## 3 Results and Discussion
The SERS spectrum of PBMCs vs. SW480 showed a significantly different profile. Notably, the SW480 cells spectrum showed a higher enhancement on the peaks at 452, 515, 669, 752, 824, 1051, 1092, 1251, 1272, and 1458 cm\({}^{-1}\). The prominent Raman peaks of the SW480 cells, compared with the PBMCs, at around 747 cm\({}^{-1}\) and 1000-1300 cm\({}^{-1}\) regions can be assigned to the molecular structures of the nucleic acids, proteins, and lipids (phospholipids) (Table 1). The higher intensities of these SERS peaks in SW480 cells demonstrated that there was an increase in the amount of proteins, as expected for cancer cells [49]. Many spectral differences between PBMCs and SW480 cells can be clearly observed, which suggests the feasibility of these substrates to differentiate between different cell populations. For example, within the field of liquid biopsy, and, more specifically, for the isolation of circulating tumor cells (CTCs) from whole blood, it is relevant to be able to distinguish between CTCs, white blood cells (WBCs), and red blood cells (RBCs) [50]. The isolation of CTCs from white blood cells and red bloods cells has been an intense field of study due to their clinical relevance [51]. In this context, being able to distinguish between different cell populations is of outmost relevance for the liquid biopsy field. These preliminary results need to be further developed, but are promising \begin{table}
\begin{tabular}{c c c c} \hline
**Raman Shift (cm\({}^{-1}\))** & **Vibrational Mode** & **Peak Assignment** & **Reference** \\ \hline
516 & S-S Disulfide stretching & Proteins & [52] \\
638–642 & C–S Stretch & Tyrosine & [53,54] \\
649 & C-C twisting mode & D-glucose, lactose & [55] \\
725–728 & \(\delta\)(C–H) & Adenine, coenzyme & [52,56] \\
758 & & Typtophan & [53] \\
823 & Ring Breathing & Tyrosine & [56], \\
813, & \(v\) (C–C–O) & L-Serine, glutathione, & [57] \\
886 & \(v\) (C–O–H) & Glutathione, d-(C)-galactosamine & [58] \\
925–935 & C–C stretching mode, & Proteins and collagen & [58] \\  & C–C \(\alpha\) helix & Phospholipids, glucose-I\({}^{\prime}\)-phosphate, & [53,58] \\
979–999 & & DNA (around 980 cm\({}^{-1}\)) & [59], \\
1046, 1048 & C–H bedding & Glycogen: C–O, C–C & [53] \\
1080–1099 & C–C stretching, & Nucleic acids, phospholipids & [58] \\
1094–1134 & C–N stretch, & Lipids, D-mannos & [53], \\  & C–C stretching & & [54] \\
1206 & Ring vibration & Tyrosine & [56] \\
1240–1260 & C–N, N–H & Amide III & [58] \\
1269 & \(\delta\) (=CH) & Phospholipids & [60] \\
1316–1329 & CH vibration, & DNA/RNA, lipids & [55], \\  & CH\({}_{2}\) twisting & DNA/RNA, lipids & [52] \\
1331 & \(v\)(C–H) & Nucleic acid bases & [57] \\  & CH\({}_{3}\)CH\({}_{2}\) & Trypothan, adenine, guanine & [52] \\  & wagging & & \\
1400–1420 & CH\({}_{3}\) & Proteins, amino acids (around 1414 cm\({}^{-1}\)) & [58] \\
1441–1445 & \(\delta\)(CH\({}_{2}\)), \(\delta\)(CH\({}_{3}\)) & Proteins, & [52], \\  & Collagen and phospholipids & [57] \\
1607 & & & \\
1625–1699 & \(\gamma\)(C=O) & Amide I (\(\alpha\)-helix), collagen, Amide I & [56, 57, 58] \\ \hline \end{tabular}
\end{table} 
Table 1: Assignment of the Raman vibrations corresponding to cell molecular components. Figure 6: Spectrum of the paper (blank), and cell lysate from healthy (PBMCs) and cancer (SW480) cells, obtained by using the developed SERS paper substrate. Each spectrum showed is the average of five spectra taken at different positions of the substrate. for the feasibility of this strategy as a potential diagnostic system. Toward this purpose, the authors plan the implementation of chemometric tools, as well as increase the number of samples to be analyzed, and eventually to validate the system with clinical samples.