10.1371/journal.pone.0008058.g013 Figure 13  Fourier-transformed infrared spectroscopy of protein-mineral nanoparticles reveals the presence of both carbonate and phosphate.
Protein-mineral nanoparticles were obtained as described in Fig. 9, by diluting BSF (A), HSA (B), or both proteins (C) into DMEM, then adding 0.3 mM each of CaCl2 and NaH2PO4, and incubating the solutions in cell culture conditions for 1 month. Calcium granules were prepared by adding either CaCl2 (D) or NaH2PO4 (E) into FBS, or a combination of both CaCl2 and NaH2PO4 (F) into HS, as described in the  Materials and Methods . NB were cultured from 10% HS (G, “HS-NB”) or 10% FBS (H and I, corresponding to “Nanons” and “DSM 5820”, respectively). The FTIR spectra of the protein-mineral nanoparticles revealed peaks similar to both calcium granules and NB as shown by the presence of phosphate peaks at 575 cm−1, 605 cm−1, 960 cm−1, and 1,000–1,150 cm−1 as well as carbonate peaks at 875 cm−1 and 1,400–1,430 cm−1. Some of the peaks corresponding to phosphate or carbonate were not detected in a few calcium granule samples such as the one shown in (D). In the various nanoparticle samples presented here, peaks corresponding to amide I, II, and III at 1,660 cm−1, 1,550 cm−1, and 1,250 cm−1, respectively, were observed and were attributed to the presence of serum proteins. Spectra for the controls CaCO3 (J), Ca3(PO4)2 (K), and HAP (L), diluted and washed in double-distilled water, were included as controls. Residual water (H2O) was observed at 1,650 cm−1 in some controls prepared in the absence of proteins (K and L); this peak could also have contributed to the intensity of the amide I peak seen at 1,660 cm−1 in the other samples shown.