*2.4. Preparation of AgNPs-PDMS SERS Substrate*

Ag colloids with different concentrations were prepared by concentrating or diluting the as-synthesized Ag colloid. In detail, the original Ag colloid (1 mL) was pipetted into centrifuge tubes and precipitated by centrifuging at 8000 rpm for 10 min (MiniSpin, Eppendorf, Hamburg, Germany). The supernatant was carefully drawn out using a pipette, and the remaining AgNPs were resuspended with 100 μL (Ag10×), 200 μL (Ag5×), 500 μL (Ag2×), 1 mL (Ag1×), and 2 mL (Ag0.5×) deionized water, respectively.

For the fabrication of AgNPs-PDMS substrate, Ag colloid (5 μL) was dropped on the amino-functionalized PDMS film. After drying under vacuum, a uniform circle of AgNPs was formed on the PDMS. The morphology of the prepared SERS substrate was characterized using a scanning electron microscope (Quanta 650FEG, FEI Co., Ltd., Hillsboro, OR, USA).

### *2.5. Optimization and Evaluation of AgNPs-PDMS SERS Substrate*

SERS activity of the prepared AgNPs-PDMS substrate was investigated by incubating with different concentrations of R6G solutions (0.1 μg L−1~1 mg L−1). SERS spectra were collected using a Raman microscope system (DXR, Thermo Fisher Scientific Inc., Waltham, MA, USA) with optimized parameters: excited at 780 nm, scanning range between 500 and 1900 cm<sup>−</sup>1, objective lens 10×, laser power 0.5 mW, integration time 2 s.

For evaluating signal uniformity, sixteen random spots on the substrate were chosen, and SERS spectra were collected after incubating with 100 μg L−<sup>1</sup> R6G solution. Time stability was also examined by collecting the SERS spectrum every 5 days. The tested substrate was stored in a sealed tube filled with nitrogen gas after each measurement.
