2.1. Substrate Fabrication

Au monometallic nanoislands substrates were fabricated using a simple method composed of two deposition steps on circular glass substrates 0.1 mm thick and 13 mm in diameter (Figure 1a). The first step involved depositing a thin layer of Au on top of the glass substrate using an electron beam evaporator. Thicknesses of 2, 7, and 15 nm of Au were chosen, with deposition rates varying from 2 Å/s for 2 and 7 nm thick to 0.5 Å/s for 15 nm thick. Our laboratory previously observed that using an electron beam evaporator with small metal thicknesses (less than 25 nm) led to an aggregation of Au particles around specific nucleation points rather than a homogeneous distribution along the whole surface. The observed color of these Au nanofilms varied with the thickness deposited, ranging from light pink at 2 nm width, dark blue at 7 nm, and dark yellow at 15 nm, suggesting different optical properties. Subsequently, a thin film of SiOx was deposited on top of the Au layer using plasma-enhanced chemical vapor deposition with Surface Technology Systems 310PC-DF capacitive plasma equipment operating at 20 W power, 300 °C temperature, and SiH₄ and N₂O as precursor gases. This SiOx deposition aimed to create a spacing layer between the Au nanoparticles and the surrounding media to avoid surface plasmon quenching. We chose deposited thicknesses of 2 and 5 nm of SiOx combined with the three Au thicknesses previously mentioned to observe possible differences during fluorescent microscopy measurements. Sample morphology characterization was performed initially using scanning electron microscopy (SEM). Images were taken using a SEM FEI VERIOS 460 with EDX (energy-dispersive X-ray spectroscopy) at 2 kV and 13 pA current. Figure 1b shows how the Au deposition acquires a nanoislands-like appearance without further thermal dewetting processes needed. Fully isolated Au nanoislands can be identified when depositing 2 nm of Au, whereas the nanoislands fuse to form broader structures when increasing up to 15 nm. EDX analysis confirmed the presence of Au in the samples. The area fraction occupied by Au nanoparticles was computed from SEM images using ImageJ (Fiji) [29]. Au thickness of 2 nm led to 44% area fraction occupied, similar to 7 nm with an area of around 48%, whereas 15 nm of Au was close to 90% area covered. Additionally, atomic force microscopy (Bruker—Dimension Icon AFM) images were taken to evaluate the 3D structure and appearance of these nanostructures (Figure 1c) in tapping mode (1 × 1 µm area). See Figure S1 in Supplementary for AFM profiles.

(a) Schematic illustration of Au nanoislands fabrication: I. Electron beam Au evaporation. II. PECVD SiOx deposition; (b) SEM topography images (secondary electrons); (c) AFM (tapping mode) images. I. 2 nm Au, 2 nm SiOx II. 2 nm Au, 5 nm SiOx III. 7 nm Au, 2 nm SiOx IV. 7 nm Au, 5 nm SiOx V. 15 nm Au, 2 nm SiOx VI. 15 nm Au, 5 nm SiOx. The maximum AFM heights (white color) for each sample are: I. 14.44 nm; II. 6.25 nm; III. 41.10 nm; IV. 44.67 nm; V. 71.96 nm; VI. 43.77 nm. The RMS roughness values are: I. 1.6 nm; II. 0.85 nm; III. 7.32 nm; IV. 6.78 nm; V. 7.25 nm; VI. 46.49 nm. The average heights are: I. 5.43 nm; II. 2.74 nm; III. 14.53 nm; IV. 20.27 nm; V. 24.30 nm; VI. 17.90 nm.