Surface-enhanced Raman scattering (SERS) is highly dependent on the adsorption of target molecules onto metallic surfaces, such as colloidal metallic nanoparticles. The selection of suitable substrates is crucial for optimizing SERS performance. Herein, we investigated the dependence of two pesticide SERS signals, thiabendazole (TBZ) and carbendazim (MBC), on both Ag nanoparticles (reduced by hydroxylamine—AgH or citrate—AgCT) and the aggregation conditions induced by adding different salts (NaCl, KCl or KNO₃). In addition to SERS experiments, in order to assess the induced aggregation of the Ag nanoparticles, UV-Vis absorption spectroscopy, dynamic light scattering (DLS) and zeta potential were employed. For AgH, the use of salts did not yield the greatest effect in the presence of TBZ, as only with the pesticide was it possible to achieve the highest aggregation and greater intensity of the SERS signal. In contrast, with the MBC pesticide, the KNO₃ salt promoted the greatest aggregation state and was crucial for obtaining the most amplified SERS signal. The thicker coating layer of AgCT prevented the adsorption of both pesticides on the surface of the nanoparticles, which was achievable using salts containing Cl⁻ ions. Additionally, to obtain the SERS signal of MBC with AgCT, besides the presence of chlorinated salts, other adjustments were necessary, such as changing both the pH of the medium (from pH 5.8 to pH 8, for which MBC is in its neutral form) and the laser lines (from 785 to 514.5 nm). These findings demonstrated that although the pesticide molecules belong to the same chemical functional group, their detection was strongly influenced by the surface of the silver nanoparticles and the salts added. This highlights the specific nuances in detection depending on the method of Ag synthesis and the nature of the aggregating agents used.

The level of aggregation and aggregate morphology of metallic nanoparticles are factors that influence the SERS signal (surface-enhanced Raman scattering), affecting reproducibility and sensitivity. This study presents a systematic evaluation of the colloidal aggregation on the SERS signal by combining transmission electron microscopy and UV-vis extinction spectroscopy. It focuses on the effect of two methods of sample preparation ("external standard method-ESM" and "standard addition method-SAM") on the SERS signal using the fungicide thiabendazole (TBZ) in Ag colloid as a probe molecule. The TBZ critical concentration (concentration for which SERS reaches the maximum intensity) was 6.0 × 10^-6 mol/L for ESM and 1.5 × 10^-6 mol/L for SAM. Besides, TBZ exhibited a sigmoid-type isotherm for ESM, indicating formation of a TBZ first layer on Ag nanoparticles at lower concentrations (Ag aggregates more compact; size 2 μm). For SAM, the TBZ first layer formation was also observed at lower concentrations (Ag aggregates more branched; 2 μm). The Ag aggregation mechanisms align with reaction-limited colloidal aggregation at lower concentrations and diffusion-limited colloidal aggregation at higher concentrations. We believe these results contribute to the SERS research field despite all of the work already done over its 50-year history.

Carbendazim (MBC), a commonly used fungicide from the benzimidazole group, was applied in this study as a probe molecule to understand the influence of sample preparation on the SERS (surface-enhanced Raman scattering) signal. We applied the external standard method (ESM), preparing fresh Ag colloid samples (reduced by hydroxylamine) for each concentration and measuring with and without potassium nitrate (KNO₃) as an aggregation-inducing salt. The impact of sample dilution before or after the addition of the salt to the Ag colloid was also explored. SERS signals were correlated with Ag colloid aggregation observed via transmission electron microscopy (TEM), UV-Vis extinction, dynamic light scattering (DLS), and zeta potential, examining diffusion-limited cluster aggregation (DLCA) and reaction-limited cluster aggregation (RLCA) mechanisms. The optimal results were achieved without KNO₃, with more compact aggregates at lower concentrations and more branched ones at higher concentrations. Dilution of the Ag colloid before salt addition enabled lower detection limits than without any dilution. No SERS signal was observed when the salt was added before dilution. These findings emphasize that a consistent relationship between aggregate morphology and the SERS signal cannot be generalized across analytes. Analyte-specific properties play a crucial role in determining optimal aggregation conditions for SERS analysis.