SERS analysis of saliva and its key components: The effects of various collection methods, sample dilution, excitation wavelengths, and enhancing substrates

Recently, human saliva has become a subject of research as an excellent material for patient-friendly diagnostics. An increasing number of diagnostic tests utilize saliva due to its easy and noninvasive collection, eliminating the patient's stress. Simultaneously, developing Surface-Enhanced Raman Scattering (SERS) spectroscopy offers new possibilities for analyzing saliva's composition. Saliva is a complex biological material; many factors influence its composition, including medication use, diseases, stress, hormone levels, diet, age, and hydration. This complexity raises the question of whether it is possible to observe and definitively attribute changes in specific substances through SERS spectra. One of the key questions we posed is how the SERS spectrum will change with an increased level of α-amylase 1 A (AMY1A), an enzyme marker of acute stress. AMY1A forms complexes with proline-rich proteins (PRP). Thus, we examined whether similar spectral changes are observed with a PRP level increase in saliva. Another focus was lysozyme C (LYZ C), a nonspecific marker of infectious diseases. We examined how increased levels of LYZ C affect SERS spectra, particularly considering its sensitivity to changes in the ionic composition of saliva and its complexation with PRP and lactoferrin (LF). Moreover, we explored whether the albumin (HSA) level, which plays a vital role in regulating osmotic pressure, influences LYZ C activity and how it is manifested in SERS. Furthermore, we investigated the effect of saliva dilution and collection methods on SERS spectra. We searched for correlations with significant components such as AMY1A, HSA, LYZ C, LF, and Poly-L-proline (PLP is an analog of PRP). We showed the role of gold (Au) and silver (Ag) substrates, comparing the spectral differences. Solving the issues is crucial for the ability of SERS techniques to detect and/or monitor biomolecules in saliva and can lead to significant advancements in noninvasive diagnostics.