A new coumarin-derived fluorescent probe (1) exhibited significant fluorescence quenching in the
presence of Cu2+ions. Other metal ions, e.g. Ag+, Ca2+, Cd2+, Co2+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+,
Ni2+, Pb2+, and Zn2+, produced only minor changes in the fluorescence of chemosensor 1. The binding
ratio of the chemosensor–Cu2+ complexes was found to be 2 : 1, according to Job plot experiments.
The association constant (Ka) for Cu2+ binding with chemosensor 1 was found to be 9.56 109M 2.
The maximum fluorescence quenching caused by Cu2+binding with chemosensor 1 occurred over a pH
range of 5–9. Moreover, fluorescence microscopy experiments showed that chemosensor 1 could be
used as a fluorescent probe for detecting Cu2+in living cells.
1 Introduction
The development of highly selective chemosensors for the recognition of environmentally and biologically important metal ions, such as Cu2+, Cd2+, Fe3+, Hg2+, Pb2+, and Zn2+, has been a key issue in research.1–5Copper, after iron and zinc, is the third most abundant essential transition metal ion in the human body, and plays vital roles in several natural processes.6 Many proteins use copper ions as their catalytic center. However, copper ions also catalyze the formation of reactive oxygen species (ROS) that can harm lipids, nucleic acids, and proteins. Several research studies have linked the cellular toxicity of copper ions to serious diseases such as Alzheimer’s disease,7 Indian childhood
cirrhosis (ICC),8prion disease,9and Menkes and Wilson diseases.10 Due to its widespread application, the copper ion is also a significant metal pollutant. The limit of copper in drinking water as set by the US Environmental Protection Agency (EPA) is 1.3 ppm (B20 mM).
The determination of copper ions in various samples has been an important topic in the area of environmental protection and food safety. Several methods have been applied for the detection of copper ions, including atomic absorption spectrometry,11inductively coupled plasma mass spectroscopy (ICPMS),12 inductively coupled plasma-atomic emission spectrometry (ICP-AES),13and voltammetry.14 Most of these methods require expensive instru-ments and are not suitable for direct assays. Recently, more attention has been focused on the development of fluorescent chemosensors for the detection of Cu2+ions.15–28
In this study, a new coumarin derivative 1 containing a phenol hydrazone moiety was designed for metal ion detection. Two components make up chemosensor 1; a coumarin moiety as a reporter, and a phenol hydrazone moiety as a metal ion chelator (Scheme 1). Binding metal ions to chemosensor 1 causes fluores-cence quenching of coumarin. The metal ions Ag+, Ca2+, Cd2+, Co2+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+were tested for metal ion binding with chemosensor 1. Cu2+was the only ion that caused significant fluorescence quenching upon binding with chemosensor 1.
2 Results and discussion
2.1 Synthesis of chemosensor 1
Chemosensor 1 was synthesized by the reaction of phenol hydrazone and 7-diethylaminocoumarin-3-aldehyde to form an imine bond between phenol hydrazone and coumarin (Scheme 1). Chemosensor 1 is light yellow and has an absorption band centered at 467 nm. Chemosensor 1 exhibits a green emission band centered at 537 nm with a quantum yield of F = 11.5%.
2.2 Cation-sensing properties
The sensing ability of chemosensor 1 was tested by mixing it with the metal ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+. Cu2+ was the only metal ion
causing a visible color change from light yellow to red and green fluorescence quenching (Fig. 1). During Cu2+ titration with chemosensor 1, the absorbance at 487 nm decreased, and a new band centered at 440 nm was formed (Fig. 2). The color change from light yellow to red revealed this 47 nm blue shift. To further evaluate the selectivity of chemosensor 1 towards various metal ions, the fluorescence spectra of chemosensor 1
Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan 300, Republic of China. E-mail: [email protected];
Fax: +886-3-5723764; Tel: +886-3-5712121 ext. 56506
†Electronic supplementary information (ESI) available:1H and13C NMR spectra of chemosensor 1, ESI mass spectra of the Cu2+–1
2complexes and the calibration curve of 1–Cu2+(10 mM) in a methanol–water solution. See DOI: 10.1039/c4nj00695j Received (in Montpellier, France)
30th April 2014, Accepted 24th June 2014 DOI: 10.1039/c4nj00695j
www.rsc.org/njc
were taken in the presence of several metal ions. However, Cu2+ was the only metal ion that caused significant fluorescence quenching (Fig. 1). During Cu2+titration with chemosensor 1, the intensity of the 537 nm emission band decreased (Fig. 2). After the addition of greater than one equivalent of Cu2+, the emission intensity reached a minimum. These observations suggest that Cu2+is the only metal ion that readily binds with
chemosensor 1, causing significant fluorescence quenching and permitting highly selective detection of Cu2+.
To study the influence of other metal ions on Cu2+binding with chemosensor 1, we performed competitive experiments with other metal ions (20 mM) in the presence of Cu2+(20 mM) (Fig. 3). The fluorescence quenching caused by the Cu2+solution with most other metal ions was similar to that caused by Cu2+ alone. This indicates that the other metal ions do not interfere significantly with the binding of chemosensor 1 to Cu2+.
In order to understand the binding stoichiometry of chemo-sensor 1–Cu2+ complexes, Job plot experiments were carried out. In Fig. 4, the emission intensity at 537 nm is plotted against the molar fraction of chemosensor 1 at a constant total concentration (20 mM). The minimum emission intensity was reached when the molar fraction was 0.67. These results indicate a 2 : 1 ratio for 1–Cu2+complexes, in which one Cu2+binds with
two chemosensor 1 molecules. The formation of a Cu2+–12
complex was also confirmed by ESI-MS, in which the peak at m/z = 788.7 indicates a 1 : 2 stoichiometry for the [Cu + 12–H]+
complex (see Fig. S4 in the ESI†). The association constant, Ka,
was evaluated graphically by plotting a2/(1 a) vs. 1/[Cu2+], where a is defined as [F F0]/[F1 F0] (Fig. 5). The data were
linearly fitted, and the Kavalue was determined from the slope
and the intercept of the line. The association constant (Ka) for
Cu2+binding in chemosensor 1 was found to be 9.56 109M 2.
The limit of detection for chemosensor 1 as a fluorescent sensor for Cu2+detection was determined from a plot of fluorescence intensity as a function of Cu2+concentration (see Fig. S5 in the ESI†). It was found that chemosensor 1 has a limit of detection of 0.27 mM, which allows for the detection of Cu2+ ions in the
micromolar concentration range.
To gain a clearer understanding of the structure of the Cu2+–12
complexes,1H NMR spectroscopy (Fig. 6) was employed. Cu2+is a paramagnetic ion which affects the NMR resonance frequency of protons that are close to the Cu2+binding site. The1H NMR spectra of chemosensor 1, recorded with increasing amounts of Cu2+, show that the proton signal (Hf, OH) at d = 11.2 ppm almost completely
disappears upon the addition of Cu2+(Fig. 6). The proton signal (He, imine) at d = 8.65 ppm became broader as Cu2+was added,
indicating that Cu2+binds to the nitrogen atom at the imine bond. Other peaks remained unchanged. These observations indicate that Cu2+ binds to chemosensor 1 through one imine nitrogen atom and one hydroxyl oxygen atom.
To elucidate the structure of the Cu2+–1
2complexes, we employed
density functional theory (DFT) calculations using the Gaussian 09 software package. Chemosensor 1 and Cu2+–12 complexes were
subjected to energy optimization using B3LYP/6-31G and B3LYP/ LANL2DZ, respectively. The global minima structure for the Cu2+–12
complex is shown in Fig. 7. The distances of Cu2+from the two oxygen atoms were 1.90 Å and 1.90 Å, and those from the two nitrogen atoms were 2.01 Å and 2.02 Å.
Scheme 1 Synthesis of chemosensor 1.
Fig. 1 Color (top) and fluorescence (bottom) changes in 1 upon the addition of various metal ions in a methanol–water (v/v = 1 : 1, 10 mM HEPES, pH 7.0)
solution.
We performed pH titration of chemosensor 1 to determine a suitable pH range for Cu2+ sensing. In Fig. 8, the emission intensities of metal-free chemosensor 1 at most pH values are high. When the pH is higher than 9, the emission intensities decrease slightly due to deprotonation of the hydroxy group in chemosensor 1. After mixing chemosensor 1 with Cu2+, the emission intensity at 537 nm suddenly decreased in the pH
range of 5.0 to 9.0. When the pH was lower than 5, the emission intensity at 537 nm increased slightly, compared to that at pH 7.0. This is due to protonation on the Schiff base, preventing the formation of the Cu2+–1
2complex.
For a chemosensor to be extensively used in the detection of specific targets, reversibility is an important issue. To explore whether the binding process of chemosensor 1 with Cu2+ is reversible, an excess amount of CN was added into a solution of chemosensor 1 with Cu2+. In Fig. 9, the emission peak at 537 nm increases significantly after addition of CN . When Cu2+was added to the system, the fluorescence of 1 was again quenched. This observation indicates the reversibility of the binding of chemosensor 1 with Cu2+.
2.3 Living cell imaging
Chemosensor 1 was also tested for living cell imaging. First, HeLa cells incubated with 1 displayed a strong fluorescence image (Fig. 10). The overlapping of the fluorescence and bright
Fig. 2 Absorption (top) and emission spectra (bottom) of 1 (10 mM) in
methanol–water (v/v = 1 : 1, 10 mM HEPES, pH 7.0) solution upon the
addition of 0–2.0 mM of Cu2+. The excitation wavelength was 467 nm.
Fig. 3 Fluorescence response of 1 (10 mM) to Cu2+(20 mM) or 20 mM of
other metal ions (the black bar) and to the mixture of other metal ions
(20 mM) with 20 mM of Cu2+(the gray bar) in methanol–water (v/v = 1 : 1,
10 mM HEPES, pH 7.0) solutions.
Fig. 4 Job plot of 1–Cu2+complexes, where the emission intensity at
537 nm is plotted against mole fraction of 1, at a constant total
concen-tration of 2.0 10 5
M in methanol–water (v/v = 1 : 1, 10 mM HEPES, pH 7.0) solutions.
Fig. 5 The plot of chemosensor 1 with Cu2+in methanol/water (v/v = 1 : 1,
10 mM HEPES, pH 7.0) solution. The excitation wavelength was 467 nm and the observed wavelength was 537 nm.
field images reveals that the fluorescence signals are localized in the intracellular area, indicating good cell membrane perme-ability of chemosensor 1. After further treatment with Cu2+, the
fluorescence vanished. These observations demonstrate effec-tive binding of chemosensor 1 with Cu2+.
3 Conclusions
In this study, a coumarin-based fluorescent chemosensor, chemosensor 1, was developed for Cu2+ detection. Significant fluorescence quenching was observed with chemosensor 1 in the presence of Cu2+ions, while other metal ions, e.g. Ag+, Ca2+, Cd2+, Co2+, Fe2+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+caused only minor changes in fluorescence intensity. The optimal pH range for Cu2+detection by chemosensor 1 is from 5–9. This coumarin-based Cu2+chemosensor serves as an effective and non-destructive probe for Cu2+detection in living cells.
4 Experimental section
4.1 Materials and instrumentation
All solvents and reagents were obtained from commercial sources and used as received without further purification. UV/Vis spectra
Fig. 6 1H NMR spectra of chemosensor 1 (10 mM) in the presence of
different amount of Cu2+in DMSO-d6.
Fig. 7 DFT-optimized structures of Cu2+–12 complexes calculated
using the B3LYP/LanL2DZ method. (Red atom, O; blue atom, N; orange atom, Cu).
Fig. 8 Fluorescence response of free chemosensor 1 (10 mM) (m) and
after addition of Cu2+(20 mM) (&) in methanol–water (v/v = 1 : 1, 10 mM
buffer, pH 3–4: PBS; pH 4.5–6: MES; pH 6.5–8.5: HEPES; pH 9–10: Tris-HCl) solution as a function of different pH values. The excitation wavelength was 467 nm.
Fig. 9 Reversibility of the interaction between 1 and Cu2+by the
intro-duction of CN to the system.
Fig. 10 Fluorescence images of HeLa cells treated with 1 and Cu(BF4)2. (left)
Bright field image; (middle) fluorescence image; and (right) merged image.
in 10 mL of methanol, and stirred overnight at room temperature. A red precipitate was formed, and the crude product was filtered, thoroughly washed with methanol to give 1. Yield: 254 mg (70%). Melting point: 196–197 1C;1H-NMR (300 MHz, DMSO-d6): d 11.23
(s, 1H), 8.90 (s, 1H), 8.65 (s, 1H), 8.54 (s, 1H), 7.67 (t, J = 7.5 Hz, 2H), 7.39 (t, J = 6.9 Hz, 1H), 6.96 (t, J = 8.1 Hz, 2H), 6.80 (dd, J = 9.0 Hz, J = 2.1 Hz, 1H), 6.64 (d, J = 1.8 Hz, 1H), 3.48 (q, J = 6.6 Hz, 4H), 1.15 (t, J = 6.6 Hz, 6H); 13C-NMR (125 MHz, DMSO-d 6): d 162.1, 160.4, 158.6, 157.2, 156.8, 152.1, 141.4, 132.9, 131.5, 131.0, 119.5, 118.3, 116.4, 111.1, 110.0, 108.0, 96.4, 44.3, 12.3; IR (KBr): 3417, 2974, 2933, 1709, 1620, 1576 cm 1; MS(EI): m/z (%) = 363 (100), 348 (26.6), 346 (37.8), 243 (38.4), 229 (59.5), 173 (87.4); HRMS (EI): m/z, calcd for C21H21N3O3 (M+): 363.1583; found:
363.1588.
4.3 Metal ion binding study by fluorescence spectroscopy Chemosensor 1 (10 mM) was added with different metal ions (20 mM). All spectra were measured in 1.0 mL methanol–water solution (v/v = 1 : 1, 10 mM HEPES, pH 7.0). The light path length of the cuvette was 1.0 cm.
For pH dependence experiments, the buffers were: pH 3–4: PBS; pH 4.5–6: MES; pH 6.5–8.5: HEPES; pH 9–10: Tris-HCl. The binding stoichiometry of 1–Cu2+complexes was determined by
Job plot experiments. The fluorescence intensity at 537 nm was plotted against molar fraction of 1 at a constant total concen-tration (20 mM) of 1 and Cu2+. The fluorescence approached a minimum intensity when the molar fraction was 0.67. These results indicate that chemosensor 1 forms a 2 : 1 complex with Cu2+. The stability constants Kaof 2 : 1 1–Cu2+complexes were
determined by the equation:31
a2/(1 a) = 1/(2KaCF[M]) (1)
where CFis the total concentration of chemosensor 1 in the
system and a is defined as the ratio between the free chemosensor 1 and the total concentration of chemosensor 1. The value "a" was obtained using eqn (2)
a = [F F0]/[F1 F0] (2)
where F is the fluorescence intensity at 537 nm at any given Cu2+ concentration, F
1is the fluorescence intensity at 537 nm
in the absence of Cu2+, F0is the maxima fluorescence intensity
at 537 nm in the presence of Cu2+. The association constant Ka
was evaluated graphically by plotting a2/(1 a) against 1/[Cu2+]. The plot a2/(1 a) vs. 1/[Cu2+] is shown in Fig. 6. Data were linearly fitted according to eqn (1) and the Ka value was
obtained from the slope of the line.
chemosensor 1 (2 mL; final concentration: 20 mM) dissolved in DMSO and incubated at 37 1C for 30 min. The treated cells were washed with PBS (3 2 mL) to remove any remaining sensor. DMEM (2 mL) was added to the cell culture, which was then treated with a 10 mM solution of Cu(BF4)2 (2 mL; final concentration:
20 mM) dissolved in sterilized PBS (pH = 7.4). The samples were incubated at 37 1C for 30 min. The culture medium was removed, and the treated cells were washed with PBS (3 2 mL) before observation. Confocal fluorescence imaging of cells was performed using 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 467 nm, and emission was collected at 517–557 nm.
4.5 Quantum chemical calculation
Quantum chemical calculations based on density functional theory (DFT) were carried out using a Gaussian 09 program. The ground-state structures of chemosensor 1 and the Cu2+–1
2
complexes were computed using the density functional theory (DFT) method with the hybrid-generalized gradient approximation (HGGA) functional B3LYP. The 6-31G basis set was assigned to nonmetal elements (C, H, N and O). For the Cu2+–12complexes, the
LANL2DZ basis set was used for Cu2+, whereas the 6-31G basis set was used for other atoms.
Acknowledgements
We gratefully acknowledge the financial support of Ministry of Science and Technology (Taiwan) and National Chiao Tung University.
Notes and references
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