Application of Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy in Human Sera: Validate the method for contributing effective strategy for the storage and preservation

The serum is one of the biological specimens that have been extensively studied as biomarkers and is recognized as an effective tool for screening and diagnosing a variety of diseases. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a practical technique utilized in investigating characteristic spectra in infectious diseases cancer, and metabolic status. However, the pre-analytical procedure as storage condition is a crucial phase that directly impacts the accuracy of the result. In this study, we intend to investigate the effects of serum stabilization under both identical and distinct storage conditions on the FTIR spectral patterns, intensity variations, and macromolecule composition. The ten serum samples were stored in 3 different conditions including 4 °C, −20°C, and −80°C for six different time intervals (7, 14, 21, 28, 56, and 84 days). The spectra were obtained by ATR-FTIR spectroscopy. The serum stabilization was studied in two investigations: one focusing on the stability of serum under identical conditions over six different time intervals, and the other examining the stability of serum stored under three distinct conditions. Principal Component Analysis (PCA) and Area Under the Peak (AUP) were used to investigate clusters in different sets of data across five spectral ranges. The macromolecule composition was identified in conjunction with the intensity measurements. The results showed that the same conditions remained stable across six different time intervals, as indicated by the consistent PCA results. In contrast, the stabilization of serum under different temperature condition showed different major intensities across the five selected positive bands, including 1781 cm⁻¹, 1740 cm⁻¹ 1727 cm⁻¹ corresponding to lipid region (1800–1700 cm⁻¹), and Amide region (1700 −1500 cm⁻¹) at 1646 cm⁻¹, and 1632 cm⁻¹, when the serum was stored over 28 days. This might involve lipid peroxidation-induced changes in the secondary structure of proteins. Our findings indicated that serum should be stored at −80°C to ensure stabilization and achieve the highest intensity. Additionally, preserving serum under consistent conditions was crucial to minimize variability and prevent external factors from affecting the results.