Atelocollagen-Templated Fabrication of Tangled Fibrous Silica

1. General Remarks X-ray photoelectron spectroscopy (XPS) was performed on a Shimadzu ESCA-3400. The binding energies were corrected by referencing the carbon 1s binding energy to 285 eV. Fourier transform infrared (FT-IR) spectra were conducted on a JASCO FT-IR 660 plus using the KBr disc method. Thermogravimetric analyses (TGA) were performed on a Seiko Instrumental EXSTAR 6000 with a heating rate of 10 °C min-1 under N2 gas flow. Scanning electron microscopy (SEM) observation was performed on a Hitachi SU8020 FESEM. Relative surface area and porosity were characterized by N2 gas (99.998% purity) adsorption/desorption isotherms using a MicrotracBEL BELSORP-mini II. Samples were dried under vacuum at 100 °C for 3 hours before measurements. The relative surface area was determined by the Brunauer-Emmet-Teller (BET) method. Circular dichroism (CD) spectra were recorded using a J820 CD spectropolarimeter (JASCO, Japan). Transmission electron microscopy (TEM) observation was performed on a JEOL JEM-2100.


General Remarks
X-ray photoelectron spectroscopy (XPS) was performed on a Shimadzu ESCA-3400.
The binding energies were corrected by referencing the carbon 1s binding energy to 285 eV.
Fourier transform infrared (FT-IR) spectra were conducted on a JASCO FT-IR 660 plus using the KBr disc method.Thermogravimetric analyses (TGA) were performed on a Seiko Instrumental EXSTAR 6000 with a heating rate of 10 °C min -1 under N 2 gas flow.
Scanning electron microscopy (SEM) observation was performed on a Hitachi SU8020 FE-SEM.Relative surface area and porosity were characterized by N 2 gas (99.998% purity) adsorption/desorption isotherms using a MicrotracBEL BELSORP-mini II.Samples were dried under vacuum at 100 °C for 3 hours before measurements.The relative surface area was determined by the Brunauer-Emmet-Teller (BET) method.Circular dichroism (CD) spectra were recorded using a J820 CD spectropolarimeter (JASCO, Japan).Transmission electron microscopy (TEM) observation was performed on a JEOL JEM-2100.

Fabrication of Collagen-templated Silica
Solutions of AC and SC-AC (0.1 wt%, 3.3 M) were prepared by dilution of the 2 wt% solution and pH was adjusted to 6.5 by addition of NaOH and HCl.EDA-HCl (67.2 and 88.8 mol) was added to AC and SC-AC solutions (3.3 M, 6 mL).The final volume of the Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B. This journal is © The Royal Society of Chemistry 2016 reaction mixture was set to 7.2 ml by adding water.Then, TEOS (0.15mL, 0.67 mmol) was added and the mixture was stirred at 30 °C for 20h.SDS-PAGE analysis of the reaction mixture was performed by the Laemmli method.The mixture was centrifuged (8,000g for 5 min) and the precipitate was washed 3 times by water and 20% ethanol.The washed precipitate was freeze-dried (Christ Alpha 1-2 LD Freeze Dryer) for 20 hours.Samples were calcined under air flux in an electric muffle furnace (ADVANTEC FUW220PA) at 500 °C for 5 hours.
To 12 mL of the 0.33 M (0.01 wt%) solution of AC, 13.4 mol of EDA-HCl was added.
The final volume of the reaction mixture was made up to 14.4 ml with water.Then, 20 l of TiBALDH solution (50% w/v) (42 mol) was added and the mixture was stirred at 4 °C for 20h.The product was collected by centrifuge (8,000g for 5 min), washed by 20% ethanol, and freeze-dried for 20 hours.

Figure S1 .
Figure S1.Structural analysis of AC and SC-AC.(a) CD spectrum of AC, (b) TEM image of AC, (c) CD spectrum of SC-AC, (d) TEM image of SC-AC.CD spectrum of AC andSC-AC solution (0.1 mg/mL) showed the peak maximum at 222 nm, derived from the triple helical structure of native collagen.7 2 L of the protein solution (0.1 mg/mL) was placed on a carbon-coated grid and then stained with EM Stainer (Nisshin EM Co., Ltd.) and observed using TEM.Both AC and SC-AC showed the fibrous structure.

Figure S3 .
Figure S3.Effect of NaCl on SC-AC-templated silica formation.The formation of silica was investigated using SC-AC and EDA in the presence or absence of 1M NaCl.(a) SDS-PAGE analysis of the reaction mixtures.(b) A photo of the solution captured, 20 h after TEOS addition corresponding to the lane 2 and 4 of SDS-PAGE.In the presence of 1 M NaCl, SC-AC was detected after the reaction (lane 2) and the formation of silica is not observed.

Figure S4 .
Figure S4.Effect of pH on AC-templated silica formation.The pH of of 0.1 % AC solution is 3.7.Neutralization of the AC solution by NaOH followed by the addition of EDA-HCl and TEOS lead to the formation of tangled fibrous silica (Fig. 3a).Without neutralization, silica with a sheet-like morphology is formed.

Figure S5 .
Figure S5.Heat denatured SC-AC templated silica formation.(a) CD spectrum of SC-AC before (dotted line) and after (solid line) heat denaturation (60 °C for 3 h).Heat-denatured SC-AC loses the positive peak at 222 nm.(b) SEM image of heat-denatured SC-AC templated silica.