Correction notice: Amyloid carbon hybrid membranes for universal water purification
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1 Correction notice: Amyloid carbon hybrid membranes for universal water purification Sreenath Bolisetty and Raffaele Mezzenga Nature Nanotechnology (2016). The version of this Supplementary Information file originally published was a non-final version containing incorrect weight per cent values in the captions of Figs S15, S16 and S17 and incorrect mass values in the last sentence of the caption of Table S1. These errors have been corrected in this file 29 January 2016.
2 SUPPLEMENTARY INFORMATION Amyloid carbon hybrid membranes for universal water purification Sreenath Bolisetty, Raffaele Mezzenga NATURE NANOTECHNOLOGY 1
3 SUPPLEMENTARY INFORMATION Table of Contents: Purification of nanoparticles dispersion Page 4 Catalysis study Page 4 Etching lithography Page 5 Operating flow and membrane parameters Page 5 Binding isotherms Page 5 Relative adsorption capacity of amyloid membranes Page 6 References page 7 Supplementary Figure S1 Page 8 Supplementary Figure S2 Page 8 Supplementary Figure S3 Page 9 Supplementary Table S1 Page 10 Supplementary Figure S4 Page 11 Supplementary Figure S5 Page 12 Supplementary Figure S6 Page 13 Supplementary Figure S7 Page 14 Supplementary Figure S8 Page 15 Supplementary Figure S9 Page 15 Supplementary Figure S10 Page 16 Supplementary Figure S11 Page 16 Supplementary Figure S12 Page 17 Supplementary Figure S13 Page 17 2 NATURE NANOTECHNOLOGY
4 SUPPLEMENTARY INFORMATION Supplementary Figure S14 Page 18 Supplementary Figure S15 Page 19 Supplementary Figure S16 Page 20 Supplementary Figure S17 Page 21 Supplementary Figure S18 Page 22 NATURE NANOTECHNOLOGY 3
5 SUPPLEMENTARY INFORMATION Purification of Nanoparticles Dispersion A high temperature thermal process was utilized to reduce the gold and palladium ions into the nanoparticles whilst converting the activated carbon as well as amyloid fibrils into ash. The ash and other impurities were removed by ultrasonication. Then, the sample, placed in crucible was mixed with excess distilled water and sonicated at 100Hz for 15 min. This resulted in the sedimentation of the gold (palladium) nanoparticles to the bottom of the vial and the ash suspended in the aqueous phase. The upper aqueous phase containing ash was removed and this washing step was repeated several times by ultra-sonication, resulting only into purified metal nanoparticles dispersion. This purified gold (palladium) nanoparticles dispersion sediments very quickly, due to the relatively large size of the nanoparticles (see Figure S4). Catalysis Study The gold and palladium nanoparticles were used for the catalytic reduction study of p- nitrophenol (PNP) to p-aminophenol (PAP). 500 µl of 0.1 mm 4-nitrophenol were mixed with 20 µl of freshly prepared solution of NaBH 4 (56 mm). 20 µl of gold and palladium nanoparticles were mixed with the PNP solution to initiate the catalytic reduction. The reduction catalysis with time was monitored using a UV-Visible spectrophotometer. The reaction is first order in the concentration of PNP (C PNP ) and is expressed by the following equation: CPNP dt kcatcpnp d / (1) where k cat is the apparent rate of catalysis. Upon integration eq. 1 becomes: ln( C t PNP / C 0 PNP ) = k cat t (2) 4 NATURE NANOTECHNOLOGY
6 SUPPLEMENTARY INFORMATION C t PNP is the concentration of PNP at time t and C 0 PNP is the initial concentration of PNP and C t PNP the ratio of / is equals to A = A 400 nm (t)/a 400 nm (0) where A 400 nm (t) and A 400 nm (0) C0 PNP are the absorption intensities of PNP at 400 nm at time t and zero, respectively. Therefore K cat can be obtained from the slope of the plot of ln(a) vs time, directly. Etching Lithography The lithography of the ETH logo on the gold was done using a Tescan Fera3 Plasma-FIB- SEM. For trimming and flattening the gold surface was done by 2 ua beam and then dis some fine polishing using 100 na. The engraving was done using the 4 na current and milling. The focused ion beam consists of Xe ions with an energy of 30 kv. Operating Flow and Membrane Parameters The typical carbon-amyloid membranes produced have an area of 1.8 cm 2 and a typical weight of 100mg. Using these membranes we process 50mL every 30 seconds, corresponding to 0.1 L/minute, but because we use simple vacuum filtration, this flows occurs at a pressure of only 1 Bar. By using an equivalent comparable pressure of 50 bars, as in most reverse osmosis applications, for water desalination for example (the osmotic pressure of seawater being 26 bar), and following the Hagen Poiseuille equation, the equivalent feeding flow using the same exchange area would be 50x0.1 L/minute = 5L/minute or 7200 L/day. Binding Isotherms Binding isotherms studies were performed by titration of fixed concentrations of β- lactoglobulin (ph 2) protein fibrils solution (350 µl of 2 wt.%) with various concentrations ranges of heavy metal ions having a final volume of 2 ml. The unbound free metal ions were removed by filtration, the concentration of bounded heavy metal ions on the fibrils were NATURE NANOTECHNOLOGY 5
7 SUPPLEMENTARY INFORMATION estimated by measuring the permeate concentrations by atomic absorption spectroscopy (Au, Hg, Pb) and Uv-Vis spectroscopy (Pd). The binding isotherms were fitted using the approach by Swillens and Motulsky 1, 2 : KK aa = [MM LL] [MM 0 MM][LL 0 LL] = [MM LL] [MM 0 MM LL][LL 0 MM LL] (eq. 3) where KK aa is the binding constant, [MM LL] is the concentration of the metal:ligand bound pair, [MM 0 MM] is the unbound metal concentration and [LL 0 LL] is the unbound ligand concentration. [MM] and [LL] are the bound metal and ligand concentration (identical to [MM LL]), respectively and [MM 0 ] and [LL 0 ] are the initial total metal and ligand concentration, respectively. This equation can be solved for [MM LL] yielding the following solution: [MM LL] = 1 2 ([MM 0] + [LL 0 ] + 1 KK aa ) 1 2 ([MM 0 ] + [LL 0 ] + 1 KK aa ) 2 4[MM 0 ][LL 0 ] (eq. 4) which allows fitting [MM LL] as a function of the titration increasing concentration [MM 0 ], with of KK aa and [LL 0 ] as fitting parameters. With the exception of Pd, eq. 4 can suitably be used to fit the experimental isotherms giving the fitting parameters in Table S1. Relative Adsorption Capacity of Amyloid Membranes In order to estimate the relative adsorption capacity of amyloids, the filter membranes were prepared first with 1 ml of 0.2 wt% β-lactoglobulin amyloid fibrils without adding any activated carbon. 50 ml of four different heavy metal salts, ([KAu(CN) 2 ], [HgCl 2 ], [Pb(C 2 H 3 O 2 ) 4 ] and [Na 2 PdCl 4 ]) having concentration of mg/ml were filtered individually by the amyloid filter membrane. The adsorption of the heavy metal ion inside the 6 NATURE NANOTECHNOLOGY
8 SUPPLEMENTARY INFORMATION membrane is estimated by measuring the solution concentration of heavy metal ions before (feeding solution) and after filtration (permeate) using absorption spectroscopy techniques. This allows resolving the adsorption efficiency per mg and per cycle for the amyloid fibrils. Additionally, this analysis was confirmed by measuring the amount of adsorbed ions independently: unbound metal ions were removed by washing with excess Milli-Q water. The adsorber membrane was then dissolved and heated in aqua regia, and then the amount of heavy metal ions strongly adsorbed to the amyloid fibrils was quantified with AAS. In a second step, we produce hybrid membranes made of amyloid fibrils and activated carbon, by maintaining identical the total weight of the amyloid fibrils in the membrane (i.e. as in the pure amyloid membranes). We then process the same volumes of feeding solutions with the same concentrations of metal ions. By difference between the adsorption in the two experiments series, we can resolve the specific adsorption efficiency per mg and per cycle of the activated carbon as well. Supplementary References Swillens, S. Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Mol Pharmacol 47, (1995). Motulsky, H. The graphpad guide to analyzing radioligand binding data. GraphPad Software, Inc., San Diego, CA. (1996). NATURE NANOTECHNOLOGY 7
9 SUPPLEMENTARY INFORMATION Figure S1. Atomic force microscopic (AFM) images of the β-lactoglobulin amyloids after high temperature treatment of the native monomer at 90 C and ph 2 for 5 h: (A) amyloid fibrils of several micrometers in contour length; (B) Higher magnification image of βlactoglobulin amyloid fibrils from which the twisting pitch can be easily identified. Figure S2. Docking poses of mercury ions at the 121-cys residue of the β-lactoglobulin protein sequence. (B) Amyloid-forming protein segment (LACQCL) docking with the mercury metal ions. (C) Amyloid forming segment (LACQCL) of the β-lactoglobulin protein generated from the zipperdb database server. 8 8 NATURE NANOTECHNOLOGY
10 SUPPLEMENTARY INFORMATION Figure S3. Metal adsorption isotherms (experimental points) fitted by eq. 4 for the four metals Au, Hg, Pb, Pd. With the exception of Pd, eq. 4 converges in all cases, yielding the fitting parameters given in Table S1. When not shown, error bars are smaller than symbols. NATURE NANOTECHNOLOGY 9
11 SUPPLEMENTARY INFORMATION Table S1. Fitting parameters using eq. 4 to fit Metal adsorption isotherms Metal ion [LL 0 ] ( M) KK aa (M -1 ) Wt adsorbed (%) Au Hg Pb Pd Fitting parameters suggest that Pb has the highest binding constant and total available binding sites from the ligand. Au has slightly lower binding constant than Hg, but more than 10 times available binding sites from the ligand. Pd shows adsorption isotherms significantly more complex than what captured by the model (eq. 4, see Figure S3), presumably due to the multiple valence of the ions. For sake of comparison, the saturation is obtained by using 3.5 mg of -lactoglobulin amyloid fibrils in 2 ml of solution, i.e. corresponding to 1000 M cysteine groups. This suggests, that at least in the case of Au and Pb, binding of metal ions occurs at other residues together with cysteine groups, bringing up the total amount of adsorbed mass to 16 and 29.5%, respectively (see right-most column in Table S1). By further considering the results presented in Figure 4C and 4B of the main manuscript, it is now possible to evaluate the contribution of amyloid and activated carbon to the total recovered gold: because -lactoglobulin amyloid fibrils can adsorb at saturation gold up to about 16% of its own weight, 5.7 mg of -lactoglobulin amyloid fibrils will contribute 0.9 mg to the total 100mg of recovered gold, with the 71.3 mg of activated carbon contributing the remaining 99.1 mg, i.e. an adsorption efficiency of more than 100% at saturation. Taken together, these results indicate the following important trends: on fresh (unsaturated) hybrid membranes, to be best used for heavy ions removal, amyloid fibrils play the major role, with a removal efficiency several orders of magnitude larger than activated carbon (see Figure 3D main manuscript), due to a stronger equilibrium binding constant. When the membranes are to be used for heavy metal recycling, however, the high porosity of activated carbon allows saturation at larger relative masses, increasing the performance in total adsorbed amount of heavy metal ions. These findings provide an important rationale for the optimal design of the membranes to meet specific requirements for either water purification or ion recycling. 10 NATURE NANOTECHNOLOGY
12 SUPPLEMENTARY INFORMATION Figure S4. Robustness of the composite membrane. (A-C) membrane can be processed into the desired shape by cutting with scissors. (D-F) Membranes withstanding folding and bending manipulation. NATURE NANOTECHNOLOGY 11
13 SUPPLEMENTARY INFORMATION Figure S5. The processing metal solutions are molecular solutions as demonstrated by a series of control experiments. (A) Processing Na 2 PdCl 4 solution after dialysis through a 7kDa dialysis membrane: despite the very low molecular cut-off, the permeate is coloured revealing the presence of ions vs nanoparticles, which would not diffuse through the dialysis membrane. (B) Filtering experiments performed on metal solutions obtained after dialysis as in panel A: the ion removal performance is identical as that on unfiltered solutions (see main manuscript), indicating the absence of nanoparticles in the feeding solutions. (C) Dynamic light scattering on 5 and 10 nm Au nanoparticles dispersion, compared to KAu(CN) 2 at the same metal concentration of 29 µm indicate a well defined correlation function in the case of nanoparticles, and only noise in the case of ions solution, indicating again the absence of nanoparticles in the feeding solution. The inset further confirms these findings by showing the low count rate in case of molecular salt solutions. (D) Noise detected through the dynamic light scattering correlation function for molecular solutions of Hg, Pb and Pd salts. 12 NATURE NANOTECHNOLOGY
14 SUPPLEMENTARY INFORMATION Figure S6. Calibration curves for concentration of heavy metal pollutants before and after filtration through the hybrid filter membranes. (A) Potassium dicyanoaurate, (B) Mercuric chloride, (C) Lead acetate, (D) Disodium tetrachloro palladate, (E) Uranyl acetate and (F) The total mass of Uranyl acetate and P-32 handled for the experiments was such to remain below the threshold of 900Bq set for acceptable radiation limits (Swiss StSV ref. 1. Jan 2014). NATURE NANOTECHNOLOGY 13
15 SUPPLEMENTARY INFORMATION Figure S7. Reusability of the filtration membrane in logarithmic-linear scale. Extrapolation by an exponential fit shows that at least 13 cycles are needed to bring down the efficiency of the membrane to 90% (open circle). 14 NATURE NANOTECHNOLOGY
16 SUPPLEMENTARY INFORMATION Figure S8. Transmission electron microscopy images of the gold nanoparticles recovered from the hybrid carbon-amyloid membrane by high temperature thermal treatment. Figure S9. (A) Transmission electron microscopy image of the gold nanoparticles prepared by chemical reduction. (B) UV-Vis absorption spectroscopy showing the typical gold nanoparticles absorption peak; in the inset the color of the solution after nanoparticles formation. NATURE NANOTECHNOLOGY 15
17 SUPPLEMENTARY INFORMATION Figure S10. Colour of the p-nitrophenol solution (yellow) changes (to colourless) after catalytic conversion using gold nanoparticles. Figure S11. Catalytic reduction of p-nitrophenolate ion, PNP to P-aminophenol PAP by extracted gold nanoparticles as compared to commercial gold nanoparticles of 50 nm at the same concentration. The higher slope in the case of extracted gold nanoparticles indicates higher catalytic efficiency. 16 NATURE NANOTECHNOLOGY
18 SUPPLEMENTARY INFORMATION Figure S12. Energy dispersive X-ray spectroscopy (EDX) measured on the recovered film confirms the presence of the elemental gold in the film. Figure S13. Atomic force microscopic images of the recovered gold nanoparticles redispersed in water by aid of 0.1 wt% amyloid fibrils. NATURE NANOTECHNOLOGY 17
19 SUPPLEMENTARY INFORMATION Figure S14. Catalytic reduction of p-nitrophenolate ion, PNP to P-aminophenol PAP by recovered palladium nanoparticles. Inset shows the colour of the dispersion of the palladium nanoparticles recovered and redispered in water. 18 NATURE NANOTECHNOLOGY
20 SUPPLEMENTARY INFORMATION Figure S15. Different metal ion removal efficiencies by composite membranes having 2 wt% β-lactoglobulin amyloid fibrils at different ph conditions. NATURE NANOTECHNOLOGY 19
21 SUPPLEMENTARY INFORMATION Figure S16. Concentration of mercuric chloride filtered for 10 consecutive cycles by the composite membrane having 7.4 wt% of whey amyloid fibrils. 20 NATURE NANOTECHNOLOGY
22 SUPPLEMENTARY INFORMATION Figure S17. Comparison of filtering efficiency of hybrid membranes having 2 wt% amyloid fibrils based on: (A) bovine serum albumin and activated carbon; (B) lysozyme and activated carbon; (C) celite and β-lactoglobulin. (D) Percentage of ion removal efficiency from various hybrid amyloid fibril membranes for the four metal ions considered. NATURE NANOTECHNOLOGY 21
23 SUPPLEMENTARY INFORMATION Figure S18. Filtration of bacteria (Lactobacillus plantarum) by hybrid amyloid and activated carbon membranes. The solutions before (square) and after filtration (circle) were incubated in agar gel. Growth of bacterial colony is found before the filtration but no growth is found in the filtered solution due to successful removal of bacteria by size exclusion (approximate bacteria size 1 µm) with the hybrid membranes. 22 NATURE NANOTECHNOLOGY
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