Supporting Information for
Supporting Information for Biological and Environmental Transformations of Copper-Based Nanomaterials Zhongying Wang1, Annette Von Dem Bussche3, Pranita K. Kabadi3, Agnes B. Kane3, Robert H. Hurt2, 4* 1
Department of Chemistry, 2School of Engineering, 3Department of Pathology and Laboratory Medicine, 4Institute for Molecular and Nanoscale Innovation, Brown University, Providence, Rhode Island 02912 *Address correspondence to [email protected]
Additional details on Visual MINTEQ 3.0 calculation Equilibrium solubility of the oxide and sulfide forms (Figure 1) The equilibrium dissolved metal ion concentration was estimated using Visual MINTEQ 3.0 from Gustafsson.1 The phases considered were TiO2, ZnO, NiO, CuO and Ag2O and the sulfides ZnS, NiS, CuS and Ag2S. The parameters for metal oxide calculation were set as follows: pH is fixed at 7; temperature is 25℃; ionic strength is to be calculated depending on components added; 1 mM Na+ and 1 mM NO3- is added as components; the metal oxide of interest is specified as infinite solid phase. The parameters for metal sulfide calculation were set as follows: pH is fixed at 7; temperature is 25℃; ionic strength is to be calculated depending on components added; 1 mM S2- is added as components; the metal sulfide of interest is specified as infinite solid phase.
Equilibrium concentration of dissolved copper in the corresponding pH conditions (Figure 2A) The equilibrium dissolved copper ion concentration was estimated using Visual MINTEQ 3.0. The parameters were set as follows: pH is fixed at 4, 5, 6, 7.4, 8, 9, respectively; temperature is 25℃; ionic strength is to be calculated depending on components added; 2.5 mM CuO (tenorite) is specified as finite solid phase. The
calculated equilibrium concentration of dissolved Cu is 2.5 mM, 2.5 mM, 4.7E-02 mM, 1.3E-04 mM, 2.1E-05 mM and 4.1E-06 mM, respectively. In the case of pH 7.4, the effect of phosphate was investigated through calculation by Visual MINTEQ 3.0. The parameters were set as follows: pH is fixed at 7.4; temperature is 25℃; ionic strength is to be calculated depending on components added; 11.9 mM phosphate (concentration in PBS buffer) is added as components; 2.5 mM CuO (tenorite) is specified as finite solid phase; specify Cu3(PO4)2(s) as possible solid phase. The calculated equilibrium concentration of dissolved Cu is 2.4E-03 mM, which is predicted to be slightly higher than the one at same pH but without consideration of buffer effect (1.3E-04 mM) because of the formation of soluble CuHPO4 complex. These calculations show that the effect of phosphate complexation is limited and the extra low equilibrium concentration of dissolved Cu is predicted with or without consideration of phosphate buffer effect.
Figure S1. Characterization of commercial CuO nanoparticles. (A) TEM image of CuO after dispersion in DI water and drying overnight. (B) FT-IR spectrum showing the limited number and intensity of peaks, which may be residue of organic ligands during synthesis process. (C) XRD spectra indicating the tenorite crystalline. (D) Size distribution of CuO dispersed in PBS buffer which indicated strong aggregation (determined by DLS).
Figure S2. (A) Time-dependent dissolution of 200 ppm CuO nanoparticles in PBS buffer (pH 7.4). Please note that the unaltered detected Cu release is caused by its extra low concentration, which can’t be distinguished by our ICP-AES. (B)The effects of various components in cell culture media on the dissolution of CuO (initial concentration 200 ppm) in PBS buffer in one day. High glucose: 4500 mg/L. Low glucose: 1000 mg/L; HEPES: 5958 mg/L; FBS: 10%; Glutamine: 20 mM. The formation of copper phosphate in the PBS buffer containing amino acids was estimated through Visual MINTEQ 3.0 calculation. The parameters were set as follows: pH was fixed at 7.4, Cu2+ 1 mM, phosphate 10 mM, and amino acid 2 mM or 10 mM. When the total concentration of amino acid is 2 mM, more than 80% of Cu exists as Cu-amino acid complex. At 10 mM, which more closely represents the total concentration of amino acid in cell culture media, all the copper form complex with amino acids. This calculation suggests that the formation of copper phosphate is limited in cell culture media containing ligands which have high affinity to copper ions.”
Figure S3. Hydroxyl radical EPR signal (DMPO spin trap) induced by 0.032 ppm Cu2+ with 1 mM glutamine in PBS buffer after 20 min, 45 min, 95 min incubation.
Figure S4. HRTEM image of sulfidated CuO nanoparticles (generated from 2.5 mM of CuO NPs incubated in 5mM Na2S solution for one day), corresponding to a CuO/sulfide molar ratio of 1:2. The lattice fringes of 0.304 nm spacing is close to the (102) plane of CuS.
Figure S5. Sulfidation of CuO NPs and the catalytic ability of CuS. (A) Depletion of 12.5 mM of sulfide in the presence of 2.5 mM CuO nanoparticles. (B) Comparison of catalytic activity of copper ions and CuO nanoparticles at equimolar concentration in sulfide oxidation reactions. (C) Comparison of catalytic ability of CuS precipitate and filtrate after separation by Amicon 3k ultrafilter. The catalytic ability of the clusters and precipitate was compared by monitoring the oxygen consumption in the presence of 2 mM sulfide solution. The curve of consumption of oxygen in the presence of precipitate is almost identical to that induced by the total, which indicated that the precipitate CuS played a significant role in catalytic oxidation of sulfide. The catalytic activity of CuS intermediate cluster may be very strong considering its ultralow concentration. (D) Comparison of catalytic production of hydroxyl radicals by sulfidated CuO at the initial Cu/S ratio at 0.5, 1 and 2 with original CuO NPs, which indicated the catalytic production of hydroxyl radicals increased after CuO sulfidation
Figure S6. Copper ions recovery after ultrafiltration by ultrafilter (Amicon Ultra-4 3k). Copper chloride solution with various concentration (approximately 0.1 ppm, 1 ppm, 10 ppm, 100 ppm) were prepared and filtrated by ultrafiltration. Their original concentration and their filtrate concentration were determined by ICP-AES. The dissolved copper ion recovery was calculated by Cfiltrate / Coriginal *100%. The same copper chloride solution supplemented with histidine ( molar ratio of Cu/hisdine 1:2) were also prepared at same concentration. These samples were treated the same as copper chloride solution samples. Blue and red color columns indicated the ion recovery of CuCl2 samples and CuCl2 supplemented with histidine. Our results indicated that copper ions retention by ultrafilters can be neglected when the concentration of dissolved copper is above tens of ppm level or the dissolved copper ion is complexed, which is the case in our study.
Figure S7. Viability of murine macrophages exposed to 5, 10, or 20 ppm of carbon black, CuO, and CuS NPs for 24 hrs. DNA content of untreated and treated cells was determined using Pico Green fluorescence as a surrogate for cell number. *p
Department of Chemistry, 2School of Engineering, 3Department of Pathology and Laboratory Medicine, 4Institute for Molecular and Nanoscale Innovation, Brown University, Providence, Rhode Island 02912 *Address correspondence to [email protected]
Additional details on Visual MINTEQ 3.0 calculation Equilibrium solubility of the oxide and sulfide forms (Figure 1) The equilibrium dissolved metal ion concentration was estimated using Visual MINTEQ 3.0 from Gustafsson.1 The phases considered were TiO2, ZnO, NiO, CuO and Ag2O and the sulfides ZnS, NiS, CuS and Ag2S. The parameters for metal oxide calculation were set as follows: pH is fixed at 7; temperature is 25℃; ionic strength is to be calculated depending on components added; 1 mM Na+ and 1 mM NO3- is added as components; the metal oxide of interest is specified as infinite solid phase. The parameters for metal sulfide calculation were set as follows: pH is fixed at 7; temperature is 25℃; ionic strength is to be calculated depending on components added; 1 mM S2- is added as components; the metal sulfide of interest is specified as infinite solid phase.
Equilibrium concentration of dissolved copper in the corresponding pH conditions (Figure 2A) The equilibrium dissolved copper ion concentration was estimated using Visual MINTEQ 3.0. The parameters were set as follows: pH is fixed at 4, 5, 6, 7.4, 8, 9, respectively; temperature is 25℃; ionic strength is to be calculated depending on components added; 2.5 mM CuO (tenorite) is specified as finite solid phase. The
calculated equilibrium concentration of dissolved Cu is 2.5 mM, 2.5 mM, 4.7E-02 mM, 1.3E-04 mM, 2.1E-05 mM and 4.1E-06 mM, respectively. In the case of pH 7.4, the effect of phosphate was investigated through calculation by Visual MINTEQ 3.0. The parameters were set as follows: pH is fixed at 7.4; temperature is 25℃; ionic strength is to be calculated depending on components added; 11.9 mM phosphate (concentration in PBS buffer) is added as components; 2.5 mM CuO (tenorite) is specified as finite solid phase; specify Cu3(PO4)2(s) as possible solid phase. The calculated equilibrium concentration of dissolved Cu is 2.4E-03 mM, which is predicted to be slightly higher than the one at same pH but without consideration of buffer effect (1.3E-04 mM) because of the formation of soluble CuHPO4 complex. These calculations show that the effect of phosphate complexation is limited and the extra low equilibrium concentration of dissolved Cu is predicted with or without consideration of phosphate buffer effect.
Figure S1. Characterization of commercial CuO nanoparticles. (A) TEM image of CuO after dispersion in DI water and drying overnight. (B) FT-IR spectrum showing the limited number and intensity of peaks, which may be residue of organic ligands during synthesis process. (C) XRD spectra indicating the tenorite crystalline. (D) Size distribution of CuO dispersed in PBS buffer which indicated strong aggregation (determined by DLS).
Figure S2. (A) Time-dependent dissolution of 200 ppm CuO nanoparticles in PBS buffer (pH 7.4). Please note that the unaltered detected Cu release is caused by its extra low concentration, which can’t be distinguished by our ICP-AES. (B)The effects of various components in cell culture media on the dissolution of CuO (initial concentration 200 ppm) in PBS buffer in one day. High glucose: 4500 mg/L. Low glucose: 1000 mg/L; HEPES: 5958 mg/L; FBS: 10%; Glutamine: 20 mM. The formation of copper phosphate in the PBS buffer containing amino acids was estimated through Visual MINTEQ 3.0 calculation. The parameters were set as follows: pH was fixed at 7.4, Cu2+ 1 mM, phosphate 10 mM, and amino acid 2 mM or 10 mM. When the total concentration of amino acid is 2 mM, more than 80% of Cu exists as Cu-amino acid complex. At 10 mM, which more closely represents the total concentration of amino acid in cell culture media, all the copper form complex with amino acids. This calculation suggests that the formation of copper phosphate is limited in cell culture media containing ligands which have high affinity to copper ions.”
Figure S3. Hydroxyl radical EPR signal (DMPO spin trap) induced by 0.032 ppm Cu2+ with 1 mM glutamine in PBS buffer after 20 min, 45 min, 95 min incubation.
Figure S4. HRTEM image of sulfidated CuO nanoparticles (generated from 2.5 mM of CuO NPs incubated in 5mM Na2S solution for one day), corresponding to a CuO/sulfide molar ratio of 1:2. The lattice fringes of 0.304 nm spacing is close to the (102) plane of CuS.
Figure S5. Sulfidation of CuO NPs and the catalytic ability of CuS. (A) Depletion of 12.5 mM of sulfide in the presence of 2.5 mM CuO nanoparticles. (B) Comparison of catalytic activity of copper ions and CuO nanoparticles at equimolar concentration in sulfide oxidation reactions. (C) Comparison of catalytic ability of CuS precipitate and filtrate after separation by Amicon 3k ultrafilter. The catalytic ability of the clusters and precipitate was compared by monitoring the oxygen consumption in the presence of 2 mM sulfide solution. The curve of consumption of oxygen in the presence of precipitate is almost identical to that induced by the total, which indicated that the precipitate CuS played a significant role in catalytic oxidation of sulfide. The catalytic activity of CuS intermediate cluster may be very strong considering its ultralow concentration. (D) Comparison of catalytic production of hydroxyl radicals by sulfidated CuO at the initial Cu/S ratio at 0.5, 1 and 2 with original CuO NPs, which indicated the catalytic production of hydroxyl radicals increased after CuO sulfidation
Figure S6. Copper ions recovery after ultrafiltration by ultrafilter (Amicon Ultra-4 3k). Copper chloride solution with various concentration (approximately 0.1 ppm, 1 ppm, 10 ppm, 100 ppm) were prepared and filtrated by ultrafiltration. Their original concentration and their filtrate concentration were determined by ICP-AES. The dissolved copper ion recovery was calculated by Cfiltrate / Coriginal *100%. The same copper chloride solution supplemented with histidine ( molar ratio of Cu/hisdine 1:2) were also prepared at same concentration. These samples were treated the same as copper chloride solution samples. Blue and red color columns indicated the ion recovery of CuCl2 samples and CuCl2 supplemented with histidine. Our results indicated that copper ions retention by ultrafilters can be neglected when the concentration of dissolved copper is above tens of ppm level or the dissolved copper ion is complexed, which is the case in our study.
Figure S7. Viability of murine macrophages exposed to 5, 10, or 20 ppm of carbon black, CuO, and CuS NPs for 24 hrs. DNA content of untreated and treated cells was determined using Pico Green fluorescence as a surrogate for cell number. *p
