Raman spectroscopic characterisation of non stimulated and stimulated human whole saliva

Human saliva is a unique biofluid which can reflect the physiopathological state of an individual. The wide spectrum of molecules present in saliva, compounded by the close association of salivary composition to serum metabolites, can provide valuable information for clinical diagnostic applications through highly sensitive vibrational spectroscopic techniques such as Raman spectroscopy. However, the nature of saliva, in terms of collection and patient-related characteristics, can be considered factors which may strongly affect the Raman spectral profile of salivary samples and disrupt the search for specific salivary biomarkers in the detection of diseases. The main objective of this study was to highlight spectral features associated with the type of collection in an intra- and inter-patient approach. Saliva was collected using both stimulated and non-stimulated approaches from 20 donors, concentrated by centrifugal filtration and further analysed using Raman spectroscopy. The methodology adopted for liquid saliva showed consistency in the qualitative analysis of the groups, confirming the reproducibility of this Raman spectroscopic approach. Using principal component analysis (PCA) and partial least squares – discriminant analysis (PLSDA), non stimulated saliva could be differentiated from stimulated saliva in both intra- and inter-patient analysis, with a classification efficiency of 77 and 87 %, respectively. The bicinchoninic acid (BCA) assay showed a similar trend in terms of total protein concentration, showing a slight increase in stimulated saliva samples. These results are valuable in the process of developing and establishing Raman spectroscopy as a novel diagnostic tool in the future as well as controlling variability, in order to determine specific spectroscopic markers related to a multifactorial disease for diagnostic or follow-up purposes.