• Raman spectroscopy;
  • collagen;
  • structure;
  • protein;
  • hydrogel

To investigate molecular effects of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EDC/N-hydroxysuccinimide (NHS), glyceraldehyde cross-linking as well as polymerization temperature and concentration on the three-dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm−1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm−1/1268 cm−1 in controls to 1247 cm−1/1283 cm−1 in glyceraldehyde-treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm−1 band and the appearance of a strong peak at 1468 cm−1 reflected a change in the motion of lysine/arginine CH2 groups. For the EDC-treated collagen hydrogels, the increased intensity of 823 cm−1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm−1 to 1271 cm−1 amide III band intensities in the EDC-modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH2 groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm−1 band corresponding to a COO stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.