## 3 Results and Discussion
Figure 5E shows a very homogeneous intensity profile of the mapped area for the selected characteristic band of the 1 NAT (1368 cm\({}^{-1}\), ring stretching) [46]. Figure 5: Results of the characterisation of the SERS substrates with the Raman model molecule 1 NAT: SERS spectra of different concentrations of 1 NAT on the surface of the SERS-paper substrate assembled with GNSs (**A**); SERS spectra of different concentrations of 1 NAT on the surface of the SERS-paper substrate assembled with Au@AgNRs (**B**); photograph of the final assembled NPs after injection of the Raman model molecule (**C**); bright field image at 5\(\times\) magnification of the self-assembled GNSs on the paper substrate (**D**); mapping obtained by plotting the SERS intensity of the characteristic band of the 1 NAT at 1368 cm\({}^{-1}\) (**E**) in the area marked by the orange square in (**D**) for the GNSs substrates. The remarkable SERS efficiency of these substrates is not only based on the ability of controlling the drying conditions to obtain homogenous assemblies, but also arises from the use of anisotropic NPs. Anisotropic morphologies provide an extra SERS enhancement due to the strong near-fields concentrated at their vertices [17]. Furthermore, the formation of hot-spots, which are areas with an extra enhancement due to the vicinity of NPs, contributed to the high efficiency of the substrate [17]. According to Figure 5A,B, the best results and most homogenous substrates were those obtained with the GNSs (for Supplementary information, see Figure S4). This fact may be due to different reasons, such as the concentration of GNSs being 20 mM, whereas the Au@AgNRs were used at a concentration of 10 mM due to stability reasons. Despite the fact that the 1 NAT signal at 1368 cm\({}^{-1}\) could still be found in the sample prepared with 10\({}^{-12}\) M concentration of 1 NAT, the reproducibility of the measurements was much lower. In Figure 5B, the average for five measurements does not always give a clear positive result (i.e., 10\({}^{-4}\) M and 10\({}^{-8}\) M) in the case of NRs. The lower homogeneity of the Au@AgNRs substrates can also be explained on the basis of the self-assembly distribution of Au@AgNRs. The application of NRs in SERS spectroscopy strongly relies on: (i) the orientation of the longitudinal and transverse bands of the NRs to the incident polarized light, (ii) to the arrangement of the nanorods with respect to each other (tip to tip, side to side, or side to tip), and (iii) to the adsorption of the molecules either on the tips or on the sides of the nanorod [12; 47]. When the longitudinal band of the nanorods is parallel to the incident light, there is a higher enhancement than when the incident light is parallel to the longitudinal band [48]. As seen in this substrate, there was no control over the orientation of the nanorods, which means the resulting substrate was less homogenous than the one with the GNSs. For the demonstration of the applicability of these substrates as biosensors in disease diagnosis and monitoring, we used the GNSs-paper substrates to analyze the lysed product of two different cell types, which are PBMCs (healthy cells) and SW480 (colorectal cancer cell line). For this purpose, ethanol and ultrasonication were used to lyse samples containing one million cells of each type. Despite it being possible to inject the lysed samples in the same way than when the 1 NAT was used, to demonstrate the flexibility of use of these substrates, aliquots of the cell lysates were casted on top of the GNSs paper assemblies after de-bonding and let dry. The average of five measurements at different positions of the paper substrate, and, in three substrates, are shown in Figure 6. The different peaks can be assigned to different biochemical components and molecular structures, such as nucleic acids, proteins, and lipids. Table 1 shows a list of the assignment of Raman characteristic peaks for cellular components based on previous studies.