Fine-tuning protocols for accurate study of nanoparticle adhesion and uptake after foliar deposition

Understanding the interactions between plant leaves and nanoparticles (NPs) is crucial for advancing both environmental and the safety of plant protection products. Recent studies showed how the fate of NPs on leaf surfaces is influenced by NP properties, plant surface, and environmental factors. Yet, inconsistent methods for exposing leaves and measuring NPs uptake hinder reproducibility and comparability across studies. This work investigates how exposure and rinsing protocols affect NPs dose control, distribution, and element remobilization from leaf surfaces. Four exposure approaches commonly used in the literature were tested: drop-deposition, spraying, dipping, and brushing. Sequential rinsing strategies were evaluated using diluted ethanol (EtOH), diluted nitric acid (HNO₃), and ultra-pure-water (UPW). Gold (Au) and copper oxide (CuO) NPs were used, as models presenting contrasting solubilities. These protocols were assessed on tomato leaves (Solanum lycopersicum var. micro-tom). Results indicate that drop-deposition yielded the most consistent NPs dosing, while others introduced variability in dose, coverage and aggregation. Regarding rinsing protocols, CuO NPs, being more soluble, were efficiently rinsed by HNO₃-2 %, whereas EtOH-3 % rinsing failed to remobilize Cu, indicating strong leaf attachment of CuO NPs to the leaf surface. Conversely, Au NPs were better rinsed with ethanol-based solvents, possibly due to solvent low polarity, lowered surface tension and/or wax disruption. These findings underline the critical impacts of exposure and rinsing methods on experimental outcomes, and how it could impair the interpretation of the fate of NPs on leaves. Tailoring rinsing solvent sequences can contribute to probe NP-leaf attachment fractions and their interaction nature. Further, this study provides an exposure and rinsing framework to better tackle specific research questions.