# Raman Spectroscopy as Noninvasive Method of Diagnosis of Pediatric Onset Inflammatory Bowel Disease
## 3 Results  
In Figure 1, we report the average Raman spectrum of fecal extract from non-IBD patients, obtained averaging the spectra collected for all the analyzed subjects, after having performed a baseline correction of each of them in order to compensate eventual technical and/or sample variations, and having normalized them to the total integrated area. The spectrum highlights some of the main typical protein vibrational modes, associated to the polypeptide backbone (amide bands) and to the aromatic and nonaromatic amino acid residue side. Based on comparison with literature [43; 44], the band detected at ~523 cm\({}^{-1}\) is associated to the v(S-S) stretching vibration. The feature at ~759 cm\({}^{-1}\) is assigned to the ring breathing vibration of tryptophan. Going on, the tyrosine doublet can be recognized in the ~830-850 cm\({}^{-1}\) range, the high peak corresponding to the phenylalanine (Phe) ring stretching mode is well evident at ~1000 cm\({}^{-1}\), and the v(C-N) stretching mode is responsible of the band at ~1153 cm\({}^{-1}\). Going to higher Raman shifts, a large and composite band is detected extending from ~1250 cm\({}^{-1}\) up to ~1750 cm\({}^{-1}\). It is the result of the overlapping of several contributions, among which we can distinguish the symmetric v(C-O)\({}_{8}\) stretching vibration of COO' groups, at ~1416 cm\({}^{-1}\), and the \(\delta\)(C-H\({}_{2}\)) bending vibration, at ~1468 cm\({}^{-1}\). Going on, the amide II band is recognized at ~1548 cm\({}^{-1}\), consisting of an out-of-phase combination of v(C-N) stretching and \(\delta\)(N-H) bending motions. Finally, the amide I vibration is well evident as a large band centered at ~1650 cm\({}^{-1}\). It is mainly due to the v(C=O) stretching mode and a small amount of out-of-phase v(C-N) stretching. The position of the amide I band will depend on the conformations of the polypeptide backbone, i.e., on the type of secondary structure, and on the intra- and intermolecular hydrogen bond of protein specimen [45]. Each type of secondary structure will have a typical v(C=O) stretching frequency, and this justifies the broadening of the band. Figure 2a,b depicts the average Raman spectra, in the 900-1200 cm\({}^{-1}\) and 1300-1800 cm\({}^{-1}\) wavenumber ranges respectively, of fecal extract from non-IBD subjects (black line), compared to those obtained from subjects within each disease class, i.e., CD (red line) and UC (green line) (the last two spectra were obtained following the same procedure described at the beginning of this section for  
Figure 1: Average Raman spectrum of non-IBD subjects, together with the main vibrational assignment. See text for details. the average Raman spectrum relative to non-IBD subjects). The spectra reported in Figure 2 were preliminarily normalized to the total integrated area. From an inspection of the figure it appears clear that, as disease occurs, an evident increasing is observed in spectral intensity of the band centered at \(\sim\)1000 cm\({}^{-1}\) and in the large band detected in the 1300-1800 cm\({}^{-1}\) range. About this, it is worth remarking that significant variations in integrated Raman intensity, as active inflammation occurs and progresses in severity, were observed in previous studies by colonoscopy-coupled fiber-optic probe-based Raman spectroscopy for inflammatory bowel disease of the colon, including ulcerative colitis and Crohn's disease [33]. The high quality of our spectra allowed us to quantitatively account spectral modifications eventually induced by the inflammatory state as a consequence of changes in the secondary structures, by focusing our attention into the amide I region. For the reasons explained above, and as already reported in literature [46; 47], this band constitutes an excellent marker to quantify the secondary structure and conformational changes of proteins, as a consequence of the role played by the amide moiety in crosslinking [48].