In this study, we investigated the spatial distribution and homogeneity of gold nanoparticles (AuNPs) on an alumina (Al2O3; AAO) substrate for potential application as surface-enhanced Raman scattering (SERS) sensors. The AuNPs were synthesized through thermal treatment at 450 °C at varying times (5, 15, 30, and 60 min), and their distribution was characterized using field-emission scanning electron microscopy (FE-SEM) and scanning transmission electron microscopy (STEM). The FE-SEM and STEM analyses revealed that the size and interparticle distance of the AuNPs were significantly influenced by the duration of thermal treatment, with shorter times promoting smaller and more closely spaced nanoparticles, and longer times resulting in larger and more dispersed particles. Raman spectroscopy, using Rhodamine 6G (R6G) as a probe molecule, was employed to evaluate the SERS enhancement provided by the AuNPs on the AAO substrate. Raman mapping (5 µm × 5 µm) was conducted on five sections of each sample, demonstrating improved homogeneity in the SERS effect across the substrate. The topological features of the AuNPs before and after R6G incubation were analyzed using atomic force microscopy (AFM), confirming the correlation between a decrease in surface roughness and an increase in R6G adsorption. The reproducibility of the SERS effect was quantified using the maximum intensity deviation (D), which was found to be below 20% for all samples, indicating good reproducibility. Among the tested conditions, the sample synthesized for 15 min exhibited the most favorable characteristics, with the smallest average nanoparticle size and interparticle distance, as well as the most consistent SERS enhancement. These findings suggest that AuNPs on AAO substrates, particularly those synthesized under the optimized condition of 15 min at 450 °C, are promising candidates for use in SERS-based sensors for detecting cancer biomarkers. This could be attributed to temperature propagation promoted at the time of synthesis. The results also provide insights into the influence of thermal treatment on the spatial distribution of AuNPs and their subsequent impact on SERS performance.
