*3.2. Optimization and Characterization of AgNPs-PDMS Substrate*

As the first step of modification, the amino functionalization process was optimized by adjusting the concentration of the APTES solution. PDMS slices were immersed in different concentrations of APTES ethanol solutions (0.002%, 0.005%, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, and 2%, Vf). After dropping Ag colloid and drying, it was observed that the minimum concentration of APTES was 0.01% for the successful formation of the circular AgNPs substrate (see Supplementary Materials, Figure S1), and it was established as the optimized condition for further experiments. Notably, by using a higher amount of APTES, the obtained AgNPs substrate was encapsulated with a thin layer of transparent coating in the SEM image (see Supplementary Materials, Figure S2), presumably caused by the self-polymerization of extra APTES [49]. In the SERS measurements, this coating material could prevent the access of target molecules to the proximity of AgNPs, resulting in poor sensitivity. Therefore, it is necessary to restrict the use of APTES to a minimum required level. Compared with a previous report, which required the treatment of PDMS with piranha solution before functionalization [41], this method simplifies the modification process and avoids the preparation and use of dangerous reagents.

After amino functionalization, the amount of AgNPs is another crucial factor to determine SERS activity. Thus, Ag colloids with different concentrations were prepared and evaluated. R6G has been extensively used as a probe molecule for SERS due to its excellent Raman properties [50]. In this study, it was also employed to investigate and optimize the performance of our substrate. As presented in Figure 2a, the SERS spectra of 100 <sup>μ</sup>g L−<sup>1</sup> R6G solution showed that PDMS modified with Ag5×, which was 5-fold concentrated from the original Ag colloid, produced the strongest SERS signal. The peak intensity at 1358 cm−<sup>1</sup> was five times higher than Ag0.5×. This might be caused by the fully packed AgNPs through the concentrating process, which generate more "hot spot" nanostructures in SERS enhancement. The enhancement factor (EF) for R6G was calculated based on the widely adopted equation [51]:

$$EF = \frac{I\_{SERS} / I\_{bulk}}{N\_{SERS} / N\_{bulk}}$$

where *ISERS* and *Ibulk* stand for peak intensities at 1358 cm−<sup>1</sup> from both SERS and Raman spectrum of R6G, respectively. *NSERS* and *Nbulk* represent the estimated number of molecules stroked by the laser on the SERS substrate and R6G powder. The calculation process is detailed in Supplementary Materials, Figure S3. As a result, the EF of the optimized substrate was estimated to be 1.64 × 105, which is sufficient to provide significant enhancement of Raman intensity from analytes adsorbed on the AgNPs-PDMS substrate [52].

In Figure 2b, SERS spectra of R6G with different concentrations are presented. The SERS intensity gradually increased with higher concentration of the tested solutions. As low as 0.1 μg L−<sup>1</sup> of R6G can be detected using the AgNPs-PDMS substrate. This result indicates it is feasible to use this substrate for semi-quantitative analysis.

After optimization, the morphology of the optimized substrate was characterized via scanning electron microscopy (SEM). From the image shown in Figure 2c, we can observe that the diameter of the circle was approximately 2 mm. The zoomed in image shows AgNPs were densely distributed on the PDMS film (see Figure 2d). The approximate distance between AgNPs could lead to strong SERS enhancement due to the generation of "hot spots" for molecules trapped between the gaps of AgNPs [53]. Moreover, the relatively even distribution of aggregated AgNPs guarantees a uniform distribution of the SERS signal, which is also vital for reliable SERS measurement. Statistical analysis of the distribution of particle size is presented in the inset of Figure 2d. The result showed AgNPs with sizes between 80 and 90 nm are dominant, which agreed well with the observation of TEM image and UV-Vis spectrum.
