## 3 Results and Discussion
This is considerably less than the usual assembly times for dip-coating (10-12 h). Additionally, the NPs are attached to the surface of the paper, and not to the top layer of the hybrid device (the PDMS), as confirmed by scanning electron microscopy imaging. Figure 4 shows the surface of the PDMS (Figure 4A-C) and of the paper (Figure 4D-F) at different magnifications after de-bonding. The concentration of NPs in the PDMS is remarkably lower than in the paper part. Furthermore, Figure 4F shows a very homogeneous distribution of the GNSs over the surface of the paper. Figure 2: Schematic representation of the fabrication of the hybrid PDMS-paper device (**A**–**C**) injection and self-assembly of the NPs (**D**) Raman model molecule or lysed cells injection (**E**) final disassembly of the paper sensor for analysis (**F**). In order to characterize the SERS capability of these new approaches for the fabrication of paper SERS substrates, the Raman probe 1 NAT was used. The 1 NAT solution was injected into the hybrid at decreasing concentrations for the GNSs and Au@AgNRs, respectively, using the 785 nm excitation line. A control experiment of the paper with the NPs but not the analyte was performed and is shown as the first spectrum of each series for both GNSs and Au@AgNRs. The spectra of the paper shows two intense peaks that are assigned to the C-O ring stretch modes of cellulose [42]. The limit of detection (LoD) of a SERS method can be defined as the lowest concentration at which a distinguished SERS spectrum from the blank can be obtained [43]. According to the method for the calculation of the limit of blank (LoB) and LoD described by [PERSON] and [PERSON] [44],  
\[\text{LoB}=\text{mean}_{\text{blank}}+1.645\ (\text{SD}_{\text{blank}}) \tag{1}\]  
Figure 4: SEM images of the assembled GNSs after system de-bonding at different magnifications on the PDMS part (**A–C**) and on the paper substrate (**D–F**). Figure 3: AutoCAD design showing the total area calculation for the controlled NP self-assembly (**A,B**) AutoCAD design of the reservoir used for NP assembly (i), PDMS-paper after NP injection (ii), paper showing the assembled NPs after de-bonding the PDMS (iii). \[\text{LoD}=\text{LoB}+1.645\ (\text{SD}_{\text{low concentration sample}}) \tag{2}\]  
the LoB based on data shown in Figure S3 is 1.6 SERS counts (a.u. ), and the LoD is 14.4 counts (a.u.). Considering the intensity at 1368 cm\({}^{-1}\) (\(I_{1368}\)) band for the lowest tested concentration (1 pM) being 48.8 \(\pm\) 7.80 counts (a.u. ), above the LoD, we may conclude that the limit of detection of this method is in the picomolar range. Alternatively, if we use the blank determination method, the LoD follows the following equation [45]. \[\text{LoD}=\text{mean}_{\text{blank}}+3(\text{SD}_{\text{blank}}) \tag{3}\]  
According to Equation (2), the LoD = 6.8 counts (a.u. ), again confirm that the LoD is in the picomolar range, since the lowest measured concentration is 10\({}^{-12}\) M and the mean signal for this concentration is 48.8 \(\pm\) 7.80 counts (a.u.). The different LoDs with the two reported methods are due to the confidence interval taken into account for each of them. For the method described by [PERSON], the confidence interval is 95%, whereas, for the blank determination method using three times the standard deviation of the blank to assess the noise influence, the confidence interval is 98 %. The latter demonstrates the potential of these substrates to be used in ultrasensitive sensing experiments. The homogeneity of the NPs assembly and SERS signal was tested by mapping a 100 \(\times\) 100 \(\upmu\)m\({}^{2}\) area (Figure 5C,D and Figure S4). Figure 5C is a macroscopic optical picture of a row of three of the resulting SERS paper substrates. Figure 5D is a microscopic white light image of the resulting substrate in which the orange defined area is that of the SERS map presented in Figure 5E.