## 2 The Label-Free Approach
The PC1 loading data and the calculation of Euclidian distance revealed that only the EFGP marker showed high similarity with the unique Raman bands of NSCLC exosomes, while the other protein markers had poor similarity. These results were strongly correlated with immunoblotting data. Combining SERS with multivariate analysis, the Raman spectral signatures of single exosomes derived from different biological sources were measured on a SERS substrate made of a gold periodic pyramid nanostructure with a graphene monolayer on the top (Figure 4) [100]. The SERS spectra of the exosomes isolated by two different methods were studied. It was found that SERS peaks of individual exosomes isolated by ultracentrifugation/filtration can be observed in the SERS spectra, whereas the peaks of the exosomes derived by a commercially available isolation kit, ExoQuick, were absent. For a test on the detection of specificity, exosomes from fetal bovine serum, human serum, and two human lung cancer cell lines were taken. All samples were distinct in their spectral characteristics, mostly by the relative peak intensities. A structured polycarbonate substrate from regular recordable disks (CD-R and DVD-R) coated with silver was used as a cost-effective platform for label-free SERS exosome analysis [101]. The substrate fabrication process is shown in Figure 5. The Raman fingerprints of hemoglobin and exosomes and the impact of the silver thickness, the polycarbonate nanostructure, and the excitation wavelength on Raman enhancement were investigated. The results demonstrated the feasibility of the proposed approach for the analysis of complex biomolecular targets. Another potential application is a Raman scanner based on optical disk drives. Figure 4: A hybrid substrate made of a gold periodic pyramid nanostructure covered with a single-layer of graphene. Reproduced with permission from [100], American Chemical Society, 2019. [PERSON] et al. implemented Raman and SERS spectroscopy to study the compositional differences between exosomes derived from ovarian carcinoma cells, cultivated at normal (21%) and reduced (1%) oxygen conditions [102]. Using a trained multivariate algorithm based on PCA and DFA the authors showed biomolecular differences between exosomes derived from normoxic and hypoxic tumor cells. The Raman spectra of parent ovarian cells showed a biomolecular composition similar to that of their corresponding exosomes. Distinct peak differences are demonstrated in Figure 6. Sensitivity and specificity of 100% were achieved by applying multivariate statistical algorithms to Raman microspectrometry, whereas poorer results were obtained when using the SERS method due to damage of the samples caused by the laser. It is worth noting that in all cited articles of this section the authors performing the SERS analysis of cancer-derived exosomes demonstrated exosome spectra only for specific types of cell lines, without extension to all exosomes. Therefore, it is particularly difficult to determine common exosome peaks from SERS spectra. The difference between exosomal cargo was provided either by statistical analysis of a spectrum data set or by recognizing specific peaks of tumor-derived exosomes, which don't overlap with the peaks of exosomes derived from healthy cell lines. Thus, examination of the general and specific unique peaks of exosomes derived from different types of both cancerous and healthy cells is an urgent need for reliable and efficient SERS diagnosis. Figure 5: (**a**–**c**) Disk-based SERS-active substrate fabrication; (**d**) sample analysis. Reproduced with permission from [101], Elsevier, 2017. Figure 6: Mean spectra from normoxic and hypoxic exosomes. Reproduced with permission from [102].