Different Gold Nanoparticles used to optimize DNA-PAINT microscopy Investigation of their properties and new applications in nanomedicine

Gold nanoparticles (AuNPs) are widely used in nanomedicine because of their biocompatibility, stability, and ability to deliver drugs in a targeted manner. Gold, being inert and nontoxic, is ideal for clinical applications. Another aspect is their ease of functionalization, that is, the ability to easily bind biological molecules, such as proteins, nucleic acids, or polymers onto their surface. Because of their optical properties, AuNPs can be used as probes in advanced imaging techniques such as Point accumulation for Imaging in Nanoscale Topography (DNA-PAINT) microscopy. This technique consists of exploiting fluorescent molecules that temporarily bind to a target, producing intermittent signals to accurately determine the location of emitters. Binding of the imager to the target occurs via short complementary DNA sequences. The “docking strand” is attached to the particles while the “imager strand” containing the fluorophore is free in the buffer. During DNA hybridization, the imager is excited by laser light and the emission is captured resulting in a super-resolution microscopy image. In this thesis, our aim was to demonstrate how the use of AuNPs in DNA-PAINT microscopy can improve the sensitivity, resolution and contrast of images, and how these improvements vary depending on the shape and size of the Nps used, in order to understand which configuration offers the best results. To achieve this goal, we developed a DNA-PAINT imaging approach using different AuNPs, analyzing each sample separately to assess its influence on imaging performance. In the future, it will be possible to analyze multiple samples simultaneously, allowing for rapid assessment of different biological conditions, such as drug response or genetic mutations. This approach, called the ‘one-pot bar-coded assay’, will use unique synthetic DNA sequences as molecular ‘barcodes’ to localize individual nanoparticles, reducing time and resources and obtaining comprehensive data in a single experiment. In our work, we focused on the formulation and conjugation of AuNPs with docking oligonucleotides, characterizing them through analytical techniques such as Dynamic Light Scattering (DLS), Zeta Potential (ZP) and UV-VIS spectroscopy. We synthesized the AuNPs and functionalized them with oligonucleotides (docking filaments). We then used two different ‘filament imagers’ (one complementary to the ‘docking filament’ and one non-complementary) to view the different interactions by DNA-PAINT microscopy and processing the data obtained with ad hoc MatLab codes. In parallel, we conducted a study on their ability to adhere to the coverslips to allow AuNPs to remain attached to it for the extended time required during DNA-PAINT microscopy. The results of our research revealed that spherical NPs proved to be the most suitable for analysis by DNA-PAINT microscopy. On the other hand, the complex morphology of star-shaped NPs created challenges in accurate signal localization, making data interpretation less straightforward than with spherical particles. Using the images obtained, we were able to visualize the interaction between the docking strand and the corresponding imager, making it possible to conduct large-scale analyses efficiently ensuring high image resolution. The ability to directly observe these interactions provided us with valuable information about the conjugation and adhesion process, allowing us to further optimize the experimental conditions to obtain more accurate and reproducible results.