Surface-enhanced Raman Scattering (SERS) Spectroscopy, combined with multivariate data analysis such as Principal Component Analysis (PCA), effectively detects subtle changes in complex biological samples. In this study, we applied SERS to identify subtle molecular changes in human saliva deposited on large nanostructured Ag and Au substrates, focusing on the influence of temperature variations ranging from 10°C to 45°C. The selected temperature intervals – 10°C (cooling technology), 23°C (laboratory temperature), 37°C (physiological temperature), 42°C (fever), and 45°C (extreme temperatures) – reflect real-world conditions that biological and medical samples may encounter during collection, storage, transport, and analysis. We aimed to determine whether saliva samples remain stable at these temperatures over four days or if significant changes occur. Furthermore, we investigated the reversibility of spectral alterations during thermal jumps, where samples were heated to 45°C and then cooled back to 10°C. To ensure reliability, we utilized a computer-controlled mapping stage and a thermostatic sample holder, allowing precise temperature control and repeated recordings at identical locations on the substrate. Attention was given to intensity changes of marker bands, including band ratios, such as the ratio of 1175 cm⁻¹ to 1005 cm⁻¹ bands (protein hydration marker), the ratio of 856 cm⁻¹ to 831 cm⁻¹ bands (hydrophobicity marker of the environment surrounding tyrosine), and the ratio of 1360 cm⁻¹ to 1340 cm⁻¹ bands (hydrophobicity marker of the environment surrounding tryptophan) at different temperatures. The protein hydration marker exhibited a progressive decrease with increasing temperature, indicating water loss from the protein environment. In contrast, the hydrophobicity markers for tyrosine and tryptophan residues showed an increasing trend, suggesting enhanced hydrophobicity and a temperature-dependent reorganization of the protein structure on the SERS-active surfaces. In addition to these markers, we monitored changes related to amino acid residue bands for each temperature during the stability tests and thermal cycling. The spectral changes were associated with water loss and the reorganization of molecules near the nanostructured plasmonic surface, indicating saliva's sensitivity to temperature conditions. Our findings emphasize the importance of maintaining proper storage conditions for saliva films on large-area substrates to preserve sample integrity and prevent the misinterpretation of temperature-induced spectral changes. This study contributes to best practices for SERS analysis of thermally sensitive materials, particularly biofluids, especially in the context of medical diagnostics.
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