM).

The development of fluorescent chemosensors for Cu²⁺ detection has been an important research topic. Because Cu²⁺ is known as a fluorescence quencher, most fluorescent chemosensors detect Cu²⁺ by the fluorescence quenching processes, which involve charge or energy transfer mechanisms.⁹ Due to sensitivity issues, fluorescent sensors with a turn-off process offer poor sensitivity for metal ion detection compared to fluorescence enhancement sensors.^10–29 This paper reports on a newly designed pyrene-based fluorescence enhancement chemosensor for Cu²⁺ that is based on photoinduced electron transfer (PET). Cu²⁺ binding with the chemosensor blocks the PET mechanism and greatly enhances the fluorescence of the pyrene moiety.

In this report, a pyrene based fluorescent probe (PHP) with hydrazinylpyridine moiety³⁰ was developed for the detection of Cu²⁺ ions. The chemosensor PHP exhibits weak fluorescence due to fluorescence quenching by photo-induced electron transfer from nitrogen lone pairs onto pyrene. The binding of a metal ion chemosensor blocks the PET mechanism, resulting in significant enhancement in pyrene fluorescence. The metal ions Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ were tested with the fluorescent probe PHP. Selectivity testing revealed that Cu²⁺ causes a visible color change in PHP, from light yellow or colorless, and a blue emission on ligation to PHP; no other tested ions produced a significant color change. Furthermore, chemosensor PHP is cell membrane permeable and can be used for the detection of Cu²⁺ in living cells.

2. Experimental

2.1 Materials and instrumentation

All solvents and reagents were obtained from commercial sources and used without further purification. UV/Vis spectra were recorded on an Agilent 8453 UV/Vis spectrometer. Fluorescence spectra measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer. NMR spectra were obtained on a Bruker DRX-300 and Agilent Unity INOVA-500 NMR spectrometer. Fluorescent images were taken on a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope.

2.2 Synthesis of (Z)-2-(2-(pyren-1-ylmethylene)hydrazinyl)pyridine

1-Pyrenecarboxaldehyde (230 mg, 1.0 mmol) and 2-hydrazinylpyridine (182 mg, 1.1 mmol) were added to a 10 mL ethanol solution. The reaction mixture was refluxed for 12 h. The resulting precipitate was collected by filtration and then purified by column chromatography (ethyl acetate : hexane = 1 : 1) to give 1 as a bright yellow solid. Yield: 336 mg (89%). Melting point: 234–236 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 11.11 (s, 1H), 9.13 (s, 1H), 8.70 (d, 1H, J = 9.3 Hz), 8.61 (d, 1H, J = 8.4 Hz), 8.31 (m, 4H), 8.17–8.21 (m, 3H), 8.10 (t, 1H, J = 7.5 Hz), 7.7 (t, 1H, J = 6.9 Hz), 7.34 (d, 1H, J = 8.4 Hz), 6.83 (t, 1H, J = 5.1 Hz); ¹³C NMR (75 MHz, DMSO-d₆): 157.4, 148.4, 138.6, 137.7, 131.5, 131.3, 130.8, 128.8, 128.7, 128.1, 128.1, 127.9, 127.0, 126.2, 125.8, 124.8, 124.5, 124.4, 122.2, 115.7, 107.0; MS(ESI): m/z = 322.2 ([M + H]⁺); HRMS (ESI): calcd for C₂₂H₁₆N₃ ([M + H]⁺) 322.1344; found 322.1345. FTIR (cm⁻¹): 3200, 2997, 1595, 1444, 1329, 1139, 994, 906, 843.

2.3 Cation selection study by fluorescence spectroscopy

PHP (10 μM) was added with different cations (1 mM). All spectra were measured in 1.0 mL acetonitrile–PBS buffer (0.01 M, pH-7.4) (v/v = 4 : 6). The light path length of cuvette was 1.0 cm.

2.4 Determination of the binding stoichiometry and the stability constants K_a of Cu²⁺ binding in chemosensor PHP

The binding stoichiometry of PHP–Cu²⁺ complexes was determined by Job plot experiments. The emission at 389 nm was plotted against molar fraction of PHP under a constant total concentration. The total concentration of sensor and Cu²⁺ ion was 200 μM. The molar fraction at maximum emission intensity represents the binding stoichiometry of the PHP–Cu²⁺ complexes. The maximum emission intensity was reached at a molar fraction of 0.5. The association constants K_a of PHP–Cu²⁺ complexes were determined by the Benesi–Hilderbrand equation:³¹

1/ΔF = 1/ΔF_sat + 1/(ΔF_satK_a[Cu²⁺]) (1)

where ΔF is the fluorescence intensity difference at 389 nm and ΔF_sat is the maximum fluorescence intensity difference at 389 nm. The association constant K_a was evaluated graphically by plotting 1/ΔF against 1/[Cu²⁺]. Data were linearly fitted according to eqn (1) and the K_a value was obtained from the slope and intercept of the line.

2.5 The pH dependence on the reaction of Cu²⁺ with PHP by fluorescence spectroscopy

PHP (10 μM) was added with Cu²⁺ (10 μM) in 1.0 mL acetonitrile–PBS buffer (0.01 M, pH-7.4) (v/v = 4 : 6). The buffers were: pH 3–4, KH₂PO₄/HCl; pH 4.5–6, KH₂PO₄/NaOH; pH 6.5–8.5, HEPES; pH 9–10, Tris–HCl.

2.6 Cell culture for RAW264.7 macrophages

The cell line RAW264.7 was provided by the Food Industry Research and Development Institute (Taiwan). RAW264.7 cells were cultured in Dulbecco's modied Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under an atmosphere of 5% CO₂. Cells were plated on 18 mm glass coverslips and allowed to adhere for 24 h.

2.7 Cytotoxicity assay

The methyl thiazolyl tetrazolium (MTT) assay was used to measure the cytotoxicity of PHP in RAW264.7 cells. RAW264.7 cells were seeded into a 96-well cell-culture plate. Various concentrations (10, 20, 30, 40, 50 μM) of PHP were added to the wells. The cells were incubated at 37 °C under 5% CO₂ for 24 h. 10 μL MTT (5 mg mL⁻¹) was added to each well and incubated at 37 °C under 5% CO₂ for 4 h. Remove the MTT solution and yellow precipitates (formazan) observed in plates were dissolved in 200 μL DMSO and 25 μL Sorenson's glycine buffer (0.1 M glycine and 0.1 M NaCl). Multiskan GO microplate reader was used to measure the absorbance at 570 nm for each well. The viability of cells was calculated according to the following equation:

Cell viability (%) = (mean of absorbance value of treatment group)/(mean of absorbance value of control group).

2.8 Fluorescence imaging of PHP

Experiments to assess Cu²⁺ uptake were performed in PBS with 10 μM CuCl₂. Treated with the cells with 2 μL of 10 mM metal ions (final concentration: 10 μM) dissolved in sterilized PBS (pH 7.4) and incubated for 30 min at 37 °C. The treated cells was washed PBS (3 × 2 mL) to remove remaining metal ions. Culture media (2 mL) was added to the cell culture, which was treated with a 10 mM solution of PHP (2 μL; final concentration: 10 μM) dissolved in DMSO. The samples were incubated at 37 °C for 30 min. The culture media was removed, and the treated cells were washed with PBS (3 × 2 mL) before observation. Confocal fluorescence imaging of cells was performed with a Leica TCS SP5 X AOBS Confocal Fluorescence Microscope (Germany), and a 63× oil-immersion objective lens was used. The cells were excited with a white light laser at 346 nm, and emission was collected at 380 ± 10 nm.

2.9 Computational methods

Quantum chemical calculations based on density functional theory (DFT) were carried out using a Gaussian 09 program. The ground-state structures of PHP and PHP–Cu²⁺ complexes were computed using the density functional theory (DFT) method with functional B3LYP. The 6-31G basis set was assigned to nonmetal elements (C, H, and N). For the PHP–Cu²⁺ complex, the LanL2DZ basis set was used for Cu²⁺, whereas the 6-31G basis set was used for other atoms.

3. Results and discussion

3.1 Synthesis of PHP

Chemosensor PHP was synthesized by the reaction of 1-pyrenecarboxaldehyde and 2-hydrazinylpyridine to form an imine bond between hydrazinylpyridine and pyrene (Scheme 1). PHP is yellow and has an absorption band at 383 nm, which is red-shifted by 50 nm from the pyrene absorption band at 335 nm. This is due to longer conjugated double bonds in chemosensor PHP. Chemosensor PHP exhibits a weak fluorescence (Φ = 0.001) compared to pyrene (Φ = 0.6–0.9). This observation can be attributed to fluorescence quenching by photoinduced electron transfer from electron lone pairs on nitrogen onto pyrene.

Scheme 1 Synthesis of PHP.

3.2 Cation-sensing properties

The sensing properties of PHP were investigated by monitoring the absorption and emission behaviors upon addition of several metal ions Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺, and Zn²⁺. Cu²⁺ caused a visible color change in PHP, from light yellow to colorless, and had a blue emission (Fig. 1); other metal ions only caused a minor change in the absorption and emission spectra (see Fig. S5 in the ESI†). During Cu²⁺ titration with PHP, the absorbance at 385 nm decreased in intensity, and a new band centered at 343 nm appeared (Fig. 2a). The color change from light yellow to colorless (Fig. 1) clearly indicates the 42 nm blue shift. The new band at 343 nm is close to the absorption band of pyrene at 335 nm. This observation suggests that Cu²⁺ binding with chemosensor PHP blocks conjugation between the double bonds, resulting in a shorter absorption wavelength. In addition, Cu²⁺ titration with chemosensor PHP results in a new emission band centered at 389 nm (Fig. 2b). After adding 1.5 molar equivalents of Cu²⁺, the emission intensity reached a maximum. The quantum yield of the new emission band was 0.56, which was 560-fold that of chemosensor PHP at 0.001. Cu²⁺ was the only metal ion of those we tested that readily binds with chemosensor PHP to yield a significant fluorescence enhancement, suggesting application for the highly selective detection of Cu²⁺ ion.

Fig. 1 (a) Color and (b) fluorescence changes of PHP (100 μM) after addition of various metal ions (100 μM) in CH₃CN/H₂O (v/v = 4 : 6, 10 mM PBS, pH 7.4) solution.

Fig. 2 (a) Absorption change and (b) fluorescence response of PHP (10 μM) to various equivalents of Cu²⁺ in CH₃CN/H₂O (v/v = 4 : 6, 10 mM PBS, pH 7.4) solution.

To understand the binding stoichiometry of the PHP–Cu²⁺ complex, Job plot experiments were carried out. In Fig. 3, the emission intensity at 389 nm is plotted against the molar fraction of chemosensor PHP at a constant total concentration of 50 μM. Maximum emission intensity was reached for a molar fraction of 0.50, indicating that one Cu²⁺ ion binds with one chemosensor PHP molecule. The formation of a PHP–Cu²⁺ complex was confirmed by ESI-MS, in which the peak at m/z = 383.05 indicates a 1 : 1 stoichiometry for the [(PHP–H⁺) + Cu²⁺] complex (see Fig. S5 in the ESI†). The association constant K_a was evaluated graphically by plotting 1/(F − F₀) against 1/[Cu²⁺] (Fig. 4). The data was linearly fit according to the Benesi–Hilderbrand equation, and the K_a value was obtained from the slope and intercept of the line. The K_a value of the PHP–Cu²⁺ complex was 1.0 × 10⁴ M⁻¹. The detection limit of PHP as a fluorescent sensor for the analysis of Cu²⁺ was determined from the plot of fluorescence intensity as a function of the concentration of Cu²⁺ (see Fig. S7 in the ESI†). PHP was found to have a detection limit of 0.04 μM, which is reasonable for the detection of micromolar concentrations of Cu²⁺.

Fig. 3 Job plot of the Cu²⁺–PHP complexes in CH₃CN/H₂O (v/v = 4 : 6, 10 mM PBS, pH 7.4) solution. The total concentration of PHP and Cu²⁺ was 50 μM. The excitation wavelength was 346 nm.

Fig. 4 Binding constant for titration of Cu²⁺ (0.1 to 1.0 eq) against ratio of fluorescence response for PHP (10 μM) in CH₃CN/H₂O (v/v = 4 : 6, 10 mM PBS, pH 7.4) solution. The excitation wavelength was 346 nm.

To further confirm the high selectivity of PHP for Cu²⁺ detection, a competitive experiment of coexisting ions was performed, in which PHP (10 μM) was examined with 40 μM of various metal ions Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺, and Zn²⁺. Cu²⁺ followed by the addition 20 μM of Cu²⁺ ions. As shown in Fig. 5, the fluorescence enhancement observed for most of the mixtures of Cu²⁺ with other metal ions was similar to that caused by Cu²⁺ alone. These observations indicated that most of the other metal ions do not interfere with the binding of PHP to Cu²⁺.

Fig. 5 Fluorescence response of PHP (10 μM) to Cu²⁺ (20 μM) or 100 μM of other metal ions (the black bar portion) and to the mixture of other metal ions (40 μM) with Cu²⁺ (20 μM) (the gray bar portion) in CH₃CN/H₂O (v/v = 4 : 6, 10 mM PBS, pH 7.4) solution.

A pH titration of PHP was carried out to determine a suitable pH range for Cu²⁺ detection. As depicted in Fig. 6, the emission intensities of metal-free PHP are very low at all pH values. After mixing PHP with Cu²⁺, the emission intensity at 389 nm increased in the pH range of 4.0–10.0. At pH < 5, the emission intensity is bigger due to higher emission from the protonation form of the PHP–Cu²⁺ complex. These observations indicated that the PHP–Cu²⁺ complex is essentially pH-insensitive over the range 4.0 to 10.0, indicating that the fluorescence of the PHP–Cu²⁺ complex is stable over a wide pH range.

Fig. 6 Fluorescence response (389 nm) of free PHP (10 μM) and after addition of Cu²⁺ (10 μM) in CH₃CN/H₂O (v/v = 4 : 6, 10 mM PBS) solutions as a function of various pH values. The excitation wavelength was 346 nm.

To understand the reversibility of the PHP–Cu²⁺ complex, a reversibility experiment was carried out with the addition of EDTA, which has a strong binding ability towards Cu²⁺. Fig. 7 shows that the introduction of EDTA can immediately decrease the emission intensity. Further addition of Cu²⁺ can restore the fluorescent state. This cycle (Cu²⁺–EDTA) can be carried out four times. This regeneration indicates that PHP can be reused with proper treatment.

Fig. 7 Reversible binding of Cu²⁺ with PHP. Fluorescence spectra of (a) PHP, (b) PHP in the presence of Cu²⁺ (10 μM), and (c) probe in the presence of Cu²⁺ (10 μM) upon addition of EDTA (10 μM).

In order to investigate Cu²⁺ binding to PHP, density functional theory (DFT) calculations were employed. Due to the 1 : 1 ligand-to-metal complex determined by Job plot, the chemosensor PHP with and without Cu²⁺ was subjected to energy optimization at the B3LYP hybrid functional with the LanL2DZ basis set. The lowest energy conformation for PHP–Cu²⁺ has one chemosensor PHP molecule binding with one Cu²⁺ ion, where the Cu²⁺ ion is bonded by two nitrogens at a distance of 2.06, and 2.03 Å, respectively (Fig. 8).

Fig. 8 DFT-optimized structures of (a) PHP and (b) PHP + Cu²⁺ complex using the B3LYP/LanL2DZ method (blue atom, N; pink atom, Cu).

To investigate the mechanism of Cu²⁺-detection, density functional theory (DFT) calculations were also employed using the Gaussian 09 software package. As shown in Fig. 9, the highest occupied molecular orbital (HOMO) of PHP (electron donor) is close to that of the fluorophore pyrene (electron acceptor); the HOMO energy level (−4.93 eV) of the binding moiety is higher than that of pyrene (−5.59 eV). Consequently, when the pyrene moiety is excited by light, electron transfer from the binding moiety to the pyrene is energetically allowed. Hence, the pyrene fluorescence is quenched by the PET process (Φ < 0.01). In contrast, upon the binding of PHP to Cu²⁺, the HOMO energy level of the binding moiety decreases to below that of pyrene; the PET process is thus forbidden and pyrene fluorescence reemerges.

Fig. 9 Energy diagram for the reaction of PHP with Cu²⁺.

3.3 Cell imaging of PHP

The potential of PHP for imaging Cu²⁺ in living cells was investigated next. First, an MTT assay with a RAW264.7 cell line was used to determine the cytotoxicity of PHP. In Fig. 10, the cellular viability was estimated to be greater than 80% after 24 h, which indicates that PHP (<30 μM) has low cytotoxicity. Cell images were further obtained using a confocal fluorescence microscope. When RAW264.7 cells were incubated with PHP (10 μM), no fluorescence was observed (Fig. 11a). After treatment with Cu²⁺, bright blue fluorescence was observed in the RAW264.7 cells (Fig. 11b). An overlay of fluorescence and bright-field images showed that the fluorescence signals were localized in the intracellular area, indicating a subcellular distribution of Cu²⁺ and good cell-membrane permeability of PHP.

Fig. 10 Cell viability values (%) estimated by an MTT assay versus incubation concentrations of PHP. RAW264.7 cells were cultured in the presence of PHP (0–50 μM) at 37 °C for 24 h.

Fig. 11 Fluorescence images of macrophage (RAW264.7) cells treated with PHP and Cu²⁺. (Left) Bright field image; (Middle) fluorescence image; and (Right) merged image (Ex. 346 nm, Em. 380–390 nm).

4. Conclusion

In conclusion, we have developed a new fluorescent probe PHP for a rapid, highly selective and sensitive response to Cu²⁺ ions over the other metal ions via a fluorescent turn-on response. An extremely suitable pH range was established for Cu²⁺ detection with PHP between 4.0–9.5. Further, the DFT calculation demonstrated a fluorescent turn-on mechanism in which the photoinduced electron transfer (PET) from the donor to pyrene is suppressed by Cu²⁺ binding. Most importantly, the chemosensor PHP can potentially be applied in fluorescence imaging of living cells. This pyrene-based chemosensor PHP has low cytotoxicity and can therefore be used for detecting Cu²⁺ in living cells.

Acknowledgements

We gratefully acknowledge the financial support of Ministry of Science and Technology (Taiwan, MOST 103-2113-M-009-005).

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra of PHP, ESI-mass of PHP and PHP + Cu²⁺, calibration curve of PHP with Cu(II). See DOI: 10.1039/c5ra05440k

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