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NBD-based Fluorescent Chemosensor for the Selective Quantification of Copper and Sulfide in Aqueous Solution and Living Cells Qingtao Menga, Yu Shib, Cuiping Wanga, Hongmin Jiaa, Xue Gaoa, Run Zhangb*, Yongfei Wanga and Zhiqiang Zhanga* 5

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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x Chemosensors play important roles in cations and anions recognition in biological, industrial, and environmental processes. Although much efforts have been made to develop artificial fluorescent receptors for Cu2+ and S2−, their applications are limited in the detection in bulk solutions. In this work, we report a novel fluorescence chemosensor (NL) based on 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorophore for the quantification of Cu2+ and S2− in single intact cell. NL features specifically binds to Cu2+ in the presence of other competing cations with observable changes in UV−vis and fluorescence spectra in HEPES buffer. The in situ generated NL-Cu2+ ensemble is able to selectively sense S2− over other anions and biothiols based on the displacement approach, given a remarkable recovery of fluorescence and UV−vis absorption spectra. The detection limits of NL to Cu2+ and NL-Cu2+ to S2− were estimated to be 1.6 nM and 0.17 μM, respectively. NL and resultant complex NL-Cu2+ exhibit low cytotoxicity, cell-membrane permeability, which capable of Cu2+ and S2− imaging and quantification in living MDA-MB-231 cells.

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Chemosensors are the molecules of abiotic origin that bind selectively and reversibly the analyte of interest with concomitant change in one or more properties of the system, 1-6 such as fluorescent, 7 color, 8 or redox potentials. 9 Among of them, fluorescent chemosensors have several advantages over the other methods due to their sensitivity, specificity and real-time monitoring with fast response time. To date, enormous amount of works have been done for the rational design of fluorescent chemosensor for ions and neutral analytes. 10-17 Copper is the third element in abundance among the essential heavy metals (next to iron and zinc) in the human body and plays an important role in various physiological processes. 18 Cu2+-containing enzymes play a significant role in different catalytic processes starting from providing energy for biochemical reactions to assisting the formation of cross-links in collagen and elastin, and thereby maintaining and repairing connective tissues related to heart and arteries. 19-20 The average concentration of blood copper in the normal group is 100–150 μg/L. 21 Notably, Cu2+ can be toxic to biological systems when levels of Cu2+ ions exceed cellular needs, due to its capability of displacing other metal ions which act as cofactor in enzyme-catalyzed reactions. 22 Thus, considerable attention has been devoted to the development of fluorescent chemosensors for highly selective and sensitive recognition of Cu2+ in aqueous and biological systems. 23–25 Sulfide (S2−) has long been known as a toxic species generated not only as a byproduct in industrial processes but also in biosystems due to microbial reduction of sulfate by anaerobic bacteria and formation of sulfur-containing amino acids in meat This journal is © The Royal Society of Chemistry [year]

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proteins. 26-28 However, exposure to high level of S2− can produce various physiological and biochemical problems including irritation in mucous membranes, unconsciousness and respiratory paralysis. 29-30 Therefore, development of quick and sensitive method for immediate S2− detection in aqueous media and in biological systems is highly required. However, anion recognition in aqueous media is still an extremely challenging task for a number of reasons, such as strong hydration nature, larger ionic radius and complicated geometries of the anions. 31 Recently, one feasible strategy for the design anions sensing system is by employing the displacement method, in which the fluorophore–metal “ensemble” is non-fluorescent due to metal ion-induced fluorescence quenching. The further addition of anions may release the fluorophores with revival of fluorescence. There are several advantages of this anion sensing method, such as improved water solubility, multiple chemosensor types and straightforward synthesis procedures. 32 It is well known that Cu2+ can quench the emission of fluorescent chemosensors conferring a non-fluorescence state to ensemble devices. 33 Sulfide is known to react with Cu2+ to form a very stable CuS species, which has a low-solubility product constant ksp = 6.3×10−36. 34 Therefore, the utilization of the higher affinity of Cu2+ towards S atom for designing S2− fluorescence chemosensor with a fluorescence turn-on response have received considerable attention since they show a greater enhancement in S2−-binding affinity than purely organic receptors. 35 However, up to now, only a limited number of S2− selective chemosensors based on Cu2+ sequestering approach in water or organic/water mix-solvent have been reported in literature. 36-40 This intrigued us to design [journal], [year], [vol], 00–00 | 1

Organic & Biomolecular Chemistry Accepted Manuscript

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In this paper, we presented a NBD-based fluorescence chemosensor (NL) by a straightforward synthetic route (Scheme 1). 41 The emission signal was selectively quenched by Cu2+ via forming a NL-Cu2+ complex, and was exclusively recovered followed by addition of S2−. This “ON–OFF–ON” response provides a convenient and practical way in detection of both Cu2+ and S2− in environmental and biological samples. The photophysical properties and recognition behaviors of NL have been investigated in detail through UV−vis absorption spectra, fluorescence spectra, confocal fluorescence images, and flow cytometry quantification in single intact cells.

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suitable pH range for the present system is pH 4.5−10, which suggests that sensor NL is suitable for application under physiological conditions. The coordination of NL with Cu2+ was first investigated by UV−vis absorption spectra in HEPES buffer. As shown in Fig. 1, NL displays an absorption band with a maximum absorbance peak at about 430 nm, which is characteristic of NBD under this condition. 45 Upon an increase in the concentration of Cu2+ (0−2 equiv), the maximum absorbance peak gradually decreased with two clear isosbestic points at 376 nm and 462 nm, which indicated that the coordination of NL to a Cu2+ center. Other competitive metal ions such as Fe3+, Hg2+, Cd2+, Pb2+, Zn2+, Ni2+, Co2+, Mn2+, Cr3+, Ag+ and Ca2+, Mg2+, Ba2+, Li+, K+, Na+ did not induce any UV−vis absorption responses (Fig. S6, ESI†). These results indicate that NL could be used as a potential candidate of chemosensor for Cu2+ with a high selectivity.

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Scheme 1. Synthesis of fluorescence chemosensor NL: (1) 1% hydrazine hydrate, CHCl3/CH3OH, rt. (2) 4-diethylaminosalicylaldehyde, C2H5OH, reflux.

of NL-Cu2+ complex is presented due to the protonation of N and O atom leading to the displacement of Cu2+. Thus, the

Results and discussion 20

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We chose 7-nitrobenz-2-oxa-1,3-diazole (NBD) as the fluorophore due to its excellent photophysical properties, such as long-wavelength absorption and emission, high fluorescence quantum yield and good cell permeability. 42 NL was facilely synthesized by a two-step reaction. 7-nitrobenz-2-oxa-1,3-diazole chloride (NBD-Cl) was treated with 1% hydrazine in chloroform/methanol (1∶1) solution under room temperature to form intermediate 1. Further reaction of 1 with 4diethylaminosalicylaldehyde in ethanol solution gave target compound NL in quantitative yield. The structure of NL was well confirmed by NMR, high resolution mass spectra and elemental analysis. NL is stable in aqueous, which is confirmed by fluorescent intensity measurements (Fig. S4, ESI†). Salicylaldehyde-based hydrazone framework is a typical structure utilized for the construction of Cu2+ fluorescent chemosensor. 43 Accordingly, we believe NL will be an excellent Cu2+ selective fluorescence sensors and its resultant complex could be used as a S2−-specific fluorescence chemosensor in aqueous solution.

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For the biological application of NL, the sensing should operate in a physiological range of pH. The pKa of NL was calculated to be 11.03 ± 0.031 according to the literature method. 44 As shown in Fig. S5, the fluorescent intensity of NL is stable at pH 4.5−10.5 without obvious florescence “ON–OFF” response. 2 | Journal Name, [year], [vol], 00–00

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Fig. 1 UV-vis absorption spectra of NL (10 μM) in the presence of increasing amount of Cu2+ (0–20 μM) in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4). 1.0

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While the pH value is more than 10.5, the deprotonating of hydroxyl leads to the fluorescent quenching due to the notorious photo-induced electron transfer (PeT) from salicylaldehyde to NBD. When pH is less than 4.5, the fluorescence enhancement

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Fig. 2 Fluorescence spectra of NL (10 μM) in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4) in the presence of different amounts of Cu2+ (0–20 μM). Insert: normalized fluorescence intensities of NL (10 μM) at 519 nm as a function of Cu2+ (0–20 μM). Excitation was performed at 430 nm.

The fluorescence titration of NL towards representative metal

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and prepare of novel Cu2+-fluorophore complex to develop of S2−-specific fluorescence chemosensor.

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conditions. Upon addition of competitive cations (20 μM) to the solution of NL (10 μM), the quenching rate of the emission intensities recorded at 519 nm (F0/F) were shown in Fig. 4. No significant changes of the fluorescence intensities occurred in the presence of Fe3+, Hg2+, Cd2+, Pb2+, Zn2+, Ni2+, Co2+, Mn2+, Cr3+, Ag+ and Ca2+, Mg2+, Ba2+, Li+, K+, Na+. The addition of 1 equiv. of Cu2+ to the above solution giving rise to drastic quenching in accordance with the addition of 20 μM of Cu2+ alone, indicating that Cu2+-specific responses were not disturbed by competitive ions. Spectra recognition of S2− by NL-Cu2+ 70 60

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Fig. 6 Normalized fluorescence responses of NL-Cu2+ (10 μM) in HEPES (THF: H2O = 3:7, 20 mM, pH = 7.4) in the presence of all kinds of analytes (20 μM): 1. S2−, 2. AcO−, 3. Cl−, 4. Br−, 5. F−, 6. HSO4−, 7. NO2−, 8. PO43−, 9. HPO42−, 10. H2PO4−, 11. SCN−, 12. Cys, 13. Hcys, 14. GSH. The intensities were recorded at 519 nm, excitation at 430 nm.

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NL showed a selective fluorescence quenching only with Cu2+ among the various metal ions under similar testing This journal is © The Royal Society of Chemistry [year]

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Fig. 4 Normalized fluorescence responses of NL (10 μM) to various cations in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4). The orange bars represent the emission changes of NL in the presence of cations of interest (all are 20 μM). The green bars represent the changes of the emission that occurs upon the subsequent addition of 20 μM of Cu2+ to the above solution. The intensities were recorded at 519 nm, excitation at 430 nm.

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Scheme 2. The proposed mechanism for S2− detection by NL-Cu2+

Then, we are encouraged to envision that the obtained fluorescence sluggish NL-Cu2+ system could be employed as a promising ensemble for fluorescence “OFF–ON” detection of S2− Journal Name, [year], [vol], 00–00 | 3

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ions and its selectivity for Cu2+ in HEPES buffer (THF: H2O = 3:7, 20 mM, pH = 7.4) were further investigated. As shown in Fig.2, NL displayed strong green fluorescence (Φ = 0.22) centred at 519 nm. Upon addition of 20 μM Cu2+, a prompt change of the emission intensity of NL (10 μM) was observed leading to more than 95% quenching of the emission maxima at 519 nm (Φ2 = 0.011), 46 which could be ascribed to the paramagnetic quenching effect of Cu2+. According to linear Benesie-Hildebrand expression, the measured fluorescence intensity [1/(F0–F)] at 519 nm varied as a function of 1/[Cu2+] in a linear relationship (R2 = 0.9949), indicating the formation of 1:1 stoichiometry between Cu2+ and NL (Fig. 3). 47 The association constant of NL with Cu2+ in HEPES buffered was accordingly calculated to be 1.589 ×105 M-1. Job's plot also reveals that Cu2+ forms a 1:1 complex with NL (Fig. S7, ESI†). In addition, the fluorescence intensity changes of NL at 519 nm exhibited a linear correlation to Cu2+ in the concentration range from 0 to 0.4 μM (R2 = 0.9964). The detection limit for Cu2+ was estimated to be 1.6 nM based on a 3σ/slope (where ‘‘slop’’ is the calibration sensitivity of the fluorescence intensity change (ΔF = F0–F) vs. [Cu2+], and “σ” is the standard deviation of the blank signal (F0) obtained without Cu2+) under this experimental conditions (Fig. S8, ESI†), 48 which is below the maximum permissive level of Cu2+ in drinking water (20 μM) set by the U. S. Environmental Protection Agency. 49

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Living-cell fluorescence imaging studies

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Fig. 7 UV-vis absorption spectra of NL-Cu2+ (10 μM) in HEPES (DMSO: H2O = 3:7, 20 mM, pH = 7.2) in the presence of all kinds of analytes (20 μM): 1. S2−, 2. AcO−, 3. Cl−, 4. Br− , 5. F−, 6. NO2−, 7. HSO4−, 8. SCN− , 9. PO43−, 10. HPO42−, 11. H2PO4−, 12. Cys, 13. Hcys, 14. GSH.

2 4 6 10 Concentration of NL (M)

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Fig. 9 Cell viability values (%) assessed using an MTT proliferation test versus incubation concentrations of NL. MDA-MB-231 cells were cultured in the presence of 0–20 μM NL at 25 °C for 20 h. Viability(%) = mean of absorbance value of treatment group/mean absorbance value of control×100%. Fluorescence

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in the absorbance maximum at 430 nm as shown in Fig. 7, which corresponding to the free chemosensor NL (Fig. S9, ESI†). The binding properties of NL-Cu2+ with S2− have been investigated by performing UV–vis titration experiments in THF/H2O (3:7, v/v). Upon the addition of increasing amounts of S2− from (0–20 μM) to the solution of NL-Cu2+ resulted in the obvious enhancement of an adsorption band centered at 420 nm to match the native NL state (Fig. 8). To gain insight into the sensing mechanism, the ESI-MS analysis was studied. The molecular-ion peak at m/z 468.0 is assignable to [NL – H + Cu + Cl + H]+ species (Fig. S10, ESI†), while subsequent addition of S2− to the above solution gives a molecular ion peak at m/z 369.1 corresponding to [NL – H]– (Fig. S11, ESI†), indicating that a displacement strategy for S2− detection (Scheme 2).

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Fig. 8 UV-vis spectra of NL-Cu2+ (10 μM) in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4) in the presence of different amounts of S2− (0–20 μM). 60

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We also examined the UV–vis changes of the NL-Cu2+ ensemble with S2−. Interestingly, the UV–Vis absorption signature of NL-Cu2+ system was unperturbed in the presence of physiological and environmental important anions and smallmolecule thiols. However, addition of S2− resulted in an increase 4 | Journal Name, [year], [vol], 00–00

Fig. 10 Confocal fluorescence images in MDA-MB-231 cells at 37 °C. (a1) MDA-MB-231 cells were stained with 10 μM NL for 20 min. (b1) NL-loaded MDA-MB-231 cells were treated with 20 μM Cu2+ for 15 min. (c1) 40 μM of S2– for another 30 min. (a2, b2, c2) and (a3, b3, c3) are the brightfield and overlay images of the MDA-MB-231 cells. λex = 405 nm, λem = 490-525, scale bars = 40 μm.

The feasibility of NL for the biological imaging application in living MDA-MB-231 cells was firstly valuated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. 50 As shown in Fig. 9. The cellular viability was estimated This journal is © The Royal Society of Chemistry [year]

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via Cu2+ displacement approach. The NL-Cu2+ solution was prepared in situ by addition of 2.0 equiv. of Cu2+ to NL (10 μM) in HEPES buffer. Subsequently, fluorescence emission and UV−vis absorption spectra changes of NL-Cu2+ upon addition of various anions were examined. As shown in Fig. 5 (a), the addition of increasing concentrations of S2− (0–2 equiv.), the fluorescence emission of NL-Cu2+ system at 519 nm was gradually recovered (Φ2 = 0.21), which is essentially identical with the emission wavelength of free NL, The detection limit of NL-Cu2+ for S2− was evaluated to be 0.17 μM based on a 3σ/slope (Fig. 5 b). 47 However, other physiological and environmental important anions, such as F−, Cl−, Br−, I−, SO32−, HSO4−, Ac−, NO3−, NO2−, CO32−, PO43−, HPO42−, H2PO4−, and small-molecule thiols, such as Cys, Hcys and GSH, induced negligible fluorescence intensity changes (Fig. 6). These results demonstrate that NL-Cu2+ has an excellent selectivity toward S2−, and the proposed Cu2+ displacement was occurred.

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to be approximately 90% after incubation with NL (20 μM) for 24 h, which demonstrates that NL has low cytotoxicity and biocompatibility to living cells. To access the feasibility of Cu2+/ S2− detection in living cells, the fluorescence imaging was recorded using confocal fluorescence microscopy. The cells were incubated with a PBS solution containing 10 μM of compound NL for 20 min at 37 oC in a 5% CO2-95% air incubator. Then the cells were washed with PBS three times and mounted on a microscope stage. As shown in Fig. 10 (a1), the strong green fluorescence of MDA-MB-231 cells after stained with NL demonstrated the good cell-membrane permeability of this sensor. Further incubation with 20 μM Cu2+, the green fluorescence of the system was disappeared (Fig. 10 (b), suggests that the sensor, NL could be used to fluorescently detect Cu2+ in the living cells. After treat the cells with sodium sulfide (40 μM) for another 30 min, the intense green fluorescence of cells was resumed (Fig. 10 (c). These experiments indicated that NL could be potentially used to monitor intracellular Cu2+ and its fluorescence-silent NLCu2+ ensemble can be used as the imaging agent for S2− in living cells.

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Compared to the microscope imaging, flow cytometry provides rapid and quantitative analysis of large numbers of cells individually.51 In this work, we also quantitatively evaluated the NL stained living MDA-MB-231 cells and its “ON−OFF−ON” response to Cu2+ and S2− through the measurement of fluorescence in 10 000 individual cells of each population by flow cytometer. Fig. 11A shows the shift of the fluorescence signal measured of living MDA-MB-231 cells treated with NL, then Cu2+, and S2−, respectively. In control experiments, the cells without staining exhibited negligible background fluorescence (Fig. 11A (a)). After incubation with 10 M NL in PBS, the fluorescence intensity of the cell population increased significantly, and 99.6% of cells stained with NL (Fig. 11A (b)). Further incubation of cells with Cu2+ for 15 min led to the completely quenching of intracellular fluorescence (Fig. 11A (c)). However, in the presence of S2−, 99.7% of cells were switched on as shown the histogram in Fig. 11A (d). As shown in Fig. 11B, the florescence “ON−OFF−ON” response of the chemosensor, NL for Cu2+ and S2− was corroborated by the quantification by mean fluorescence of the MDA-MB-231 cells during the incubation (Fig. S12, ESI†).

Conclusions This journal is © The Royal Society of Chemistry [year]

In conclusion, we have developed a new NBD-based fluorescent chemosensor (NL) for Cu2+ detection in mixed aqueous media (THF: H2O = 3:7, HEPES buffered, pH = 7.4). Interaction of NL with Cu2+ led to fluorescence intensity completely quenching through a 1:1 binding mode. The detection limit of NL for Cu2+ was estimated to be 1.6 nM based on a 3σ/slope. The quenched fluorescence of the in situ generated NL-Cu2+ ensemble could be recovered upon the addition of S2−, realizing the detection of S2− by utilizing Cu2+ displacement approach with a detection limit of 0.17 μM. The MTT assay determined that NL exhibits low cytotoxicity toward living MDA-MB-231 cells. Confocal microscopy imaging suggested that NL has potential as a powerful tool for the imaging of Cu2+ and S2− in living cells. In addition, NL could be used to screen intracellular Cu2+ and S2− by flow cytometry in a rapid, sensitive, and quantitative fashion.

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Fig. 11 (A) Flow cytometric analysis of MDA-MB-231 cells loaded with NL and “ON−OFF−ON” response to Cu2+ and S2−. (a): control group, MDA-MB-231 cells only; (b): MDA-MB-231 stained with 10 M NL for 20 min; (c): NL loaded MDA-MB-231 cells treated with Cu2+ (20 M) for 15 min; (d): cells incubated with 40 M S2- for another 30 min. (B) Mean fluorescence per MDA-MB-231 cell of (a), (b), (c), and (d) incubation conditions.

DOI: 10.1039/C4OB02178A

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All reagents and solvents were of AR grade and used without further purification unless otherwise noted. 7-nitrobenz-2-oxa1,3-diazole chloride (NBD-Cl), hydrazine hydrate and 4diethylaminosalicylaldehyde were purchased from Sinopharm Chemical Reagent Co., Ltd. (China); Fresh stock solution of metal ions (nitrate salts, 20 mM) and anions (as sodium salts, 20 mM) in H2O were prepared for further experiments. 1 H-NMR and 13C-NMR spectra were recorded with a AVANCE500MHZ spectrometer (BRUKER) with chemical shifts reported as ppm (in DMSO, TMS as internal standard). High resolution mass spectra were recorded on TripleTOF® 4600 spectrometer (AB Sciex , USA). ESI mass spectra (ESI-MS) were recorded on a HP1100LC/MSD spectrometer. The elemental analyses of C, H, N and O were performed on a Vario EL III elemental analyzer. Fluorescence spectra were determined with LS 55 luminescence spectrometer (Perkin Elmer, USA). The absorption spectra were measured with a Lambda 900 UV/VIS/NIR spectrophotometer (Perkin Elmer, USA). Fluorescent images were acquired on an Olympus Fluoview FV 1000 IX81 inverted confocal laser-scanning microscope with an objective lens (×20). The excitation wavelength was 405 nm. Flow cytometry analysis was recorded on a BD FACSAria II flow cytometer with a laser at 405 nm. The data were analysed with Flowing software. General procedures of spectra detection

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Stock solutions of NL was prepared in HEPES aqueous buffer (THF: H2O = 3:7, 20 mM, pH = 7.4). Excitation wavelength for NL was 430 nm. Before spectroscopic measurements, the solution was freshly prepared by diluting the high concentration stock solution to corresponding solution (10 μM). Each time a 3 mL solution of chenosensor was filled in a quartz cell of 1 cm optical path length, and different stock solutions of cations were added into the quartz cell gradually by using a micro-syringe. The volume of cationic stock solution added was less than 100 μL with the purpose of keeping the total volume of testing solution without obvious change. NL-Cu2+ stock solution for S2− detection was prepared by addition of 2.0 equiv. of Cu2+ to NL (10 μM) solution in HEPES buffer (THF: H2O = 3:7, 20 mM, pH = 7.4). Journal Name, [year], [vol], 00–00 | 5

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Quantum yield measurement

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Fluorescence quantum yield was determined using optically matching solutions of rhodamine B (Φf = 0.69 in ethanol) as standard at an excitation wavelength of 550 nm and the quantum yield is calculated using the equation (1).52

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7-nitrobenz-2-oxa-1,3-diazole chloride (NBD-Cl) (100 mg, 0.5 mmol) was dissolved in chloroform (50 mL). A 1% hydrazine solution (0.77 mL hydrazine in 50 mL methanol) was then added to the solution and allowed to stir at room temperature for 1 h. A yellow-brown precipitate of 1 was formed and isolated without further purification in quantitative yield. 54 To a solution of 1 (0.195 g, 1 mmol) in 20 mL ethanol, 4diethyllaminosalicylaldhyde (0.193 g, 1 mmol) in 10 mL ethanol was added at room temperature. The stirred reaction mixture was heated to reflux for 6 h to yield a brown black precipitate. After cooling to room temperature, the precipitate was washed with ethanol and dried under vacuum to obtain NL in 80% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.60 (s, 1H), 8.67 (d, J = 7.9 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 6.33 (dd, J = 9.0 Hz, 1H), 6.07 (s, J = 2.3 Hz, 1H), 3.40 (q, J = 7.0 Hz, 4H), 1.12 (t, J = 7.0 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 163.34, 153.75, 149.54, 143.34, 135.52, 133.94, 132.44, 130.46, 128.76, 111.17, 104.42, 95.89, 44.08, 12.38. TOF-MS: [NL + H]+ calcd for C17H19N6O4, 371.1462; found, 371.1453. Anal. Calcd for: C, 55.13; H, 4.90; N, 22.69; O, 17.28 Found: C, 55.28; H, 4.96; N, 22.87; O, 17.49. Cytotoxicity assay

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The cytotoxicity of NL toward the human breast carcinoma cell

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mean of absorbance value of treatment group  100% mean absorbance value of control

(3)

Confocal fluorescence imaging in live cells 70

(2)

Where F and F0 represent the fluorescence emission of NL in the presence and absence of Cu2+, respectively, Fmin is the saturated emission of NL in the presence of excess amount of Cu2+; [Cu2+] is the concentration of Cu2+ ion added, and Ka is the binding constant.

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line MDA-MB-231 has been measured by methyl thiazolyl tetrazolium (MTT) assay. 55 MDA-MB-231 cells were seeded at a density of 5 × 104 cells/mL in a 96-well microassay culture plate and growth 24 h at 37 oC in a 5% CO2 incubator. Different concentrations of NL were added with fresh culture medium into the wells. Control wells were prepared by the addition of culture medium, and wells containing culture media without cells were used as blanks. After incubated at 37 oC in a 5% CO2 incubator for 24 h, RPMI-1640 was removed and cells were washed with PBS for three times. Then, 100 μL, 0.5 mg/mL MTT solution in PBS was added to each well, and the cells were further incubated for 4 h. The excess MTT solution was then carefully removed from wells, and the formed formazan was dissolved in 100 μL of DMSO (dimethyl sulfoxide). The optical density of each well was then measured at a wavelength of 590 nm using a microplate reader (Bio-Rad, xMark). The following formula was used to calculate the viability of cell growth:

Association constant calculation: Generally, for the formation of 1:1 complexation species formed by the chemosensor compound and the guest cations, the BenesiHildebrand equation used is as follow. 53

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MDA-MB-231 cells incubated with NL (10 μΜ, in PBS medium) for 20 minutes at 37 ºC in a CO2 incubator (95% relative humidity, 5% CO2). After washing with PBS three times, MDAMB-231 cells were supplemented with Cu2+ (20 μΜ) for 15 minutes, followed incubated with S2− (40 μΜ) for another 30 min. After removal of the remaining compounds, the cells were imaged by confocal microscope. Flow cytometry analysis

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MDA-MB-231 cells at a density of 1×105 cells/mL were cultured into a six-chamber culture well for 24 h in an incubator, and then NL (10 M) was added with serum free fresh RPMI-1640 medium, this addition was followed by further incubation for 20 min. After removal of growth medium, washed with PBS for three times, the cells were treated with PBS containing 20 M Cu2+ for 15 min. Then, the Cu2+ treated cells were incubated with 40 M S2− for another 30 min. All cells were detached from the well using trypsin-EDTA solution and washed three times with PBS for the flow cytometry analysis. Cells incubated with RPMI1640 for 24 h were used as the controls for all experiments.

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This work is supported by the National Natural Science Foundation of China (No. 21301011), the State Key Laboratory of Fine Chemicals (KF1305) and Program for Liaoning Excellent Talents in University (No. LR2014009). Dr. R. Zhang wish to acknowledge Macquarie University Research Fellowship Scheme (MQRF-1487520).

Notes and references a

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School of Chemical Engineering, University of Science and Technology Liaoning, Anshan, 114044, China. Email: [email protected]; Tel: +86-421-5928009. b Department of Chemistry and Biomolecular Sciences Faculty of Science, Macquarie University, Sydney NSW, 2109, Australia. Email: [email protected]. Tel: +61 (2) 9850 1175.

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NBD-based fluorescent chemosensor for the selective quantification of copper and sulfide in an aqueous solution and living cells.

Chemosensors play important roles in cation and anion recognition in biological, industrial, and environmental processes. Although many efforts have b...
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