- Email: [email protected]
A visible-light-excited europium(III) complex-based luminescent probe for visualizing copper ions and hydrogen sulfide in living cells
Optical Materials 75 (2018) 243e251
Contents lists available at ScienceDirect
Optical Materials journal homepage: www.elsevier.com/locate/optmat
A visible-light-excited europium(III) complex-based luminescent probe for visualizing copper ions and hydrogen sulfide in living cells Yiren Wang a, Huan Wang a, Mei Yang a, Jingli Yuan b, Jing Wu a, * a b
School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian, 116029, China State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, 116024, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2017 Received in revised form 19 October 2017 Accepted 21 October 2017
Development of visible-light-excited lanthanide (III) complex-based luminescent probes is highly appealing due to their superiority of less damage to the living biosystems over the conventional UVlight-excited ones. In this work, a visible-light-excited europium (III) complex-based luminescent probe, BPED-BHHCT-Eu3þ-BPT, has been designed and synthesized by conjugating the Cu2þ-binding N,Nb-diketone ligand 4,40 bis(2-pyridylmethyl)ethanediamine (BPED) to a tetradentate 00 00 00 00 00 00 00 00 00 00 bis(1 ,1 ,1 ,2 ,2 ,3 ,3 -heptafluoro-4 ,6 -hexanedione-6 -yl)chlorosulfo-o-terphenyl (BHHCT) and coordinating with a coligand 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine) (BPT) for the time-gated luminescence detection of Cu2þ ions and hydrogen sulfide (H2S) in living cells. BPED-BHHCTEu3þ-BPT exhibited a sharp excitation peak at 407 nm and a wide excitation window extending to beyond 460 nm. Upon its reaction with Cu2þ ions, the luminescence of BPED-BHHCT-Eu3þ-BPT was efficiently quenched, which could be reversibly restored by the addition of H2S due to the strong affinity between Cu2þ ions and H2S. The “on-off-on” type luminescence behavior of BPED-BHHCT-Eu3þ-BPT towards Cu2þ ions and H2S enabled the sensing of the two species with high sensitivity and selectivity. The performances of BPED-BHHCT-Eu3þ-BPT for visualizing intracellular Cu2þ ions and H2S were investigated, and the results have demonstrated the practical applicability of the probe for molecular imaging of cells. © 2017 Published by Elsevier B.V.
Keywords: Europium(III) complex Visible-light-excitation Luminescent probe Copper ions Hydrogen sulfide
1. Introduction Trace elements (e.g., Mn, Fe, Co, Ni, Cu, Zn, Se, Mo, I) and macroelements (e.g., Na, Mg, K, Ca, P, Cl, S) are essential to support various biochemical processes for human life [1]. As an example of trace elements, copper ion (Cu2þ) plays an important role in various physiological processes of organism, such as haemopoiesis, iron absorption, and enzyme-catalyzed and redox reactions [2]. As a typical macroelement, sulfur is mainly contained in amino acid (cysteine, methionine, etc.) and vitamin (biotin, thiamine, etc.). Currently, hydrogen sulfide (H2S), generated from cysteine or homocysteine in biosystems [3,4], has been considered as the third endogenous signaling gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO) [5]. H2S also plays critical roles in diverse physiological processes including apoptosis, blood pressure regulation, neuromodulation, and inhibition of insulin signaling
* Corresponding author. E-mail address: [email protected] (J. Wu). https://doi.org/10.1016/j.optmat.2017.10.030 0925-3467/© 2017 Published by Elsevier B.V.
[6,7]. Increasing evidences have shown that the abnormal amounts of Cu2þ ions and H2S will bring about health problems [8]. For example, excess Cu2þ ions can disrupt protein activity and induce tissue necrosis [9], which is related to Parkinson's disease [10] and Wilson's disease [11]. The abnormal production of H2S is also associated with many diseases such as diabetes [12], chronic kidney disease [13], hypertension [14], and Alzheimer's disease [3]. Therefore, methods for the detection of Cu2þ ions and H2S in biosystems, especially in living cells, are highly demanded for their physiological and pathological research [15]. To date, several traditional analytical methods have been adopted to detect Cu2þ ions and H2S in various samples, including colorimetry [16], UVevis spectroscopy [17] and fluorescence spectrometry [18] for Cu2þ ions, and electrochemistry [19], chemiluminescence [20], gas chromatography [21] and sulfide precipitation [22] for H2S. These methods usually require post-mortem and destruction of samples, which are not suitable for the realtime monitoring of Cu2þ ions and H2S in living biosystems. In addition, the low concentration of Cu2þ ions and high activity of H2S also limit the usability of these methods. Recently, the optical
244
Y. Wang et al. / Optical Materials 75 (2018) 243e251
imaging technique with the use of luminescent probes provides an appropriate approach for in vivo detecting biomolecules due to its high sensitivity, high spatial resolution, and non-destructive detection [23,24]. A number of luminescent probes for Cu2þ ions have been designed utilizing its strong luminescence quenching ability [18,25,26]. Meanwhile, various luminescent probes for H2S have also been developed based on its special properties such as strong reducing ability [27,28], quencher (e.g., Cu2þ, Hg2þ) removal [29,30] and nucleophilic reaction [31,32]. However, the major drawback of these organic fluorophore-based or ruthenium(II) complex-based probes is their susceptibility to photobleaching and strong biological autofluorescence. As an improvement, lanthanide(III) complex-based luminescent probes (mainly Eu3þ and Tb3þ complexes) have attracted great interest due to their ideal luminescent properties including large Stokes shift, long luminescence lifetime, sharp emission profile and good photostability [33,34]. In particular, the super long-lived luminescence enables their usages in the time-gated detection technique, which can effectively eliminate the autofluorescence interference and contribute to improve the sensitivity and selectivity to the target species in complicated biosystems [35,36]. A few lanthanide(III) complex-based luminescent probes have been reported for the detection of Cu2þ ions [37,38] and H2S [38,39]. However, a key problem of all these lanthanide(III) complex-based probes is that their excitation windows are limited to the UV region (<380 nm), which will cause excitation phototoxicity to some UV light-sensitive living biosamples and be unfavorable for the photostability of probes. Therefore, the development of visible-lightexcited lanthanide complex(III)-based probes is urgently required for the pursuit of their wider bioassay applications. Herein, we developed a unique visible-light-excited Eu3þ complex, BPED-BHHCT-Eu3þ-BPT, in which the ligand BPED-BHHCT was synthesized by conjugating a Cu2þ-binding moiety N,N-bis(2pyridylmethyl)ethanediamine (BPED) to a tetradentate b-diketone 4,40 -bis(100,100,100,200 ,200 ,300 ,300 -heptafluoro-400 ,600 -hexane- dione-600 -yl) chlorosulfo-o-terphenyl (BHHCT), and the coligand 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine (BPT) was introduced to achieve the visible-light excitation of the Eu3þ complex. BPED-BHHCT-Eu3þ-BPT exhibited a strong visible excitation peak at 407 nm, whose corresponding fluorescence emission at 611 nm could be selectively quenched by Cu2þ ions due to the coordination of Cu2þ with the BPED moiety and the resulting intramolecular photo-induced electron transfer (PET). The trapped BPED group could be released by the strong affinity between the coordinated Cu2þ and H2S, which resulted in the formation of CuS precipitate and restored the luminescence of the quenched probe. The decrease of luminescence intensity at 611 nm was linearly correlated with the Cu2þ concentration, and the reverse luminescence enhancement also showed a good linearity against the H2S concentration. Therefore, BPED-BHHCT-Eu3þ-BPT can be used as a luminescent probe for the time-gated luminescence detection of Cu2þ ions and H2S in aqueous media. To examine the applicability of the new probe for bioimaging, the BPED-BHHCT-Eu3þ-BPTloaded HepG2 cells were prepared, and the exogenous Cu2þ ions and H2S in the cells were imaged on a true-color time-gated luminescence microscope. The results demonstrated the practical utility of the new probe for visualizing intracellular Cu2þ ions and H2S. Scheme 1 shows the structure of BPED-BHHCT-Eu3þ-BPT and its luminescence response reaction with Cu2þ ion and H2S. 2. Experimental 2.1. Materials and physical measurements The tetradentate b-diketone BHHCT [40], BPT [41] and BPED [42]
were synthesized according to the previously reported methods. Triton X-100 was purchased from Acros Organics. Dichloromethane (CH2Cl2) was used after appropriate distillation and purification. Cultured HepG2 cells were obtained from Dalian Medical University. The isotonic saline solution consisting of 140 mmol NaCl, 10 mmol glucose and 3.5 mmol KCl was prepared in our laboratory. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification. 1 H NMR spectrum was measured on a Bruker Avance spectrometer (400 MHz). Mass spectrum was recorded on a HP1100LC/ MSD electrospray ionization mass spectrometer (ESI-MS). Elemental analysis was carried out on a Vario-EL analyser. Absorption spectra were measured on a Perkin-Elmer Lambda 35 UVevis spectrometer. Time-gated luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer with the following conditions: delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation slit, 10 nm; and emission slit, 10 nm. Luminescence lifetimes were measured on an FS5 spectrofluorometer of Edinburgh Instruments. Relative luminescence quantum yields of BPED-BHHCT-Eu3þ-BPT and its reaction product with Cu2þ, Cu2þ-BPED-BHHCT-Eu3þ-BPT, were measured with the Eu3þ complex, Eu3þ-(40 -phenyl-2,20 :60 ,200 -terpyridine-6,600 -diyl) bis-(methylene-nitrilo)tetrakis(acetate), as a reference (F ¼ 0.16) [43]. All bright-field and luminescence imaging measurements were carried out on a laboratory-use true-color time-gated luminescence microscope [44].
2.2. Synthesis of BPED-BHHCT BPED (35 mg, 0.12 mmol) was dissolved in dichloromethane CH2Cl2 (6 mL), and then BHHCT (100 mg, 0.12 mmol), triethylamine (0.15 mL) and dimethylaminopyridine (2.9 mg, 0.024 mmol) were added to the solution. The reaction mixture was stirred at room temperature in the dark for 5 days. After the solvent was evaporated, the residue was washed with diluted hydrochloric acid (1.0 M, 80 mL), and then dissolved in CH2Cl2 (200 mL). The CH2Cl2 solution was washed three times with 120 mL of water and dried with Na2SO4. After evaporation, the ligand BPED-BHHCT was obtained as yellow solid (120.5 mg, 89% yield). 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 1.25 (s, 1H), 1.80 (m, 4H), 3.98e4.01 (d, 4H; J ¼ 12.0 Hz), 6.56 (d, 2H; J ¼ 5.2 Hz), 7.04 (m, 4H), 7.23e7.21 (d, 4H; J ¼ 8.0 Hz), 7.77e7.86 (d, 8H; J ¼ 12.0 Hz), 8.61 (m, 4H). ESI-MS (m/ z): 1011.3 ([M þ H]þ, 100%, calcd 1011.2). Elemental analysis calcd (%) for C44H33F14N4O6.5S (BPED-BHHCT$0.5H2O): C, 51.82; H, 3.26; N, 5.49; found (%): C, 52.28; H, 3.19; N, 5.54.
2.3. Preparation of the stock solution of BPED-BHHCT-Eu3þ-BPT The stock solution of the complex BPED-BHHCT-Eu3þ-BPT was prepared by in situ mixing equiv of BPED-BHHCT (0.5 mmol), BPT (1 mmol) and EuCl3$6H2O (0.5 mmol) in ethanol. The obtained stock solution was stored at 4 C and properly diluted with 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 before use.
2.4. Luminescence response of BPED-BHHCT-Eu3þ-BPT towards various metal ions The reactions of BPED-BHHCT-Eu3þ-BPT with various metal ions were performed in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 with the same concentration of BPED-BHHCT-Eu3þBPT. The solutions were stirred at room temperature for 30 min, and then subjected to time-gated luminescence measurements.
Y. Wang et al. / Optical Materials 75 (2018) 243e251
245
Scheme 1. Reaction mechanism for the luminescence response of BPED-BHHCT-Eu3þ-BPT towards Cu2þ ion and H2S.
2.5. Luminescence response of Cu2þ-BPED-BHHCT-Eu3þ-BPT towards various anions The Cu2þ-BPED-BHHCT-Eu3þ-BPT solution was prepared by reacting BPED-BHHCT-Eu3þ-BPT (1 mM) with Cu2þ ions (2 mM) at room temperature for 30 min in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. After the addition of various anions, the solutions were stirred for another 45 min, and then subjected to time-gated luminescence measurements. 2.6. Luminescence imaging of Cu2þ ions and H2S in HepG2 cells HepG2 cells, cultured on a 35 mm glass-bottom culture dish (f 20 mm) in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin, were washed three times with the isotonic saline solution, and then incubated with the isotonic saline solution containing BPED-BHHCT-Eu3þ-BPT (5 mM) and 0.001% cremophor C40 for 1 h. After being washed three times with the isotonic saline solution, the BPED-BHHCT-Eu3þ-BPTloaded cells were further incubated with different concentrations of Cu2þ ions (1 and 5 mM) and cremophor C40 (0.001%) for 30 min. The Cu2þ-BPED-BHHCT-Eu3þ-BPT-loaded cells were washed three times with the isotonic saline solution, and then subjected to luminescence imaging measurements on the microscope (excitation filter, 380e420 nm; dichroic mirror, 400 nm; emission filter, >590 nm). The time-gated luminescence imaging measurements were carried out with the following conditions: delay time, 33 ms;; gate time, 1.0 ms; exposure time, 1.1 s. The steady-state luminescence imaging measurements were carried out with an exposure time of 1.1 s. The Cu2þ-BPED-BHHCT-Eu3þ-BPT-loaded cells, prepared by incubating the BPED-BHHCT-Eu3þ-BPT-loaded cells in the isotonic
saline solution containing Cu2þ ions (15 mM) and cremophor C40 (0.001%) for 30 min, were washed three times with the isotonic saline solution, and then incubated with different concentrations of H2S (50 and 100 mM) and cremophor C40 (0.001%) for another 45 min. After being washed three times with the isotonic saline solution, the cells were subjected to luminescence imaging measurements with the above-mentioned conditions. 2.7. MTT assay The cytotoxicity of BPED-BHHCT-Eu3þ-BPT to HepG2 cells was measured by the MTT assay [45]. HepG2 cells, cultured in RPMI1640 medium, were washed with the isotonic saline solution and then incubated with the isotonic saline solution containing different concentrations of BPED-BHHCT-Eu3þ-BPT at 37 C in a 5% CO2/95% air incubator for 24 h. The culture solutions were removed, and the cells were further incubated in the isotonic saline solution containing 455 mg/mL of MTT for another 4 h. After removal of the supernatants, the cells were dissolved in DMSO (100 mL), and then the absorbances at 490 nm were measured on an Infinite M200 Pro microplate reader. 3. Results and discussion 3.1. Design, synthesis and characterization of the Eu3þ complexbased probe To date, although several lanthanide(III) complex-based luminescent probes for Cu2þ ions and H2S have been developed, the optical windows of these probes are limited to the UV region. It is desirable to extend the excitation wavelength towards the visible region to minimize the effects of excitation phototoxicity on the
246
Y. Wang et al. / Optical Materials 75 (2018) 243e251
Scheme 2. Reaction pathway for the synthesis of BPED-BHHCT-Eu3þ-BPT.
living biosamples. Recently, a few visible-light-excited ternary Еu3þ complexes have been synthesized by introducing a dipyrazolyltriazine-derivative coligand to a Еu3þ-b-diketonate complex [33,41,46]. On the basis of these findings, it is greatly possible for the design of visible-light-excited Еu3þ complex-based luminescent probes by coordinating a dipyrazolyltriazine derivative to the Еu3þ-b-diketonate complex that is preferentially modified with an analyte-recognition moiety. In this work, by the modification of a tetradentate b-diketone ligand BHHCT with a Cu2þ-specifically reactive moiety BPED and the common coordination of BPT, we designed a novel visible-lightexcited Eu3þ complex-based luminescent probe, BPED-BHHCTEu3þ-BPT, for detecting Cu2þ ions and H2S. The ligand BPED-BHHCT and the complex BPED-BHHCT-Eu3þ-BPT were synthesized by the reaction procedures as shown in Scheme 2 and confirmed by 1H NMR, ESI-MS and elemental analyses (Fig. S1eS2 in Supplementary material). To reveal the coordination relationship between Eu3þ and the ligands (BPED-BHHCT and BPT), the Job's plotting analyses were carried out in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2%
Triton X-100. Firstly, the binding stoichiometry of BPT to Eu3þ was explored. As shown in Fig. 1A, the Job's plot exhibited a maximum value at approximate 0.66 molecular fraction, which proves that the reaction between Eu3þ and BPT has a coordination ratio of 1:2. Then the binding stoichiometry of BPED-BHHCT to Eu(BPT)2 was evaluated. As shown in Fig. 1B, the Job's plot showed a maximum value at 0.5 molecular fraction, indicating that the reaction between BPED-BHHCT and Eu(BPT)2 has a coordination ratio of 1:1. These results demonstrate that the main species of the probe in aqueous solution at micromolar concentrations should be Eu(BPED-BHHCT) (BPT)2. The photophysical properties of BPED-BHHCT-Eu3þ-BPT and its reaction product with Cu2þ, Cu2þ-BPED-BHHCT-Eu3þ-BPT, were investigated systematically in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 (Table S1 in Supplementary material and Fig. 2). As shown in Fig. 2B, BPED-BHHCT-Eu3þ-BPT and Cu2þ-BPED-BHHCT-Eu3þ-BPT exhibited the same excitation spectrum profiles with two maximum excitation wavelengths at 337 nm and 407 nm, which agreed well with their absorption peaks
Fig. 1. (A) Job's plot of the reaction between BPT and Eu3þ in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 (the total concentration of BPT and Eu3þ was kept at 10 mM); (B) Job's plot of the reaction between Eu(BPT)2 and BPED-BHHCT in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 (the total concentration of Eu(BPT)2 and BPED-BHHCT was kept at 5 mM).
Y. Wang et al. / Optical Materials 75 (2018) 243e251
247
Fig. 2. (A) UVevis absorption spectra of BPED-BHHCT-Eu3þ-BPT (8 mM, red line) and Cu2þ-BPED-BHHCT-Eu3þ-BPT (8 mM, black line) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100; (B) Time-gated excitation (lem ¼ 611 nm) and emission (lex ¼ 407 nm) spectra of BPED-BHHCT-Eu3þ-BPT (1 mM, red line) and Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM, black line) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effects of pH on the luminescence intensities of BPED-BHHCT-Eu3þ-BPT (a) and Cu2þ-BPED-BHHCT-Eu3þ-BPT (b).
(Fig. 2A). Under the light excitation of 407 nm, the two Eu3þ complexes displayed a typical Eu3þ emission pattern with a main peak at approximate 611 nm (5D0/7F2 transition of Eu3þ ion) (Fig. 2B). There was no remarkable difference for the molar absorption coefficients of the two complexes, which were determined to be 5.8 104 cm1 M1 and 5.6 104 cm1 M1 for BPED-BHHCTEu3þ-BPT and Cu2þ-BPED-BHHCT-Eu3þ-BPT, respectively. However, it is noteworthy that BPED-BHHCT-Eu3þ-BPT was strongly luminescent with a wide excitation window extending beyond 460 nm, a high quantum yield (F ¼ 61.3%) and a long luminescence lifetime (t ¼ 560 ms)). After the reaction with Cu2þ, the luminescence of BPED-BHHCT-Eu3þ-BPT was efficiently quenched with a 125-fold decrease in luminescence quantum yield (F ¼ 0.49%) and a more than 2-fold decrease in luminescence lifetime (t ¼ 240 ms)). The luminescence quenching of BPED-BHHCT-Eu3þ-BPT by Cu2þ ions was attributed to the intramolecular photo-induced electron transfer (PET) process from the b-diketone-Eu3þ-BPT moiety to the BPED-Cu2þ moiety [47e49], which interrupted the luminescence emission of Eu3þ ion. These results suggest that BPED-BHHCT-Eu3þBPT can be used as a turn-off luminescent probe for the detection of Cu2þ ions. To investigate the effects of pH on the reaction of the probe with Cu2þ, the luminescence intensities of BPED-BHHCT-Eu3þ-BPT and Cu2þ-BPED-BHHCT-Eu3þ-BPT were determined in 0.05 M Tris-HCl
Fig. 4. (A) Time-gated excitation (lem ¼ 611 nm) and emission (lex ¼ 407 nm) spectra of BPED-BHHCT-Eu3þ-BPT (1 mM) in the presence of different concentrations of Cu2þ in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. The concentrations of Cu2þ are 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, 2.0, 2.3, 2.5, 2.7 and 3 mM; (B) Dose-dependent luminescence response of BPED-BHHCT-Eu3þ-BPT (1 mM) to Cu2þ.
248
Y. Wang et al. / Optical Materials 75 (2018) 243e251
buffer containing 0.2% Triton X-100 with different pHs. As shown in Fig. 3, the luminescence intensity of BPED-BHHCT-Eu3þ-BPT had a small change in the range of pH 5e8 with a maximum value at pH 7.5, and a distinct luminescence decrease was observed at pH < 5 or pH > 8. The pH-dependent luminescence enhancement of BPEDBHHCT-Eu3þ-BPT in acidic pH range might be due to the structural transformation of the b-diketone moiety and the deprotonation of the BPED moiety in the complex, while the pH-dependent luminescence decline in basic pH range is considered to be attributed to the hydrolysis of Eu3þ. However, in the buffers with a pH range of 3e10, the luminescence intensity of Cu2þ-BPED-BHHCT-Eu3þ-BPT was almost pH-independent. Thus, BPED-BHHCT-Eu3þ-BPT can be used as an efficient probe in weakly acidic, neutral and weakly basic buffers. 3.2. Quantitative detection of Cu2þ ions and H2S in aqueous media Fig. 5. Luminescence intensity responses of BPED-BHHCT-Eu3þ-BPT (1 mM) to various metal ions (5 mM) in the presence and absence of Cu2þ (3 mM) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. Metal ions: (a) Cd2þ, (b) Mn2þ, (c) Zn2þ, (d) Ca2þ, (e) Mg2þ, (f) Fe3þ, (g) Co2þ, (h) Agþ, (i) Ni2þ, (j) Pb2þ, (k) Fe2þ, (l) Kþ, (m) Naþ, (n) blank.
To evaluate the applicability of BPED-BHHCT-Eu3þ-BPT as a time-gated luminescence probe for the quantitative detection of Cu2þ ions, the time-gated excitation and emission spectra of the probe in the presence of different concentrations of Cu2þ ions were
Fig. 6. (A) Time-gated excitation (lem ¼ 612 nm) and emission (lex ¼ 407 nm) spectra of Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM) in the presence of different concentrations of H2S in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. The H2S concentrations are 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mM; (B) Dose-dependent luminescence response of Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM) to H2S.
Fig. 7. Luminescence intensity responses of Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM) to various anions (100 mM) in the presence and absence of sulfide ion (100 mM) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. Anions: (a) H2PO 4 , (b) HCO3 , (c) 3 2 4 B2O2 7 , (d) F , (e) CH3COO , (f) P7O10 , (g) Cl , (h) CO3 , (i) I , (j) NO2 , (k) NO3 , (l) P2O7 , 2 2 (m) ClO 3 , (n) SO4 , (o) S2O3 , (p) blank.
recorded in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. As shown in Fig. 4A, upon the reaction with different concentrations of Cu2þ ions, the luminescence intensities of the probe gradually decreased up to a 44-fold luminescence quenching. By plotting the luminescence intensity change against Cu2þ concentration, a good linearity correlation that can be expressed as I0I ¼ 342.8[Cu2þ]þ22.2 (I0 and I are the luminescence intensities of the probe in the absence and presence of Cu2þ, respectively) was obtained in the range of 0.2e2.0 mM (Fig. 4B). The detection limit for Cu2þ ions, calculated as the concentration corresponding to triple standard deviations of the background signal, is 0.038 mM. The luminescence response specificity of BPED-BHHCT-Eu3þBPT towards Cu2þ ions was also examined. Fig. 5 shows the luminescence intensities (611 nm) of BPED-BHHCT-Eu3þ-BPT after reacting with Cu2þ and other metal ions in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. Upon its reaction with 5.0 equiv of metal ions, such as Fe3þ, Co2þ, Agþ, Ni2þ, Pb2þ, and those that exist at much higher cellular concentrations including Naþ, Kþ, Mg2þ and Ca2þ, the luminescence intensity of the probe showed a negligible change. Although an obvious luminescence intensity change was produced upon addition of 5.0 equiv of Zn2þ, Cd2þ,
Y. Wang et al. / Optical Materials 75 (2018) 243e251
Mn2þ and Fe2þ, whereas a nearly 100% luminescence quenching was observed after the probe reacted with 3.0 equiv of Cu2þ. Furthermore, it is notable that all the chosen metal ions had almost no influences on the luminescence response of the probe to Cu2þ ions. These results demonstrate the highly specific response of BPED-BHHCT-Eu3þ-BPT towards Cu2þ ions. Due to the strong affinity between H2S and Cu2þ, the coordinated Cu2þ in the complex Cu2þ-BPED-BHHCT-Eu3þ-BPT would be captured by H2S to form CuS precipitate, and thus Cu2þ-BPEDBHHCT-Eu3þ-BPT could be a time-gated luminescence probe for H2S. Fig. 6A shows the time-gated excitation and emission spectra of Cu2þ-BPED-BHHCT-Eu3þ-BPT in the presence of different concentrations of H2S in the buffer. Upon the addition of H2S, the luminescence intensities (612 nm) of Cu2þ-BPED-BHHCT-Eu3þ-BPT gradually increased up to a 35-fold luminescence enhancement. Moreover, the emission enhancement of the Cu2þ-BPED-BHHCTEu3þ-BPT solution showed an excellent linear relationship in the H2S concentration range of 1e50 mM, which can be expressed as II0 ¼ 3.96[H2S]þ2.02 (Fig. 6B, I and I0 are the luminescence intensities of Cu2þ-BPED-BHHCT-Eu3þ-BPT in the presence and absence of H2S, respectively). The detection limit for H2S is 0.22 mM, which is 1.9 times lower than that of the reported dipicolylamine (DPA)-Cu(II) containing fluorescein sensor for H2S [50]. Thus, a high sensitivity can be achieved using Cu2þ-BPED-BHHCT-Eu3þ-BPT as a time-gated probe for the H2S quantification. The luminescence response selectivity of Cu2þ-DPA-BHHCT3þ Eu -BPT towards H2S was investigated in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. As shown in Fig. 7, the luminescence intensity of Cu2þ-BPED-BHHCT-Eu3þ-BPT did not respond 2 to 100 M equiv of various anions including SO2 4 , S2O3 , H2PO4 , 3 2 2 P2O4 7 , P7O10 , HCO3 , CO3 , NO3 , NO2 , F , Cl , I , ClO3 , B2O7 , CH3COO, whereas a remarkable luminescence enhancement was observed in the presence of H2S. In addition, the competition experiment results showed that more than 85% luminescence intensity revivals were achieved in the presence of H2S and any
249
chosen interfering anion (100 molar equiv). These results indicate that Cu2þ-BPED-BHHCT-Eu3þ-BPT has great potential to be used for the sensing of H2S in biosystems. 3.3. Luminescence imaging detection of Cu2þ ions and H2S in living cells Before investigating the sensing behavior of BPED-BHHCT-Eu3þBPT to intracellular Cu2þ ions and H2S, the toxicity of BPED-BHHCTEu3þ-BPT was examined by using the MTT assay method [44]. As shown in Fig. S3, the cell viabilities still remained at nearly 100% after incubation with different concentrations of BPED-BHHCTEu3þ-BPT for 24 h, which demonstrated that the cytotoxicity of BPED-BHHCT-Eu3þ-BPT was very low. The feasibility of BPED-BHHCT-Eu3þ-BPT for the detection of 2þ Cu ions and H2S in living HepG2 cells was further investigated. As shown in Fig. 8, after incubating with BPED-BHHCT-Eu3þ-BPT (5 mM), the cells exhibited bright red steady-state and time-gated luminescence, indicating that the probe could enter into the cells by an ordinary co-incubation method. After the BPED-BHHCT-Eu3þBPT-loaded HepG2 cells were treated with Cu2þ ions, the intracellular red signals were greatly weakened with the increase of Cu2þ concentration and completely disappeared till the Cu2þ concentration reaching to 15 mM, which confirmed the formation of Cu2þBPED-BHHCT-Eu3þ-BPT inside the cells. It is also clearly shown that the time-gated luminescence images of the cells displayed higher sensitivity and selectivity than the steady-state ones because the strong blue autofluorescence from the cells was thoroughly eliminated by the time-gated detection mode. After the cells were incubated with BPED-BHHCT-Eu3þ-BPT (5 mM) for 1 h and Cu2þ ions (15 mM) for another 30 min, the Cu2þ-BPED-BHHCT-Eu3þ-BPTloaded cells were further incubated with different concentrations of H2S, and remarkable intracellular red luminescence enhancement was observed with the increased H2S. These results demonstrate that BPED-BHHCT-Eu3þ-BPT can be easily transferred into
Fig. 8. Bright-field (above), steady-state (middle), and time-gated (below) luminescence images of Cu2þ ions and H2S in HepG2 cells. The cells were incubated with BPED-BHHCTEu3þ-BPT (5 mM) for 1 h (A), and then with 1 mM (B) and 5 mM (C) of Cu2þ ions for 30 min. The cells were incubated with BPED-BHHCT-Eu3þ-BPT (5 mM) for 1 h and Cu2þ ions (15 mM) for another 30 min, and then with 50 mM (D) and 100 mM (E) of H2S for 45 min. Scale bar: 10 mm.
250
Y. Wang et al. / Optical Materials 75 (2018) 243e251
living cells for specifically visualizing intracellular Cu2þ ions and H2S. 4. Conclusion In summary, by incorporating a BPED binding moiety and a dipyrazolyltriazine-derivative coligand into a Eu3þ-b-diketonate complex, a unique visible-light-excited Eu3þ complex-based luminescent probe, BPED-BHHCT-Eu3þ-BPT, has been successfully developed for the time-gated luminescence detection of Cu2þ ions and H2S in living cells. This probe can specifically recognize Cu2þ ions, resulting from remarkable luminescence quenching, which can be reversibly restored upon the addition of H2S. Compared with the previously reported luminescent probes for Cu2þ ions and H2S, the new probe has the advantages of visible excitation wavelength, wide excitation window, high quantum yield, high sensitivity and selectivity, and the applicability for the time-gated measurement. The results of luminescence imaging by monitoring Cu2þ ions and H2S in living cells demonstrated the utility of the probe for in vivo Cu2þ ions and H2S detection. The new visible-light-excited timegated luminescence probe provides a novel strategy for visualizing Cu2þ ions and H2S in cells and biological tissues, which would be a useful tool for evaluating the pathogenic roles of Cu2þ ions and H2S in various biosystems. Acknowledgements We acknowledge the financial support from the Undergraduate Training Programs for Innovation and Entrepreneurship of Liaoning Province (Grant No. 201510165033). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.optmat.2017.10.030. References [1] Y.H. Li, H. Wang, J.H. Li, J. Zheng, X.H. Xu, R.H. Yang, Simultaneous intracellular b-D-glucosidase and phosphodiesterase I activities measurements based on a triple-signaling fluorescent probe, Anal. Chem. 83 (2011) 1268e1274. [2] K.P. Carter, A.M. Young, A.E. Palmer, Fluorescent sensors for measuring metal ions in living systems, Chem. Rev. 114 (2014) 4564e4601. [3] K. Eto, T. Asada, K. Arima, T. Makifuch, H. Kimura, Brain hydrogen sulfide is severely decreased in Alzheimer's disease, Biochem. Biophys. Res. Commun. 293 (2002) 1485e1488. [4] L. Wei, Z.T. Zhu, Y.Y. Li, L. Yi, Z. Xi, A highly selective and fast-response fluorescent probe for visualization of enzymatic H2S production in vitro and in living cells, Chem. Commun. 51 (2015) 10463e10466. [5] Z.Q. Guo, S. Park, J. Yoon, I. Shin, Recent progress in the development of nearinfrared fluorescent probes for bioimaging applications, Chem. Soc. Rev. 43 (2014) 16e29. [6] Y. Kaneko, Y. Kimura, H. Kimura, I. Niki, L-cysteine inhibits insulin release from the pancreatic beta-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitte, Diabetes 55 (2006) 1391e1397. [7] A.K. Mustafa, G. Sikka, S.K. Gazi, J. Steppan, S.M. Jung, A.K. Bhunia, V.M. Barodka, F.K. Gazi, R.K. Barrow, R. Wang, L.M. Amzel, D.E. Berkowitz, S.H. Snyder, Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels, Circ. Res. 109 (2011) 1259e1268. [8] M. Colon, J.L. Todoli, M. Hidalgo, M. Iglesias, Development of novel and sensitive methods for the determination of sulfide in aqueous samples by hydrogen sulfide generation-inductively coupled plasma-atomic emission spectroscopy, Anal. Chim. Acta 609 (2008) 160e168. [9] B. Liu, H. Wang, T.S. Wang, Y.Y. Bao, F.F. Du, J. Tian, Q.B. Li, R. Bai, A new ratiometric ESIPT sensor for detection of palladium species in aqueous solution, Chem. Commun. 48 (2012) 2867e2869. [10] J.G. Huang, M. Tang, M. Liu, M. Zhou, Z.G. Liu, Y. Cao, M.Y. Zhu, S.G. Liu, W.B. Zeng, Development of a fast responsive and highly sensitive fluorescent probe for Cu2þ ion and imaging in living cells, Dyes Pigment. 107 (2014) 1e8. [11] D.J. Waggoner, T.B. Bartnikas, J.D. Gitlin, The role of copper in neurodegenerative disease, Neurobiol. Dis. 6 (1999) 221e230. [12] W. Yang, G.D. Yang, X.M. Jia, L.Y. Wu, R. Wang, Activation of KATP channels by H2S in rat insulin-secretingcells and the underlying mechanisms, J. Physiol.
569 (2005) 519e531. [13] A.F. Perna, D. Ingrosso, Low hydrogen sulphide and chronic kidney disease: a dangerous liaison, Transplant 27 (2012) 486e493. [14] G.D. Yang, L.Y. Wu, B. Jiang, W. Yang, J.S. Qi, K. Cao, Q.H. Meng, A.K. Mustafa, W.T. Mu, S.M. Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine g-Lyase, Science 322 (2008) 587e590. [15] F.P. Hou, L. Huang, P.X. Xi, J. Cheng, X.F. Zhao, G.Q. Xie, Y.J. Shi, F.J. Cheng, X.J. Yao, D.C. Bai, Z.J. Zeng, A retrievable and highly selective fluorescent probe for monitoring sulfide and imaging in living cells, Inorg. Chem. 51 (2012) 2454e2460. [16] Y. Fu, Q.C. Feng, X.J. Jiang, H. Xu, M. Li, S.Q. Zeng, New fluorescent sensor for Cu(2þ) and S(2-) in 100% aqueous solution based on displacement approach, Dalton Trans. 43 (2014) 5815e5822. nez, R. Martínez-Ma n ~ ez, F. Sanceno n, J.V. Ros-Lis, A. Benito, J. Soto, [17] D. Jime A new chromo-chemodosimeter selective for sulfide anion, J. Am. Chem. Soc. 125 (2003) 9000e9001. [18] R.V. Rathod, S. Bera, M. Singh, D. Mondal, A colorimetric and fluorometric investigation of Cu(II) ion in aqueous medium with a fluorescein-based chemosensor, RSC Adv. 6 (2016) 34608e34615. [19] Y. Jin, H. Wu, Y. Tian, L.H. Chen, J.J. Cheng, S.P. Bi, Indirect determination of sulfide at ultratrace levels in natural waters by flow injection on-line sorption in a knotted reactor coupled with hydride generation atomic fluorescence spectrometry, Anal. Chem. 79 (2007) 7176e7181. [20] F. Maya, J.M. Estela, V. Cerd a, Improving the chemiluminescence-based determination of sulphide in complex environmental samples by using a new, automated multi-syringe flow injection analysis system coupled to a gas diffusion unit, Anal. Chim. Acta 601 (2007) 87e94. [21] J. Radford-Knoery, G.A. Cutter, Determination of carbonyl sulfide and hydrogen sulfide species in natural waters using specialized collection procedures and gas chromatography with flame photometric detection, Anal. Chem. 65 (1993) 976e982. [22] E.G. Lynn, R.C. Austin, Hydrogen sulfide in the pathogenesis of atherosclerosis and its therapeutic potential, Clin. Pharmacol. 4 (2011) 97e108. [23] O. Thoumine, H. Ewers, M. Heine, L. Groc, R. Frischknecht, G. Giannone, C. Poujol, P. Legros, B. Lounis, L. Cognet, D. Choquet, Interplay between adhesion turnover and cytoskeleton dynamics in the control of growth cone migration, Chem. Rev. 108 (2008) 1565e1587. [24] Y.M. Yang, Q. Zhao, W. Feng, F.Y. Li, Luminescent chemodosimeters for bioimaging, Chem. Rev. 113 (2013) 192e270. [25] Y.B. Ding, Y.Y. Tang, W.H. Zhu, Y.S. Xie, Fluorescent and colorimetric ion probes based on conjugated oligopyrroles, Chem. Soc. Rev. 44 (2015) 1101e1112. [26] M.M. Guo, P.Y. Dong, Y. Feng, X.G. Xi, R. Shao, X.H. Tian, B.H. Zhang, M.Z. Zhu, X.M. Meng, A two-photon fluorescent probe for biological Cu (Ⅱ) and PPi detection in aqueous solution and in vivo, Biosens. Bioelectron 90 (2017) 276e282. [27] A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc. 133 (2011) 10078e10080. [28] H.J. Peng, Y.F. Cheng, C.F. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, A fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood, Angew. Chem. Int. Ed. 50 (2011) 9672e9675. [29] R. Zhang, X.J. Yu, Y.J. Yin, Z.Q. Ye, G.L. Wang, J.L. Yuan, Development of a heterobimetallic Ru(II)-Cu(II) complex for highly selective and sensitive luminescence sensing of sulfide anions, Anal. Chim. Acta 691 (2011) 83e88. [30] Z.Q. Ye, X. An, B. Song, W.Z. Zhang, Z.C. Dai, J.L. Yuan, A novel dinuclear ruthenium(II)-copper(II) complex-based luminescent probe for hydrogen sulfide, Dalton Trans. 43 (2014) 13055e13060. [31] X.W. Cao, W.Y. Lin, K.B. Zheng, L.W. He, A near-infrared fluorescent turn-on probe for fluorescence imaging of hydrogen sulfide in living cells based on thiolysis of dinitrophenyl ether, Chem. Commun. 48 (2012) 10529e10531. [32] Y.C. Chen, C.C. Zhu, Z.H. Yang, J.J. Chen, Y.F. He, Y. Jiao, W.J. He, L. Qiu, J.J. Cen, Z.J. Guo, A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angew. Chem. Int. Ed. 125 (2013) 1732e1735. [33] J. Wu, Z.J. Ye, G.L. Wang, D.Y. Jin, J.L. Yuan, Y.F. Guan, J. Piper, Visible-lightsensitized highly luminescent europium nanoparticles: preparation and application for time-gated luminescence bioimaging, J. Mater. Chem. 19 (2009) 1258e1264. [34] Z.Q. Ye, G.L. Wang, J.X. Chen, X.Y. Fu, W.Z. Zhang, J.L. Yuan, Development of a novel terbium chelate-based luminescent chemosensor for time-resolved luminescence detection of intracellular Zn2þ ions, Biosens. Bioelectron. 26 (2010) 1043e1048. [35] J.C.G. Bünzli, Lanthanide luminescence for biomedical analyses and imaging, Chem. Rev. 110 (2010) 2729e2755. [36] M.C. Heffern, L.M. Matosziuk, T.J. Meade, Lanthanide probes for bioresponsive imaging, Chem. Rev. 114 (2014) 4496e4539. [37] B.K. McMahon, T. Gunnlaugsson, Lanthanide luminescence sensing of copper and mercury ions using an iminodiacetate-based Tb(III)-cyclen chemosensor, Tetrahedron Lett. 51 (2010) 5406e5410. [38] Y.W. Yip, G.L. Law, W.T. Wong, A highly selective on-off-on responsive lanthanide(III) based probe for recognition of copper and hydrogen sulfide, Dalton Trans. 45 (2016) 928e935. [39] Z.C. Dai, L. Tian, B. Song, Z.Q. Ye, X.L. Liu, J.L. Yuan, Ratiometric time-gated luminescence probe for hydrogen sulfide based on lanthanide complexes,
Y. Wang et al. / Optical Materials 75 (2018) 243e251 Anal. Chem. 86 (2014) 11883e11889. [40] J.L. Yuan, K. Matsumoto, A new tetradentate b-diketonate-europium chelate that can be covalently bound to proteins for time-resolved fluoroimmunoassay, Anal. Chem. 70 (1998) 596e601. [41] F.M. Xue, Y. Ma, L.M. Fu, R. Hao, G.S. Shao, M.S. Tang, A europium complex with enhanced long-wavelength sensitized luminescent properties, Phys. Chem. Chem. Phys. 12 (2010) 3195e3202. [42] Y.H. Chiu, J.W. Canary, Stability and acidity constants for ternary ligand-zinchydroxo complexes of tetradentate tripodal tigands, Inorg. Chem. 42 (2003) 5107e5116. [43] A. Juriset, V. Balzani, F. Barigelletti, S. Campagna, P. Belser, A. Zelewsky, Cheminform abstract: Ru(II) polypyridine complexes: photophysics, photochemistry, electrochemistry, and chemiluminescence, Coord. Chem. Rev. 84 (1988) 85e277. [44] L.X. Zhang, X.L. Zheng, W. Deng, Y.Q. Lu, S. Lechevallier, Z.Q. Ye, E.M. Goldys, J.M. Dawes, J.A. Piper, J.L. Yuan, M. Verelst, D.Y. Jin, Practical implementation, characterization and applications of a multi-colour time-gated luminescence microscope, Sci. Rep. 4 (2014) 6597. [45] U. Schatzschneider, J. Niesel, I. Ott, R. Gust, H. Alborzinia, S. Wӧlfl, Cellular uptake, cytotoxicity, and metabolic profiling of human cancer cells treated
[46]
[47]
[48]
[49]
[50]
251
with ruthenium(II) polypyridyl complexes [Ru(bpy)2(N-N)]Cl2 with N-N¼bpy, phen, dpq, dppz, and dppn, Chem. Med. Chem. 3 (2008) 1104e1109. H. Zhang, L. Tian, R.Y. Zhang, Z.Q. Ye, J.L. Yuan, Preparation of visible-lightexcited luminescence enhancement solutions for time-resolved luminescence detection of europium biolabel, Analyst 137 (2012) 4502e4508. C.F. Chow, M.H.W. Lam, W.Y. Wong, A heterobimetallic ruthenium(II) -copper(II) donor-acceptor complex as a chemodosimetric ensemble for selective cyanide detection, Inorg. Chem. 43 (2004) 8387e8393. N. Isoda, Y. Torii, T. Okada, M. Misoo, H. Yokoyama, N. Ikeda, M. Nojiri, S. Suzuki, K. Yamaguchi, The first example of photochemical reduction of nitrite into nitrogen monoxide by a dinuclear Ru(II)-Cu(II) complex and photoinduced intramolecular electron transfer reaction between Ru(II) and Cu(II) moieties, Dalton Trans. 46 (2009) 10175e10177. C.P. Myers, J.R. Miller, M.E. Williams, Impacts of the location and number of [Cu(bpy)2]2þ cross-links on the emission photodynamics of [Ru(bpy)3]2þ with pendant oligo(aminoethylglycine) chains, J. Am. Chem. Soc. 131 (2009) 15291e15300. M.G. Choi, S. Cha, H. Lee, H.L. Jeon, S.K. Chang, Sulfide-selective chemosignaling by a Cu2þ complex of dipicolylamine appended fluorescein, Chem. Commun. 47 (2009) 7390e7392.
Contents lists available at ScienceDirect
Optical Materials journal homepage: www.elsevier.com/locate/optmat
A visible-light-excited europium(III) complex-based luminescent probe for visualizing copper ions and hydrogen sulfide in living cells Yiren Wang a, Huan Wang a, Mei Yang a, Jingli Yuan b, Jing Wu a, * a b
School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian, 116029, China State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, 116024, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2017 Received in revised form 19 October 2017 Accepted 21 October 2017
Development of visible-light-excited lanthanide (III) complex-based luminescent probes is highly appealing due to their superiority of less damage to the living biosystems over the conventional UVlight-excited ones. In this work, a visible-light-excited europium (III) complex-based luminescent probe, BPED-BHHCT-Eu3þ-BPT, has been designed and synthesized by conjugating the Cu2þ-binding N,Nb-diketone ligand 4,40 bis(2-pyridylmethyl)ethanediamine (BPED) to a tetradentate 00 00 00 00 00 00 00 00 00 00 bis(1 ,1 ,1 ,2 ,2 ,3 ,3 -heptafluoro-4 ,6 -hexanedione-6 -yl)chlorosulfo-o-terphenyl (BHHCT) and coordinating with a coligand 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine) (BPT) for the time-gated luminescence detection of Cu2þ ions and hydrogen sulfide (H2S) in living cells. BPED-BHHCTEu3þ-BPT exhibited a sharp excitation peak at 407 nm and a wide excitation window extending to beyond 460 nm. Upon its reaction with Cu2þ ions, the luminescence of BPED-BHHCT-Eu3þ-BPT was efficiently quenched, which could be reversibly restored by the addition of H2S due to the strong affinity between Cu2þ ions and H2S. The “on-off-on” type luminescence behavior of BPED-BHHCT-Eu3þ-BPT towards Cu2þ ions and H2S enabled the sensing of the two species with high sensitivity and selectivity. The performances of BPED-BHHCT-Eu3þ-BPT for visualizing intracellular Cu2þ ions and H2S were investigated, and the results have demonstrated the practical applicability of the probe for molecular imaging of cells. © 2017 Published by Elsevier B.V.
Keywords: Europium(III) complex Visible-light-excitation Luminescent probe Copper ions Hydrogen sulfide
1. Introduction Trace elements (e.g., Mn, Fe, Co, Ni, Cu, Zn, Se, Mo, I) and macroelements (e.g., Na, Mg, K, Ca, P, Cl, S) are essential to support various biochemical processes for human life [1]. As an example of trace elements, copper ion (Cu2þ) plays an important role in various physiological processes of organism, such as haemopoiesis, iron absorption, and enzyme-catalyzed and redox reactions [2]. As a typical macroelement, sulfur is mainly contained in amino acid (cysteine, methionine, etc.) and vitamin (biotin, thiamine, etc.). Currently, hydrogen sulfide (H2S), generated from cysteine or homocysteine in biosystems [3,4], has been considered as the third endogenous signaling gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO) [5]. H2S also plays critical roles in diverse physiological processes including apoptosis, blood pressure regulation, neuromodulation, and inhibition of insulin signaling
* Corresponding author. E-mail address: [email protected] (J. Wu). https://doi.org/10.1016/j.optmat.2017.10.030 0925-3467/© 2017 Published by Elsevier B.V.
[6,7]. Increasing evidences have shown that the abnormal amounts of Cu2þ ions and H2S will bring about health problems [8]. For example, excess Cu2þ ions can disrupt protein activity and induce tissue necrosis [9], which is related to Parkinson's disease [10] and Wilson's disease [11]. The abnormal production of H2S is also associated with many diseases such as diabetes [12], chronic kidney disease [13], hypertension [14], and Alzheimer's disease [3]. Therefore, methods for the detection of Cu2þ ions and H2S in biosystems, especially in living cells, are highly demanded for their physiological and pathological research [15]. To date, several traditional analytical methods have been adopted to detect Cu2þ ions and H2S in various samples, including colorimetry [16], UVevis spectroscopy [17] and fluorescence spectrometry [18] for Cu2þ ions, and electrochemistry [19], chemiluminescence [20], gas chromatography [21] and sulfide precipitation [22] for H2S. These methods usually require post-mortem and destruction of samples, which are not suitable for the realtime monitoring of Cu2þ ions and H2S in living biosystems. In addition, the low concentration of Cu2þ ions and high activity of H2S also limit the usability of these methods. Recently, the optical
244
Y. Wang et al. / Optical Materials 75 (2018) 243e251
imaging technique with the use of luminescent probes provides an appropriate approach for in vivo detecting biomolecules due to its high sensitivity, high spatial resolution, and non-destructive detection [23,24]. A number of luminescent probes for Cu2þ ions have been designed utilizing its strong luminescence quenching ability [18,25,26]. Meanwhile, various luminescent probes for H2S have also been developed based on its special properties such as strong reducing ability [27,28], quencher (e.g., Cu2þ, Hg2þ) removal [29,30] and nucleophilic reaction [31,32]. However, the major drawback of these organic fluorophore-based or ruthenium(II) complex-based probes is their susceptibility to photobleaching and strong biological autofluorescence. As an improvement, lanthanide(III) complex-based luminescent probes (mainly Eu3þ and Tb3þ complexes) have attracted great interest due to their ideal luminescent properties including large Stokes shift, long luminescence lifetime, sharp emission profile and good photostability [33,34]. In particular, the super long-lived luminescence enables their usages in the time-gated detection technique, which can effectively eliminate the autofluorescence interference and contribute to improve the sensitivity and selectivity to the target species in complicated biosystems [35,36]. A few lanthanide(III) complex-based luminescent probes have been reported for the detection of Cu2þ ions [37,38] and H2S [38,39]. However, a key problem of all these lanthanide(III) complex-based probes is that their excitation windows are limited to the UV region (<380 nm), which will cause excitation phototoxicity to some UV light-sensitive living biosamples and be unfavorable for the photostability of probes. Therefore, the development of visible-lightexcited lanthanide complex(III)-based probes is urgently required for the pursuit of their wider bioassay applications. Herein, we developed a unique visible-light-excited Eu3þ complex, BPED-BHHCT-Eu3þ-BPT, in which the ligand BPED-BHHCT was synthesized by conjugating a Cu2þ-binding moiety N,N-bis(2pyridylmethyl)ethanediamine (BPED) to a tetradentate b-diketone 4,40 -bis(100,100,100,200 ,200 ,300 ,300 -heptafluoro-400 ,600 -hexane- dione-600 -yl) chlorosulfo-o-terphenyl (BHHCT), and the coligand 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,5-triazine (BPT) was introduced to achieve the visible-light excitation of the Eu3þ complex. BPED-BHHCT-Eu3þ-BPT exhibited a strong visible excitation peak at 407 nm, whose corresponding fluorescence emission at 611 nm could be selectively quenched by Cu2þ ions due to the coordination of Cu2þ with the BPED moiety and the resulting intramolecular photo-induced electron transfer (PET). The trapped BPED group could be released by the strong affinity between the coordinated Cu2þ and H2S, which resulted in the formation of CuS precipitate and restored the luminescence of the quenched probe. The decrease of luminescence intensity at 611 nm was linearly correlated with the Cu2þ concentration, and the reverse luminescence enhancement also showed a good linearity against the H2S concentration. Therefore, BPED-BHHCT-Eu3þ-BPT can be used as a luminescent probe for the time-gated luminescence detection of Cu2þ ions and H2S in aqueous media. To examine the applicability of the new probe for bioimaging, the BPED-BHHCT-Eu3þ-BPTloaded HepG2 cells were prepared, and the exogenous Cu2þ ions and H2S in the cells were imaged on a true-color time-gated luminescence microscope. The results demonstrated the practical utility of the new probe for visualizing intracellular Cu2þ ions and H2S. Scheme 1 shows the structure of BPED-BHHCT-Eu3þ-BPT and its luminescence response reaction with Cu2þ ion and H2S. 2. Experimental 2.1. Materials and physical measurements The tetradentate b-diketone BHHCT [40], BPT [41] and BPED [42]
were synthesized according to the previously reported methods. Triton X-100 was purchased from Acros Organics. Dichloromethane (CH2Cl2) was used after appropriate distillation and purification. Cultured HepG2 cells were obtained from Dalian Medical University. The isotonic saline solution consisting of 140 mmol NaCl, 10 mmol glucose and 3.5 mmol KCl was prepared in our laboratory. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification. 1 H NMR spectrum was measured on a Bruker Avance spectrometer (400 MHz). Mass spectrum was recorded on a HP1100LC/ MSD electrospray ionization mass spectrometer (ESI-MS). Elemental analysis was carried out on a Vario-EL analyser. Absorption spectra were measured on a Perkin-Elmer Lambda 35 UVevis spectrometer. Time-gated luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer with the following conditions: delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation slit, 10 nm; and emission slit, 10 nm. Luminescence lifetimes were measured on an FS5 spectrofluorometer of Edinburgh Instruments. Relative luminescence quantum yields of BPED-BHHCT-Eu3þ-BPT and its reaction product with Cu2þ, Cu2þ-BPED-BHHCT-Eu3þ-BPT, were measured with the Eu3þ complex, Eu3þ-(40 -phenyl-2,20 :60 ,200 -terpyridine-6,600 -diyl) bis-(methylene-nitrilo)tetrakis(acetate), as a reference (F ¼ 0.16) [43]. All bright-field and luminescence imaging measurements were carried out on a laboratory-use true-color time-gated luminescence microscope [44].
2.2. Synthesis of BPED-BHHCT BPED (35 mg, 0.12 mmol) was dissolved in dichloromethane CH2Cl2 (6 mL), and then BHHCT (100 mg, 0.12 mmol), triethylamine (0.15 mL) and dimethylaminopyridine (2.9 mg, 0.024 mmol) were added to the solution. The reaction mixture was stirred at room temperature in the dark for 5 days. After the solvent was evaporated, the residue was washed with diluted hydrochloric acid (1.0 M, 80 mL), and then dissolved in CH2Cl2 (200 mL). The CH2Cl2 solution was washed three times with 120 mL of water and dried with Na2SO4. After evaporation, the ligand BPED-BHHCT was obtained as yellow solid (120.5 mg, 89% yield). 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 1.25 (s, 1H), 1.80 (m, 4H), 3.98e4.01 (d, 4H; J ¼ 12.0 Hz), 6.56 (d, 2H; J ¼ 5.2 Hz), 7.04 (m, 4H), 7.23e7.21 (d, 4H; J ¼ 8.0 Hz), 7.77e7.86 (d, 8H; J ¼ 12.0 Hz), 8.61 (m, 4H). ESI-MS (m/ z): 1011.3 ([M þ H]þ, 100%, calcd 1011.2). Elemental analysis calcd (%) for C44H33F14N4O6.5S (BPED-BHHCT$0.5H2O): C, 51.82; H, 3.26; N, 5.49; found (%): C, 52.28; H, 3.19; N, 5.54.
2.3. Preparation of the stock solution of BPED-BHHCT-Eu3þ-BPT The stock solution of the complex BPED-BHHCT-Eu3þ-BPT was prepared by in situ mixing equiv of BPED-BHHCT (0.5 mmol), BPT (1 mmol) and EuCl3$6H2O (0.5 mmol) in ethanol. The obtained stock solution was stored at 4 C and properly diluted with 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 before use.
2.4. Luminescence response of BPED-BHHCT-Eu3þ-BPT towards various metal ions The reactions of BPED-BHHCT-Eu3þ-BPT with various metal ions were performed in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 with the same concentration of BPED-BHHCT-Eu3þBPT. The solutions were stirred at room temperature for 30 min, and then subjected to time-gated luminescence measurements.
Y. Wang et al. / Optical Materials 75 (2018) 243e251
245
Scheme 1. Reaction mechanism for the luminescence response of BPED-BHHCT-Eu3þ-BPT towards Cu2þ ion and H2S.
2.5. Luminescence response of Cu2þ-BPED-BHHCT-Eu3þ-BPT towards various anions The Cu2þ-BPED-BHHCT-Eu3þ-BPT solution was prepared by reacting BPED-BHHCT-Eu3þ-BPT (1 mM) with Cu2þ ions (2 mM) at room temperature for 30 min in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. After the addition of various anions, the solutions were stirred for another 45 min, and then subjected to time-gated luminescence measurements. 2.6. Luminescence imaging of Cu2þ ions and H2S in HepG2 cells HepG2 cells, cultured on a 35 mm glass-bottom culture dish (f 20 mm) in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin, were washed three times with the isotonic saline solution, and then incubated with the isotonic saline solution containing BPED-BHHCT-Eu3þ-BPT (5 mM) and 0.001% cremophor C40 for 1 h. After being washed three times with the isotonic saline solution, the BPED-BHHCT-Eu3þ-BPTloaded cells were further incubated with different concentrations of Cu2þ ions (1 and 5 mM) and cremophor C40 (0.001%) for 30 min. The Cu2þ-BPED-BHHCT-Eu3þ-BPT-loaded cells were washed three times with the isotonic saline solution, and then subjected to luminescence imaging measurements on the microscope (excitation filter, 380e420 nm; dichroic mirror, 400 nm; emission filter, >590 nm). The time-gated luminescence imaging measurements were carried out with the following conditions: delay time, 33 ms;; gate time, 1.0 ms; exposure time, 1.1 s. The steady-state luminescence imaging measurements were carried out with an exposure time of 1.1 s. The Cu2þ-BPED-BHHCT-Eu3þ-BPT-loaded cells, prepared by incubating the BPED-BHHCT-Eu3þ-BPT-loaded cells in the isotonic
saline solution containing Cu2þ ions (15 mM) and cremophor C40 (0.001%) for 30 min, were washed three times with the isotonic saline solution, and then incubated with different concentrations of H2S (50 and 100 mM) and cremophor C40 (0.001%) for another 45 min. After being washed three times with the isotonic saline solution, the cells were subjected to luminescence imaging measurements with the above-mentioned conditions. 2.7. MTT assay The cytotoxicity of BPED-BHHCT-Eu3þ-BPT to HepG2 cells was measured by the MTT assay [45]. HepG2 cells, cultured in RPMI1640 medium, were washed with the isotonic saline solution and then incubated with the isotonic saline solution containing different concentrations of BPED-BHHCT-Eu3þ-BPT at 37 C in a 5% CO2/95% air incubator for 24 h. The culture solutions were removed, and the cells were further incubated in the isotonic saline solution containing 455 mg/mL of MTT for another 4 h. After removal of the supernatants, the cells were dissolved in DMSO (100 mL), and then the absorbances at 490 nm were measured on an Infinite M200 Pro microplate reader. 3. Results and discussion 3.1. Design, synthesis and characterization of the Eu3þ complexbased probe To date, although several lanthanide(III) complex-based luminescent probes for Cu2þ ions and H2S have been developed, the optical windows of these probes are limited to the UV region. It is desirable to extend the excitation wavelength towards the visible region to minimize the effects of excitation phototoxicity on the
246
Y. Wang et al. / Optical Materials 75 (2018) 243e251
Scheme 2. Reaction pathway for the synthesis of BPED-BHHCT-Eu3þ-BPT.
living biosamples. Recently, a few visible-light-excited ternary Еu3þ complexes have been synthesized by introducing a dipyrazolyltriazine-derivative coligand to a Еu3þ-b-diketonate complex [33,41,46]. On the basis of these findings, it is greatly possible for the design of visible-light-excited Еu3þ complex-based luminescent probes by coordinating a dipyrazolyltriazine derivative to the Еu3þ-b-diketonate complex that is preferentially modified with an analyte-recognition moiety. In this work, by the modification of a tetradentate b-diketone ligand BHHCT with a Cu2þ-specifically reactive moiety BPED and the common coordination of BPT, we designed a novel visible-lightexcited Eu3þ complex-based luminescent probe, BPED-BHHCTEu3þ-BPT, for detecting Cu2þ ions and H2S. The ligand BPED-BHHCT and the complex BPED-BHHCT-Eu3þ-BPT were synthesized by the reaction procedures as shown in Scheme 2 and confirmed by 1H NMR, ESI-MS and elemental analyses (Fig. S1eS2 in Supplementary material). To reveal the coordination relationship between Eu3þ and the ligands (BPED-BHHCT and BPT), the Job's plotting analyses were carried out in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2%
Triton X-100. Firstly, the binding stoichiometry of BPT to Eu3þ was explored. As shown in Fig. 1A, the Job's plot exhibited a maximum value at approximate 0.66 molecular fraction, which proves that the reaction between Eu3þ and BPT has a coordination ratio of 1:2. Then the binding stoichiometry of BPED-BHHCT to Eu(BPT)2 was evaluated. As shown in Fig. 1B, the Job's plot showed a maximum value at 0.5 molecular fraction, indicating that the reaction between BPED-BHHCT and Eu(BPT)2 has a coordination ratio of 1:1. These results demonstrate that the main species of the probe in aqueous solution at micromolar concentrations should be Eu(BPED-BHHCT) (BPT)2. The photophysical properties of BPED-BHHCT-Eu3þ-BPT and its reaction product with Cu2þ, Cu2þ-BPED-BHHCT-Eu3þ-BPT, were investigated systematically in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 (Table S1 in Supplementary material and Fig. 2). As shown in Fig. 2B, BPED-BHHCT-Eu3þ-BPT and Cu2þ-BPED-BHHCT-Eu3þ-BPT exhibited the same excitation spectrum profiles with two maximum excitation wavelengths at 337 nm and 407 nm, which agreed well with their absorption peaks
Fig. 1. (A) Job's plot of the reaction between BPT and Eu3þ in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 (the total concentration of BPT and Eu3þ was kept at 10 mM); (B) Job's plot of the reaction between Eu(BPT)2 and BPED-BHHCT in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100 (the total concentration of Eu(BPT)2 and BPED-BHHCT was kept at 5 mM).
Y. Wang et al. / Optical Materials 75 (2018) 243e251
247
Fig. 2. (A) UVevis absorption spectra of BPED-BHHCT-Eu3þ-BPT (8 mM, red line) and Cu2þ-BPED-BHHCT-Eu3þ-BPT (8 mM, black line) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100; (B) Time-gated excitation (lem ¼ 611 nm) and emission (lex ¼ 407 nm) spectra of BPED-BHHCT-Eu3þ-BPT (1 mM, red line) and Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM, black line) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effects of pH on the luminescence intensities of BPED-BHHCT-Eu3þ-BPT (a) and Cu2þ-BPED-BHHCT-Eu3þ-BPT (b).
(Fig. 2A). Under the light excitation of 407 nm, the two Eu3þ complexes displayed a typical Eu3þ emission pattern with a main peak at approximate 611 nm (5D0/7F2 transition of Eu3þ ion) (Fig. 2B). There was no remarkable difference for the molar absorption coefficients of the two complexes, which were determined to be 5.8 104 cm1 M1 and 5.6 104 cm1 M1 for BPED-BHHCTEu3þ-BPT and Cu2þ-BPED-BHHCT-Eu3þ-BPT, respectively. However, it is noteworthy that BPED-BHHCT-Eu3þ-BPT was strongly luminescent with a wide excitation window extending beyond 460 nm, a high quantum yield (F ¼ 61.3%) and a long luminescence lifetime (t ¼ 560 ms)). After the reaction with Cu2þ, the luminescence of BPED-BHHCT-Eu3þ-BPT was efficiently quenched with a 125-fold decrease in luminescence quantum yield (F ¼ 0.49%) and a more than 2-fold decrease in luminescence lifetime (t ¼ 240 ms)). The luminescence quenching of BPED-BHHCT-Eu3þ-BPT by Cu2þ ions was attributed to the intramolecular photo-induced electron transfer (PET) process from the b-diketone-Eu3þ-BPT moiety to the BPED-Cu2þ moiety [47e49], which interrupted the luminescence emission of Eu3þ ion. These results suggest that BPED-BHHCT-Eu3þBPT can be used as a turn-off luminescent probe for the detection of Cu2þ ions. To investigate the effects of pH on the reaction of the probe with Cu2þ, the luminescence intensities of BPED-BHHCT-Eu3þ-BPT and Cu2þ-BPED-BHHCT-Eu3þ-BPT were determined in 0.05 M Tris-HCl
Fig. 4. (A) Time-gated excitation (lem ¼ 611 nm) and emission (lex ¼ 407 nm) spectra of BPED-BHHCT-Eu3þ-BPT (1 mM) in the presence of different concentrations of Cu2þ in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. The concentrations of Cu2þ are 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, 2.0, 2.3, 2.5, 2.7 and 3 mM; (B) Dose-dependent luminescence response of BPED-BHHCT-Eu3þ-BPT (1 mM) to Cu2þ.
248
Y. Wang et al. / Optical Materials 75 (2018) 243e251
buffer containing 0.2% Triton X-100 with different pHs. As shown in Fig. 3, the luminescence intensity of BPED-BHHCT-Eu3þ-BPT had a small change in the range of pH 5e8 with a maximum value at pH 7.5, and a distinct luminescence decrease was observed at pH < 5 or pH > 8. The pH-dependent luminescence enhancement of BPEDBHHCT-Eu3þ-BPT in acidic pH range might be due to the structural transformation of the b-diketone moiety and the deprotonation of the BPED moiety in the complex, while the pH-dependent luminescence decline in basic pH range is considered to be attributed to the hydrolysis of Eu3þ. However, in the buffers with a pH range of 3e10, the luminescence intensity of Cu2þ-BPED-BHHCT-Eu3þ-BPT was almost pH-independent. Thus, BPED-BHHCT-Eu3þ-BPT can be used as an efficient probe in weakly acidic, neutral and weakly basic buffers. 3.2. Quantitative detection of Cu2þ ions and H2S in aqueous media Fig. 5. Luminescence intensity responses of BPED-BHHCT-Eu3þ-BPT (1 mM) to various metal ions (5 mM) in the presence and absence of Cu2þ (3 mM) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. Metal ions: (a) Cd2þ, (b) Mn2þ, (c) Zn2þ, (d) Ca2þ, (e) Mg2þ, (f) Fe3þ, (g) Co2þ, (h) Agþ, (i) Ni2þ, (j) Pb2þ, (k) Fe2þ, (l) Kþ, (m) Naþ, (n) blank.
To evaluate the applicability of BPED-BHHCT-Eu3þ-BPT as a time-gated luminescence probe for the quantitative detection of Cu2þ ions, the time-gated excitation and emission spectra of the probe in the presence of different concentrations of Cu2þ ions were
Fig. 6. (A) Time-gated excitation (lem ¼ 612 nm) and emission (lex ¼ 407 nm) spectra of Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM) in the presence of different concentrations of H2S in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. The H2S concentrations are 0, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mM; (B) Dose-dependent luminescence response of Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM) to H2S.
Fig. 7. Luminescence intensity responses of Cu2þ-BPED-BHHCT-Eu3þ-BPT (1 mM) to various anions (100 mM) in the presence and absence of sulfide ion (100 mM) in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. Anions: (a) H2PO 4 , (b) HCO3 , (c) 3 2 4 B2O2 7 , (d) F , (e) CH3COO , (f) P7O10 , (g) Cl , (h) CO3 , (i) I , (j) NO2 , (k) NO3 , (l) P2O7 , 2 2 (m) ClO 3 , (n) SO4 , (o) S2O3 , (p) blank.
recorded in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. As shown in Fig. 4A, upon the reaction with different concentrations of Cu2þ ions, the luminescence intensities of the probe gradually decreased up to a 44-fold luminescence quenching. By plotting the luminescence intensity change against Cu2þ concentration, a good linearity correlation that can be expressed as I0I ¼ 342.8[Cu2þ]þ22.2 (I0 and I are the luminescence intensities of the probe in the absence and presence of Cu2þ, respectively) was obtained in the range of 0.2e2.0 mM (Fig. 4B). The detection limit for Cu2þ ions, calculated as the concentration corresponding to triple standard deviations of the background signal, is 0.038 mM. The luminescence response specificity of BPED-BHHCT-Eu3þBPT towards Cu2þ ions was also examined. Fig. 5 shows the luminescence intensities (611 nm) of BPED-BHHCT-Eu3þ-BPT after reacting with Cu2þ and other metal ions in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. Upon its reaction with 5.0 equiv of metal ions, such as Fe3þ, Co2þ, Agþ, Ni2þ, Pb2þ, and those that exist at much higher cellular concentrations including Naþ, Kþ, Mg2þ and Ca2þ, the luminescence intensity of the probe showed a negligible change. Although an obvious luminescence intensity change was produced upon addition of 5.0 equiv of Zn2þ, Cd2þ,
Y. Wang et al. / Optical Materials 75 (2018) 243e251
Mn2þ and Fe2þ, whereas a nearly 100% luminescence quenching was observed after the probe reacted with 3.0 equiv of Cu2þ. Furthermore, it is notable that all the chosen metal ions had almost no influences on the luminescence response of the probe to Cu2þ ions. These results demonstrate the highly specific response of BPED-BHHCT-Eu3þ-BPT towards Cu2þ ions. Due to the strong affinity between H2S and Cu2þ, the coordinated Cu2þ in the complex Cu2þ-BPED-BHHCT-Eu3þ-BPT would be captured by H2S to form CuS precipitate, and thus Cu2þ-BPEDBHHCT-Eu3þ-BPT could be a time-gated luminescence probe for H2S. Fig. 6A shows the time-gated excitation and emission spectra of Cu2þ-BPED-BHHCT-Eu3þ-BPT in the presence of different concentrations of H2S in the buffer. Upon the addition of H2S, the luminescence intensities (612 nm) of Cu2þ-BPED-BHHCT-Eu3þ-BPT gradually increased up to a 35-fold luminescence enhancement. Moreover, the emission enhancement of the Cu2þ-BPED-BHHCTEu3þ-BPT solution showed an excellent linear relationship in the H2S concentration range of 1e50 mM, which can be expressed as II0 ¼ 3.96[H2S]þ2.02 (Fig. 6B, I and I0 are the luminescence intensities of Cu2þ-BPED-BHHCT-Eu3þ-BPT in the presence and absence of H2S, respectively). The detection limit for H2S is 0.22 mM, which is 1.9 times lower than that of the reported dipicolylamine (DPA)-Cu(II) containing fluorescein sensor for H2S [50]. Thus, a high sensitivity can be achieved using Cu2þ-BPED-BHHCT-Eu3þ-BPT as a time-gated probe for the H2S quantification. The luminescence response selectivity of Cu2þ-DPA-BHHCT3þ Eu -BPT towards H2S was investigated in 0.05 M Tris-HCl buffer of pH 7.5 containing 0.2% Triton X-100. As shown in Fig. 7, the luminescence intensity of Cu2þ-BPED-BHHCT-Eu3þ-BPT did not respond 2 to 100 M equiv of various anions including SO2 4 , S2O3 , H2PO4 , 3 2 2 P2O4 7 , P7O10 , HCO3 , CO3 , NO3 , NO2 , F , Cl , I , ClO3 , B2O7 , CH3COO, whereas a remarkable luminescence enhancement was observed in the presence of H2S. In addition, the competition experiment results showed that more than 85% luminescence intensity revivals were achieved in the presence of H2S and any
249
chosen interfering anion (100 molar equiv). These results indicate that Cu2þ-BPED-BHHCT-Eu3þ-BPT has great potential to be used for the sensing of H2S in biosystems. 3.3. Luminescence imaging detection of Cu2þ ions and H2S in living cells Before investigating the sensing behavior of BPED-BHHCT-Eu3þBPT to intracellular Cu2þ ions and H2S, the toxicity of BPED-BHHCTEu3þ-BPT was examined by using the MTT assay method [44]. As shown in Fig. S3, the cell viabilities still remained at nearly 100% after incubation with different concentrations of BPED-BHHCTEu3þ-BPT for 24 h, which demonstrated that the cytotoxicity of BPED-BHHCT-Eu3þ-BPT was very low. The feasibility of BPED-BHHCT-Eu3þ-BPT for the detection of 2þ Cu ions and H2S in living HepG2 cells was further investigated. As shown in Fig. 8, after incubating with BPED-BHHCT-Eu3þ-BPT (5 mM), the cells exhibited bright red steady-state and time-gated luminescence, indicating that the probe could enter into the cells by an ordinary co-incubation method. After the BPED-BHHCT-Eu3þBPT-loaded HepG2 cells were treated with Cu2þ ions, the intracellular red signals were greatly weakened with the increase of Cu2þ concentration and completely disappeared till the Cu2þ concentration reaching to 15 mM, which confirmed the formation of Cu2þBPED-BHHCT-Eu3þ-BPT inside the cells. It is also clearly shown that the time-gated luminescence images of the cells displayed higher sensitivity and selectivity than the steady-state ones because the strong blue autofluorescence from the cells was thoroughly eliminated by the time-gated detection mode. After the cells were incubated with BPED-BHHCT-Eu3þ-BPT (5 mM) for 1 h and Cu2þ ions (15 mM) for another 30 min, the Cu2þ-BPED-BHHCT-Eu3þ-BPTloaded cells were further incubated with different concentrations of H2S, and remarkable intracellular red luminescence enhancement was observed with the increased H2S. These results demonstrate that BPED-BHHCT-Eu3þ-BPT can be easily transferred into
Fig. 8. Bright-field (above), steady-state (middle), and time-gated (below) luminescence images of Cu2þ ions and H2S in HepG2 cells. The cells were incubated with BPED-BHHCTEu3þ-BPT (5 mM) for 1 h (A), and then with 1 mM (B) and 5 mM (C) of Cu2þ ions for 30 min. The cells were incubated with BPED-BHHCT-Eu3þ-BPT (5 mM) for 1 h and Cu2þ ions (15 mM) for another 30 min, and then with 50 mM (D) and 100 mM (E) of H2S for 45 min. Scale bar: 10 mm.
250
Y. Wang et al. / Optical Materials 75 (2018) 243e251
living cells for specifically visualizing intracellular Cu2þ ions and H2S. 4. Conclusion In summary, by incorporating a BPED binding moiety and a dipyrazolyltriazine-derivative coligand into a Eu3þ-b-diketonate complex, a unique visible-light-excited Eu3þ complex-based luminescent probe, BPED-BHHCT-Eu3þ-BPT, has been successfully developed for the time-gated luminescence detection of Cu2þ ions and H2S in living cells. This probe can specifically recognize Cu2þ ions, resulting from remarkable luminescence quenching, which can be reversibly restored upon the addition of H2S. Compared with the previously reported luminescent probes for Cu2þ ions and H2S, the new probe has the advantages of visible excitation wavelength, wide excitation window, high quantum yield, high sensitivity and selectivity, and the applicability for the time-gated measurement. The results of luminescence imaging by monitoring Cu2þ ions and H2S in living cells demonstrated the utility of the probe for in vivo Cu2þ ions and H2S detection. The new visible-light-excited timegated luminescence probe provides a novel strategy for visualizing Cu2þ ions and H2S in cells and biological tissues, which would be a useful tool for evaluating the pathogenic roles of Cu2þ ions and H2S in various biosystems. Acknowledgements We acknowledge the financial support from the Undergraduate Training Programs for Innovation and Entrepreneurship of Liaoning Province (Grant No. 201510165033). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.optmat.2017.10.030. References [1] Y.H. Li, H. Wang, J.H. Li, J. Zheng, X.H. Xu, R.H. Yang, Simultaneous intracellular b-D-glucosidase and phosphodiesterase I activities measurements based on a triple-signaling fluorescent probe, Anal. Chem. 83 (2011) 1268e1274. [2] K.P. Carter, A.M. Young, A.E. Palmer, Fluorescent sensors for measuring metal ions in living systems, Chem. Rev. 114 (2014) 4564e4601. [3] K. Eto, T. Asada, K. Arima, T. Makifuch, H. Kimura, Brain hydrogen sulfide is severely decreased in Alzheimer's disease, Biochem. Biophys. Res. Commun. 293 (2002) 1485e1488. [4] L. Wei, Z.T. Zhu, Y.Y. Li, L. Yi, Z. Xi, A highly selective and fast-response fluorescent probe for visualization of enzymatic H2S production in vitro and in living cells, Chem. Commun. 51 (2015) 10463e10466. [5] Z.Q. Guo, S. Park, J. Yoon, I. Shin, Recent progress in the development of nearinfrared fluorescent probes for bioimaging applications, Chem. Soc. Rev. 43 (2014) 16e29. [6] Y. Kaneko, Y. Kimura, H. Kimura, I. Niki, L-cysteine inhibits insulin release from the pancreatic beta-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitte, Diabetes 55 (2006) 1391e1397. [7] A.K. Mustafa, G. Sikka, S.K. Gazi, J. Steppan, S.M. Jung, A.K. Bhunia, V.M. Barodka, F.K. Gazi, R.K. Barrow, R. Wang, L.M. Amzel, D.E. Berkowitz, S.H. Snyder, Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels, Circ. Res. 109 (2011) 1259e1268. [8] M. Colon, J.L. Todoli, M. Hidalgo, M. Iglesias, Development of novel and sensitive methods for the determination of sulfide in aqueous samples by hydrogen sulfide generation-inductively coupled plasma-atomic emission spectroscopy, Anal. Chim. Acta 609 (2008) 160e168. [9] B. Liu, H. Wang, T.S. Wang, Y.Y. Bao, F.F. Du, J. Tian, Q.B. Li, R. Bai, A new ratiometric ESIPT sensor for detection of palladium species in aqueous solution, Chem. Commun. 48 (2012) 2867e2869. [10] J.G. Huang, M. Tang, M. Liu, M. Zhou, Z.G. Liu, Y. Cao, M.Y. Zhu, S.G. Liu, W.B. Zeng, Development of a fast responsive and highly sensitive fluorescent probe for Cu2þ ion and imaging in living cells, Dyes Pigment. 107 (2014) 1e8. [11] D.J. Waggoner, T.B. Bartnikas, J.D. Gitlin, The role of copper in neurodegenerative disease, Neurobiol. Dis. 6 (1999) 221e230. [12] W. Yang, G.D. Yang, X.M. Jia, L.Y. Wu, R. Wang, Activation of KATP channels by H2S in rat insulin-secretingcells and the underlying mechanisms, J. Physiol.
569 (2005) 519e531. [13] A.F. Perna, D. Ingrosso, Low hydrogen sulphide and chronic kidney disease: a dangerous liaison, Transplant 27 (2012) 486e493. [14] G.D. Yang, L.Y. Wu, B. Jiang, W. Yang, J.S. Qi, K. Cao, Q.H. Meng, A.K. Mustafa, W.T. Mu, S.M. Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine g-Lyase, Science 322 (2008) 587e590. [15] F.P. Hou, L. Huang, P.X. Xi, J. Cheng, X.F. Zhao, G.Q. Xie, Y.J. Shi, F.J. Cheng, X.J. Yao, D.C. Bai, Z.J. Zeng, A retrievable and highly selective fluorescent probe for monitoring sulfide and imaging in living cells, Inorg. Chem. 51 (2012) 2454e2460. [16] Y. Fu, Q.C. Feng, X.J. Jiang, H. Xu, M. Li, S.Q. Zeng, New fluorescent sensor for Cu(2þ) and S(2-) in 100% aqueous solution based on displacement approach, Dalton Trans. 43 (2014) 5815e5822. nez, R. Martínez-Ma n ~ ez, F. Sanceno n, J.V. Ros-Lis, A. Benito, J. Soto, [17] D. Jime A new chromo-chemodosimeter selective for sulfide anion, J. Am. Chem. Soc. 125 (2003) 9000e9001. [18] R.V. Rathod, S. Bera, M. Singh, D. Mondal, A colorimetric and fluorometric investigation of Cu(II) ion in aqueous medium with a fluorescein-based chemosensor, RSC Adv. 6 (2016) 34608e34615. [19] Y. Jin, H. Wu, Y. Tian, L.H. Chen, J.J. Cheng, S.P. Bi, Indirect determination of sulfide at ultratrace levels in natural waters by flow injection on-line sorption in a knotted reactor coupled with hydride generation atomic fluorescence spectrometry, Anal. Chem. 79 (2007) 7176e7181. [20] F. Maya, J.M. Estela, V. Cerd a, Improving the chemiluminescence-based determination of sulphide in complex environmental samples by using a new, automated multi-syringe flow injection analysis system coupled to a gas diffusion unit, Anal. Chim. Acta 601 (2007) 87e94. [21] J. Radford-Knoery, G.A. Cutter, Determination of carbonyl sulfide and hydrogen sulfide species in natural waters using specialized collection procedures and gas chromatography with flame photometric detection, Anal. Chem. 65 (1993) 976e982. [22] E.G. Lynn, R.C. Austin, Hydrogen sulfide in the pathogenesis of atherosclerosis and its therapeutic potential, Clin. Pharmacol. 4 (2011) 97e108. [23] O. Thoumine, H. Ewers, M. Heine, L. Groc, R. Frischknecht, G. Giannone, C. Poujol, P. Legros, B. Lounis, L. Cognet, D. Choquet, Interplay between adhesion turnover and cytoskeleton dynamics in the control of growth cone migration, Chem. Rev. 108 (2008) 1565e1587. [24] Y.M. Yang, Q. Zhao, W. Feng, F.Y. Li, Luminescent chemodosimeters for bioimaging, Chem. Rev. 113 (2013) 192e270. [25] Y.B. Ding, Y.Y. Tang, W.H. Zhu, Y.S. Xie, Fluorescent and colorimetric ion probes based on conjugated oligopyrroles, Chem. Soc. Rev. 44 (2015) 1101e1112. [26] M.M. Guo, P.Y. Dong, Y. Feng, X.G. Xi, R. Shao, X.H. Tian, B.H. Zhang, M.Z. Zhu, X.M. Meng, A two-photon fluorescent probe for biological Cu (Ⅱ) and PPi detection in aqueous solution and in vivo, Biosens. Bioelectron 90 (2017) 276e282. [27] A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc. 133 (2011) 10078e10080. [28] H.J. Peng, Y.F. Cheng, C.F. Dai, A.L. King, B.L. Predmore, D.J. Lefer, B. Wang, A fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood, Angew. Chem. Int. Ed. 50 (2011) 9672e9675. [29] R. Zhang, X.J. Yu, Y.J. Yin, Z.Q. Ye, G.L. Wang, J.L. Yuan, Development of a heterobimetallic Ru(II)-Cu(II) complex for highly selective and sensitive luminescence sensing of sulfide anions, Anal. Chim. Acta 691 (2011) 83e88. [30] Z.Q. Ye, X. An, B. Song, W.Z. Zhang, Z.C. Dai, J.L. Yuan, A novel dinuclear ruthenium(II)-copper(II) complex-based luminescent probe for hydrogen sulfide, Dalton Trans. 43 (2014) 13055e13060. [31] X.W. Cao, W.Y. Lin, K.B. Zheng, L.W. He, A near-infrared fluorescent turn-on probe for fluorescence imaging of hydrogen sulfide in living cells based on thiolysis of dinitrophenyl ether, Chem. Commun. 48 (2012) 10529e10531. [32] Y.C. Chen, C.C. Zhu, Z.H. Yang, J.J. Chen, Y.F. He, Y. Jiao, W.J. He, L. Qiu, J.J. Cen, Z.J. Guo, A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angew. Chem. Int. Ed. 125 (2013) 1732e1735. [33] J. Wu, Z.J. Ye, G.L. Wang, D.Y. Jin, J.L. Yuan, Y.F. Guan, J. Piper, Visible-lightsensitized highly luminescent europium nanoparticles: preparation and application for time-gated luminescence bioimaging, J. Mater. Chem. 19 (2009) 1258e1264. [34] Z.Q. Ye, G.L. Wang, J.X. Chen, X.Y. Fu, W.Z. Zhang, J.L. Yuan, Development of a novel terbium chelate-based luminescent chemosensor for time-resolved luminescence detection of intracellular Zn2þ ions, Biosens. Bioelectron. 26 (2010) 1043e1048. [35] J.C.G. Bünzli, Lanthanide luminescence for biomedical analyses and imaging, Chem. Rev. 110 (2010) 2729e2755. [36] M.C. Heffern, L.M. Matosziuk, T.J. Meade, Lanthanide probes for bioresponsive imaging, Chem. Rev. 114 (2014) 4496e4539. [37] B.K. McMahon, T. Gunnlaugsson, Lanthanide luminescence sensing of copper and mercury ions using an iminodiacetate-based Tb(III)-cyclen chemosensor, Tetrahedron Lett. 51 (2010) 5406e5410. [38] Y.W. Yip, G.L. Law, W.T. Wong, A highly selective on-off-on responsive lanthanide(III) based probe for recognition of copper and hydrogen sulfide, Dalton Trans. 45 (2016) 928e935. [39] Z.C. Dai, L. Tian, B. Song, Z.Q. Ye, X.L. Liu, J.L. Yuan, Ratiometric time-gated luminescence probe for hydrogen sulfide based on lanthanide complexes,
Y. Wang et al. / Optical Materials 75 (2018) 243e251 Anal. Chem. 86 (2014) 11883e11889. [40] J.L. Yuan, K. Matsumoto, A new tetradentate b-diketonate-europium chelate that can be covalently bound to proteins for time-resolved fluoroimmunoassay, Anal. Chem. 70 (1998) 596e601. [41] F.M. Xue, Y. Ma, L.M. Fu, R. Hao, G.S. Shao, M.S. Tang, A europium complex with enhanced long-wavelength sensitized luminescent properties, Phys. Chem. Chem. Phys. 12 (2010) 3195e3202. [42] Y.H. Chiu, J.W. Canary, Stability and acidity constants for ternary ligand-zinchydroxo complexes of tetradentate tripodal tigands, Inorg. Chem. 42 (2003) 5107e5116. [43] A. Juriset, V. Balzani, F. Barigelletti, S. Campagna, P. Belser, A. Zelewsky, Cheminform abstract: Ru(II) polypyridine complexes: photophysics, photochemistry, electrochemistry, and chemiluminescence, Coord. Chem. Rev. 84 (1988) 85e277. [44] L.X. Zhang, X.L. Zheng, W. Deng, Y.Q. Lu, S. Lechevallier, Z.Q. Ye, E.M. Goldys, J.M. Dawes, J.A. Piper, J.L. Yuan, M. Verelst, D.Y. Jin, Practical implementation, characterization and applications of a multi-colour time-gated luminescence microscope, Sci. Rep. 4 (2014) 6597. [45] U. Schatzschneider, J. Niesel, I. Ott, R. Gust, H. Alborzinia, S. Wӧlfl, Cellular uptake, cytotoxicity, and metabolic profiling of human cancer cells treated
[46]
[47]
[48]
[49]
[50]
251
with ruthenium(II) polypyridyl complexes [Ru(bpy)2(N-N)]Cl2 with N-N¼bpy, phen, dpq, dppz, and dppn, Chem. Med. Chem. 3 (2008) 1104e1109. H. Zhang, L. Tian, R.Y. Zhang, Z.Q. Ye, J.L. Yuan, Preparation of visible-lightexcited luminescence enhancement solutions for time-resolved luminescence detection of europium biolabel, Analyst 137 (2012) 4502e4508. C.F. Chow, M.H.W. Lam, W.Y. Wong, A heterobimetallic ruthenium(II) -copper(II) donor-acceptor complex as a chemodosimetric ensemble for selective cyanide detection, Inorg. Chem. 43 (2004) 8387e8393. N. Isoda, Y. Torii, T. Okada, M. Misoo, H. Yokoyama, N. Ikeda, M. Nojiri, S. Suzuki, K. Yamaguchi, The first example of photochemical reduction of nitrite into nitrogen monoxide by a dinuclear Ru(II)-Cu(II) complex and photoinduced intramolecular electron transfer reaction between Ru(II) and Cu(II) moieties, Dalton Trans. 46 (2009) 10175e10177. C.P. Myers, J.R. Miller, M.E. Williams, Impacts of the location and number of [Cu(bpy)2]2þ cross-links on the emission photodynamics of [Ru(bpy)3]2þ with pendant oligo(aminoethylglycine) chains, J. Am. Chem. Soc. 131 (2009) 15291e15300. M.G. Choi, S. Cha, H. Lee, H.L. Jeon, S.K. Chang, Sulfide-selective chemosignaling by a Cu2þ complex of dipicolylamine appended fluorescein, Chem. Commun. 47 (2009) 7390e7392.