Supporting information A Cyanine-modified Nanosystem for in vivo ...
Supporting information A Cyanine-modified Nanosystem for in vivo Upconversion Luminescence Bioimaging of Methylmercury Yi Liu, Min Chen, Tianye Cao, Yun Sun, Chunyan Li, Qian Liu, Tianshe Yang, Liming Yao, Wei Feng, Fuyou Li*
Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Institutes of Biomedical Sciences & Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China
Materials and instruments All reagents and chemicals were procured from commercial sources and used without further purification. 2,3,3-Trimethylbenzoindolenine, 10-bromoundecanoic acid 1,2-dichlorobenzene, POCl3, cyclohexanone, n-butanol, Ethylenediamine, benzene, phenyl-4-isothiocyanate were obtained from Alfa Aesar, and were used without further purification. Rare earth oxides Y 2O3 (99.999%), Yb2O3 (99.999%), Er2O3 (99.999%), and Tm2O3 (99.999%) were purchased from Shanghai Yuelong New Materials Co. Ltd. Oleic acid (OA) (>90%) and octadecene (ODE) were purchased from Alfa Aesar Ltd. All other chemical reagents of analytical grade were used directly without further purification. RECl3 were prepared with the literature method. DMF and deionized water was used to prepare all aqueous solutions. Solutions of MeHg+, Cu2+, Zn2+, and Fe2+ ions were prepared from their chlorate salts; solutions of Ag+, Ni2+, Cd2+, Pb2+, Co2+ were prepared from their nitrate salts. S1
The 1H NMR spectra were recorded on a Bruker spectrometer at 400 MHz. All chemical shifts are reported in the standard δ notation of parts per million (ppm). MALDI-TOF MS were measured on a Voyager DE-STR. UV-Vis absorption spectra were recorded on a Shimadzu 3000 spectrophotometer. Upconversion luminescence (UCL) emission spectra were measured on an Edinburgh FLS920 luminescence spectrometer with an external 3 W adjustable 980 nm semiconductor laser. In our case, all power densities of CW 980 nm excitation for the UCL measurements were fixed at 45 W/cm2. FT-IR spectra were measured using an IR Prestige-21 spectrometer (Shimadzu) from samples in KBr pellets. X-ray powder diffraction (XRD) measurements were performed on a Bruker D8 diffractometer at a scanning rate of 1o/min in the 2θ range of 10-90o, with graphite monochromated Cu Kα radiation (λ=1.5406 nm). Transmission electron microscope (TEM) messages were collected on a JEM 2010 operating at an acceleration voltage of 200 kV. The as-prepared samples were dispersed in cyclohexane and dripped water onto a copper grid for the TEM tests.
S2
Experimental Section
Scheme S1. Synthetic routine of hCy7
Synthesis of compound 2
1
: 2,3,3-Trimethylbenzoindolenine (6.3 g, 30.0 mmol) and
10-bromoundecanoic (8.0 g, 30.0 mmol) were dissolved in toluene (40 mL). The mixture was stirred at 100 °C for 20 h leading to needle like crystals. The reaction was cooled to room temperature. Product was filtered, and washed with ether and dried to give 10.6 g (90%) of 2 as a solid. 1H NMR (400 MHz, CDCl3) δ 8.24 – 7.93 (m, 3H), 7.79 – 7.64 (m, 3H), 3.71 (q, J = 7.0 Hz, 2H), 2.30 (t, J = 7.4 Hz, 6H), 1.85 (br, 3H), 1.26 (br, 10H); MS (MALDI-TOF-MS): calcd. For C26H36NO2+ 394.27 [M]+; found 393.39 [M-H]+. Synthesis of compound 4 2: A solution of POCl3 (37 mL, 397 mmol) in DCM (35 mL) was slowly added to an ice-cooled solution of DMF (40 mL, 516 mmol) in DCM (40 mL). After the addition was finished, cyclohexanone (10 g, 100 mmol) was added in via syringe. The resulted reaction mixture was refluxed for 2 h. The mixture was then cooled in ice. Water (200 mL), pre-cooled to 0°C was added slowly while the mixture was stirred. The mixture was stirred for 30 min. DCM layer was collected and the water layer was extracted with additional DCM. The DCM
S3
solutions were combined, passed through the MgSO4 column, concentrated on a rotavapor and treated with pentane (200 mL) to give 4.68g (27%) of 4 as yellow crystalline solid to reserve in the cool temperture. 1H NMR (400 MHz, CDCl3): δ2.46 (t, J = 6.2 Hz, 4H), 1.75 – 1.68 (m, 2H).; MS (MALDI-TOF-MS): calcd. For C8H9ClO2 172.03 [M]+; found 172.16 [M]+. Synthesis of compound 5: Into a flask attached with Dean-Stark trap and a condenser were added 1-hydroxycarbonylethyl-2,3,3-trimethylbenzoindoleninium bromide 2 (7.9 g, 20 mmol), freshly prepared 2-chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene 4 (1.7 g, 10 mmol), n-butanol (200 mL) and benzene (20 mL). The mixture was heated to 120 °C for 24h, resulting in a green solution. Solvents were removed on a rotavapor. Residue was washed with hexane/EtOAc and eluted with DCM/MeOH from a silica gel plug to give the crude product. Further chromatography on a silica gel column with gradient Hexane/EtOAc-DCM/MeOH solvent system led to 14.7 g (80%) of 5 as dark green solid. 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 14.2 Hz, 2H), 8.15 (d, J = 8.5 Hz, 2H), 7.99 – 7.96 (m, 4H), 7.66 – 7.61 (m, 2H), 7.52 – 17.46 (m, 4H), 6.30 (d, J = 14.2 Hz, 2H), 4.35 (t, J = 7.2 Hz, 4H), 4.07 (t, J = 6.7 Hz, 4H), 2.79 (t, J = 5.9 Hz, 4H), 2.28 (t, J = 7.5 Hz, 4H), 2.04 (s, 12H), 1.97 – 1.88 (m, 4H), 1.64 – 1.56 (m, 8H), 1.53 – 1.46 (m, 4H), 1.43 – 1.34 (m, 8H), 1.33 – 1.21 (m, 18H), 0.93 (t, J = 7.4 Hz, 6H). MS (MALDI-TOF-MS): calcd. For C68H92ClN2O4+ 1035.67 [M]+; found 1035.10 [M]+. Synthesis of compound 7 3: Ethylenediamine (30.0 g, 0.5 mol) was dissolved in ethanol (30 mL), then phenyl-4-isothiocyanate (6) (6.8 g, 50 mmol) in ethanol (30 mL) was added dropwise to the mixture on an ice bath. The mixture was then stirred at room temperature for 5 h, and resulting in a large white solid. Then the mixture was filtrated to afford a white solid (6.2 g). 1H NMR (400 MHz, CDCl3) δ8.04 (br, 1H), 7.43 (t, J = 7.4 Hz, 2H), 7.36 – 7.28 (m, 3H), 6.83 (br, 1H), 3.74-3.65 (m,
S4
2H), 2.94 (t, J = 5.6 Hz 2H), 1.29 (br, 2H); MS (MALDI-TOF-MS): calcd. For C9H13N3S 195.1 [M]+; found 194.3 [M-H]+. Synthesis of compound hCy7: Compound 5 (1.11 g, 1.0 mmol) and compound 7 (0.39 g, 2.0 mmol) were dissolved in anhydrous DMF (50 mL). The mixture was stirred at 85 ºC for 12 h under an argon atmosphere. The solvent was removed under reduced pressure and then the crude product was purified by silica gel chromatography with dichloromethane/methanol (20:1) to afford the desired product as a deep blue solid (40%). 1H NMR (400 MHz, CDCl3) δ 10.08 (br, 1H), 9.63 (br, 1H), 8.57 (br, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.90 – 7.80 (m, 3H), 7.78 (d, J = 12.7 Hz, 2H), 7.50 (t, J = 8.0 Hz 2H), 7.36 (t, J = 8.0 Hz 2H), 7.32 (t, J = 8.0 Hz 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.11 (t, J = 8.0 Hz 1H), 5.62 (d, J = 13.0 Hz, 2H), 4.13 – 4.05 (m, 8H), 3.91 – 3.85 (m, 4H), 2.52 (t, J = 5.8 Hz, 4H), 1.94 (s, 12H), 1.91 – 1.85 (m, 2H), 1.82 – 1.75 (m, 4H), 1.44 – 1.32 (m, 40H), 0.92 (t, J = 7.3 Hz, 6H); MS (MALDI-TOF-MS): calcd. For C77H104N5O4S+ 1194.74 [M]+; found 1194.77 [M]+.
The MeHg+ solution was added into the blue solution hCy7, the mixted solution was stirred at room temperature for 1 min. The colour of the solution changed into green to form the compound hCy7’. MS (MALDI-TOF-MS): calcd. For C77H102N5O4+ 1160.79 [M]+; found 1160.85 [M]+.
S5
Synthesis of Oleic acid (OA) coated UCNPs (denoted as OA-UCNPs). OA-UCNPs were prepared by a modified solvothermal process according to the reported method.4 YCl3 (0.78 mmol), YbCl3 (0.20 mmol), ErCl3 (0.016 mmol) and TmCl3 (0.004 mmol) were mixed with 8 mL oleic acid and 15 mL octadecene (ODE) in a 50 mL flask. The solution was heated to 160 °C to form a homogeneous solution, and then cooled down to room temperature. 8 mL methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was slowly added into the flask and stirred for 45 minutes. Subsequently, the solution was slowly heated and degassed at 120 °C for 30 minutes to remove methanol, and then heated to 300 °C and maintained for 1h under N2 protection. After the solution was cooled naturally, nanoparticles were precipitated from the solution with ethanol, and washed with ethanol/cyclohexane (9:1, v/v) for three times. Assembly of P-PEG and hCy7 or hCy7’ (denoted as hCy7-UCNPs or P-PEG-hCy7’-UCNPs). hCy7-UCNPs were prepared according to our previous method.5 The chloroform solutions of complex hCy7 or hCy7’ (5 mg in total) were added to the prepared OA-UCNPs (10 mg) in a 10 mL round-bottomed flask. Initially, the nanoparticles were dispersed in the chloroform by ultrasonication, and then the mixture was stirred for 4 h at room temperature to obtain a homogeneous phase. Furthermore, the amphiphilic polymer (P-PEG, 10 mg) was added into the mixture, the mixture was stirred overnight at room temperature. The mixture was centrifuged (14000rpm, 8 min every time in 20 ˚C), and the collected solid was repeatedly washed with water. The precipitate could be redispersed in deionized water. Procedures for metallic cation sensing: Stock solutions of the metal ions (2.5 mM) were prepared in H2O. A stock solution of hCy7-UCNPs (containing 10 μM hCy7) was prepared in H2O. The sensing of hCy7-UCNPs to MeHg+ was performed by adding the MeHg+ stock solution by
S6
means of a micro-pipette to 2 mL solution of hCy7-UCNPs. Test samples for selectivity experiments were prepared by adding appropriate amounts of anions stock solution with a similar procedure. In competition experiments, MeHg+ was added to solutions containing hCy7-UCNPs and the other metal ions of interest. All test solutions were stirred for 20 min under room-temperature. For all upconversion luminescence (UCL) measurements, excitation was fixed at 980 nm, and UCL emission was collected from 400 to 850 nm. Cell culture: The cell lines HeLa were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The HeLa cells were grown in MEM (modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) at 37 oC and 5% CO2. HeLa cells were planted on 14 mm glass coverslips and allowed to adhere for 24 h. Laser-scanning upconversion luminescence microscopy (LSUCLM) imaging: Experiments to assess MeHg+ uptake were performed over 20 h in the same medium supplemented with 50 μM MeHg+. Before the experiments, HeLa cells were washed with PBS buffer, and then the cells were incubated with 5 μM hCy7-UCNPs in PBS for 1 h at 37 oC. Cell imaging was then carried out after washing the cells with PBS. Laser-scanning upconversion luminescence microscopy (LSUCLM) imaging was performed with an OLYMPUS FV1000 scanning unit.6 Cells loaded with OA-NIR-UCNPs were excited by a CW laser at 980 nm (Connet Fiber Optics, China) with the focused power of ~14 mW. UCL emission was collected from 500 to 560 nm, and 600 to 700 nm. Cytotoxicity of hCy7-UCNPs: In vitro cytotoxicity was measured by performing methyl thiazolyl tetrazolium (MTT) assays on the HeLa cells. Cells were seeded into a 96-well cell culture plate at 5×104/well, under 100% humidity, and were cultured at 37 ˚C and 5% CO2 for 24 h; different concentrations of hCy7-UCNPs (0, 12.5, 25, 50, 100, 200, 400 and 800 μg/mL, diluted in RPMI
S7
1640) were then added to the wells. The cells were subsequently incubated for 24 h or 48 h at 37 ˚C under 5% CO2. Then, MTT (10 mL; 5 mg/mL) was added to each well and the plate was incubated for an additional 4 h at 37 ˚C under 5% CO2. After the addition of 10% sodium dodecyl sulfate (SDS, 100 mL/well), the assay plate was allowed to stand at room-temperature for 12 h. The optical density OD570 value (Abs.) of each well, with background subtraction at 690 nm, was measured by means of a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) *100%. Upconversion luminescence in vivo imaging: In vivo and ex vivo upconversion luminescence imaging was performed with a modified upconversion luminescence in vivo imaging system designed by our group.7 In this system, two external 0-5 W adjustable CW 980 nm lasers (Connet Fiber Optics, China) were used as the excitation sources and an Andor DU897 EMCCD as the signal collector. Images of luminescent signals were analyzed with Kodak Molecular Imaging Software. UCL signals were collected at 800 ±12 nm. LRET efficiency: The LRET efficiency was deduced from the upconversion emission spectra of Ir1-UCNPs by the following equation. E = 1 – UCLhCy7-UCNPs / UCLOA-UCNPs
S8
Scheme S2. The possible mechanism of the sensing reaction between MeHg+ and hCy7.
S9
Figure S1. The 1H NMR spectra in the down-field of hCy7 (left) and 5 (right) in the CDCl3.
S10
Figure S2. The MALDI-TOF-MS spectrum of hCy7.
S11
Figure S3. The MALDI-TOF-MS spectrum of hCy7’.
S12
Figure S4. (a) Change in the absorption spectra of 10 μM hCy7 in EtOH/H2O (4:1, v/v) upon gradual addition of MeHg+ ions (from 0 to 6 eq.). (b) The ratio (A845 nm/A670 nm) of absorbance at 845 nm to 670 nm of hCy7 upon addition of MeHg+. (c) The color change of the hCy7 EtOH/H2O (4:1, v/v) upon addition of MeHg+.
S13
short-chain analogues
HOMO
LUMO
Cy7
Cy7’
Figure S5. Molecular orbital plots (LUMO and HOMO) of the short-chain analogues of the dye Cy7 by DFT calculations.
S14
Figure S6. The absorbance spectra (a) and ratio (A845 nm/A670 nm) of absorbance at 845 nm to 670 nm of the hCy7 (10 µM) in EtOH/H2O (4:1, v/v), in the presence of various representative metal cations (50 µM).
S15
Figure S7. The EDXA of the OA-UCNPs (a) and hCy7-UCNPs (b).
S16
Figure S8. The FTIR spectra of the OA-UCNPs, hCy7, P-PEG, and hCy7-UCNPs.
S17
Figure S9. The 1H NMR of the hCy7-UCNPs in D2O. The appearance of the chemical shifts both in the low field (6.75 and 7.15 ppm) assigned to the aromatic protons of hCy7 and in the high field 3.50 ppm) attributed to the protons (-CH2-O-) in the polymer P-PEG
S18
Figure S10. The concentration of hCy7 loaded in the hCy7-UCNPs was calculated using the absorption spectroscopy technique. (a) Absorption spectra of the hCy7 with different concentrations of 1-14 µM. (b) The absorbance at 670 nm as a function of hCy7 concentration. The hCy7 content (red point) of hCy7-UCNPs (0.06 mg/mL) was determined to be 13.6%.
S19
Figure S11. The absorption spectra (a) and emission spectra (b) of Cy7-UCNPs and hCy7-UCNPs in aqueous solution (ex = 980 nm).
S20
Figure S12. The ratio (A845 nm/A670 nm) of absorbance at 670 nm and 845 nm versus addition of MeHg+ in the hCy7-UCNPs.
S21
Figure S13. (a) The ratio (UCL660 nm/UCL800 nm) of upconversion luminescence intensities at 660 to 800 nm of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S22
Figure S14. (a) The ratio (UCL660 nm/UCL540 nm) of upconversion luminescence intensities at 660 and 540 nm of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S23
Figure S15. (a) The ratio (UCL800 nm/UCL540 nm) of upconversion luminescence intensities at 660 and 540 nm of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S24
Figure S16. (a) Upconversion luminescence intensities at 800 nm (UCL800 nm) of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S25
Figure S17. (a) Upconversion luminescence intensities at 660 nm (UCL660 nm) of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S26
Figure S18. (a) The absorbance spectra of hCy7-UCNPs in the solution upon gradual addition of various metal ions. (b) The ratio (A845 nm/A670 nm) of absorbance at 845 nm and 670 nm in the presence of various representative metal ions.
S27
Figure S19. The UCL emission spectra of hCy7-UCNPs (0.05 mg mL-1) in the solution upon gradual addition of various metal ions (25 µM).
S28
Figure S20. The competition experiments were carried out by adding MeHg+ (20 µM) to solutions of hCy7-UCNPs (0.05 mg mL-1) in the presence of other cations (25 µM).
S29
Figure S21. In vitro cell viability of HeLa and KB cells incubated with hCy7-UCNPs at different concentration for 48 hours.
S30
Figure S22. Time-dependent change in absorbance at 670 nm of hCy7-UCNPs in aqueous solution upon addition of MeHg+ ion (0, 4, or 8 eq.).
S31
Figure S23. (a) Change in absorption spectra of hCy7-UCNPs upon gradual addition of Hg2+; (b) Reaction kinetics curves of hCy7-UCNPs upon addition of Hg2+.
REFERENCES 1. Zhang, Z. R.; Achilefu, S. Org. Lett. 2004, 6, 2067. 2. Reynolds, G. A.; Drexhage, K. H. J. Org. Chem. 1997, 42, 85. 3. Tilley, J. W.; Levitan, P.; Kierstead, R. W. J. Med. Chem. 1980, 23, 1387. 4. Liu, Q.; Sun, Y.; Li, C. G.; Zhou, J.; Li, C. Y.; Yang, T. S.; Zhang, X. Z.; Yi, T.; Wu, D. M.; Li, F. Y. ACS Nano 2011, 5, 3146. 5. Yao, L. M.; Zhou J.; Liu, J. L.; Feng, W.; Li, F. Y. Adv. Fun. Mater. 2012, 22, 2667. 6. Yu, M. X.; Li, F. Y.; Chen, Z. G.; Hu, H.; Zhan, C.; Yang, H.; Huang, C. H. Anal. Chem. 2009, 81, 930. 7. Xiong, L. Q.; Chen, Z. G.; Tian, Q. W.; Cao, T. Y.; Xu, C. J.; Li, F. Y. Anal. Chem. 2009, 81, 8687.
S32
Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Institutes of Biomedical Sciences & Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China
Materials and instruments All reagents and chemicals were procured from commercial sources and used without further purification. 2,3,3-Trimethylbenzoindolenine, 10-bromoundecanoic acid 1,2-dichlorobenzene, POCl3, cyclohexanone, n-butanol, Ethylenediamine, benzene, phenyl-4-isothiocyanate were obtained from Alfa Aesar, and were used without further purification. Rare earth oxides Y 2O3 (99.999%), Yb2O3 (99.999%), Er2O3 (99.999%), and Tm2O3 (99.999%) were purchased from Shanghai Yuelong New Materials Co. Ltd. Oleic acid (OA) (>90%) and octadecene (ODE) were purchased from Alfa Aesar Ltd. All other chemical reagents of analytical grade were used directly without further purification. RECl3 were prepared with the literature method. DMF and deionized water was used to prepare all aqueous solutions. Solutions of MeHg+, Cu2+, Zn2+, and Fe2+ ions were prepared from their chlorate salts; solutions of Ag+, Ni2+, Cd2+, Pb2+, Co2+ were prepared from their nitrate salts. S1
The 1H NMR spectra were recorded on a Bruker spectrometer at 400 MHz. All chemical shifts are reported in the standard δ notation of parts per million (ppm). MALDI-TOF MS were measured on a Voyager DE-STR. UV-Vis absorption spectra were recorded on a Shimadzu 3000 spectrophotometer. Upconversion luminescence (UCL) emission spectra were measured on an Edinburgh FLS920 luminescence spectrometer with an external 3 W adjustable 980 nm semiconductor laser. In our case, all power densities of CW 980 nm excitation for the UCL measurements were fixed at 45 W/cm2. FT-IR spectra were measured using an IR Prestige-21 spectrometer (Shimadzu) from samples in KBr pellets. X-ray powder diffraction (XRD) measurements were performed on a Bruker D8 diffractometer at a scanning rate of 1o/min in the 2θ range of 10-90o, with graphite monochromated Cu Kα radiation (λ=1.5406 nm). Transmission electron microscope (TEM) messages were collected on a JEM 2010 operating at an acceleration voltage of 200 kV. The as-prepared samples were dispersed in cyclohexane and dripped water onto a copper grid for the TEM tests.
S2
Experimental Section
Scheme S1. Synthetic routine of hCy7
Synthesis of compound 2
1
: 2,3,3-Trimethylbenzoindolenine (6.3 g, 30.0 mmol) and
10-bromoundecanoic (8.0 g, 30.0 mmol) were dissolved in toluene (40 mL). The mixture was stirred at 100 °C for 20 h leading to needle like crystals. The reaction was cooled to room temperature. Product was filtered, and washed with ether and dried to give 10.6 g (90%) of 2 as a solid. 1H NMR (400 MHz, CDCl3) δ 8.24 – 7.93 (m, 3H), 7.79 – 7.64 (m, 3H), 3.71 (q, J = 7.0 Hz, 2H), 2.30 (t, J = 7.4 Hz, 6H), 1.85 (br, 3H), 1.26 (br, 10H); MS (MALDI-TOF-MS): calcd. For C26H36NO2+ 394.27 [M]+; found 393.39 [M-H]+. Synthesis of compound 4 2: A solution of POCl3 (37 mL, 397 mmol) in DCM (35 mL) was slowly added to an ice-cooled solution of DMF (40 mL, 516 mmol) in DCM (40 mL). After the addition was finished, cyclohexanone (10 g, 100 mmol) was added in via syringe. The resulted reaction mixture was refluxed for 2 h. The mixture was then cooled in ice. Water (200 mL), pre-cooled to 0°C was added slowly while the mixture was stirred. The mixture was stirred for 30 min. DCM layer was collected and the water layer was extracted with additional DCM. The DCM
S3
solutions were combined, passed through the MgSO4 column, concentrated on a rotavapor and treated with pentane (200 mL) to give 4.68g (27%) of 4 as yellow crystalline solid to reserve in the cool temperture. 1H NMR (400 MHz, CDCl3): δ2.46 (t, J = 6.2 Hz, 4H), 1.75 – 1.68 (m, 2H).; MS (MALDI-TOF-MS): calcd. For C8H9ClO2 172.03 [M]+; found 172.16 [M]+. Synthesis of compound 5: Into a flask attached with Dean-Stark trap and a condenser were added 1-hydroxycarbonylethyl-2,3,3-trimethylbenzoindoleninium bromide 2 (7.9 g, 20 mmol), freshly prepared 2-chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene 4 (1.7 g, 10 mmol), n-butanol (200 mL) and benzene (20 mL). The mixture was heated to 120 °C for 24h, resulting in a green solution. Solvents were removed on a rotavapor. Residue was washed with hexane/EtOAc and eluted with DCM/MeOH from a silica gel plug to give the crude product. Further chromatography on a silica gel column with gradient Hexane/EtOAc-DCM/MeOH solvent system led to 14.7 g (80%) of 5 as dark green solid. 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 14.2 Hz, 2H), 8.15 (d, J = 8.5 Hz, 2H), 7.99 – 7.96 (m, 4H), 7.66 – 7.61 (m, 2H), 7.52 – 17.46 (m, 4H), 6.30 (d, J = 14.2 Hz, 2H), 4.35 (t, J = 7.2 Hz, 4H), 4.07 (t, J = 6.7 Hz, 4H), 2.79 (t, J = 5.9 Hz, 4H), 2.28 (t, J = 7.5 Hz, 4H), 2.04 (s, 12H), 1.97 – 1.88 (m, 4H), 1.64 – 1.56 (m, 8H), 1.53 – 1.46 (m, 4H), 1.43 – 1.34 (m, 8H), 1.33 – 1.21 (m, 18H), 0.93 (t, J = 7.4 Hz, 6H). MS (MALDI-TOF-MS): calcd. For C68H92ClN2O4+ 1035.67 [M]+; found 1035.10 [M]+. Synthesis of compound 7 3: Ethylenediamine (30.0 g, 0.5 mol) was dissolved in ethanol (30 mL), then phenyl-4-isothiocyanate (6) (6.8 g, 50 mmol) in ethanol (30 mL) was added dropwise to the mixture on an ice bath. The mixture was then stirred at room temperature for 5 h, and resulting in a large white solid. Then the mixture was filtrated to afford a white solid (6.2 g). 1H NMR (400 MHz, CDCl3) δ8.04 (br, 1H), 7.43 (t, J = 7.4 Hz, 2H), 7.36 – 7.28 (m, 3H), 6.83 (br, 1H), 3.74-3.65 (m,
S4
2H), 2.94 (t, J = 5.6 Hz 2H), 1.29 (br, 2H); MS (MALDI-TOF-MS): calcd. For C9H13N3S 195.1 [M]+; found 194.3 [M-H]+. Synthesis of compound hCy7: Compound 5 (1.11 g, 1.0 mmol) and compound 7 (0.39 g, 2.0 mmol) were dissolved in anhydrous DMF (50 mL). The mixture was stirred at 85 ºC for 12 h under an argon atmosphere. The solvent was removed under reduced pressure and then the crude product was purified by silica gel chromatography with dichloromethane/methanol (20:1) to afford the desired product as a deep blue solid (40%). 1H NMR (400 MHz, CDCl3) δ 10.08 (br, 1H), 9.63 (br, 1H), 8.57 (br, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.90 – 7.80 (m, 3H), 7.78 (d, J = 12.7 Hz, 2H), 7.50 (t, J = 8.0 Hz 2H), 7.36 (t, J = 8.0 Hz 2H), 7.32 (t, J = 8.0 Hz 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.11 (t, J = 8.0 Hz 1H), 5.62 (d, J = 13.0 Hz, 2H), 4.13 – 4.05 (m, 8H), 3.91 – 3.85 (m, 4H), 2.52 (t, J = 5.8 Hz, 4H), 1.94 (s, 12H), 1.91 – 1.85 (m, 2H), 1.82 – 1.75 (m, 4H), 1.44 – 1.32 (m, 40H), 0.92 (t, J = 7.3 Hz, 6H); MS (MALDI-TOF-MS): calcd. For C77H104N5O4S+ 1194.74 [M]+; found 1194.77 [M]+.
The MeHg+ solution was added into the blue solution hCy7, the mixted solution was stirred at room temperature for 1 min. The colour of the solution changed into green to form the compound hCy7’. MS (MALDI-TOF-MS): calcd. For C77H102N5O4+ 1160.79 [M]+; found 1160.85 [M]+.
S5
Synthesis of Oleic acid (OA) coated UCNPs (denoted as OA-UCNPs). OA-UCNPs were prepared by a modified solvothermal process according to the reported method.4 YCl3 (0.78 mmol), YbCl3 (0.20 mmol), ErCl3 (0.016 mmol) and TmCl3 (0.004 mmol) were mixed with 8 mL oleic acid and 15 mL octadecene (ODE) in a 50 mL flask. The solution was heated to 160 °C to form a homogeneous solution, and then cooled down to room temperature. 8 mL methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was slowly added into the flask and stirred for 45 minutes. Subsequently, the solution was slowly heated and degassed at 120 °C for 30 minutes to remove methanol, and then heated to 300 °C and maintained for 1h under N2 protection. After the solution was cooled naturally, nanoparticles were precipitated from the solution with ethanol, and washed with ethanol/cyclohexane (9:1, v/v) for three times. Assembly of P-PEG and hCy7 or hCy7’ (denoted as hCy7-UCNPs or P-PEG-hCy7’-UCNPs). hCy7-UCNPs were prepared according to our previous method.5 The chloroform solutions of complex hCy7 or hCy7’ (5 mg in total) were added to the prepared OA-UCNPs (10 mg) in a 10 mL round-bottomed flask. Initially, the nanoparticles were dispersed in the chloroform by ultrasonication, and then the mixture was stirred for 4 h at room temperature to obtain a homogeneous phase. Furthermore, the amphiphilic polymer (P-PEG, 10 mg) was added into the mixture, the mixture was stirred overnight at room temperature. The mixture was centrifuged (14000rpm, 8 min every time in 20 ˚C), and the collected solid was repeatedly washed with water. The precipitate could be redispersed in deionized water. Procedures for metallic cation sensing: Stock solutions of the metal ions (2.5 mM) were prepared in H2O. A stock solution of hCy7-UCNPs (containing 10 μM hCy7) was prepared in H2O. The sensing of hCy7-UCNPs to MeHg+ was performed by adding the MeHg+ stock solution by
S6
means of a micro-pipette to 2 mL solution of hCy7-UCNPs. Test samples for selectivity experiments were prepared by adding appropriate amounts of anions stock solution with a similar procedure. In competition experiments, MeHg+ was added to solutions containing hCy7-UCNPs and the other metal ions of interest. All test solutions were stirred for 20 min under room-temperature. For all upconversion luminescence (UCL) measurements, excitation was fixed at 980 nm, and UCL emission was collected from 400 to 850 nm. Cell culture: The cell lines HeLa were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The HeLa cells were grown in MEM (modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) at 37 oC and 5% CO2. HeLa cells were planted on 14 mm glass coverslips and allowed to adhere for 24 h. Laser-scanning upconversion luminescence microscopy (LSUCLM) imaging: Experiments to assess MeHg+ uptake were performed over 20 h in the same medium supplemented with 50 μM MeHg+. Before the experiments, HeLa cells were washed with PBS buffer, and then the cells were incubated with 5 μM hCy7-UCNPs in PBS for 1 h at 37 oC. Cell imaging was then carried out after washing the cells with PBS. Laser-scanning upconversion luminescence microscopy (LSUCLM) imaging was performed with an OLYMPUS FV1000 scanning unit.6 Cells loaded with OA-NIR-UCNPs were excited by a CW laser at 980 nm (Connet Fiber Optics, China) with the focused power of ~14 mW. UCL emission was collected from 500 to 560 nm, and 600 to 700 nm. Cytotoxicity of hCy7-UCNPs: In vitro cytotoxicity was measured by performing methyl thiazolyl tetrazolium (MTT) assays on the HeLa cells. Cells were seeded into a 96-well cell culture plate at 5×104/well, under 100% humidity, and were cultured at 37 ˚C and 5% CO2 for 24 h; different concentrations of hCy7-UCNPs (0, 12.5, 25, 50, 100, 200, 400 and 800 μg/mL, diluted in RPMI
S7
1640) were then added to the wells. The cells were subsequently incubated for 24 h or 48 h at 37 ˚C under 5% CO2. Then, MTT (10 mL; 5 mg/mL) was added to each well and the plate was incubated for an additional 4 h at 37 ˚C under 5% CO2. After the addition of 10% sodium dodecyl sulfate (SDS, 100 mL/well), the assay plate was allowed to stand at room-temperature for 12 h. The optical density OD570 value (Abs.) of each well, with background subtraction at 690 nm, was measured by means of a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) *100%. Upconversion luminescence in vivo imaging: In vivo and ex vivo upconversion luminescence imaging was performed with a modified upconversion luminescence in vivo imaging system designed by our group.7 In this system, two external 0-5 W adjustable CW 980 nm lasers (Connet Fiber Optics, China) were used as the excitation sources and an Andor DU897 EMCCD as the signal collector. Images of luminescent signals were analyzed with Kodak Molecular Imaging Software. UCL signals were collected at 800 ±12 nm. LRET efficiency: The LRET efficiency was deduced from the upconversion emission spectra of Ir1-UCNPs by the following equation. E = 1 – UCLhCy7-UCNPs / UCLOA-UCNPs
S8
Scheme S2. The possible mechanism of the sensing reaction between MeHg+ and hCy7.
S9
Figure S1. The 1H NMR spectra in the down-field of hCy7 (left) and 5 (right) in the CDCl3.
S10
Figure S2. The MALDI-TOF-MS spectrum of hCy7.
S11
Figure S3. The MALDI-TOF-MS spectrum of hCy7’.
S12
Figure S4. (a) Change in the absorption spectra of 10 μM hCy7 in EtOH/H2O (4:1, v/v) upon gradual addition of MeHg+ ions (from 0 to 6 eq.). (b) The ratio (A845 nm/A670 nm) of absorbance at 845 nm to 670 nm of hCy7 upon addition of MeHg+. (c) The color change of the hCy7 EtOH/H2O (4:1, v/v) upon addition of MeHg+.
S13
short-chain analogues
HOMO
LUMO
Cy7
Cy7’
Figure S5. Molecular orbital plots (LUMO and HOMO) of the short-chain analogues of the dye Cy7 by DFT calculations.
S14
Figure S6. The absorbance spectra (a) and ratio (A845 nm/A670 nm) of absorbance at 845 nm to 670 nm of the hCy7 (10 µM) in EtOH/H2O (4:1, v/v), in the presence of various representative metal cations (50 µM).
S15
Figure S7. The EDXA of the OA-UCNPs (a) and hCy7-UCNPs (b).
S16
Figure S8. The FTIR spectra of the OA-UCNPs, hCy7, P-PEG, and hCy7-UCNPs.
S17
Figure S9. The 1H NMR of the hCy7-UCNPs in D2O. The appearance of the chemical shifts both in the low field (6.75 and 7.15 ppm) assigned to the aromatic protons of hCy7 and in the high field 3.50 ppm) attributed to the protons (-CH2-O-) in the polymer P-PEG
S18
Figure S10. The concentration of hCy7 loaded in the hCy7-UCNPs was calculated using the absorption spectroscopy technique. (a) Absorption spectra of the hCy7 with different concentrations of 1-14 µM. (b) The absorbance at 670 nm as a function of hCy7 concentration. The hCy7 content (red point) of hCy7-UCNPs (0.06 mg/mL) was determined to be 13.6%.
S19
Figure S11. The absorption spectra (a) and emission spectra (b) of Cy7-UCNPs and hCy7-UCNPs in aqueous solution (ex = 980 nm).
S20
Figure S12. The ratio (A845 nm/A670 nm) of absorbance at 670 nm and 845 nm versus addition of MeHg+ in the hCy7-UCNPs.
S21
Figure S13. (a) The ratio (UCL660 nm/UCL800 nm) of upconversion luminescence intensities at 660 to 800 nm of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S22
Figure S14. (a) The ratio (UCL660 nm/UCL540 nm) of upconversion luminescence intensities at 660 and 540 nm of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S23
Figure S15. (a) The ratio (UCL800 nm/UCL540 nm) of upconversion luminescence intensities at 660 and 540 nm of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S24
Figure S16. (a) Upconversion luminescence intensities at 800 nm (UCL800 nm) of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S25
Figure S17. (a) Upconversion luminescence intensities at 660 nm (UCL660 nm) of hCy7-UCNPs versus addition of MeHg+ concentration. (b) The analysis of detected limit of hCy7-UCNPs for detection MeHg+.
S26
Figure S18. (a) The absorbance spectra of hCy7-UCNPs in the solution upon gradual addition of various metal ions. (b) The ratio (A845 nm/A670 nm) of absorbance at 845 nm and 670 nm in the presence of various representative metal ions.
S27
Figure S19. The UCL emission spectra of hCy7-UCNPs (0.05 mg mL-1) in the solution upon gradual addition of various metal ions (25 µM).
S28
Figure S20. The competition experiments were carried out by adding MeHg+ (20 µM) to solutions of hCy7-UCNPs (0.05 mg mL-1) in the presence of other cations (25 µM).
S29
Figure S21. In vitro cell viability of HeLa and KB cells incubated with hCy7-UCNPs at different concentration for 48 hours.
S30
Figure S22. Time-dependent change in absorbance at 670 nm of hCy7-UCNPs in aqueous solution upon addition of MeHg+ ion (0, 4, or 8 eq.).
S31
Figure S23. (a) Change in absorption spectra of hCy7-UCNPs upon gradual addition of Hg2+; (b) Reaction kinetics curves of hCy7-UCNPs upon addition of Hg2+.
REFERENCES 1. Zhang, Z. R.; Achilefu, S. Org. Lett. 2004, 6, 2067. 2. Reynolds, G. A.; Drexhage, K. H. J. Org. Chem. 1997, 42, 85. 3. Tilley, J. W.; Levitan, P.; Kierstead, R. W. J. Med. Chem. 1980, 23, 1387. 4. Liu, Q.; Sun, Y.; Li, C. G.; Zhou, J.; Li, C. Y.; Yang, T. S.; Zhang, X. Z.; Yi, T.; Wu, D. M.; Li, F. Y. ACS Nano 2011, 5, 3146. 5. Yao, L. M.; Zhou J.; Liu, J. L.; Feng, W.; Li, F. Y. Adv. Fun. Mater. 2012, 22, 2667. 6. Yu, M. X.; Li, F. Y.; Chen, Z. G.; Hu, H.; Zhan, C.; Yang, H.; Huang, C. H. Anal. Chem. 2009, 81, 930. 7. Xiong, L. Q.; Chen, Z. G.; Tian, Q. W.; Cao, T. Y.; Xu, C. J.; Li, F. Y. Anal. Chem. 2009, 81, 8687.
S32
