2016 Volume 64 Issue 1 Pages 27-33
We report a novel ratiometric and reversible fluorescent probe for Cd2+ detection utilizing a 6-(dimethylamino)quinaldine derivative as the fluorophore and a 2-hydrazinopyridine derivative as Cd2+ chelator. This ratiometric fluorescent probe possesses favorable photophysical properties. It shows a large (55 nm) red-shift from 515 nm to 570 nm in the emission spectrum. Moreover, this probe also exhibits an excellent linear relationship of fluorescence intensity ratio (F570/F515) (R2=0.989) vs. Cd2+ concentration in the range of 0–10 µM at physiological pH, which can serve as a “quantitative detecting” probe for Cd2+. Utilizing this sensitive and selective probe, we have successfully detected Cd2+ in living cells.
Cadmium, one of the most toxic metals, has been widely used in industry and agriculture.1) It can be accumulated by soil, plants and other organisms which will easily lead to serious environment contamination and health problems.2) Excessive exposure to cadmium will cause imbalance in cellular processes, resulting in various diseases including cancers3) and neurodegenerative diseases.4–6) For example, cadmium can induce lipid peroxidation and effect on antioxidant enzyme in bean7); exposure to Cd2+ at concentrations above 1 µM will inhibit DNA synthesis and cell division8); 2.5 µM of CdCl2 exposure on laboratory rats could significantly elevate the prostatic tumour incidence; and a 20 or 40 µM dosage would strongly induce leydig cell tumour incidence.9) Therefore, the development of simple, sensitive and reliable analytical methods for Cd2+ quantification in environmental samples and in the live biological samples is very important and highly desirable.
Several conventional methods such as atomic absorption10) and inductively coupled plasma (ICP) atomic emission spectroscopy11) have been developed for cadmium detection with good sensitivity. However, these methods required special equipment and complicated sample pretreatment, which were unsuitable for utilizing in real-time and in vivo application. Fluorescent probes for detecting metal ions and small molecules appear to have more advantages due to its operational simplicity, cost effectiveness, high sensitivity and selectivity. More importantly, these are suitable for utilizing in living cells or tissues for real-time detection.12) Recently, much attention has been paid to develop fluorescent probes for detection cadmium. To the best of our knowledge, several fluorescent probes for cadmium have been reported.13–18) In these probes, however, their practical application is still limited as their poor water solubility (need more than 30% organic solvent as co-solution) or need complicated synthesis steps to obtain these probes. Many of these probes can not reduce the fluorescence interference of Zn2+ due to the similar coordination properties of Zn2+ and Cd2+.19,20) As a result, only few of them have been utilized for in vivo imaging. Moreover, many of these reported probes detect cadmium via the variety of fluorescent intensity,14,21–26) which limits its application in quantitative analysis of Cd2+ in vivo, as the strong autofluorescence of biomolecules background would lead to low signal-to-noise ratio. Through monitoring the fluorescence intensity ratios at two different wavelengths before and after the coordination event, the target concentration can be quantitatively determined by ratiometric fluorescent probe without the influence of artifacts in principle.27) A few of ratiometric fluorescent probes have been reported recently for Cd2+, but there are still need for complicated synthesis process to obtain them,28–30) which increase the cost in practice. To date, it is still urgently needed to develop novel Cd2+ probes based on ratiometric fluorescent method with more simple synthesis process, high sensitivity and good selectivity for the detection of cadmium in in vitro and in vivo.
Herein, a novel ratiometric Cd2+ probe, NJ1Cd has been designed and synthesized as a 6-dimethylamino substituted quinoline-based Schiff’s base. We utilized 6-dimethylamino group substituted quinoline skeleton as the fluorophore as its favorable photophysical properties, good water solubility and low cytotoxicity.30,31) Meanwhile, 2-hydrazinopyridine was introduced into quinoline skeleton used as Cd2+ chelator which will enhance its sensitivity and selectivity for cadmium detection. Therefore, we successfully obtained a new ratiometric fluorescent chemosensor for cadmium, which displays a large Stocks shift (143 nm, Fig. S1) and exhibits excellent fluorescence sensing ability for Cd2+ in aqueous media and in living cells.
The preparation of the fluorescent probe (NJ1Cd) and the possible coordination mechanism of NJ1Cd toward Cd2+ were shown in Chart S1 and Chart 1, respectively. The synthesis is quite straightforward, NJ1Cd can be easily synthesized by a simple condensation reaction of 6-(dimethylamino)quinaldine-2-carbaldehyde and 2-hydrazinopyridine in EtOH. The final probe was characterized by NMR spectroscopy and mass spectrometry (see the supporting information). Therefore, this simple probe is composed of a 6-dimethylamino group substituted quinoline moiety and a 2-hydrazinopyridine unit. The reason for utilizing 6-dimethylamino substituted quinoline skeleton is that it can function as an efficient fluorophore group and an electron donor in a push–pull system,30) 2-hydrazinopyridine moiety was used as a cadmium receptor and the electron acceptor in the push–pull system that could enhance the affinity of probe for cadmium. Upon addition of cadmium, we anticipate that the potential metal ion binding unit of probe will interact with metal ion that will inhibit the C=N isomerization of this Schiff’s-base fluorescent probe, meanwhile, the intramolecular charge transfer will happen between the electron donor and the electron acceptor within this push–pull system, resulting in a red shift emission in fluorescence spectra,32) which may provide a ratiometric detection of cadmium.
With this novel probe in hand, we first investigated its absorption and emission spectra in phosphate buffered saline (PBS) buffer (pH 7.4, 0.25% dimethyl sulfoxide (DMSO), 25°C). As described in Fig. 1, the free NJ1Cd showed two absorption peaks at 337 nm and 383 nm respectively (Fig. 1a). When titrated by Cd2+ (10 eq.), the 337 nm band decreased gradually accompanied by a red-shift to 349 nm. The 383 nm band also decreased gradually with a red-shift to 425 nm. Free NJ1Cd in PBS buffer displayed two excitation bands centered at 338 nm and 372 nm and one emission band at 515 nm (Fig. S1). Its quantum yield was determined as 0.088 with quinine sulfate in 0.5 M H2SO4 as the reference.33)
Further, we evaluated NJ1Cd (5 µM) with Cd2+ (50 µM) in PBS buffer (pH 7.4) at 25°C for different time. The time-lapse assay showed the complex of NJ1Cd and Cd2+only need 2 min to react completely (Fig. S2). Cd2+ titration leads to the distinct emission red-shift from 515 nm to 570 nm (Fig. 1b). The emission ratio at 570 and 515 nm (F570/F515) increases upon the addition of an increasing amount of Cd2+ (0–60 µM) (Fig. 2a, Fig. S3). Significantly, the ratio enhancement at 570 nm and 515 nm is linear with regard to the concentration of Cd2+ even at low µM level (R2=0.989) (Fig. 2b). Additionally, the limit of detection of NJ1Cd was calculated to be 0.26 µM, which is more sensitive than the previously reported probes (PBQ, 0.566 µM; CYP-1, 3.1 µM; CYP-2, 2.3 µM).18,34) The results indicate that NJ1Cd is sensitive to Cd2+ and can be potentially used to quantitatively detect Cd2+ concentration.
λex=375 nm. Each value is represented as the mean±standard deviation from triplicate measurements.
We then examined the reversibility of the fluorescence response of NJ1Cd for cadmium via adding ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetic acid (NTA) into the detection system (Fig. 3, Fig. S4). After the emission of the fluorescent spectra red-shift caused by the addition of Cd2+, the following treatment with 100 µM EDTA would recover the fluorescent spectra to the level before Cd2+ addition. The second cycle of the sequential addition of the other 50 µM Cd2+, the red-shift response from 515 nm to 570 nm reappeared and then disappeared again while adding another 100 µM EDTA. These results indicated that the fluorescence response of NJ1Cd to cadmium is reversible.
Cd2+ and EDTA were added alternatively into the solution of NJ1Cd (5 µM) in PBS buffer (pH 7.4, 0.25% DMSO). λex=375 nm.
The fluorescence responses of NJ1Cd to various representative metal ions were investigated in PBS buffer. As shown in Fig. S5, a larger red-shift from 515 nm to 570 nm and the obvious enhancement of fluorescent intensity ratio (F570/F515) was observed with addition of Cd2+. However, the addition of other metal ions such as Na+, K+, Ca2+, Mg2+, Al3+, Fe2+, Fe3+, Cr3+, Mn2+, Co2+, Ni2+, Ag+, Cu+, Pb2+, Pd2+, Cu2+ and Hg2+ had almost no changes or little effect on the ratio (F570/F515) of NJ1Cd. More importantly, Zn2+ also only induced a slight change of emission intensity at 515 nm, but it had little effect on the enhancement of the F570/F515 ratio (Fig. 4). Competition experiments were conducted in the presence of Cd2+ mixed with other metal ions in PBS buffer (Fig. S6). Most metal ions showed no significant variation in the ratio fluorescence intensity, whereas Fe2+, Cr3+ and Mn2+ slightly quenched the fluorescence and Co2+, Ni2+, Cu+, Cu2+ quenched the fluorescence. A similar phenomenon was previously reported,35) but the influence of these ions in vivo can be neglected because of their low concentration.36–38) Fluorescent pH titration of NJ1Cd and NJ1Cd/Cd2+ complex showed the relatively stable ratio from pH 6.4 to 8.3, which warrants its application in physiological dtection (Fig. S7).
Excitation: 375 nm. S=free probe.
To understand why the Cd2+-coordinated complex exhibits a red shift in the fluorescence spectra, we have optimized the structures of the first singlet excited state (S1) of NJ1Cd and the Cd2+-coordinated complex with time-dependent density functional theory (TDDFT).39) The optimized structures are shown in Fig. 5. TDDFT calculations have been carried out at the 6–31G(d, p) level, using the M06-2X40) functional in Gaussian 09 package.41) The emission energies of the S1 states of both molecules were calculated with the same functional at the 6–311++G(d, p) level. The results are listed in Table 1.
| System | Wavelength (nm) | Oscillator strength | Assignment |
|---|---|---|---|
| NJ1Cd | 397 | 0.7281 | HOMO–LUMO (98%) |
| The Cd2+-coordinated complex | 537 | 0.3258 | HOMO–LUMO (98%) |
Our calculations show that the S1–S0 transition in NJ1Cd and the Cd2+-coordinated complex are corresponding to the highest occupied molecular orbital (HOMO)—lowest unoccupied molecular orbital (LUMO) transition. For the molecule NJ1Cd, both HOMO and LUMO are quite delocalized on the whole molecule (Fig. 6a). The calculated fluorescence emission wavelength is 397 nm and the oscillator strength is 0.7281. For the Cd2+-coordinated complex, the HOMO is localized around the quinoline fragment and the LUMO is localized around two C=N fragments. The LUMO–HOMO transition is a charge transfer (CT) transition (Fig. 6b). The fluorescence emission wavelength is 537 nm and the oscillator strength is 0.3258. A comparison of fluorescence emission wavelengths in two molecules reveals that the red shift for the fluorescence emission induced by Cd2+ is 140 nm. This result is in qualitative agreement with the observed experimental red shift (60 nm).
Cell cytotoxicity assays were conducted using Hela cells to test the cytotoxicity of NJ1Cd. As shown in Fig. 7, the cell viability remains more than 90% after treated with 5 µM NJ1Cd for 24 h. The result indicated that NJ1Cd is almost no cytotoxicity for long period incubation at low concentration and should be safe for cell imaging.
To further investigate the biological application of NJ1Cd, confocal microscopy experiments were performed. HeLa cells incubated with 5 µM NJ1Cd for 30 min and washed in PBS buffer at 37°C showed clear intracellular fluorescence in the overlap image collected at 500–520 nm and 570–590 nm, indicating that NJ1Cd is cell-permeable (Figs. 8a, d). When the cells were treated with 50 µM CdCl2 at 37°C for 30 min, an obvious fluorescence decrease in the cyan channel and strong fluorescence in the yellow channel was detected, displaying the increased Cd2+ concentration (Figs. 8b, e) but, when the cells treated with 100 µM EDTA and 5 µM NJ1Cd for 30 min followed by adding 50 µM CdCl2, the above phenomenon did not appear, which indicates that EDTA chelates Cd2+ (Figs. 8c, f). The overlap images show the variation of fluorescence intensity when adding CdCl2. The data obtained from HeLa cells demonstrate that NJ1Cd is able to visualize the alteration of intracellular Cd2+ through the overlap images, which is obviously superior to intensity-based images of the sole emission channel.
(a and d) HeLa cells incubated with NJ1Cd (5 µM) for 30 min; (b and e) NJ1CD-stained cells exposed to 50 µM CdCl2 for 30 min; (c and f) HeLa cells incubated with NJ1Cd (5 µM) and EDTA (100 µM) for 30 min, followed by being exposed to 50 µM CdCl2 for 30 min. (a–c) Bright-field images. (d–f) Overlap of fluorescence images with emission collected at 500–520 nm and 570–590 nm. Scale bar: 20 µm.
In conclusion, we have successfully developed a novel quinoline-based ratiometric fluorescent probe, NJ1Cd which can discriminate Cd2+ from other ions based on a large Cd2+-induced emission red shift (55 nm). Moreover, it is a reversible probe which makes it more competitive in practice. NJ1Cd is sensitive to Cd2+ and can be potentially used to quantitatively detect Cd2+ concentration in 2 min. Confocal microscopy experiments showed it is suitable for detecting Cd2+ in vivo. The simple synthesis process of NJ1Cd makes it suitable for practice application. Ongoing efforts are focused on developing more sensitive and selective fluorescent probes for optical imaging Cd2+ in living cells, tissues and animals as well as the cadmium related environmental studies.
All chemicals were purchased from commercial suppliers and used without further purification. The stock solutions of metal ions for fluorescence discrimination were prepared from ZnSO4·7H2O, MnCl2·4H2O, Ca(NO3)2·4H2O, CuSO4·5H2O, MgSO4·7H2O, FeSO4·7H2O, AlCl3, KCl, NaCl, AgNO3, CuCl, CdCl2, CrCl3, PbCl2, NiSO4·6H2O, FeCl3, CoSO4·7H2O, Hg(NO3)2 using deionized water. The 1H-NMR and 13C-NMR spectra were recorded on a Bruker DRX-400 with tetramethylsilane (TMS) as internal standard in CDCl3. High resolution mass spectra was performed by Mass Spectrometry Facility at Nanjing University. Fluorescence measurements were performed on an Hitachi Fluorescence Spectrophotometer F-7000. All absorption measurements were recorded on a Shimadzu UV-2550 spectrophotometer. All imaging experiments were performed on a fixed cell DSU spinning confocal microscope (Olympus). The pH measurements were carried out on a UB-7 pH meter (Denver).
General Procedure for AnalysisA stock solution of NJ1Cd (5 mM) was prepared in DMSO, which then diluted to the required concentration for measurement. The stock solutions of various cations were prepared with double distilled H2O. All fluorescence measurements were carried out at room temperature. The samples were excited at 375 nm with the excitation and emission slit widths set at 5.0 nm. The emission spectrum was scanned from 385 nm to 700 nm at 1200 nm/min. The photomultiplier voltage was set at 550 V.
Cytotoxicity Assays5-Dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously reported to test the cytotoxic effect of the probe in cells.42) Hela cells were passed and plated to ca. 70% confluence in 96-well paltes 24 h before treatment. Prior to NJ1Cd treatment, Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum was removed and replaced with fresh DMEM, and aliquots of NJ1Cd stock solutions (5 mM DMSO) were added to obtain final concentrations of 5, 10, 30 µM respectively. The treated cells were incubated for 24 h at 37°C, under 5% CO2. Subsquently, cells were treated with 5 mg/mL MTT and incubated for an additional 4 h (37°C, 5% CO2). Then the cells were dissolved in DMSO, and the absorbance at 570 nm was recorded. The cell viability (%) was calculated according to the following equation: Cell viability%=OD570 (sample)/OD570 (control)×100, where OD570 (sample) represents the optical density of the wells treated with various concentrations of NJ1Cd and OD570 (control) represents that of the wells treated with DMEM plus 10% fetal bovine serum. The present cell survival values is related to untreated control cells.
Cell Culture and Fluorescence ImagingHeLa cells were grown up in DMEM medium with 10% fetal bovine serum/penicillin/streptomycin in a 5% CO2 atmosphere at 37°C. Cells were then seeded on Coverglass-Bottom confocal dish and continuously incubated at 37°C in a 5% CO2 atmosphere for 24 h. After removing the incubation media and rinsed with 1×PBS for three times, the cells were stained with NJ1Cd by incubating the cells in 5 µM NJ1Cd solution with or without 100 µM EDTA for 30 min, respectively. Then the cells were washed three times with PBS and imaged with confocal microscope. For the imaging of HeLa cells with exogenous Cd2+, the exogenous Cd2+ was introduced by incubating the cells with 50 µM CdCl2 solution for another 0.5 h (prepared by diluting 50 mM CdCl2 stock solution with 1× PBS). The cells were rinsed with 1× PBS and imaged. The confocal imaging was carried out with laser confocal microscope with a 60× oil-immersion objective using a dual-channel mode excited at 405 nm, and the band paths are 500–520 and 570–590 nm, respectively. The imaging of HeLa cells was finished by Olympus FV10-ASW, and captured using Slidebook software. For all experiments, solution of NJ1Cd was prepared in DMSO (5 mM) and diluted into DMEM to the desired working concentration (5 µM, 0.1% DMSO). NJ1Cd was diluted into DMEM to the desired working concentrations (50 µM) from a 5 mM stock solution.
Synthesis of NJ1CdDimethyl-[2-(pyridin-2-yl-hydrazonomethyl)-quinolin-6-yl]-amine6-(Dimethylamino)quinaldine-2-carbaldehyde was prepared according to the reported procedures.43) 2-Hydrazinopyridine was purchased from J&K Scientific Ltd.
To a stirred solution of 6-(dimethylamino)quinaldine-2-carbaldehyde (90 mg, 0.45 mmol) in ethanol (30 mL) was added 2-hydrazinopyridine (63 mg, 0.58 mmol). The reaction mixture was refluxed for 7 h. After the mixture was cooled to room temperature, the solvent was removed by evaporation in vacuo. The resulting residue was purified by flash column chromatography on silica gel to afford pure NJ1Cd as a yellow powder (28 mg, 21%) (petroleum ether–EtOAc=1 : 2, v/v. Rf=0.5). 1H-NMR (400 MHz, CDCl3) δ: 8.90 (s, 1H), 8.21 (dd, J=4.9, 0.9 Hz, 1H), 8.05 (d, J=8.7 Hz, 1H), 8.01 (s, 1H), 7.98–7.89 (m, 2H), 7.71–7.62 (m, 1H), 7.47 (t, J=10.2 Hz, 1H), 7.40–7.32 (m, 1H), 6.89–6.76 (m, 2H), 3.12 (d, J=6.3 Hz, 6H). 13C-NMR (100 MHz, CDCl3) δ: 156.32, 150.18, 148.80, 147.69, 141.62, 140.51, 138.16, 134.20, 129.70, 129.57, 119.17, 118.06, 116.16, 107.59, 105.17, 40.63. High resolution (HR)-MS (time-of-flight (TOF)-MS-electron ionization (EI)+): Calcd for [M]+ 291.1484. Found 291.1478.
This work is financially supported by Grants of the National Natural Science Foundation of China (21302094), Jiangsu Province large scientific instruments shared services platform (BZ201307), the Jiangsu Natural Science Foundation (BK20130552), and the Research Fund for the Doctoral Program of Higher Education of China (20130091120036).
The authors declare no conflict of interest.
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