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A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols
SAA-117770; No of Pages 8 Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols Duobin Chao a,⁎, Yaping Pan b, Xue-Wang Gao c a
School of Materials Science and Chemical Engineering, Ningbo University, Zhejiang 315211, China School of Petroleum and Chemical Engineering, Dalian University of Technology, Panjin, Liaoning 124221, China Key Laboratory of Photochemical Conversion and Optoelectronic Materials Technical Institute of Physics and Chemistry, University of Chinese Academy of Sciences Chinese Academy of Sciences, Beijing 100190, China
b c
a r t i c l e
i n f o
Article history: Received 25 August 2019 Received in revised form 25 October 2019 Accepted 4 November 2019 Available online xxxx Keywords: TADF Donor–Acceptor Sensor Copper Biothiol
a b s t r a c t A new long-lived Donor–Acceptor (D–A) fluorophore based on carbazolyl dicyanobenzene was developed as an ON–OFF–ON multifunctional fluorescent probe 1 for sequential detection of Cu2+ and biothiols (Cys, Hcy and GSH). The fluorescence of probe 1 can be significantly and selectively quenched by Cu2+. Meanwhile, the fluorescence lifetime decreased from 2.1 μs to 18.5 ns. The limit of detection was determined to be 33.6 nM. Upon addition of biothiols (Cys, Hcy and GSH), the generated ensemble 1-Cu2+ displayed a “turn-on” fluorescent response at 555 nm and an obvious recovery in fluorescence lifetime and UV–vis absorption within 1 min. The limit of detection for Cys, Hcy and GSH were calculated by fluorescence titration experiments to be 0.19, 0.21 and 0.29 μM, respectively. The ensemble 1-Cu2+ was further successfully applied in bioimaging. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The identification of transition metal ions has attracted significant interest in recent years due to its crucial roles in environmental and biological systems [1,2]. Cu2+ is one of the most abundant and indispensable metal elements, playing important roles in many biological systems, and metabolic processes [3]. However, the disordered level of Cu2+ may destroy the balance of reactive oxygen species, and in turn exhibit serious toxicity, [4] resulting in many serious health problems, such as Wilson's, Parkinson's, and Alzheimer's disease [5–10]. Besides, a large amount of Cu2+ in life and industry could also cause serious environmental pollution. Therefore, monitoring Cu2+ in the human body and the environment is an urgent task. On the other hand, biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play important roles in many biological processes, like the cellular antioxidant defense and the growth of tissues in living system [11–13]. It has been reported that irregular level of biothiols is related to some diseases. Cys deficiency may cause many syndromes, such as retarded growth, hair depigmentation, lethargy, ⁎ Corresponding author. E-mail address: [email protected] (D. Chao).
liver damage, muscle and fat loss [14,15]. GSH is an important tripeptide in many biochemical processes, and GSH level in the cellular is associated with several diseases, such as leucocyte loss and HIV infection [16,17]. High levels of Hcy in human plasma are risk factors for disorders including cardiovascular and Alzheimer's diseases, neural tube defects and mental disorders [18,19]. Therefore, it is of great interest to develop efficient methods to detect biothiols in biological systems. Up to date, a variety of methods have been established to detect Cu2 + and biothiols (Cys, Hcy and GSH), including inductively coupled plasma mass spectrometry, high-performance liquid chromatography, electrochemical assays and fluorescence spectrometry [20–23]. Among these methods, fluorescence spectrometry has been recognized as one of the most promising techniques, owing to its high sensitivity, selectivity, simplicity of operation and short response time [24–27]. The reported fluorescent probes for the detection of biothiols were designed with various mechanisms, such as Michael addition reaction, [28–30] cyclization reaction, [31,32] cleavage reaction, [33–35] and others [36,37]. Recently, much attention has been paid to indicator displacement assays (IDA) between biothiols and Cu2+ complex [38,39]. The probe designed by this method can simultaneously detect Cu2+ and biothiols. The procedure relies on the fluorescence quenching property of Cu2+ and high affinity between Cu2+ and biothiols. That is to say, the
https://doi.org/10.1016/j.saa.2019.117770 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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addition of biothiols induced the Cu2+ to detach from the ensemble, releasing the probe and leading to the change of fluorescence. Taking advantage of this strategy, various luminescent probes were obtained with different kinds of fluorophores, such as coumarin, [40,41] imidazole, [42] benzothiazole derivative, [43] fluorescein [44]. However, these fluorophores are mostly limited by short fluorescence lifetimes (in the nanosecond range) and may display poor performance in fluorescence lifetime imaging. In contrast, long-lived organic fluorophores are helpful for avoiding the interference of autofluorescence background and damage of cells in biological systems, which could also be good candidates in fluorescence lifetime imaging and detection [45]. Thus, to develop organic fluorophores with long-lived is a matter of interest. In 2012, Adachi's group reported thermally activated delayed fluorescence (TADF) organic compounds for realizing organic lightemitting diodes [45]. Thermally activated delayed fluorescence (TADF) molecules undergo efficient reverse intersystem crossing (RISC) from a triplet excited state to a singlet state, so these molecules generally exhibit long fluorescence lifetimes. At present, thermally activated delayed fluorescence (TADF) molecules, such as carbazolyl dicyanobenzene (CDCBs) derivatives, which comprises carbazole as the electron donor and dicyanobenzene as the electron acceptor, are mainly used in OLED, [46] biosensing [47] and photocatalytic [48,49]. The long-lived D–A fluorophores based on carbazolyl dicyanobenzene (CDCB) is an inspiration for us to design fluorescent probes. Therefore, we designed and synthesized a long-lived probe 1 with D–A fluorophores using the distinguished properties of carbazolyl dicyanobenzene (CDCB) for the efficient recognition of Cu2+ and biothiols, in which carbazole and dicyanobenzene were used as the electron donor and the electron acceptor, respectively (Scheme 1). Terpyridine groups were introduced as a recognition group due to good coordination ability. The interaction between the metal and the ligand results in a unique structure that the final structure does not possess [50]. In this work, in the presence of Cu2+, the fluorescence of probe 1 was selectively quenched and the fluorescence lifetime also decreased. Due to the high affinity between Cu2+ and biothiols, the spectral signals and fluorescence lifetime were restored after the addition of biothiols. Ensemble 1-Cu2+ has also been successfully applied in bioimaging. To the best of our knowledge, this is the first report
Scheme 1. Synthesis of fluorescent probe 1.
regarding the use of carbazolyl dicyanobenzene (CDCB) derivatives as long-lived probe for efficiently detecting biothiols and Cu2+. 2. Experimental section 2.1. Materials and instruments All solvents and starting materials were purchased from commercial suppliers and used with no further purification. 1H NMR and 13C NMR spectra were recorded by a Bruker Avance 500 MHz spectrometer at room temperature. High–resolution mass spectra (HRMS) were measured by a Thermofisher Q–Exactive instrument equipped with an ESI ion source. UV–vis absorption spectra were recorded with AOE instrument UV–1800PC spectrophotometer. Fluorescence spectra studies were carried out with Lengguang F97PRO spectrophotometer. Transient photoluminescence decay spectra were recorded on K287 Full-featured steady-state/transient fluorescence spectrometer. Confocal Laser Scanning Microscope (CLSM) experiments were carried out using ZEISS LSM 510. 2.2. Synthesis 2.2.1. Synthesis of Cz–tpy Tpy1 and Tpy2 were synthesized according to previous procedure, [51] as shown in Scheme 1. Tpy2 (1 mmol), K2CO3 (2 mmol) and 9H-carbazol-4-ol (1.5 mmol) were dissolved in 5 mL anhydrous dimethylformamide (DMF). The mixture was stirred at 75 °C for 24 h under nitrogen atmosphere. After cooling to room temperature, the solution was poured into 300 mL cold water and filtered. The crude product was isolated by silica gel column chromatography using CH 2Cl2 /CH3 OH, (100:1, v/v) as eluent to produce a white solid. Yield: 50%. 1H NMR (500 MHz, DMSO–d6), δ (ppm): 11.31 (s, 1H), 8.78 (d, J = 4.6 Hz, 4H), 8.69 (d, J = 7.9 Hz, 2H), 8.21 (d, J = 7.8 Hz, 1H), 8.05 (dd, J = 8.8, 7.2 Hz, 4H), 7.83 (d, J = 7.9 Hz, 2H), 7.54 (ddd, J = 7.6, 4.7, 1.2 Hz, 2H), 7.48 (d, J = 8.1 Hz, 1H), 7.38–7.30 (m, 2H), 7.20–7.14 (m, 1H), 7.12 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 5.48 (s, 2H). 13C NMR (125 MHz, DMSO–d 6), δ (ppm): 156.20, 155.42, 154.98, 149.83, 149.64, 141.67, 139.46, 137.97, 137.36, 128.83, 127.65, 126.93, 125.11, 125.04, 122.67, 122.12, 121.44, 119.24, 118.41, 112.06, 110.96, 104.67, 101.52, 69.36. HRMS (m/z): found 527.1843 for [M + Na] + (calcd. for C34H24N4NaO: 527.1842). 2.2.2. Synthesis of 1 To a stirred solution of Cz–tpy (1 mmol) in dehydrated THF at room temperature under a nitrogen atmosphere, NaH (4 mmol) was added. After stirring for 30 min, 2,4,5,6-tetrafluoroisophthalonitrile (0.2 mmol) was added to the above solution. The reaction mixture stirred at room temperature for 18 h. The reaction was quenched with water (2 mL). The mixture was concentrated under reduced pressure, extracted with CH2Cl2, dried over Na2SO4 and evaporated in vacuo to obtain the crude product. The crude product was isolated by alumina column chromatography using n-hexane/dichloromethane as eluent to produce orange solid. Yield: 45%. m.p. 200.3 °C. 1H NMR (500 MHz, DMSO–d6), δ (ppm): 8.77 (d, J = 9.4 Hz, 4H), 8.74–8.56 (m, 18H), 8.37 (d, J = 7.8 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.09–7.61 (m, 30H), 7.58–7.08 (m, 23H), 6.99–6.70 (m, 6H), 6.54 (q, J = 7.5, 6.3 Hz, 1H), 5.59 (s, 2H), 5.32 (s, 4H), 5.17 (t, J = 8.6 Hz, 2H). 13C NMR (125 MHz, DMSO–d6). δ (ppm): 156.17, 156.04, 155.95, 155.40, 155.35, 155.33, 154.42, 149.79, 149.70, 149.34, 146.43, 140.69, 139.69, 138.97, 138.74, 138.73, 138.66, 137.91, 137.80, 137.78, 137.73, 137.53, 137.28, 129.02, 128.73, 128.65, 128.30, 127.70, 127.43, 127.42, 127.22, 124.99, 124.89, 123.01, 122.67, 122.06, 121.40, 121.33, 121.28, 118.40, 118.26, 118.18, 117.67, 113.10, 112.88, 112.59, 111.34, 104.92, 104.68, 63.04, 52.48, 45.87, 40.49, 40.42, 40.33, 40.25, 40.16, 40.08, 39.99, 39.83, 39.66, 39.49. HRMS (m/z): found 2175.7148 for [M + K]+ (calcd. for C144H92N18KO+ 4 : 2175.7186).
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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Elemental Anal. Calcd. for C144H92N18O4: C, 80.88; H, 4.34; N, 11.79. Found: C, 80.62; H, 4.53; N, 11.27. 2.2.3. Measurement procedures A stock solution of 1 (0.5 mM) was prepared in DMSO and stored in dark at 4 °C. The solution of 1 was appropriately diluted to be (1.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) for all spectral measurements. The solutions of various metal ions and anions were prepared in purified water. 1-Cu2+ solution for the detection of biothiols and other amino acids was prepared by addition of 5.0 equiv. Cu2+ to 1 (1.5 μM) solution in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1 v/v). 3. Results and discussion 3.1. Spectroscopic studies of 1 in the presence of Cu2+ Firstly, to verify the detection behaviour of 1 toward Cu2+, the fluorescence titration experiments were conducted. As shown in Fig. 1a, 1 displayed a remarkable emission band at 555 nm (λex = 400 nm) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Due to the strong bonding ability of the terpyridine units toward Cu2+, the fluorescence intensity of probe 1 (1.5 μM) continuously decreased with increasing concentrations of Cu2+ (0–7.5 μM). The quenching effect can be attributed to effective combination between 1 and paramagnetic Cu2+ [52,53]. Moreover, the fluorescence intensity of probe 1 was linearly related to Cu2+ in the concentration range from 0.3 to 1.8 μM (R2 = 0.99) (Fig. 1b). The limit of detection of 1 toward Cu2+ reached 33.6 nM based on a 3σ/k, which is much lower than the limit specified by the World Health Organization (WHO) guideline for drinking water (30 μM) [54,55]. Then, the interaction between 1 and Cu2+ was investigated by the UV–vis absorption spectra in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). As shown in Fig. 2, 1 exhibited strong absorption bands at 325 nm and 282 nm, while a weak band at around 379 nm was observed (Inset of Fig. 2), which could be attributed to the π − π* transition and intramolecular charge transition (ICT), respectively. Upon addition of Cu2+ (0–7.5 μM), the absorption at 282 nm gradually decreased, accompanied by the emergence of new absorption bands around 349 nm. The new absorption peak at 349 nm could be ascribed to ligand-metal charge transfer (LMCT), [56] which indicated the formation of ensemble of 1 and Cu2+, destinated as 1-Cu2+. To investigate the selectivity of probe 1, the fluorescence response of 1 in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) toward various metal cations including Cu2+, Zn2+, Cd2+, Mn2+, Al3+, Ag+, Hg2+, Mg2+, Co2+, Ni2+, Fe3+, Cr3+, Ca2+, Pb2+, Fe2+ was detected. As shown in Fig. 3, compared with other cations, only Cu2+ displayed significant fluorescence quenching effect toward 1 at 555 nm (Fig. 3a),
Fig. 2. Absorption spectra of 1 (1.5 μM) in the presence of different amounts of Cu2+ (0–7.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Inset: the absorption spectra of 1 (1.5 μM).
and the solution containing Cu2+ showed no emission under UV light (Fig. 3b). In addition, as shown in Fig. S1, in the presence of various interfering ions, it does not affect the response of probe 1 toward Cu2+. These results indicate that compound 1 has high selectivity toward Cu2+. Additionally, the influence of pH on the detection of Cu2+ is shown in Fig. 4. The emission intensity of 1 remained stable over a broad pH range of 6–10. After coordination with Cu2+, weak fluorescence from pH 3 to 10 was noticed. This result indicates that probe 1 can successfully recognize Cu2+ over a wide pH range. Furthermore, time-dependent fluorescence experiments of 1 toward Cu2+ was also investigated. As depicted in Fig. S2, upon the addition of Cu2+, the fluorescence intensity at 555 nm was quickly decreased within a few seconds. This result indicates the probe 1 can rapidly recognize Cu2+ at room temperature. 3.2. Spectroscopic studies of ensemble 1-Cu2+ to biothiols Considering that Cu2+ has a high affinity toward the amino acid containing thiol group, [57] we tested the possibility of ensemble 1-Cu2+ as a turn-on fluorescent probe for the detection of biothiols via indicator displacement assays (IDA). Firstly, the fluorescence response of 1-Cu2 + to a series of analytes, including biothiols (Cys, Hcy and GSH), S2− and other amino acids (His, Thr, Ser, Pro, Glu, Tyr, Trp, Gly, Lys) were
Fig. 1. (a) Fluorescence emission spectra of 1 (1.5 μM) in the presence of different amounts of Cu2+ (0–7.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm); (b) Linear relationship of fluorescence intensity with concentration of Cu2+.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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Fig. 3. (a) Fluorescence emission spectra of 1 (1.5 μM) and (b) the change of the color under 365 nm UV lamp in the presence of Cu2+ (7.5 μM) and other various metal ions (15 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm).
investigated by fluorescence spectroscopy (Fig. 5 and Fig. S3a). Only Cys, Hcy and GSH could significantly enhance the fluorescence intensity of 1Cu2+ at 555 nm. In the presence of other interfering ions, 1-Cu2+ still has strong selectivity for biothiols. The corresponding photographs of 1-Cu2+ in the presence of various analytes under a 365 nm UV lamp were also collected, respectively (Fig. S3b). The results demonstrate that 1-Cu2+ can selectively discriminate biothiols among a variety of analytes. Next, the relationship between the fluorescence intensity of 1-Cu2+ and the concentration of biothiols (Cys, Hcy and GSH) was studied by the titration experiment in Fig. 6. Upon the addition of Cys (0–7.5 μM), the fluorescence intensity at 555 nm was gradually increased. Moreover, the fluorescence intensity of 1-Cu2+ was linear correlation to Cys concentration from 1 to 7.5 μM, and gave the limit of detection to be 0.19 μM based on a 3σ/k. In addition, similar results were obtained, when the same amount of Hcy and GSH were added to the solution of 1Cu2+, respectively. The limits of detection of 1-Cu2+ for Hcy and GSH were calculated to be 0.21 μM and 0.29 μM. These results indicate that 1-Cu2+ can quantitatively detect biothiols with high sensitivity. The above displacement mechanism was further confirmed by UV– vis absorption spectra. Upon addition of Cys (0–7.5 μM) to the solution of 1-Cu2+ (1.5 μM), the absorption band at 349 nm gradually faded, accompanied by an enhancement of absorption at 282 nm (Fig. 7). The
Fig. 4. Fluorescence intensity of probe 1 (1.5 μM) at 555 nm with (red line) and without (black line) Cu2+ (7.5 μM) under different pH. λex = 400 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
final absorption spectrum is the same as that before the addition of Cu2+ (Inset of Fig. 7). This phenomenon demonstrated that the recovery of UV–vis absorption spectra was ascribed to the demetallation of 1-Cu2 + , induced by biothiols, which released free probe 1. Meanwhile, the fluorescence intensity of 1-Cu2+ reached maximum within 1 min in the presence of Cys and remained stable for 10 min (Fig. S4). The reversibility of 1-Cu2+ in the detection of biothiols was demonstrated by an alternate cycle experiment with titration of 1-Cu2+ with Cu2+ and Cys. As shown in Fig. 8, the fluorescence intensity of 1-Cu2+ was recovered following the addition of further Cys. This reversible cycle could be repeated at least 4 times. This result indicates that ensemble 1-Cu2+ can be developed as a turn-on and reversible chemosensor for the detection of biothiols. 3.3. Fluorescence lifetime studies As an important parameter, fluorescence lifetime can directly reflect the nature of the environment around the fluorophore and long-lived organic molecular probes can avoid the use of precious transitionmetal complexes [58,59]. Transient photoluminescence decay spectra at different stages were measured at 25 °C in air. As shown in Fig. 9, the fluorescence lifetime of 1 is calculated to be 2.1 μs, which is much longer than previous organic probes for Cu2+ [41,42]. We suppose that the relatively long lifetime is ascribed to TADF effect [60]. After adding Cu2+, the lifetime of 1 was significantly shortened (18.5 ns) in
Fig. 5. Fluorescence spectra of 1-Cu2+ (1.5 μM) in the presence of various analytes (7.5 μM) at 555 nm in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm).
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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Fig. 6. Fluorescence spectra of 1-Cu2+ (1.5 μM) in the presence of 0–7.5 μM (a) Cys, (c) Hcy, (e) GSH in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm); (b) Linear relationship of fluorescence intensity with concentration of (b) Cys, (d) Hcy, (f) GSH.
Fig. 7. Absorption spectra of 1-Cu2+ (1.5 μM) in the presence of 0–7.5 μM Cys in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Inset: absorption spectra before the addition of Cu2+.
Fig. 8. Fluorescent intensity of 1-Cu2+ (1.5 μM) at 555 nm (λex = 400 nm) in CH3CN/ HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) upon the alternate addition of Cys/Cu2+.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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comparison with its lifetime prior to binding with Cu2+ (2.1 μs), suggesting its potential application in lifetime-based sensing. Upon the addition of Cys, free probe 1 could be released, resulting in a recovery in fluorescence lifetime which was calculated to be 0.742 μs. These results indicate that 1 can be used as a long-lived molecular probe to detect Cu2 + and biothiols. 3.4. Sensing mechanism
Fig. 9. Transient photoluminescence decay spectra of 1.5 μM of 1 upon addition of Cu2+ and Cys in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) in air. λex = 400 nm. λem = 555 nm.
Based on spectroscopic results and previous studies [61,62], we proposed a possible sensing mechanism of 1 toward Cu2+ and biothiols (Scheme 2). Probe 1 exhibits strong emission with long fluorescence lifetime, while the emission turned off in the presence of Cu2+, due to the combination between terpyridine units and Cu2+, generating ensemble 1-Cu2+. The fluorescence quenching of 1 is mainly attributed to ligand-metal charge transfer (LMCT) [56]. The HRMS of 1 after the addition of Cu2+ was further carried out to prove the sensing mechanism (Fig. S5). The three new signal peaks of m/z = 632.0863, 810.1438 and 1166.2726 were derived from the [1 + 4Cu + 4Cl]4+, [1 + 3Cu + 3Cl]3+, [1 + 2Cu + 2Cl]2+ species formed by the combination of probe 1 and Cu2+ at different stoichiometric ratios (1:4, 1:3, 1:2), respectively. The ensemble 1-Cu2+ show strong emission with long lifetime again in the presence of biothiols, since biothiols have a high affinity with Cu2+. As a result, 1-Cu2+ can be demetallized by Cys, Hcy and GSH, releasing probe 1. 3.5. Application in cell imaging
Scheme 2. Possible mechanism for the detection of Cu2+ and biothiols.
Fluorescence microscopy imaging experiments was performed on the feasibility of 1-Cu2+ in detecting biothiols in HeLa cells. As shown in Fig. 10, HeLa cells were incubated with 1.5 μM 1-Cu2+ solution for 30 min at 37 °C (a–c). Then the cells were washed three times with PBS buffer, and exhibited green fluorescence. However, in the control experiment, the cells were first treated with NEM (a thiol scavenger) for 30 min, then being treated with 1-Cu2+ solution, and the cells were observed to have weak emission (d–f). These results indicate that 1-Cu2+ can be used for the detection of biothiols in cells. However, the cytotoxicity needs to be further investigated in detail for more applications.
Fig. 10. Confocal fluorescence images of HeLa cells at 37 °C. (a–c) HeLa cells incubated with 1.5 μM 1-Cu2+ for 30 min; (b) brightfield image of a; (c) merged image of a and b; (d–f) Hela cells were pre-incubated with 1 mM NEM for 30 min and then treated with 1.5 μM 1-Cu2+ for another 30 min; (e) brightfield image of d; (f) merged image d and e.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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4. Conclusions In conclusion, a new long-lived organic fluorescent probe 1, bearing a TADF core and terpyridine fragments, has been synthesized for the detection of Cu2+ and biothiols. The fluorescence intensity of probe 1 can be selectively quenched by Cu2+ and subsequently recovered in the presence of biothiols, displaying an ON–OFF–ON switching process. Meanwhile, the fluorescence lifetime (2.1 μs) decreased significantly upon addition of Cu2+ (18.5 ns) due to the formation of ensemble 1Cu2+. However, a longer lifetime of 0.742 μs is found in the presence of biothiols for the solution containing 1-Cu2+. Our results show organic TADF compounds have promising applications in lifetime-based sensing, in spite of a common sensing process.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work has been supported by the National Natural Science Foundation of China (No. 21605013 and No. 21702213) and K. C. Wong MagnaFund in Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117770.
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A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols Duobin Chao a,⁎, Yaping Pan b, Xue-Wang Gao c a
School of Materials Science and Chemical Engineering, Ningbo University, Zhejiang 315211, China School of Petroleum and Chemical Engineering, Dalian University of Technology, Panjin, Liaoning 124221, China Key Laboratory of Photochemical Conversion and Optoelectronic Materials Technical Institute of Physics and Chemistry, University of Chinese Academy of Sciences Chinese Academy of Sciences, Beijing 100190, China
b c
a r t i c l e
i n f o
Article history: Received 25 August 2019 Received in revised form 25 October 2019 Accepted 4 November 2019 Available online xxxx Keywords: TADF Donor–Acceptor Sensor Copper Biothiol
a b s t r a c t A new long-lived Donor–Acceptor (D–A) fluorophore based on carbazolyl dicyanobenzene was developed as an ON–OFF–ON multifunctional fluorescent probe 1 for sequential detection of Cu2+ and biothiols (Cys, Hcy and GSH). The fluorescence of probe 1 can be significantly and selectively quenched by Cu2+. Meanwhile, the fluorescence lifetime decreased from 2.1 μs to 18.5 ns. The limit of detection was determined to be 33.6 nM. Upon addition of biothiols (Cys, Hcy and GSH), the generated ensemble 1-Cu2+ displayed a “turn-on” fluorescent response at 555 nm and an obvious recovery in fluorescence lifetime and UV–vis absorption within 1 min. The limit of detection for Cys, Hcy and GSH were calculated by fluorescence titration experiments to be 0.19, 0.21 and 0.29 μM, respectively. The ensemble 1-Cu2+ was further successfully applied in bioimaging. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The identification of transition metal ions has attracted significant interest in recent years due to its crucial roles in environmental and biological systems [1,2]. Cu2+ is one of the most abundant and indispensable metal elements, playing important roles in many biological systems, and metabolic processes [3]. However, the disordered level of Cu2+ may destroy the balance of reactive oxygen species, and in turn exhibit serious toxicity, [4] resulting in many serious health problems, such as Wilson's, Parkinson's, and Alzheimer's disease [5–10]. Besides, a large amount of Cu2+ in life and industry could also cause serious environmental pollution. Therefore, monitoring Cu2+ in the human body and the environment is an urgent task. On the other hand, biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play important roles in many biological processes, like the cellular antioxidant defense and the growth of tissues in living system [11–13]. It has been reported that irregular level of biothiols is related to some diseases. Cys deficiency may cause many syndromes, such as retarded growth, hair depigmentation, lethargy, ⁎ Corresponding author. E-mail address: [email protected] (D. Chao).
liver damage, muscle and fat loss [14,15]. GSH is an important tripeptide in many biochemical processes, and GSH level in the cellular is associated with several diseases, such as leucocyte loss and HIV infection [16,17]. High levels of Hcy in human plasma are risk factors for disorders including cardiovascular and Alzheimer's diseases, neural tube defects and mental disorders [18,19]. Therefore, it is of great interest to develop efficient methods to detect biothiols in biological systems. Up to date, a variety of methods have been established to detect Cu2 + and biothiols (Cys, Hcy and GSH), including inductively coupled plasma mass spectrometry, high-performance liquid chromatography, electrochemical assays and fluorescence spectrometry [20–23]. Among these methods, fluorescence spectrometry has been recognized as one of the most promising techniques, owing to its high sensitivity, selectivity, simplicity of operation and short response time [24–27]. The reported fluorescent probes for the detection of biothiols were designed with various mechanisms, such as Michael addition reaction, [28–30] cyclization reaction, [31,32] cleavage reaction, [33–35] and others [36,37]. Recently, much attention has been paid to indicator displacement assays (IDA) between biothiols and Cu2+ complex [38,39]. The probe designed by this method can simultaneously detect Cu2+ and biothiols. The procedure relies on the fluorescence quenching property of Cu2+ and high affinity between Cu2+ and biothiols. That is to say, the
https://doi.org/10.1016/j.saa.2019.117770 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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addition of biothiols induced the Cu2+ to detach from the ensemble, releasing the probe and leading to the change of fluorescence. Taking advantage of this strategy, various luminescent probes were obtained with different kinds of fluorophores, such as coumarin, [40,41] imidazole, [42] benzothiazole derivative, [43] fluorescein [44]. However, these fluorophores are mostly limited by short fluorescence lifetimes (in the nanosecond range) and may display poor performance in fluorescence lifetime imaging. In contrast, long-lived organic fluorophores are helpful for avoiding the interference of autofluorescence background and damage of cells in biological systems, which could also be good candidates in fluorescence lifetime imaging and detection [45]. Thus, to develop organic fluorophores with long-lived is a matter of interest. In 2012, Adachi's group reported thermally activated delayed fluorescence (TADF) organic compounds for realizing organic lightemitting diodes [45]. Thermally activated delayed fluorescence (TADF) molecules undergo efficient reverse intersystem crossing (RISC) from a triplet excited state to a singlet state, so these molecules generally exhibit long fluorescence lifetimes. At present, thermally activated delayed fluorescence (TADF) molecules, such as carbazolyl dicyanobenzene (CDCBs) derivatives, which comprises carbazole as the electron donor and dicyanobenzene as the electron acceptor, are mainly used in OLED, [46] biosensing [47] and photocatalytic [48,49]. The long-lived D–A fluorophores based on carbazolyl dicyanobenzene (CDCB) is an inspiration for us to design fluorescent probes. Therefore, we designed and synthesized a long-lived probe 1 with D–A fluorophores using the distinguished properties of carbazolyl dicyanobenzene (CDCB) for the efficient recognition of Cu2+ and biothiols, in which carbazole and dicyanobenzene were used as the electron donor and the electron acceptor, respectively (Scheme 1). Terpyridine groups were introduced as a recognition group due to good coordination ability. The interaction between the metal and the ligand results in a unique structure that the final structure does not possess [50]. In this work, in the presence of Cu2+, the fluorescence of probe 1 was selectively quenched and the fluorescence lifetime also decreased. Due to the high affinity between Cu2+ and biothiols, the spectral signals and fluorescence lifetime were restored after the addition of biothiols. Ensemble 1-Cu2+ has also been successfully applied in bioimaging. To the best of our knowledge, this is the first report
Scheme 1. Synthesis of fluorescent probe 1.
regarding the use of carbazolyl dicyanobenzene (CDCB) derivatives as long-lived probe for efficiently detecting biothiols and Cu2+. 2. Experimental section 2.1. Materials and instruments All solvents and starting materials were purchased from commercial suppliers and used with no further purification. 1H NMR and 13C NMR spectra were recorded by a Bruker Avance 500 MHz spectrometer at room temperature. High–resolution mass spectra (HRMS) were measured by a Thermofisher Q–Exactive instrument equipped with an ESI ion source. UV–vis absorption spectra were recorded with AOE instrument UV–1800PC spectrophotometer. Fluorescence spectra studies were carried out with Lengguang F97PRO spectrophotometer. Transient photoluminescence decay spectra were recorded on K287 Full-featured steady-state/transient fluorescence spectrometer. Confocal Laser Scanning Microscope (CLSM) experiments were carried out using ZEISS LSM 510. 2.2. Synthesis 2.2.1. Synthesis of Cz–tpy Tpy1 and Tpy2 were synthesized according to previous procedure, [51] as shown in Scheme 1. Tpy2 (1 mmol), K2CO3 (2 mmol) and 9H-carbazol-4-ol (1.5 mmol) were dissolved in 5 mL anhydrous dimethylformamide (DMF). The mixture was stirred at 75 °C for 24 h under nitrogen atmosphere. After cooling to room temperature, the solution was poured into 300 mL cold water and filtered. The crude product was isolated by silica gel column chromatography using CH 2Cl2 /CH3 OH, (100:1, v/v) as eluent to produce a white solid. Yield: 50%. 1H NMR (500 MHz, DMSO–d6), δ (ppm): 11.31 (s, 1H), 8.78 (d, J = 4.6 Hz, 4H), 8.69 (d, J = 7.9 Hz, 2H), 8.21 (d, J = 7.8 Hz, 1H), 8.05 (dd, J = 8.8, 7.2 Hz, 4H), 7.83 (d, J = 7.9 Hz, 2H), 7.54 (ddd, J = 7.6, 4.7, 1.2 Hz, 2H), 7.48 (d, J = 8.1 Hz, 1H), 7.38–7.30 (m, 2H), 7.20–7.14 (m, 1H), 7.12 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 5.48 (s, 2H). 13C NMR (125 MHz, DMSO–d 6), δ (ppm): 156.20, 155.42, 154.98, 149.83, 149.64, 141.67, 139.46, 137.97, 137.36, 128.83, 127.65, 126.93, 125.11, 125.04, 122.67, 122.12, 121.44, 119.24, 118.41, 112.06, 110.96, 104.67, 101.52, 69.36. HRMS (m/z): found 527.1843 for [M + Na] + (calcd. for C34H24N4NaO: 527.1842). 2.2.2. Synthesis of 1 To a stirred solution of Cz–tpy (1 mmol) in dehydrated THF at room temperature under a nitrogen atmosphere, NaH (4 mmol) was added. After stirring for 30 min, 2,4,5,6-tetrafluoroisophthalonitrile (0.2 mmol) was added to the above solution. The reaction mixture stirred at room temperature for 18 h. The reaction was quenched with water (2 mL). The mixture was concentrated under reduced pressure, extracted with CH2Cl2, dried over Na2SO4 and evaporated in vacuo to obtain the crude product. The crude product was isolated by alumina column chromatography using n-hexane/dichloromethane as eluent to produce orange solid. Yield: 45%. m.p. 200.3 °C. 1H NMR (500 MHz, DMSO–d6), δ (ppm): 8.77 (d, J = 9.4 Hz, 4H), 8.74–8.56 (m, 18H), 8.37 (d, J = 7.8 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.09–7.61 (m, 30H), 7.58–7.08 (m, 23H), 6.99–6.70 (m, 6H), 6.54 (q, J = 7.5, 6.3 Hz, 1H), 5.59 (s, 2H), 5.32 (s, 4H), 5.17 (t, J = 8.6 Hz, 2H). 13C NMR (125 MHz, DMSO–d6). δ (ppm): 156.17, 156.04, 155.95, 155.40, 155.35, 155.33, 154.42, 149.79, 149.70, 149.34, 146.43, 140.69, 139.69, 138.97, 138.74, 138.73, 138.66, 137.91, 137.80, 137.78, 137.73, 137.53, 137.28, 129.02, 128.73, 128.65, 128.30, 127.70, 127.43, 127.42, 127.22, 124.99, 124.89, 123.01, 122.67, 122.06, 121.40, 121.33, 121.28, 118.40, 118.26, 118.18, 117.67, 113.10, 112.88, 112.59, 111.34, 104.92, 104.68, 63.04, 52.48, 45.87, 40.49, 40.42, 40.33, 40.25, 40.16, 40.08, 39.99, 39.83, 39.66, 39.49. HRMS (m/z): found 2175.7148 for [M + K]+ (calcd. for C144H92N18KO+ 4 : 2175.7186).
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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Elemental Anal. Calcd. for C144H92N18O4: C, 80.88; H, 4.34; N, 11.79. Found: C, 80.62; H, 4.53; N, 11.27. 2.2.3. Measurement procedures A stock solution of 1 (0.5 mM) was prepared in DMSO and stored in dark at 4 °C. The solution of 1 was appropriately diluted to be (1.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) for all spectral measurements. The solutions of various metal ions and anions were prepared in purified water. 1-Cu2+ solution for the detection of biothiols and other amino acids was prepared by addition of 5.0 equiv. Cu2+ to 1 (1.5 μM) solution in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1 v/v). 3. Results and discussion 3.1. Spectroscopic studies of 1 in the presence of Cu2+ Firstly, to verify the detection behaviour of 1 toward Cu2+, the fluorescence titration experiments were conducted. As shown in Fig. 1a, 1 displayed a remarkable emission band at 555 nm (λex = 400 nm) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Due to the strong bonding ability of the terpyridine units toward Cu2+, the fluorescence intensity of probe 1 (1.5 μM) continuously decreased with increasing concentrations of Cu2+ (0–7.5 μM). The quenching effect can be attributed to effective combination between 1 and paramagnetic Cu2+ [52,53]. Moreover, the fluorescence intensity of probe 1 was linearly related to Cu2+ in the concentration range from 0.3 to 1.8 μM (R2 = 0.99) (Fig. 1b). The limit of detection of 1 toward Cu2+ reached 33.6 nM based on a 3σ/k, which is much lower than the limit specified by the World Health Organization (WHO) guideline for drinking water (30 μM) [54,55]. Then, the interaction between 1 and Cu2+ was investigated by the UV–vis absorption spectra in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). As shown in Fig. 2, 1 exhibited strong absorption bands at 325 nm and 282 nm, while a weak band at around 379 nm was observed (Inset of Fig. 2), which could be attributed to the π − π* transition and intramolecular charge transition (ICT), respectively. Upon addition of Cu2+ (0–7.5 μM), the absorption at 282 nm gradually decreased, accompanied by the emergence of new absorption bands around 349 nm. The new absorption peak at 349 nm could be ascribed to ligand-metal charge transfer (LMCT), [56] which indicated the formation of ensemble of 1 and Cu2+, destinated as 1-Cu2+. To investigate the selectivity of probe 1, the fluorescence response of 1 in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) toward various metal cations including Cu2+, Zn2+, Cd2+, Mn2+, Al3+, Ag+, Hg2+, Mg2+, Co2+, Ni2+, Fe3+, Cr3+, Ca2+, Pb2+, Fe2+ was detected. As shown in Fig. 3, compared with other cations, only Cu2+ displayed significant fluorescence quenching effect toward 1 at 555 nm (Fig. 3a),
Fig. 2. Absorption spectra of 1 (1.5 μM) in the presence of different amounts of Cu2+ (0–7.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Inset: the absorption spectra of 1 (1.5 μM).
and the solution containing Cu2+ showed no emission under UV light (Fig. 3b). In addition, as shown in Fig. S1, in the presence of various interfering ions, it does not affect the response of probe 1 toward Cu2+. These results indicate that compound 1 has high selectivity toward Cu2+. Additionally, the influence of pH on the detection of Cu2+ is shown in Fig. 4. The emission intensity of 1 remained stable over a broad pH range of 6–10. After coordination with Cu2+, weak fluorescence from pH 3 to 10 was noticed. This result indicates that probe 1 can successfully recognize Cu2+ over a wide pH range. Furthermore, time-dependent fluorescence experiments of 1 toward Cu2+ was also investigated. As depicted in Fig. S2, upon the addition of Cu2+, the fluorescence intensity at 555 nm was quickly decreased within a few seconds. This result indicates the probe 1 can rapidly recognize Cu2+ at room temperature. 3.2. Spectroscopic studies of ensemble 1-Cu2+ to biothiols Considering that Cu2+ has a high affinity toward the amino acid containing thiol group, [57] we tested the possibility of ensemble 1-Cu2+ as a turn-on fluorescent probe for the detection of biothiols via indicator displacement assays (IDA). Firstly, the fluorescence response of 1-Cu2 + to a series of analytes, including biothiols (Cys, Hcy and GSH), S2− and other amino acids (His, Thr, Ser, Pro, Glu, Tyr, Trp, Gly, Lys) were
Fig. 1. (a) Fluorescence emission spectra of 1 (1.5 μM) in the presence of different amounts of Cu2+ (0–7.5 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm); (b) Linear relationship of fluorescence intensity with concentration of Cu2+.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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Fig. 3. (a) Fluorescence emission spectra of 1 (1.5 μM) and (b) the change of the color under 365 nm UV lamp in the presence of Cu2+ (7.5 μM) and other various metal ions (15 μM) in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm).
investigated by fluorescence spectroscopy (Fig. 5 and Fig. S3a). Only Cys, Hcy and GSH could significantly enhance the fluorescence intensity of 1Cu2+ at 555 nm. In the presence of other interfering ions, 1-Cu2+ still has strong selectivity for biothiols. The corresponding photographs of 1-Cu2+ in the presence of various analytes under a 365 nm UV lamp were also collected, respectively (Fig. S3b). The results demonstrate that 1-Cu2+ can selectively discriminate biothiols among a variety of analytes. Next, the relationship between the fluorescence intensity of 1-Cu2+ and the concentration of biothiols (Cys, Hcy and GSH) was studied by the titration experiment in Fig. 6. Upon the addition of Cys (0–7.5 μM), the fluorescence intensity at 555 nm was gradually increased. Moreover, the fluorescence intensity of 1-Cu2+ was linear correlation to Cys concentration from 1 to 7.5 μM, and gave the limit of detection to be 0.19 μM based on a 3σ/k. In addition, similar results were obtained, when the same amount of Hcy and GSH were added to the solution of 1Cu2+, respectively. The limits of detection of 1-Cu2+ for Hcy and GSH were calculated to be 0.21 μM and 0.29 μM. These results indicate that 1-Cu2+ can quantitatively detect biothiols with high sensitivity. The above displacement mechanism was further confirmed by UV– vis absorption spectra. Upon addition of Cys (0–7.5 μM) to the solution of 1-Cu2+ (1.5 μM), the absorption band at 349 nm gradually faded, accompanied by an enhancement of absorption at 282 nm (Fig. 7). The
Fig. 4. Fluorescence intensity of probe 1 (1.5 μM) at 555 nm with (red line) and without (black line) Cu2+ (7.5 μM) under different pH. λex = 400 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
final absorption spectrum is the same as that before the addition of Cu2+ (Inset of Fig. 7). This phenomenon demonstrated that the recovery of UV–vis absorption spectra was ascribed to the demetallation of 1-Cu2 + , induced by biothiols, which released free probe 1. Meanwhile, the fluorescence intensity of 1-Cu2+ reached maximum within 1 min in the presence of Cys and remained stable for 10 min (Fig. S4). The reversibility of 1-Cu2+ in the detection of biothiols was demonstrated by an alternate cycle experiment with titration of 1-Cu2+ with Cu2+ and Cys. As shown in Fig. 8, the fluorescence intensity of 1-Cu2+ was recovered following the addition of further Cys. This reversible cycle could be repeated at least 4 times. This result indicates that ensemble 1-Cu2+ can be developed as a turn-on and reversible chemosensor for the detection of biothiols. 3.3. Fluorescence lifetime studies As an important parameter, fluorescence lifetime can directly reflect the nature of the environment around the fluorophore and long-lived organic molecular probes can avoid the use of precious transitionmetal complexes [58,59]. Transient photoluminescence decay spectra at different stages were measured at 25 °C in air. As shown in Fig. 9, the fluorescence lifetime of 1 is calculated to be 2.1 μs, which is much longer than previous organic probes for Cu2+ [41,42]. We suppose that the relatively long lifetime is ascribed to TADF effect [60]. After adding Cu2+, the lifetime of 1 was significantly shortened (18.5 ns) in
Fig. 5. Fluorescence spectra of 1-Cu2+ (1.5 μM) in the presence of various analytes (7.5 μM) at 555 nm in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm).
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
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Fig. 6. Fluorescence spectra of 1-Cu2+ (1.5 μM) in the presence of 0–7.5 μM (a) Cys, (c) Hcy, (e) GSH in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) at 555 nm (λex = 400 nm); (b) Linear relationship of fluorescence intensity with concentration of (b) Cys, (d) Hcy, (f) GSH.
Fig. 7. Absorption spectra of 1-Cu2+ (1.5 μM) in the presence of 0–7.5 μM Cys in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v). Inset: absorption spectra before the addition of Cu2+.
Fig. 8. Fluorescent intensity of 1-Cu2+ (1.5 μM) at 555 nm (λex = 400 nm) in CH3CN/ HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) upon the alternate addition of Cys/Cu2+.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
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comparison with its lifetime prior to binding with Cu2+ (2.1 μs), suggesting its potential application in lifetime-based sensing. Upon the addition of Cys, free probe 1 could be released, resulting in a recovery in fluorescence lifetime which was calculated to be 0.742 μs. These results indicate that 1 can be used as a long-lived molecular probe to detect Cu2 + and biothiols. 3.4. Sensing mechanism
Fig. 9. Transient photoluminescence decay spectra of 1.5 μM of 1 upon addition of Cu2+ and Cys in CH3CN/HEPES buffer (10 mM, pH = 7.4, 1:1, v/v) in air. λex = 400 nm. λem = 555 nm.
Based on spectroscopic results and previous studies [61,62], we proposed a possible sensing mechanism of 1 toward Cu2+ and biothiols (Scheme 2). Probe 1 exhibits strong emission with long fluorescence lifetime, while the emission turned off in the presence of Cu2+, due to the combination between terpyridine units and Cu2+, generating ensemble 1-Cu2+. The fluorescence quenching of 1 is mainly attributed to ligand-metal charge transfer (LMCT) [56]. The HRMS of 1 after the addition of Cu2+ was further carried out to prove the sensing mechanism (Fig. S5). The three new signal peaks of m/z = 632.0863, 810.1438 and 1166.2726 were derived from the [1 + 4Cu + 4Cl]4+, [1 + 3Cu + 3Cl]3+, [1 + 2Cu + 2Cl]2+ species formed by the combination of probe 1 and Cu2+ at different stoichiometric ratios (1:4, 1:3, 1:2), respectively. The ensemble 1-Cu2+ show strong emission with long lifetime again in the presence of biothiols, since biothiols have a high affinity with Cu2+. As a result, 1-Cu2+ can be demetallized by Cys, Hcy and GSH, releasing probe 1. 3.5. Application in cell imaging
Scheme 2. Possible mechanism for the detection of Cu2+ and biothiols.
Fluorescence microscopy imaging experiments was performed on the feasibility of 1-Cu2+ in detecting biothiols in HeLa cells. As shown in Fig. 10, HeLa cells were incubated with 1.5 μM 1-Cu2+ solution for 30 min at 37 °C (a–c). Then the cells were washed three times with PBS buffer, and exhibited green fluorescence. However, in the control experiment, the cells were first treated with NEM (a thiol scavenger) for 30 min, then being treated with 1-Cu2+ solution, and the cells were observed to have weak emission (d–f). These results indicate that 1-Cu2+ can be used for the detection of biothiols in cells. However, the cytotoxicity needs to be further investigated in detail for more applications.
Fig. 10. Confocal fluorescence images of HeLa cells at 37 °C. (a–c) HeLa cells incubated with 1.5 μM 1-Cu2+ for 30 min; (b) brightfield image of a; (c) merged image of a and b; (d–f) Hela cells were pre-incubated with 1 mM NEM for 30 min and then treated with 1.5 μM 1-Cu2+ for another 30 min; (e) brightfield image of d; (f) merged image d and e.
Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770
D. Chao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
4. Conclusions In conclusion, a new long-lived organic fluorescent probe 1, bearing a TADF core and terpyridine fragments, has been synthesized for the detection of Cu2+ and biothiols. The fluorescence intensity of probe 1 can be selectively quenched by Cu2+ and subsequently recovered in the presence of biothiols, displaying an ON–OFF–ON switching process. Meanwhile, the fluorescence lifetime (2.1 μs) decreased significantly upon addition of Cu2+ (18.5 ns) due to the formation of ensemble 1Cu2+. However, a longer lifetime of 0.742 μs is found in the presence of biothiols for the solution containing 1-Cu2+. Our results show organic TADF compounds have promising applications in lifetime-based sensing, in spite of a common sensing process.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work has been supported by the National Natural Science Foundation of China (No. 21605013 and No. 21702213) and K. C. Wong MagnaFund in Ningbo University. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117770.
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Please cite this article as: D. Chao, Y. Pan and X.-W. Gao, A long-lived Donor–Acceptor fluorescent probe for sequential detection of Cu2+ and biothiols, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117770