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Fluorescent norbornene for sequential detection of mercury and biothiols
Dyes and Pigments 172 (2020) 107872
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Fluorescent norbornene for sequential detection of mercury and biothiols Qing Guo a b
a,1
, Yang Zhang
b,1
a
a,∗
, Zhi-Hong Lin , Qian-Yong Cao , Yong Chen
T
b,∗∗
Department of Chemistry, Nanchang University, Nanchang, 330031, PR China Institute for Advanced Study, Nanchang University, Nanchang, 330031, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Norbornene Mercury(II) recognition Biothiols recognition Ratiometric sensing
A pyrene functionalized norbornene (1) and its homopolymer (P1), which was obtained by the ring opening metathesis polymerization (ROMP) method, have been designed and synthesized. Both 1 and P1 exhibit fluorescence ratiometric sensing of Hg2+ ions in Tris-HCl buffer solution. In addition, the in situ obtained Hg(II) complexes 1-Hg2+ and P1–Hg2+ can sequentially detect biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) via restoration their original fluorescence. In addition, probe 1 has been successfully used for detection of Hg2+ and Cys in living cells.
1. Introduction Mercury is a well-known toxic substance. Mercury pollution poses a serious threat to human health, the environment and the ecosystem [1,2]. When it accumulated in organisms through the food chain, mercury ions can inactivate enzymes and affect the normal metabolism of cells through binding with the sulfhydryl groups of proteins, which can cause irreversible damage to liver, brain organs and cardiovascular diseases [3‒7]. Therefore, the sensitive and rapid detection of Hg(II) in environmental and biological systems is particularly important. So far, several techniques for detection of mercury ions have been developed, such as atomic absorption spectroscopy (AAS), high performance liquid chromatography (HPLC), inductively coupled plasma mass spectrometry (ICP-MS) and electrochemical methods [8‒13]. These conventional techniques often require expensive devices and a lot of manipulation time. Recently, man-made fluorescent sensors have attracted much attention because the sensitivity and simplicity of the fluorescence technique, and ease of carrying out on-site testing [14‒16]. Thus, many fluorescent sensors with different flurophores (e.g. rhodamine, pyrene, naphthalimide, boron dipyrromethene, dansyl groups) and metal ions binding sites have been established for sensing and detection of Hg2+ in the environment and biosystems [17‒27]. However, these reproted Hg2+ sensors have drawbacks such as interference from other metal ions, a turn-off response, and poor solubility in aqueous solution [17,24,28‒30]. It still remains a challenge to design new Hg2+ sensors with high selectivity and sensitivity. Biothiols, including glutathione (GSH), cysteine (Cys), and
homocysteine (Hcy), are involved in many important physiological processes in organisms [31‒34]. The abnormal levels of biothiols can cause a number of diseases on living system. For example, the defect of Cys leads to cardiovascular disease and some skin diseases. Therefore, it is especially important to selectively identify Cys and other thiol-containing amino acids in biosystems. To date, a number of fluorescent probes for the detection of biothiols have been constructed through different strategies, including competitive displacement assays, metalligand interactions, and specific reactions between probes and biothiols [35‒39]. Among them, the coordination metal complexes via the displacement approach, particularly the Hg(II) and Cu(II) complexes, have attracted much attention because the strong binding ability of sulfur atom toward these two thiophilic metals [40‒47]. Regarding the intrinsic fluorescence quenching effect of Hg(II) and Cu(II) ions, a “turnon” fluorescence sensor can be easily obtained via the strong interaction between Hg(II) and biothiol, in which displaces the organic ligand with resorting its original fluorescence. It is well known that the present sensors for recognition of Hg(II) thiols are most limited to small molecules. Polymer anchored sensor systems are still relatively rare. The polymer based sensors are regarded to show some advantages over the small organic probes, such as signal amplification and better water solubility [48‒49]. In this context, here in we report a pyrene-functionalized norbornene 1 and its homopolymer P1 for sequential recognition of Hg(II) and biothiols in aqueous solution and biosystem. Norbornene is a good polymer backbone, and has been widely used for construction various polymer sensors for anion, metal ion and small molecule [50‒54]. We find both monomer 1
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q.-Y. Cao), [email protected] (Y. Chen). 1 These two authors contributed equally to this work. ∗∗
https://doi.org/10.1016/j.dyepig.2019.107872 Received 3 August 2019; Received in revised form 7 September 2019; Accepted 7 September 2019 Available online 08 September 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
Dyes and Pigments 172 (2020) 107872
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Scheme 1. Synthesis route of monomer 1 and its homopolymer P1.
and homopolymer P1 show a good ratiometric response toward Hg(II) over other metal ions, and the in situ obtained Hg(II) complexes can be used as a secondary sensor for biothiols. The biological application of probes for the detection of Hg(II) and Cys in living cells is discussed. The binding mechanism between probes and guests has been detailedly investigated by UV–vis, emission spectra and 1H NMR titrations.
generation catalyst (34 mg, 0.04 mmol) were dissolved in dichloromethane (5 mL). This mixture stirred for 4 h at room temperature. Then ethyl-vinyl ether (1 mL) was added to quench the polymerization. The off white polymer P1 (140 mg, 70%) solid was then obtained by precipitating in cold ether and concentrated under vacuum. FT IR (KBr) ʋmax (cm−1): 3007, 2942, 1604, 1575, 1476, 1396, 1280, 1044, 785, 722. 1H NMR (400 MHz, DMSO‑d6): δ 8.87 (s, 1H), 8.42 (s, 8H), 7.09 (s, 2H), 5.10 (s, 2H), 4.32 (s, 2H), 3.93 (s, 2H), 3.26 (s, 3H), 2.67 (s, 1H), 2.34 (s, 1H), 1.58 (s, 3H), 1.23 (s, 1H). GPC (THF, polystyrene standards): Mn = 35500, Mw = 35800, and PDI = 1.01.
2. Experimental section 2.1. General reagents and instrumentations
2.4. Calculation of the association constants and limit of detection (LOD)
All the chemical reagents and solvents are analytic pure and used without further purification. The two precursors N-(2-azidoethyl) pyrene-1-sulfonamide (7) and N-(prop-2-ynyl)bicyclo[2.2.1]hept-5ene-2-carboxamide (4) were prepared by the reported method [51,55]. The 1H and 13C NMR spectra were recorded on a Bruker AVANCE 400 NMR instrument in DMSO‑d6 solution. The chemical shifts are reported as δ values (ppm) relative to TMS. The UV–vis spectra were recorded on a PerkinElmer LAMBDA 35 UV/Vis system. The emission spectra were measured on a Hitach F-4500 Fluorescence spectrophotometer.
The binding constants for 1-Hg2+ and P1–Hg2+ complexes were obtained from the fluorescence titration data. According to the BenesiHildebrand method, the equation for 2:1 host:guest complexes is given below [51,56]:
1 1 1 + = I − I0 I ′ − I0 K (I ′ − I0)[M ]2 In the equations, I0 is the intensity of fluorescence of 1 and P1 without Hg2+, I is the intensity with a particular concentration of Hg2+, I′ is the intensity of the fully complexed form at the highest concentration of Hg2+, and K is the binding constant. The LODs were obtained via the following equation:
2.2. Synthesis of probe 1 (N-((1-(2-(pyrene-1-sulfonamido)ethyl)-1H1,2,3-triazol-4-yl)- methyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide) Under nitrogen, compounds 4 (175 mg, 1 mmol), 7 (350 mg, 1 mmol), CuSO4·5H2O (26.6 mg, 0.15 mmol) and L-ascorbic acid sodium (40 mg, 0.2 mmol) were added into DMF (3 mL) solvent in a round bottom flask. The mixture was stirred at room temperature for 5 h. Then the reaction mixture was poured into cold deionized water (50 mL) to get the precipitate. The pure yellow compound 1 (475 mg, 90% yield) was obtained by silica gel chromatograph using CH2Cl2/MeOH (95:5, v/v) as the eluent. mp 187 °C. FT IR (KBr) ʋmax (cm−1): 3006, 2945, 1605, 1578, 1475, 1396, 1284, 1040, 985, 786, 734. 1H NMR (400 MHz, DMSO‑d6): δ 8.83 (d, J = 9.5 Hz, 1H), 8.60 (d, J = 8.4 Hz, 1H), 8.46–8.29 (m, 5H), 8.25–8.15 (m, 2H), 7.30 (s, 1H), 6.41 (s, 1H), 6.30 (s, 1H), 6.09 (t, J = 4.4 Hz, 1H), 5.83–5.74 (m, 1H), 4.28 (t, J = 5.8 Hz, 2H), 3.99 (q, J = 8.3, 5.8 Hz, 2H), 3.35 (q, J = 6.0 Hz, 2H), 3.02 (s, 1H), 2.81 (s, 1H), 2.76 (dd, J = 9.2, 4.6 Hz, 1H), 1.83–1.72 (m, 1H), 1.33–1.26 (m, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 137.2, 132.6, 130.9, 130.5, 130.0, 127.6, 127.5, 127.3, 127.1, 124.6, 123.7, 123.4, 49.8, 49.4, 46.0, 43.6, 42.8, 42.5, 34.5, 28.8. TOF-HRMS (m/z): Calcd. for C29H27N5O3S 526.1907 (M + H+), found 526.1912.
LOD = 3δ/k Where the δ represents five blank measurements of average standard deviation, and k represents the slope of linear fitting curve. 3. Results and discussion The synthesis rout of the targets probes 1 and P1 was shown in Scheme 1. With pyrene and 5-norbornene-2-carboxylic acid as the starting materials, the pyrenyl appended azide 7 and the norbornene functionalized alkyne 4 were first synthesized by the literature method. With the CuSO4⋅5H2O/sodium ascorbate as the catalysts, click reaction of 7 and 4 gave probe 1 in high yield (90%) in DMF solution [57,58]. The homopolymer P1 was then obtained by ring-opening metathesis polymerization (ROMP) of monomer 1 using of Grubbs 2 catalyst [59]. The structure of 1 and P1 was confirmed by 1H NMR and FT IR spectra, and also by 1H NMR, 13C NMR, FT IR spectroscopy and ESI mass spectra for 1. Thermal property of the homopolymer P1 was examined by thermogravimetric analysis (TGA) (Fig. S1). Polymer P1 possesses high thermal stability with decomposition temperatures (temperature at 5% weight loss) above 282 °C, higher than that of monomer 1.
2.3. Synthesis of P1 Under nitrogen, monomer 1 (200 mg, 0.4 mmol) and Grubbs 2ed 2
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The recognition ability of 1 and P1 toward various metal ions (K+, Na+, Ca2+, Pb2+, Ba2+, Fe3+, Cu2+, Cr3+, Co2+, Mg2+, Zn2+, Ag+, Cd2+, Hg2+, Fe2+) was then investigated by fluorescence spectra in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. Both 1 and P1 show typical pyrene-based absorption band at about 352 nm (ε = 5.1 × 104 mol−1 dm3•cm−1 for 1, and 3.9 × 104 mol−1 dm3•cm−1 for P1). With excitation at 365 nm by a UV hand-held lamp, the two probes exhibit similar strong blue fluorescence. However, the emission spectra are different. Probe 1 gives only a strong pyrene-based monomer emission at 384 nm, while P1 shows both a strong monomer emission at 384 nm, and a weak excimer emission centered at 490 nm, which may originate from the π-π stacking of the adjacent pyrene units of polymer P1. Actually, we found P1 adopts nona-aggregations in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. The mean hydrated diameter of P1 was obtained to be 375 nm by the dynamic light scattering experiments (Fig. S2). The Stokes shift of 1 and P1 is determined to be 30 nm. In addition, the relative fluorescence quantum yield was calculated using quinine sulfate as a reference standard, with the values 0.45 of 1, and 0.18 of P1, respectively. Upon the addition of equal amount (10 equiv) of various metal ions, the absorption of 1 and P1 barely changed (Fig. S3). However, 1 and P1 show a significant fluorescence response toward Hg(II) over other metal ions (Fig. 1). Upon binding with Hg(II), the monomer probe 1 exhibits a new excimer emission at 490 nm with quenching the monomer emission at 384 nm, while the homopolymer P1 shows only a decrease in the monomer emission at 384 nm. An obviously emission color change from blue to green was observed for both 1 and P1 after coordination with Hg(II). These phenomena indicated that 1 and P1 can be used as ratiometric fluorescence sensors of Hg(II). Fig. 2 shows the detailed emission changes of 1 toward Hg(II). Upon titration of Hg(II) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution, the pyrene-based monomer emission of 1 at 384 nm gradually decrease, with increasing a new pyrene excimer band at 490 nm. The formation pyrene excimer emission implies that two probes 1 interact with Hg(II) ion with stacking the pyrene fluorophores. An obvious isoemissive point at 461 nm was observed, indicating the formation of a 2:1 stoichiometry complex species during the interaction. This process is saturated about 10 equivalents Hg(II), with the I490nm/I384nm ration changing from 0.011 to 0.323. The binding constant between 1 and Hg(II) was obtained via the emission titration data. The binding stoichiometry of 1
with Hg(II) was established by using a Job's plot between the mole fraction and relative intensity changes at 384 nm. We observed that the fluorescence intensity went through maxima at molar fraction of 0.7, which indicates that the sensor 1 forms a 2:1 stoichiometry complex with Hg(II) (Fig. S4). According to the emission titration data, the binding constant for 1-Hg(II) complex was found to be 2.66 × 108 M−2 (Fig. S5), using the Benesi-Hildebrand equation. The titration experiments also showed that the luminescence intensity of 1 increased linearly with the concentration of Hg2+ in 10–50 μM at 384 nm. The detection limit was calculated to be 35.8 nM based on 3δ/k (δ = 1.12, k = 9.35 × 107) method (Fig. S5), indicating probe 1 gives a high sensitivity toward Hg2+. Similarly, the emission titration of P1 toward Hg(II) was also carried out (Fig. S6). The binding constant between P1 and Hg(II) was found to be 1.16 × 109 M−2 (Fig. S7), with and the detection limit of 38.3 nM. Thus, the single molecular probe of 1 still shows a good sensing ability toward Hg2+ after anchoring in the polymer chain. Next the competition experiments of probes 1 and P1 toward Hg(II) in the presence of other tested metal ions were performed (Fig. 3a and Figs. S8a). The presence of the other miscellaneous competitive metal ions scarcely induced any change in the emission response compared to Hg(II) alone. Furthermore, the sensing ability of 1 and P1 toward Hg(II) at different pH values was also monitored (Figs. 3b and S8b). Both probes showed satisfactory sensing response over a wide pH range from 2 to 10. Therefore, the current probes can detect Hg(II) in physiological conditions, which could be the advantageous property for further cellbased studies. Regarding the thiophilic property of the Hg2+ ions, and the key roles of the biothiols in biological systems, we think that the in situ 1Hg2+ and P1–Hg2+ complexes can be used as secondary sensors for biothiols. The fluorescence response of the 1-Hg2+ and P1–Hg2+ complexes toward 17 different amino acids (free, Ala, Asp, Arg, His, Leu, Lys, Hcy, Cys, GSH, Gly, Glu, Met, Phe, Ser, Thr, Trp, Val) was shown in Fig. 4 and Figs. S9. It was found that both 1-Hg2+ and P1–Hg2+ show a good fluorescence response toward Hcy, Cys and GSH. Upon addition these three thio-containing amino acids, the original fluorescence of 1 and P1 was restored, with the color changing form weak green to strong blue emission (Fig. S10). Obviously, the ligand displacement reaction happens upon addition of these biothios, as the following equation: 1/P1–Hg2+ + biothiols → 1/P1 + biothiols-Hg2+. Fig. 1. (a) The emission color changes of 1 and P1 before and after the addition of 10 equivalents metal ions with exciting at 365 nm by a UV hand-held lamp; (b) and (c). Emission spectra of 1 (b) and P1 (c) Before and after the addition of 10 equivalent of various metal ions in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution, c = 10 μM, λexi = 355 nm.
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Fig. 2. (a) The fluorescence spectra changes of 1 (10 μM) with the increasing the concentration of Hg(II) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. (b) The fluorescence intensity ratio I490nm/I384nm of 1 versus the equivalents of Hg(II) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. Fig. 3. (a) Bar diagram showing the emission ratio I490nm/I384nm of 1 (10 μM) upon the addition of 10 equivalents other metal anions (black bar, 1-none, 2K+, 3-Na+, 4-Ca2+, 5-Pb2+, 6-Ba2+, 7-Fe3+, 8Cu2+, 9-Cr3+, 10-Co2+, 11-Mg2+, 12-Zn2+, 13-Ag+, 14-Cd2+, 15-Fe2+) and then 10 equivalent Hg(II) (red bar). (b) The relative fluorescence changes of 1 (10 μM) at 384 nm upon the addition of 10 equivalents of Hg(II) at different pH values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
S12). Similar sigmoidal curves of fluorescence intensity against these biothios concentration were also observed, with the low LOD values range from 21 nM to 75 nM (Table S1, Fig. S13‒S14). To reveal the binding mechanism between host and guest, the 1H NMR spectra of 1 before and after addition of Hg(II) in D2O-DMSO (1:9, v/v) solution were investigated (Fig. 6). In D2O-DMSO solution, the triazole ring proton Ha gives a large down-field shift from 7.42 ppm to 7.95 ppm upon binding with Hg(II), indicating that the triazole N atom, together with the sulfonamide oxygen donor, play a key role for binding with the Hg(II) center [60]. In addition, an obviously up-field shift was observed in pyrene protons in 8.3–8.9 ppm, which may be attributed to the π-π stacking interaction of the intermolecular pyrenyl for coordination, as confirmed by the emission titration results. Upon further addition of Cys, the 1H NMR spectra of 1 were almost fully revived, indicating the displacement of probe 1 from the 1-Hg2+ complex. Low toxicity is a very important indicator for fluorescent probes used in biological systems. Therefore, we used the standard MTT assay
These remarkable changes in fluorescence spectra indicate that 1 and P1 can be used as a potential tool for sensitive detecting biothiols over other amino acids. Fig. 5 shows the detailed emission changes of 1-Hg(II) toward Cys for an example. The in situ 1-Hg(II) complex was obtained by mixing 100 μM Hg2+ with 10 μM 1 in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. The monomer emission of 1-Hg(II) at 380 nm was increased with decreasing the excimer emission at 480 nm by the incremental addition of Hg2+. The fluorescence changes reached a maximum plateau at around 100 μM Cys, indicating a full displacement Hg2+ from 1Hg(II) complex. The plot of fluorescence intensity against Cys concentration showed a sigmoidal curve, with an obvious fluorescence response beginning at 40 μM of Cys. A linear response was found from 65 μM to 90 μM at 384 nm, and the detection limit was measured as low as 21 nM based on 3δ/k (δ = 1.12, k = 1.63 × 108) method. The emission titrations of 1-Hg2+ toward GSH and Hcy (Fig. S11), and P1–Hg2+ toward Cys, GSH and Hcy were also investigated (Fig.
Fig. 4. (a) Emission spectra of 1-Hg(II) complex (10 μM) before and after the addition of 10 equivalent of various amino acids in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. (b) Bar diagram showing the change of the emission intensity recorded at 384 nm upon addition of various amino acids of monomer 1-Hg(II) complex. 1:free, 2: Ala, 3: Asp, 4: Arg, 5: His, 6:Leu, 7:Lys, 8:Hcy, 9:Cys, 10:GSH, 11:Gly, 12:Glu, 13:Met, 14:Phe, 15:Ser, 16:Thr, 17:Trp, 18:Val.
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Fig. 5. (a) The change of fluorescence emission spectra of monomer 1-Hg(II) complex after the addition of various amounts of Cys (10 μM) in DMSOTris (1:9, v/v, pH = 7.4) buffer solution, (b) the fluorescence intensity ratio of I384nm/I490nm after the addition of various equivalents of Cys (10 μM) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution.
to study the cytotoxicity of probe 1. HepG-2 liver cancer cells were selected and incubated with the compound 1 at concentrations ranging from 0 to 40 μM for 24 h (Fig. 7). More than 80% of the cells survived even the concentration reach 40 μM, demonstrating that compound 1 is low cytotoxic to cells under the experimental conditions. Due to the good biocompatibility, we used monomer 1 to detect Hg (II) and Cys in living cells (Fig. 8). The HepG-2 cells were first treated by N-methylmaleimide (1 mM) to avoid the interference from intracellular thiols [61]. Then the HepG-2 cells were co-stained with probe 1 (10 μM) and commercially available dye Mito-Tracker red (100 nM), and incubated for 30 min (37 °C) in a CO2 incubator (95% relative humidity with 5% CO2). The brightness at the blue channel (410–450 nm) was observed in the cytoplasm and cell membrance. Thus, probe 1 penetrates well into cells and has a good imaging capability. After addition of Hg(II) and incubating for further 30 min, the fluorescence signal at the blue channel diminished with growing the fluorescence at the green channel (450–500 nm). When 10 μM Cys was added, the original blue fluorescence of probe 1 was recovered due to the replacement of Hg(II) by Cys in living cells. Meanwhile the dye Mito-Tracker red light channel used for the control experiment showed almost no change. Thus, probe 1 was successfully demonstrated for the fluorescence imaging of intracellular Hg(II), and the resulting compound 1-Hg(II) complex served to detect the thiol-containing amino acids in living cells.
Fig. 7. Cell viability results after incubation of HEPG-2 cells with various concentration of monomer 1 in aqueous solution.
4. Conclusions To summarize, we have designed a pyrene-functionalized norbornene 1 and its homopolymer P1 for sequential recognition of Hg2+ ions and biothiols. In Tris-HCl buffer solution, both 1 and P1 show an fluorescence ratiometric sensing of Hg2+ ions, with decreasing the pyrene-based emission at 380 nm, and increasing a new band at 480 nm. The 1H NMR titrations reveal that the triazole N atom and the sulfonamide oxygen donor of the probe play a key role for binding with
Fig. 6. The partial 1H NMR change of 1 sequential addition of Hg(II) and Cys in D2O-DMSO‑d6 (1:9, v/v) solution. 5
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Fig. 8. Fluorescence images for liver cancer HEPG-2 cells with 1 and Mito-Tracker red. (a) 10 μM of 1 and 100 nM of Mito-Tracker red, (b) 10 μM of 1 and the addition of 10 μM Hg(II), (c) 10 μM of probe 1 and the addition of 10 μM Hg(II) and the addition of 50 μM Cys. The HepG-2 cells were pretreated by N-methylmaleimide. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Hg(II) center. In addition, the in situ obtained Hg(II) complexes of 1Hg2+ and P1–Hg2+ can be used as a secondary sensor for detecting thio-containing amino acids with low LODs, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The addition of these amino acids can restore the fluorescence of probes for the ligands displacement mechanism. Furthermore, probe 1 has been successfully used for fluorescence imaging of intracellular Hg2+ and Cys in living cells.
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Conflicts of interest The authors declared that they have no conflicts of interest to this work. Acknowledgments This work was supported by the National Natural Science Foundation of China (nos. 21762028 and 21462027). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107872. References [1] Järup L. Hazards of heavy metal contamination. Br Med Bull 2003;68:167–82. [2] Boening DW. Ecological effects, transport, and fate of mercury: a general review. Chemosphere 2000;40:1335–51. [3] Clarkson TW, Magos L, Myers GJ. The toxicology of mercury—current exposures and clinical manifestations. J Med 2003;349:1731–7. [4] Reus IS, Bando I, Andres D, Cascales M. Relationship between expression of HSP70 and metallothionein and oxidative stress during mercury chloride induced acute liver injury in rats. J Biochem Mol Toxicol 2003;17:161–8. [5] Azevedo BF, Furieri LB, Pecanha FM, Wiggers GA, Vassallo PF, Simoes MR, Fiorim J, Batista PR, Fioresi M, Rossoni L, Stefanon I, Alonso MJ, Salaices M, Vassallo1 DV. Toxic effects of mercury on the cardiovascular and central nervous systems. BioMed Res Int 2012:1‒12. [6] Genchi G, Sinicropi MS, Carocci A, Lauria G, Catalano A. Mercury exposure and heart diseases. Int J Environ Res Public Health 2017;14:74. [7] Virtanena JK, Rissanena TH, Voutilainena S, Tuomainen TP. Mercury as a risk factor
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Fluorescent norbornene for sequential detection of mercury and biothiols Qing Guo a b
a,1
, Yang Zhang
b,1
a
a,∗
, Zhi-Hong Lin , Qian-Yong Cao , Yong Chen
T
b,∗∗
Department of Chemistry, Nanchang University, Nanchang, 330031, PR China Institute for Advanced Study, Nanchang University, Nanchang, 330031, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Norbornene Mercury(II) recognition Biothiols recognition Ratiometric sensing
A pyrene functionalized norbornene (1) and its homopolymer (P1), which was obtained by the ring opening metathesis polymerization (ROMP) method, have been designed and synthesized. Both 1 and P1 exhibit fluorescence ratiometric sensing of Hg2+ ions in Tris-HCl buffer solution. In addition, the in situ obtained Hg(II) complexes 1-Hg2+ and P1–Hg2+ can sequentially detect biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) via restoration their original fluorescence. In addition, probe 1 has been successfully used for detection of Hg2+ and Cys in living cells.
1. Introduction Mercury is a well-known toxic substance. Mercury pollution poses a serious threat to human health, the environment and the ecosystem [1,2]. When it accumulated in organisms through the food chain, mercury ions can inactivate enzymes and affect the normal metabolism of cells through binding with the sulfhydryl groups of proteins, which can cause irreversible damage to liver, brain organs and cardiovascular diseases [3‒7]. Therefore, the sensitive and rapid detection of Hg(II) in environmental and biological systems is particularly important. So far, several techniques for detection of mercury ions have been developed, such as atomic absorption spectroscopy (AAS), high performance liquid chromatography (HPLC), inductively coupled plasma mass spectrometry (ICP-MS) and electrochemical methods [8‒13]. These conventional techniques often require expensive devices and a lot of manipulation time. Recently, man-made fluorescent sensors have attracted much attention because the sensitivity and simplicity of the fluorescence technique, and ease of carrying out on-site testing [14‒16]. Thus, many fluorescent sensors with different flurophores (e.g. rhodamine, pyrene, naphthalimide, boron dipyrromethene, dansyl groups) and metal ions binding sites have been established for sensing and detection of Hg2+ in the environment and biosystems [17‒27]. However, these reproted Hg2+ sensors have drawbacks such as interference from other metal ions, a turn-off response, and poor solubility in aqueous solution [17,24,28‒30]. It still remains a challenge to design new Hg2+ sensors with high selectivity and sensitivity. Biothiols, including glutathione (GSH), cysteine (Cys), and
homocysteine (Hcy), are involved in many important physiological processes in organisms [31‒34]. The abnormal levels of biothiols can cause a number of diseases on living system. For example, the defect of Cys leads to cardiovascular disease and some skin diseases. Therefore, it is especially important to selectively identify Cys and other thiol-containing amino acids in biosystems. To date, a number of fluorescent probes for the detection of biothiols have been constructed through different strategies, including competitive displacement assays, metalligand interactions, and specific reactions between probes and biothiols [35‒39]. Among them, the coordination metal complexes via the displacement approach, particularly the Hg(II) and Cu(II) complexes, have attracted much attention because the strong binding ability of sulfur atom toward these two thiophilic metals [40‒47]. Regarding the intrinsic fluorescence quenching effect of Hg(II) and Cu(II) ions, a “turnon” fluorescence sensor can be easily obtained via the strong interaction between Hg(II) and biothiol, in which displaces the organic ligand with resorting its original fluorescence. It is well known that the present sensors for recognition of Hg(II) thiols are most limited to small molecules. Polymer anchored sensor systems are still relatively rare. The polymer based sensors are regarded to show some advantages over the small organic probes, such as signal amplification and better water solubility [48‒49]. In this context, here in we report a pyrene-functionalized norbornene 1 and its homopolymer P1 for sequential recognition of Hg(II) and biothiols in aqueous solution and biosystem. Norbornene is a good polymer backbone, and has been widely used for construction various polymer sensors for anion, metal ion and small molecule [50‒54]. We find both monomer 1
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (Q.-Y. Cao), [email protected] (Y. Chen). 1 These two authors contributed equally to this work. ∗∗
https://doi.org/10.1016/j.dyepig.2019.107872 Received 3 August 2019; Received in revised form 7 September 2019; Accepted 7 September 2019 Available online 08 September 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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Scheme 1. Synthesis route of monomer 1 and its homopolymer P1.
and homopolymer P1 show a good ratiometric response toward Hg(II) over other metal ions, and the in situ obtained Hg(II) complexes can be used as a secondary sensor for biothiols. The biological application of probes for the detection of Hg(II) and Cys in living cells is discussed. The binding mechanism between probes and guests has been detailedly investigated by UV–vis, emission spectra and 1H NMR titrations.
generation catalyst (34 mg, 0.04 mmol) were dissolved in dichloromethane (5 mL). This mixture stirred for 4 h at room temperature. Then ethyl-vinyl ether (1 mL) was added to quench the polymerization. The off white polymer P1 (140 mg, 70%) solid was then obtained by precipitating in cold ether and concentrated under vacuum. FT IR (KBr) ʋmax (cm−1): 3007, 2942, 1604, 1575, 1476, 1396, 1280, 1044, 785, 722. 1H NMR (400 MHz, DMSO‑d6): δ 8.87 (s, 1H), 8.42 (s, 8H), 7.09 (s, 2H), 5.10 (s, 2H), 4.32 (s, 2H), 3.93 (s, 2H), 3.26 (s, 3H), 2.67 (s, 1H), 2.34 (s, 1H), 1.58 (s, 3H), 1.23 (s, 1H). GPC (THF, polystyrene standards): Mn = 35500, Mw = 35800, and PDI = 1.01.
2. Experimental section 2.1. General reagents and instrumentations
2.4. Calculation of the association constants and limit of detection (LOD)
All the chemical reagents and solvents are analytic pure and used without further purification. The two precursors N-(2-azidoethyl) pyrene-1-sulfonamide (7) and N-(prop-2-ynyl)bicyclo[2.2.1]hept-5ene-2-carboxamide (4) were prepared by the reported method [51,55]. The 1H and 13C NMR spectra were recorded on a Bruker AVANCE 400 NMR instrument in DMSO‑d6 solution. The chemical shifts are reported as δ values (ppm) relative to TMS. The UV–vis spectra were recorded on a PerkinElmer LAMBDA 35 UV/Vis system. The emission spectra were measured on a Hitach F-4500 Fluorescence spectrophotometer.
The binding constants for 1-Hg2+ and P1–Hg2+ complexes were obtained from the fluorescence titration data. According to the BenesiHildebrand method, the equation for 2:1 host:guest complexes is given below [51,56]:
1 1 1 + = I − I0 I ′ − I0 K (I ′ − I0)[M ]2 In the equations, I0 is the intensity of fluorescence of 1 and P1 without Hg2+, I is the intensity with a particular concentration of Hg2+, I′ is the intensity of the fully complexed form at the highest concentration of Hg2+, and K is the binding constant. The LODs were obtained via the following equation:
2.2. Synthesis of probe 1 (N-((1-(2-(pyrene-1-sulfonamido)ethyl)-1H1,2,3-triazol-4-yl)- methyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide) Under nitrogen, compounds 4 (175 mg, 1 mmol), 7 (350 mg, 1 mmol), CuSO4·5H2O (26.6 mg, 0.15 mmol) and L-ascorbic acid sodium (40 mg, 0.2 mmol) were added into DMF (3 mL) solvent in a round bottom flask. The mixture was stirred at room temperature for 5 h. Then the reaction mixture was poured into cold deionized water (50 mL) to get the precipitate. The pure yellow compound 1 (475 mg, 90% yield) was obtained by silica gel chromatograph using CH2Cl2/MeOH (95:5, v/v) as the eluent. mp 187 °C. FT IR (KBr) ʋmax (cm−1): 3006, 2945, 1605, 1578, 1475, 1396, 1284, 1040, 985, 786, 734. 1H NMR (400 MHz, DMSO‑d6): δ 8.83 (d, J = 9.5 Hz, 1H), 8.60 (d, J = 8.4 Hz, 1H), 8.46–8.29 (m, 5H), 8.25–8.15 (m, 2H), 7.30 (s, 1H), 6.41 (s, 1H), 6.30 (s, 1H), 6.09 (t, J = 4.4 Hz, 1H), 5.83–5.74 (m, 1H), 4.28 (t, J = 5.8 Hz, 2H), 3.99 (q, J = 8.3, 5.8 Hz, 2H), 3.35 (q, J = 6.0 Hz, 2H), 3.02 (s, 1H), 2.81 (s, 1H), 2.76 (dd, J = 9.2, 4.6 Hz, 1H), 1.83–1.72 (m, 1H), 1.33–1.26 (m, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 137.2, 132.6, 130.9, 130.5, 130.0, 127.6, 127.5, 127.3, 127.1, 124.6, 123.7, 123.4, 49.8, 49.4, 46.0, 43.6, 42.8, 42.5, 34.5, 28.8. TOF-HRMS (m/z): Calcd. for C29H27N5O3S 526.1907 (M + H+), found 526.1912.
LOD = 3δ/k Where the δ represents five blank measurements of average standard deviation, and k represents the slope of linear fitting curve. 3. Results and discussion The synthesis rout of the targets probes 1 and P1 was shown in Scheme 1. With pyrene and 5-norbornene-2-carboxylic acid as the starting materials, the pyrenyl appended azide 7 and the norbornene functionalized alkyne 4 were first synthesized by the literature method. With the CuSO4⋅5H2O/sodium ascorbate as the catalysts, click reaction of 7 and 4 gave probe 1 in high yield (90%) in DMF solution [57,58]. The homopolymer P1 was then obtained by ring-opening metathesis polymerization (ROMP) of monomer 1 using of Grubbs 2 catalyst [59]. The structure of 1 and P1 was confirmed by 1H NMR and FT IR spectra, and also by 1H NMR, 13C NMR, FT IR spectroscopy and ESI mass spectra for 1. Thermal property of the homopolymer P1 was examined by thermogravimetric analysis (TGA) (Fig. S1). Polymer P1 possesses high thermal stability with decomposition temperatures (temperature at 5% weight loss) above 282 °C, higher than that of monomer 1.
2.3. Synthesis of P1 Under nitrogen, monomer 1 (200 mg, 0.4 mmol) and Grubbs 2ed 2
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The recognition ability of 1 and P1 toward various metal ions (K+, Na+, Ca2+, Pb2+, Ba2+, Fe3+, Cu2+, Cr3+, Co2+, Mg2+, Zn2+, Ag+, Cd2+, Hg2+, Fe2+) was then investigated by fluorescence spectra in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. Both 1 and P1 show typical pyrene-based absorption band at about 352 nm (ε = 5.1 × 104 mol−1 dm3•cm−1 for 1, and 3.9 × 104 mol−1 dm3•cm−1 for P1). With excitation at 365 nm by a UV hand-held lamp, the two probes exhibit similar strong blue fluorescence. However, the emission spectra are different. Probe 1 gives only a strong pyrene-based monomer emission at 384 nm, while P1 shows both a strong monomer emission at 384 nm, and a weak excimer emission centered at 490 nm, which may originate from the π-π stacking of the adjacent pyrene units of polymer P1. Actually, we found P1 adopts nona-aggregations in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. The mean hydrated diameter of P1 was obtained to be 375 nm by the dynamic light scattering experiments (Fig. S2). The Stokes shift of 1 and P1 is determined to be 30 nm. In addition, the relative fluorescence quantum yield was calculated using quinine sulfate as a reference standard, with the values 0.45 of 1, and 0.18 of P1, respectively. Upon the addition of equal amount (10 equiv) of various metal ions, the absorption of 1 and P1 barely changed (Fig. S3). However, 1 and P1 show a significant fluorescence response toward Hg(II) over other metal ions (Fig. 1). Upon binding with Hg(II), the monomer probe 1 exhibits a new excimer emission at 490 nm with quenching the monomer emission at 384 nm, while the homopolymer P1 shows only a decrease in the monomer emission at 384 nm. An obviously emission color change from blue to green was observed for both 1 and P1 after coordination with Hg(II). These phenomena indicated that 1 and P1 can be used as ratiometric fluorescence sensors of Hg(II). Fig. 2 shows the detailed emission changes of 1 toward Hg(II). Upon titration of Hg(II) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution, the pyrene-based monomer emission of 1 at 384 nm gradually decrease, with increasing a new pyrene excimer band at 490 nm. The formation pyrene excimer emission implies that two probes 1 interact with Hg(II) ion with stacking the pyrene fluorophores. An obvious isoemissive point at 461 nm was observed, indicating the formation of a 2:1 stoichiometry complex species during the interaction. This process is saturated about 10 equivalents Hg(II), with the I490nm/I384nm ration changing from 0.011 to 0.323. The binding constant between 1 and Hg(II) was obtained via the emission titration data. The binding stoichiometry of 1
with Hg(II) was established by using a Job's plot between the mole fraction and relative intensity changes at 384 nm. We observed that the fluorescence intensity went through maxima at molar fraction of 0.7, which indicates that the sensor 1 forms a 2:1 stoichiometry complex with Hg(II) (Fig. S4). According to the emission titration data, the binding constant for 1-Hg(II) complex was found to be 2.66 × 108 M−2 (Fig. S5), using the Benesi-Hildebrand equation. The titration experiments also showed that the luminescence intensity of 1 increased linearly with the concentration of Hg2+ in 10–50 μM at 384 nm. The detection limit was calculated to be 35.8 nM based on 3δ/k (δ = 1.12, k = 9.35 × 107) method (Fig. S5), indicating probe 1 gives a high sensitivity toward Hg2+. Similarly, the emission titration of P1 toward Hg(II) was also carried out (Fig. S6). The binding constant between P1 and Hg(II) was found to be 1.16 × 109 M−2 (Fig. S7), with and the detection limit of 38.3 nM. Thus, the single molecular probe of 1 still shows a good sensing ability toward Hg2+ after anchoring in the polymer chain. Next the competition experiments of probes 1 and P1 toward Hg(II) in the presence of other tested metal ions were performed (Fig. 3a and Figs. S8a). The presence of the other miscellaneous competitive metal ions scarcely induced any change in the emission response compared to Hg(II) alone. Furthermore, the sensing ability of 1 and P1 toward Hg(II) at different pH values was also monitored (Figs. 3b and S8b). Both probes showed satisfactory sensing response over a wide pH range from 2 to 10. Therefore, the current probes can detect Hg(II) in physiological conditions, which could be the advantageous property for further cellbased studies. Regarding the thiophilic property of the Hg2+ ions, and the key roles of the biothiols in biological systems, we think that the in situ 1Hg2+ and P1–Hg2+ complexes can be used as secondary sensors for biothiols. The fluorescence response of the 1-Hg2+ and P1–Hg2+ complexes toward 17 different amino acids (free, Ala, Asp, Arg, His, Leu, Lys, Hcy, Cys, GSH, Gly, Glu, Met, Phe, Ser, Thr, Trp, Val) was shown in Fig. 4 and Figs. S9. It was found that both 1-Hg2+ and P1–Hg2+ show a good fluorescence response toward Hcy, Cys and GSH. Upon addition these three thio-containing amino acids, the original fluorescence of 1 and P1 was restored, with the color changing form weak green to strong blue emission (Fig. S10). Obviously, the ligand displacement reaction happens upon addition of these biothios, as the following equation: 1/P1–Hg2+ + biothiols → 1/P1 + biothiols-Hg2+. Fig. 1. (a) The emission color changes of 1 and P1 before and after the addition of 10 equivalents metal ions with exciting at 365 nm by a UV hand-held lamp; (b) and (c). Emission spectra of 1 (b) and P1 (c) Before and after the addition of 10 equivalent of various metal ions in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution, c = 10 μM, λexi = 355 nm.
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Fig. 2. (a) The fluorescence spectra changes of 1 (10 μM) with the increasing the concentration of Hg(II) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. (b) The fluorescence intensity ratio I490nm/I384nm of 1 versus the equivalents of Hg(II) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. Fig. 3. (a) Bar diagram showing the emission ratio I490nm/I384nm of 1 (10 μM) upon the addition of 10 equivalents other metal anions (black bar, 1-none, 2K+, 3-Na+, 4-Ca2+, 5-Pb2+, 6-Ba2+, 7-Fe3+, 8Cu2+, 9-Cr3+, 10-Co2+, 11-Mg2+, 12-Zn2+, 13-Ag+, 14-Cd2+, 15-Fe2+) and then 10 equivalent Hg(II) (red bar). (b) The relative fluorescence changes of 1 (10 μM) at 384 nm upon the addition of 10 equivalents of Hg(II) at different pH values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
S12). Similar sigmoidal curves of fluorescence intensity against these biothios concentration were also observed, with the low LOD values range from 21 nM to 75 nM (Table S1, Fig. S13‒S14). To reveal the binding mechanism between host and guest, the 1H NMR spectra of 1 before and after addition of Hg(II) in D2O-DMSO (1:9, v/v) solution were investigated (Fig. 6). In D2O-DMSO solution, the triazole ring proton Ha gives a large down-field shift from 7.42 ppm to 7.95 ppm upon binding with Hg(II), indicating that the triazole N atom, together with the sulfonamide oxygen donor, play a key role for binding with the Hg(II) center [60]. In addition, an obviously up-field shift was observed in pyrene protons in 8.3–8.9 ppm, which may be attributed to the π-π stacking interaction of the intermolecular pyrenyl for coordination, as confirmed by the emission titration results. Upon further addition of Cys, the 1H NMR spectra of 1 were almost fully revived, indicating the displacement of probe 1 from the 1-Hg2+ complex. Low toxicity is a very important indicator for fluorescent probes used in biological systems. Therefore, we used the standard MTT assay
These remarkable changes in fluorescence spectra indicate that 1 and P1 can be used as a potential tool for sensitive detecting biothiols over other amino acids. Fig. 5 shows the detailed emission changes of 1-Hg(II) toward Cys for an example. The in situ 1-Hg(II) complex was obtained by mixing 100 μM Hg2+ with 10 μM 1 in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. The monomer emission of 1-Hg(II) at 380 nm was increased with decreasing the excimer emission at 480 nm by the incremental addition of Hg2+. The fluorescence changes reached a maximum plateau at around 100 μM Cys, indicating a full displacement Hg2+ from 1Hg(II) complex. The plot of fluorescence intensity against Cys concentration showed a sigmoidal curve, with an obvious fluorescence response beginning at 40 μM of Cys. A linear response was found from 65 μM to 90 μM at 384 nm, and the detection limit was measured as low as 21 nM based on 3δ/k (δ = 1.12, k = 1.63 × 108) method. The emission titrations of 1-Hg2+ toward GSH and Hcy (Fig. S11), and P1–Hg2+ toward Cys, GSH and Hcy were also investigated (Fig.
Fig. 4. (a) Emission spectra of 1-Hg(II) complex (10 μM) before and after the addition of 10 equivalent of various amino acids in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution. (b) Bar diagram showing the change of the emission intensity recorded at 384 nm upon addition of various amino acids of monomer 1-Hg(II) complex. 1:free, 2: Ala, 3: Asp, 4: Arg, 5: His, 6:Leu, 7:Lys, 8:Hcy, 9:Cys, 10:GSH, 11:Gly, 12:Glu, 13:Met, 14:Phe, 15:Ser, 16:Thr, 17:Trp, 18:Val.
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Fig. 5. (a) The change of fluorescence emission spectra of monomer 1-Hg(II) complex after the addition of various amounts of Cys (10 μM) in DMSOTris (1:9, v/v, pH = 7.4) buffer solution, (b) the fluorescence intensity ratio of I384nm/I490nm after the addition of various equivalents of Cys (10 μM) in DMSO-Tris (1:9, v/v, pH = 7.4) buffer solution.
to study the cytotoxicity of probe 1. HepG-2 liver cancer cells were selected and incubated with the compound 1 at concentrations ranging from 0 to 40 μM for 24 h (Fig. 7). More than 80% of the cells survived even the concentration reach 40 μM, demonstrating that compound 1 is low cytotoxic to cells under the experimental conditions. Due to the good biocompatibility, we used monomer 1 to detect Hg (II) and Cys in living cells (Fig. 8). The HepG-2 cells were first treated by N-methylmaleimide (1 mM) to avoid the interference from intracellular thiols [61]. Then the HepG-2 cells were co-stained with probe 1 (10 μM) and commercially available dye Mito-Tracker red (100 nM), and incubated for 30 min (37 °C) in a CO2 incubator (95% relative humidity with 5% CO2). The brightness at the blue channel (410–450 nm) was observed in the cytoplasm and cell membrance. Thus, probe 1 penetrates well into cells and has a good imaging capability. After addition of Hg(II) and incubating for further 30 min, the fluorescence signal at the blue channel diminished with growing the fluorescence at the green channel (450–500 nm). When 10 μM Cys was added, the original blue fluorescence of probe 1 was recovered due to the replacement of Hg(II) by Cys in living cells. Meanwhile the dye Mito-Tracker red light channel used for the control experiment showed almost no change. Thus, probe 1 was successfully demonstrated for the fluorescence imaging of intracellular Hg(II), and the resulting compound 1-Hg(II) complex served to detect the thiol-containing amino acids in living cells.
Fig. 7. Cell viability results after incubation of HEPG-2 cells with various concentration of monomer 1 in aqueous solution.
4. Conclusions To summarize, we have designed a pyrene-functionalized norbornene 1 and its homopolymer P1 for sequential recognition of Hg2+ ions and biothiols. In Tris-HCl buffer solution, both 1 and P1 show an fluorescence ratiometric sensing of Hg2+ ions, with decreasing the pyrene-based emission at 380 nm, and increasing a new band at 480 nm. The 1H NMR titrations reveal that the triazole N atom and the sulfonamide oxygen donor of the probe play a key role for binding with
Fig. 6. The partial 1H NMR change of 1 sequential addition of Hg(II) and Cys in D2O-DMSO‑d6 (1:9, v/v) solution. 5
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Fig. 8. Fluorescence images for liver cancer HEPG-2 cells with 1 and Mito-Tracker red. (a) 10 μM of 1 and 100 nM of Mito-Tracker red, (b) 10 μM of 1 and the addition of 10 μM Hg(II), (c) 10 μM of probe 1 and the addition of 10 μM Hg(II) and the addition of 50 μM Cys. The HepG-2 cells were pretreated by N-methylmaleimide. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Hg(II) center. In addition, the in situ obtained Hg(II) complexes of 1Hg2+ and P1–Hg2+ can be used as a secondary sensor for detecting thio-containing amino acids with low LODs, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH). The addition of these amino acids can restore the fluorescence of probes for the ligands displacement mechanism. Furthermore, probe 1 has been successfully used for fluorescence imaging of intracellular Hg2+ and Cys in living cells.
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